Engineered adenosine kinase variants

Abstract

The present disclosure provides engineered adenosine kinase polypeptides, and recombinant polynucleotides encoding the engineered adenosine kinases. The present disclosure further provides method of using the engineered adenosine kinases.

Description

Docket No. CX10-257WO3 ENGINEERED ADENOSINE KINASE VARIANTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Prov. Pat. Appln. Ser. No.63 / 661,424, filed June 18, 2024, and U.S. Prov. Pat. Appln. Ser. No.63 / 589,818, filed October 12, 2023, both of which are incorporated by reference in their entireties, for all purposes. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10- 257WO3_ST26.xml, created on October 9, 2024, with a file size of 4,113,546 bytes, is part of the specification and is hereby incorporated by reference herein in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to engineered adenosine kinase variants, polynucleotides encoding the engineered adenosine kinase variants, and uses of the engineered enzymes. BACKGROUND

[0004] Adenosine kinase catalyzes the phosphorylation of adenosine (Ado) to nucleoside monophosphate (5’-AMP) using ATP as a phosphate donor, and is found ubiquitously in eukaryotes. Adenosine kinase is a key step in the purine nucleotide salvage pathway involved in maintaining the proper levels of cellular nucleotides. Phosphorylation by adenosine kinase keeps the intracellular levels of adenosine low and maintains the homeostasis of concentration gradient across the cell membrane, which makes the cytosol a reservoir for extracellular adenosine kinase under physiological conditions.

[0005] In practical applications, adenosine kinase is used in enzyme cascade reactions beginning from nucleoside substrates for ATP regeneration or production of synthetic nucleotides. Although, enzyme cascades offer the opportunity to deal with unstable or toxic intermediates and used to shift the equilibrium of thermodynamically unfavorable reactions to increase the reaction yield, use of enzyme cascades requires adaptation and optimization to circumvent incompatibility issues between the individual enzymatic reactions. SUMMARY

[0006] The present disclosure provides engineered adenosine kinases for use in transformation of nucleoside substrate to the corresponding nucleoside monophosphate. In some embodiments, the adenosine kinases are engineered to have an improved property compared to a reference adenosine kinase.Docket No. CX10-257WO3

[0007] In one aspect, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808, and 1818-2124 or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808, and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0008] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to a reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0009] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0010] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to theDocket No. CX10-257WO3 reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0011] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4- 1142, 1194-1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0012] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q, 43E / K / R / S / V, 46I, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93A, 94M / N, 100G, 101F / W, 102I, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 190I, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V,Docket No. CX10-257WO3 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M, 294L, 295A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 109 / 111, 104 / 111, 38, 40, 111, 55 / 316, 258, 106 / 111, 103 / 111, 121, 207, 316, 43, 254, 233, 130, 249, 107 / 111, 253, 210, 136 / 249, 208, 34, 211, 308, 181, 248, 56, 89, 67, 111 / 121 / 233 / 316, 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316 / 320, 53 / 86 / 111 / 121 / 169 / 181 / 211 / 233 / 248 / 308 / 316 / 320, or 53 / 77 / 79 / 86 / 111 / 121 / 169 / 170 / 181 / 211 / 233 / 234 / 248 / 308 / 316 / 320, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2 wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0017] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2,Docket No. CX10-257WO3 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0019] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0020] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124.

[0021] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.Docket No. CX10-257WO3

[0022] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0023] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 27, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 276, 287, 288, 291, 292, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0024] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0025] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or to the reference sequence corresponding toDocket No. CX10-257WO3 SEQ ID NO: 90, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0026] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0027] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 111 / 233 / 316, 43 / 111 / 233 / 316, 109 / 181 / 316, 109 / 316, 111 / 233, 109 / 233 / 316, 38 / 208 / 211 / 308, 111 / 181 / 233 / 316, 109 / 111 / 233 / 316, 111 / 181 / 233 / 239 / 316, 111 / 233 / 239 / 316, 109 / 111 / 181 / 233 / 316, 111 / 181 / 316, 43 / 233, 109 / 111 / 316, 181 / 233, 34 / 211 / 248 / 249, 34 / 38 / 248 / 249, 34 / 308, 233 / 316, 43 / 233 / 316, 181 / 316, 34 / 249, 43 / 181 / 233 / 316, 34 / 38 / 103 / 116 / 253, 34, 40 / 43 / 109 / 181 / 233 / 316, 34 / 38 / 208 / 253, 103 / 249 / 253 / 308, 34 / 38 / 116 / 208, 38 / 249, 34 / 38 / 116 / 211 / 248 / 253 / 308, 43 / 109 / 316, 116 / 253, 43 / 111 / 181 / 316, 34 / 38 / 308, 109 / 111 / 181, 111 / 130 / 233, 233, 34 / 38 / 116 / 308, 109 / 233, 40 / 43 / 109 / 111 / 181 / 233 / 316, 109 / 130 / 181 / 233, 34 / 38 / 253, 253, 34 / 38 / 103 / 208 / 249, 103 / 208 / 210 / 253, 130 / 181 / 233 / 316, 109 / 111 / 130 / 181 / 233, 34 / 38 / 210 / 211 / 253 / 303, 103 / 116 / 208, 34 / 116 / 248 / 252 / 253, 34 / 38 / 103 / 210 / 211 / 249, 40 / 233 / 316, 34 / 38, 109 / 130 / 233, 111 / 130 / 181 / 233 / 316, 34 / 38 / 249 / 253, 34 / 38 / 208 / 248, 40 / 109 / 111 / 181 / 233 / 316, 130 / 316, 40 / 316, 38 / 116, 34 / 38 / 116, 38 / 116 / 208 / 210 / 249 / 253, 40 / 130 / 181 / 233 / 316, 103 / 248 / 249, or 103 / 248, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0028] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or to the reference sequence corresponding to SEQ ID NO: 254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0029] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0030] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 77 / 111 / 170 / 211 / 236, 77 / 111 / 211, 77 / 211, 53 / 77 / 170 / 211 / 320, 53 / 77 / 211 / 234 / 320, 77 / 79 / 211 / 316, 77 / 111 / 234 / 236 / 308, 111 / 170 / 211 / 234 / 236 / 248, 111 / 169 / 170 / 234 / 236 / 248, 77 / 211 / 248 / 320, 53 / 169 / 170 / 234 / 320, 111 / 211 / 234 / 236 / 248, 77, 53 / 77 / 111 / 170 / 248, 211 / 234 / 248, 77 / 86 / 170 / 211 / 234 / 308 / 320, 77 / 86, 53 / 111 / 170, 77 / 79 / 111, 53 / 77 / 111 / 169 / 236, 77 / 211 / 234, 211, 53 / 77, 86 / 169 / 234, 53 / 211 / 234 / 248, 111 / 169 / 170 / 236 / 248, 77 / 79 / 211, 77 / 79 / 169 / 170, 77 / 79 / 86 / 169 / 236, 79 / 211 / 248, 79 / 211, 53 / 86 / 211 / 234 / 236, 79 / 170 / 211, 308, 53 / 211, 169 / 234 / 236, 77 / 79 / 169 / 234 / 236, 53 / 77 / 79, 77 / 111, 53 / 234 / 236, 169 / 170, 53 / 77 / 79 / 169, 53 / 86 / 234 / 236, 53 / 169 / 234 / 236, 53 / 234,77 / 79 / 86 / 170 / 211 / 234 / 236 / 308 / 320, 237, 249, 20, 234, 254, 56, 287, 317, 299, 229, 250, 295, 144 / 255, 291, 181, 233, 248, 231, 251, 128, 156, 247, 288, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0031] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or to the reference sequence corresponding to SEQ ID NO: 370, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0032] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0033] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 299, 21, 113, 152, 169 / 255, 251,Docket No. CX10-257WO3 294, 255, 202 / 211, 31, 129, 252, 27 / 64, 128, 248, 234, 288, 20, 111, 112, 181, 119, 64, 155, 256, 254, 292, 265, 177, 232, 120, 183, 234 / 252, 181 / 250 / 252 / 299, 119 / 181 / 234 / 250 / 299, 129 / 234 / 252, 119 / 129 / 234, 119 / 181 / 234 / 250 / 256 / 299, 119 / 181 / 250 / 252, 119 / 181 / 234 / 299, 234 / 250 / 252, 119 / 181 / 252 / 299, 129 / 181 / 234 / 299, 56 / 121 / 229, 181 / 255 / 299, 119 / 299, 119 / 250 / 255 / 299, 80 / 119 / 252, 119 / 129, 119 / 129 / 250, 181 / 234 / 252 / 299, 181 / 250 / 299, 252 / 299, 119 / 181 / 299, 119 / 129 / 299, 119 / 129 / 234 / 252, 119 / 129 / 234 / 250 / 299, 119 / 181 / 234 / 252, 129 / 234 / 255 / 299, 181 / 299, 250 / 252, 250 / 255 / 299, 119 / 234 / 299, 119 / 181 / 250, 56 / 237, 250 / 299, 119 / 234 / 255 / 256 / 299, 56 / 229 / 254, 119 / 129 / 181, 56 / 121 / 295 / 317, 119 / 181 / 250 / 255 / 256, 229, 250, 119 / 234, 20 / 229 / 237 / 317, 181 / 250, 119 / 255, 121 / 156 / 229, 56 / 121 / 237, 237 / 317, 56 / 295, 56 / 121 / 229 / 236 / 254 / 295, 56 / 254, 119 / 250 / 299, 229 / 237 / 295, 20 / 229 / 317, 229 / 295, 156 / 229, 56 / 121 / 229 / 237 / 295, 237 / 254, 20 / 56, 56 / 156, 56 / 156 / 254 / 295, 20 / 56 / 229, 119 / 129 / 181 / 234, 237, 56 / 156 / 229 / 237, 20 / 56 / 102 / 229, 119 / 181 / 234, 20 / 229, 56 / 121 / 156 / 237, 56 / 156 / 229, 20 / 56 / 156 / 229 / 237 / 295, 20 / 56 / 121 / 237 / 317, 156, 156 / 237 / 295, 20 / 56 / 121, 119 / 181, 20 / 56 / 229 / 254, 56, 121 / 254, 20 / 295, 255 / 256, 56 / 121 / 229 / 254, or 119 / 129 / 234 / 255 / 256, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0034] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO: 734, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0035] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0036] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 56 / 229, 21 / 56, 181, 181 / 211 / 251, 181 / 211, 21 / 121 / 299, 229, 56, 56 / 169, 311, 52, 170, 31 / 181 / 211, 167, 303, 222, 43, 83, 113 / 181 / 211, 251, 187, 218, 181 / 251, 195, 121 / 229, 107, 141, 31 / 181, 307, 100, 169, 86, 90, 164, 310, 276, 35, 56 / 134, 211, 50, 41, 121 / 169 / 229, 74, 225, 106, 121, 31 / 113 / 181, 191, 190, 171, 168, 31 / 181 / 211 / 294,Docket No. CX10-257WO3 73, 31 / 251, 31, 196, 89, 197, 242, 51, 103, 101, 147, 40, 146, 243, 56 / 121 / 229, or 31 / 211, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0037] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or to the reference sequence corresponding to SEQ ID NO: 896, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0038] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1196-1344, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1196- 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0039] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 52 / 169, 52 / 141 / 169, 52, 41, 169, 51 / 52 / 211, 52 / 141 / 187, 51 / 52, 141, 41 / 103, 311, 167, 41 / 311, 133, 207, 14, 34, 241, 33, 27, 206, 217, 239, 13, 246, 162, 108, 160, 245, 92, 163, 104, 157, 215, 80, 54, 240, 210, 23, 53, 87, 188, 151, 29, 258, 238, 244, 192, 91, or 30, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0040] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or to the reference sequence corresponding to SEQ ID NO: 1196, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0041] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1346-1364, orDocket No. CX10-257WO3 to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1346- 1364, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0042] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 24, 234 / 236, 251 / 252, 119, 234, or 231 / 234, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0043] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or to the reference sequence corresponding to SEQ ID NO: 1366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0044] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1366-1410, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1366- 1410, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0045] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 182, 121, 249, 129, 152, 237, 155, 121 / 129 / 207 / 237 / 249, 121 / 129 / 207 / 249, 27 / 121 / 207, 27 / 121 / 207 / 249, 121 / 207, 121 / 237, 121 / 249, 121 / 207 / 252, 27 / 121 / 129 / 207, 27 / 121 / 129 / 237, 207, 182 / 207, 121 / 129, or 121 / 237 / 252, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0046] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or to the reference sequence corresponding to SEQ ID NO: 1382, wherein the amino acid sequence comprises one or more substitutions relative toDocket No. CX10-257WO3 the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0047] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1412-1564, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1412- 1564, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0048] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 314, 221, 170, 40, 49, 73, 194, 195, 147, 218, 216, 90, 89, 42, 211, 106, 171, 93, 296, 100, 243, 172, 51, 271, 90 / 171 / 191, 90 / 271, 90 / 191, 90 / 147 / 171 / 271, 191, 51 / 90, 187 / 211, 171 / 191, 170 / 211, 49 / 51, 51 / 171 / 191 / 271, 40 / 211, 106 / 187 / 211, 211 / 221, 40 / 170 / 211, 49 / 51 / 90 / 147 / 171, 51 / 90 / 171, 51 / 171, 49 / 51 / 191, 49 / 51 / 90 / 147, 49 / 51 / 171 / 271, 106 / 211 / 221, 187 / 221, 49 / 171 / 191 / 314, 51 / 191 / 271, 51 / 90 / 171 / 191 / 271, 52 / 221, 90 / 171 / 191 / 271, 191 / 271, 170 / 221, , 50 / 52, 50 / 52 / 106 / 211, 50 / 211 / 308, 50 / 52 / 106, 40 / 50 / 187 / 211 / 308, 50 / 52 / 106 / 170 / 187 / 308, 40 / 52 / 170, 50 / 106, 50 / 221 / 308, 49 / 51 / 90 / 271, 40 / 52 / 170 / 211, 187, 50 / 211 / 221 / 308, or 40 / 50 / 52 / 221, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0049] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or to the reference sequence corresponding to SEQ ID NO: 1464, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0050] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1566-1578, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1566- 1578, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.Docket No. CX10-257WO3

[0051] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 317, 113, 21, 155, 126, 155, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0052] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or to the reference sequence corresponding to SEQ ID NO: 1568, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0053] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1580-1648, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1580- 1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0054] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 246, 74, 185, 244, 164, 276, 43, 163, 217, 31, 43 / 276, 185 / 187 / 276, 43 / 187 / 222 / 276, 185 / 187 / 222 / 276, 187 / 276, 185 / 276, 164 / 222, 164 / 187 / 222 / 276, 43 / 222 / 276, 164 / 187 / 222, 222, 43 / 164 / 185 / 187 / 222, 222 / 276, 239, or 53, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0055] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or to the reference sequence corresponding to SEQ ID NO: 1616, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0056] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1650-1742, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1650- 1742, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0057] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 74 / 239 / 244 / 246, 31 / 74 / 207 / 239 / 244 / 246, 31 / 246, 74 / 244, 31 / 74, 53 / 74 / 239 / 244 / 246, 31 / 74 / 207 / 244 / 246, 31 / 74 / 163 / 239 / 244, 31 / 74 / 239 / 244 / 246, 31 / 244 / 246, 31 / 53 / 164, 31 / 239 / 244, 239 / 246, 163 / 164 / 207 / 239 / 244 / 246, 31 / 74 / 163 / 246, 244, 31 / 239 / 244 / 246, 31 / 53 / 239 / 244 / 246, 31 / 207 / 244 / 246, 31, 31 / 53 / 74 / 239 / 246, 31 / 74 / 207, 31 / 163 / 207 / 244, 31 / 53 / 244, 31 / 163, 31 / 53 / 239 / 246, 74 / 207 / 239 / 244, 31 / 163 / 164 / 207 / 239 / 244 / 246, 74 / 207, 31 / 163 / 207 / 239 / 244, 31 / 74 / 163 / 164 / 207 / 246, 31 / 53 / 207 / 239, 207, 53 / 163 / 207 / 246, 31 / 53 / 74 / 163 / 207 / 246, 31 / 163 / 239 / 246 / 308, 31 / 74 / 207 / 239 / 308, or 163 / 207 / 246, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0058] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or to the reference sequence corresponding to SEQ ID NO: 1668, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0059] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1744-1808, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1744- 1808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0060] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 312, 305, 246, 89, 33, 218, 147, 27 / 218 / 305 / 312, 27 / 35 / 106 / 218 / 312, 27 / 218 / 312, 27 / 312, 27 / 305 / 312, 27 / 106 / 218 / 312, 27 / 218, 27 / 106 / 312, 35 / 305, 27 / 144 / 312, 27 / 218 / 305, 35 / 312, 27 / 106 / 218 / 305 / 312, 27 / 35 / 106 / 312, 106 / 218 / 305, 106 / 218 / 312, 27 / 106 / 305, 27 / 35 / 94 / 106 / 305 / 312, 35 / 218 / 312, 218 / 312, 27 / 106 / 218,Docket No. CX10-257WO3 35 / 106 / 312, or 27 / 106, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0061] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or to the reference sequence corresponding to SEQ ID NO: 1766, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0062] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1818-1870, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1818- 1870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0063] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 34, 112, 126, 128, 155, 179, 183, 229, 236, 248, 253, 254, 292, 293, or 299, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0064] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or to the reference sequence corresponding to SEQ ID NO: 1826, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0065] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1872-1936, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1872- 1936, wherein the amino acid sequence comprises one or more substitutions relative to the referenceDocket No. CX10-257WO3 sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0066] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 14, 38, 46, 108, 146, 211, 243, 275, 303, 311, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0067] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 94, 106, 108, 194, 274, 303, 307, or 315, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0068] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 112 / 183 / 292 / 299, 112 / 292, 126 / 299, 155, 155 / 183 / 299, 155 / 299, 183 / 255 / 299, 183 / 299, 229 / 299, 255 / 299, and 292, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0069] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or to the reference sequence corresponding to SEQ ID NO: 1918, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0070] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1938-2010, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1938- 2010, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0071] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 26, 27, 28, 29, 31, 33, 35, 42, 136, 212, 218, 237, 239, 245, 246, 252, 276, or 316, or combinations thereof, wherein the amino acid positions are relative toDocket No. CX10-257WO3 the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0072] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or to the reference sequence corresponding to SEQ ID NO: 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0073] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2012-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2012- 2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0074] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 26 / 27, 26 / 27 / 28, 26 / 27 / 28 / 31, 26 / 27 / 28 / 245, 26 / 28 / 31, 27, 27 / 28, 27 / 28 / 29 / 126, 27 / 29, 27 / 29 / 126, 27 / 29 / 237, 27 / 29 / 245, 27 / 31 / 126 / 245, 27 / 31 / 155, 27 / 126, 27 / 126 / 155, 27 / 126 / 237, 27 / 126 / 237 / 245, 27 / 237, 27 / 245, 28, 28 / 31, 28 / 31 / 239, 28 / 42 / 126 / 245, 28 / 42 / 245, 28 / 239, 29 / 31, 29 / 31 / 126 / 239, 30 / 31 / 33 / 35 / 126 / 218, 30 / 31 / 33 / 126 / 276 / 316, 30 / 33 / 35, 31 / 33 / 218 / 276, 31 / 126, 31 / 126 / 155, 33, 33 / 126 / 239, 42, 126, 126 / 155, 126 / 155 / 237, 126 / 155 / 239 / 276, 126 / 155 / 239 / 316, 126 / 239, 126 / 239 / 316, 126 / 245, 126 / 316, 155 / 276 / 316, 155 / 316, 218, 218 / 239 / 316, 239, 245, 252 / 276, and 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0075] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.Docket No. CX10-257WO3

[0076] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0077] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0078] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0079] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or wherein the amino acid sequence comprises SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0080] In some embodiments, the engineered adenosine kinase has adenosine kinase activity and at least one improved property as compared to a reference adenosine kinase. In some embodiments, the engineered adenosine kinase has an improved property selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine, iv) increased activity on 2-fluoro modified nucleosides, and v) increased activity on 2-O-methyl modified nucleosides, or any combinations of i), ii), iii), iv), and v), compared to a reference adenosine kinase.Docket No. CX10-257WO3 In some embodiments, the reference adenosine kinase has an amino acid sequence corresponding to residue 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or an amino acid sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942. In some embodiments, the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 2, or an amino acid sequence corresponding to SEQ ID NO: 2.

[0081] In some embodiments, the engineered adenosine kinase is purified or provided as a purified preparation. In some embodiments, the engineered adenosine kinase is immobilized on a substrate.

[0082] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase described herein.

[0083] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, 1941, or 2013, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, 1941, or 2013, wherein the recombinant polynucleotide encodes an adenosine kinase.

[0084] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123 or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123 wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

[0085] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide has preferred codons for expression in a host cell. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized.

[0086] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-963 of SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, 1941, or 2013, or comprises SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, 1941, or 2013.

[0087] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141,Docket No. CX10-257WO3 1193-1807, and 1817-2123, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123.

[0088] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered adenosine kinase described herein. In some embodiments, the expression vector comprises a recombinant polynucleotide operably linked to a control sequence.

[0089] In a further aspect, the present disclosure provides a host cell comprising an expression vector for expression of an engineered adenosine kinase. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0090] In a further aspect, the present disclosure provides a method of producing an engineered adenosine kinase polypeptide, comprising culturing a host cell under suitable culture conditions such that the engineered adenosine kinase polypeptide is produced. In some embodiments, the method further comprises recovering or isolating the engineered adenosine kinase polypeptide from the culture and / or host cells. In some embodiments, the method further comprises purifying the engineered adenosine kinase polypeptide.

[0091] In another aspect, the present disclosure provides a composition comprising an engineered adenosine kinase described herein. In some embodiments, the composition is a solution or a lyophilizate. In some embodiments, the composition further comprises one or more of buffer, a nucleoside substrate, and a phosphate donor. In some embodiments, the nucleoside substrate comprises a modified nucleoside substrate. In some embodiments, the composition further comprises another enzyme, for example, an adenylate kinase, acetate kinase, 3’-O-kinase, and / or pyruvate oxidase.

[0092] In another aspect, the engineered adenosine kinases are used in the conversion of a nucleoside substrate to the corresponding nucleoside monophosphate. In some embodiments, a method of converting a nucleoside to corresponding nucleoside monophosphate (NMP) comprises contacting a nucleoside with an engineered adenosine kinase described herein in the presence of a suitable phosphate donor under suitable reaction conditions to convert the nucleoside to the corresponding nucleoside monophosphate (NMP). In some embodiments, the nucleoside substrate comprises a modified nucleoside substrate. In some embodiments, the modified nucleoside substrate comprises a modified sugar moiety and / or nucleobase.

[0093] In some embodiments, the phosphate donor is a nucleoside triphosphate, also referred to as phosphate donor NTP. In some embodiments, the phosphate donor NTP is adenosine-5’-gamma- thiotriphosphate (ATP ^S) or deoxyadenosine-5’-gamma-thiotriphosphate (dATP ^S), thereby resulting in product nucleoside 5’-O-thiophosphate (NMP-S). In some embodiments, the phosphate donor NTP has the same nucleoside structure of the nucleoside substrate.Docket No. CX10-257WO3 DETAILED DESCRIPTION

[0094] The present disclosure provides engineered adenosine kinase enzyme, recombinant polynucleotide encoding the engineered adenosine kinases, and methods of using the engineered adenosine kinases. In some embodiments, are engineered to have an improved property compared to a reference adenosine kinase. Abbreviations and Definitions

[0095] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0096] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide.

[0097] Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0098] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0099] “About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

[0100] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0101] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0102] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0103] “Adenosine kinase,” “AdoK,” or “Adk,” refers to an enzyme that catalyzes the phosphorylation of adenosine (A or ADO) to adenosine-5’-monophosphate (AMP). In someDocket No. CX10-257WO3 embodiments, adenosine kinase is classified in EC 2.7.1.20. Although the primary substrate is adenosine, adenosine kinases as used herein include enzymes that act on other nucleosides.

[0104] “Creatine kinase” refers to an enzyme that catalyzes the reversible interconversion of creatine:ATP to creatine phosphate:ADP.

[0105] “Polyphosphate kinase” refers to an enzyme that catalyzes the transfer of phosphate group(s) from high-energy, phosphate-donating molecules, such as polyphosphate (PolyPn), to specific substrates / molecules. Two families of polyphosphate kinases, PPKK1 and PPK2, are known. PPK1s preferentially synthesize polyphosphate from NTP and the corresponding reverse reaction, and PPK2s preferentially consume polyphosphate to phosphorylate nucleoside mono- or diphosphates, and the corresponding reverse reactions. In some embodiments, polyphosphate kinase includes enzymes classified in EC 2.7.4.1.

[0106] “Acetate kinase (“AcK”) refers to enzymes that are capable of catalyzing the phosphorylation of nucleoside diphosphates or analogues thereof, to nucleoside triphosphates or the corresponding analogues, using acetyl phosphate or another phosphoryl group donor. Acetate kinases as used herein includes naturally occurring, wild-type enzymes or engineered enzymes. In some embodiments, acetate kinases are naturally occurring, wild-type basic metabolic enzymes found primarily in prokaryotes that catalyze the following reaction: acetate + ATP ↔acetyl phosphate + ADP. Acetyl phosphate is an intermediate in the formation of acetyl-CoA. In some embodiments, acetate kinases are derived from the naturally occurring, wild-type enzymes.

[0107] “Adenylate kinase,” “AdyK,” or “Ayk” refers to an enzyme that catalyzes the interconversion of ATP, ADP, and AMP through transfer of phosphoryl groups. In some embodiments, adenylate kinase also includes enzymes capable of catalyzing the interconversion of NTP, NDP and NMP. In some embodiments, adenylate kinase are enzymes classified in EC 2.7.3.4.

[0108] “Pyruvate oxidase” or “Pox” refers to an enzyme that catalyzes the catalyzes the reaction between pyruvate, inorganic phosphate, and oxygen to generate acetyl phosphate and H2O2. In some embodiments, pyruvate oxidase include enzymes classified in EC 1.2.3.3.

[0109] “Catalase” refers to an enzyme that converts hydrogen peroxide (H2O2) to H2O and O2. Catalase can be used to remove residual hydrogen peroxide in applications where hydrogen peroxide is present or is a product in a process. In some embodiments, catalases includes enzymes classified in EC 1.11.1.6.

[0110] “Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.Docket No. CX10-257WO3

[0111] “Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0112] “Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about ^-carbon (C ^). For example, whereas “Ala” designates alanine without specifying the configuration about the ^-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the ^-carbon and lower case letters designate amino acids in the D-configuration about the ^-carbon. For example, “A” designates L-alanine and “a” designates D- alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0113] “Polynucleotide” and “nucleic acid’ refer to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’ deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo- and 2’ deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0114] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’-Docket No. CX10-257WO3 deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.

[0115] “Nucleobase” or “base” refers to those naturally occurring and synthetic heterocyclic moieties commonly known to those who utilize nucleic acid or polynucleotide technology to thereby generate polymers that can hybridize to polynucleotides in a sequence-specific manner. Non-limiting examples of nucleobases include, among others, adenine, cytosine, guanine, thymine, uracil, 5- propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, pseudoisouridine, 2- thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).

[0116] “Nucleoside” refers to glycosylamines comprising a nucleobase, and a 5-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term “nucleotide” refers to the glycosylamines comprising a nucleobase, a 5-carbon sugar, and one or more phosphate groups, as further described herein. In some embodiments, nucleosides can be phosphorylated by kinases to produce nucleotides.

[0117] “Nucleoside monophosphate” or “NMP” refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a phosphate moiety at the 5’-position. In some embodiments herein, “nucleoside monophosphate” is abbreviated as “NMP”. Non-limiting examples of nucleoside monophosphates include cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), and inosine monophosphate (IMP). In some embodiments, “nucleoside monophosphate” may refer to a non-natural nucleoside monophosphate. The terms “nucleoside” and “nucleotide” may be used interchangeably in some contexts.

[0118] “Nucleoside diphosphate” or “NDP” refers to glycosylamines comprising a nucleobase, a 5- carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) moiety at the 5’- position. In some embodiments herein, “nucleoside diphosphate” is abbreviated as “NDP”. Non- limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP). In some embodiments, “nucleoside diphosphate” may refer to a non-natural nucleoside diphosphate. The terms “nucleoside” and “nucleotide” may be used interchangeably in some contexts.

[0119] “Nucleoside triphosphate” or “NTP” refers to glycosylamines comprising a nucleobase, a 5- carbon sugar (e.g., ribose or deoxyribose), and a triphosphate moiety at the 5’-position. In some embodiments herein, “nucleoside triphosphate” is abbreviated as “NTP”. Non-limiting examples of nucleoside triphosphates include cytidine triphosphate (CTP), uridine triphosphate (UTP), adenosineDocket No. CX10-257WO3 triphosphate (ATP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), and inosine triphosphate (ITP). In some embodiments, “nucleoside triphosphate” may refer to a non-natural nucleoside triphosphate. The terms “nucleoside” and “nucleotide” may be used interchangeably in some contexts.

[0120] “Modified” or “non-natural” in context of a nucleoside or nucleotide refers to a nucleoside or nucleotide that has been altered to a non-naturally occurring nucleoside or nucleotide. In some embodiments, modifications to nucleoside or nucleotides include modifications to the base, sugar moiety, and / or phosphate. In some embodiments, the common modifications of the 2’-position of the sugar moiety with fluoro (F) or -O-CH3is denoted by “fN” and “mN”, respectively, where N denotes the nucleobase on the nucleoside or nucleotide. In some embodiments, the presence of a 5’- thiophosphate is denoted by “*N”.

[0121] “Locked nucleoside” or “locked nucleotide” refers to nucleoside or nucleotide, respectively, in which the ribose moiety is modified with a bridge connecting the 2’ oxygen and 4’ carbon (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735–8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138–1142). In some embodiments, the bridge is a methylene or ethylene bridge. In some embodiments, the ribose moiety of the locked nucleoside or locked nucleotide is in the C3’-endo (beta-D) or C2’-endo (alpha-L) conformation.

[0122] “Biocatalysis,” “biocatalytic,” “biotransformation,” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0123] “Wild-type” and “naturally occurring” refer to the form found in nature. For example, a wild- type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0124] “Recombinant,” “engineered,” “variant,” “non-natural,” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, which has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0125] “Percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated byDocket No. CX10-257WO3 determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted by any suitable method, including, but not limited to the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA., 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucl. Acids Res., 1977, 25(17):3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10,Docket No. CX10-257WO3 and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0126] “Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0127] “Reference sequence” refers to a defined sequence used as a basis for a sequence and / or activity comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0128] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, theDocket No. CX10-257WO3 comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0129] “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered adenosine kinase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0130] “Amino acid difference”, “residue difference” and “substitution” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X20 as compared to SEQ ID NO: 2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 20 of SEQ ID NO: 2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a leucine at position 20, then a “residue difference at position X20 as compared to SEQ ID NO: 2” refers to an amino acid substitution of any residue other than leucine at the position of the polypeptide corresponding to position 20 of SEQ ID NO: 2. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in the Tables presented in the Examples), the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a “-“. In some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino residueDocket No. CX10-257WO3 in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0131] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “ / ” (e.g., X20F / X20V, X20F / V, or 20F / V).

[0132] “Amino acid substitution set” or “substitution set” refers to a group acid substitutions in a polypeptide sequence, as compared to a reference sequence. A substitution set can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant adenosine kinases listed in the Tables provided in the Examples. In these substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., F109R;P111A) or slash (“ / ”; e.g., F109R / P111A or 109R / 111A).

[0133] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0134] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.Docket No. CX10-257WO3

[0135] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered adenosine kinase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated by “-“ in amino acid sequences.

[0136] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids to the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0137] “Functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered adenosine kinase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0138] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). The recombinant adenosine kinase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant adenosine kinase polypeptides can be an isolated polypeptide.

[0139] “Substantially pure polypeptide” or “purified protein” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. However, in some embodiments, the composition comprising adenosine kinase comprises adenosine kinase that is less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure adenosine kinase composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified toDocket No. CX10-257WO3 essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant adenosine kinase polypeptides are substantially pure polypeptide compositions.

[0140] “Improved enzyme property” refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered adenosine kinase polypeptides that exhibit an improvement in any enzyme property as compared to a reference adenosine kinase polypeptide and / or a wild-type adenosine kinase polypeptide, and / or another engineered adenosine kinase polypeptide. Thus, the level of “improvement” can be determined and compared between various adenosine kinase polypeptides, including wild-type, as well as engineered adenosine kinases. Improved properties include, but are not limited, to such properties as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In additional embodiments, the term is used in reference to the at least one improved property of adenosine kinase enzymes. In some embodiments, the present invention provides adenosine kinase polypeptides that exhibit an improvement in any enzyme property as compared to a reference adenosine kinase polypeptide and / or a wild-type adenosine kinase polypeptide, and / or another engineered adenosine kinase polypeptide. Thus, the level of “improvement” can be determined and compared between various adenosine kinase polypeptides, including wild-type, as well as engineered adenosine kinases.

[0141] “Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzyme) as compared to the reference enzyme. In some embodiments, the terms refer to an improved property of engineered adenosine kinase polypeptides provided herein, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of adenosine kinase) as compared to the reference adenosine kinase enzyme. In some embodiments, the terms are used in reference to improved adenosine kinase enzymes provided herein. Exemplary methods to determine enzyme activity of the engineered adenosine kinases of the present invention are provided in the Examples. Any property relating to enzyme activity may beDocket No. CX10-257WO3 affected, including the classical enzyme properties of Km, Vmaxor kcat, changes of which can lead to increased enzymatic activity. For example, improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold, 5-fold, 10- fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring adenosine kinase or another engineered adenosine kinase from which the adenosine kinase polypeptides were derived.

[0142] “Conversion” refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an adenosine kinase polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time.

[0143] “Stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The Tmvalues for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et al., Proc. Natl. Acad. Sci. USA., 1962, 48:1390; Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al., Proc. Natl. Acad. Sci. USA., 1986, 83:9373-9377; Kierzek et al., Biochem., 1986, 25:7840-7846; Rychlik et al., Nucl. Acids Res., 1990, 18:6409-6412 (erratum, Nucl. Acids Res., 1991, 19:698); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (2001); Suggs et al., in “Developmental Biology Using Purified Genes,” Brown et al. [eds.], pp.683-693, Academic Press, Cambridge, MA (1981); and Wetmur, Crit. Rev. Biochem. Mol. Biol., 1991, 26:227- 259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered adenosine kinase enzyme of the present invention.

[0144] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5×Docket No. CX10-257WO3 Denhart's solution, 5×SSPE, 0.2% SDS at 42 °C, followed by washing in 0.2×SSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of onlynucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42 °C, followed by washing in 0.1×SSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w / v) SDS at 65 °C and washing in 0.1x SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0145] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0146] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the adenosine kinase enzymes may be codon optimized for optimal production in the host organism selected for expression.

[0147] “Preferred,” “optimal,” and “high codon usage bias” codons when used alone or in combination refer(s) interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics ComputerDocket No. CX10-257WO3 Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., Nucl. Acids Res., 1994, 222437-46; and Wright, Gene, 1990, 87:23- 29). Codon usage tables are available for many different organisms (See e.g., Wada et al., Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Duret, et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p.2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences- CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0148] “Control sequence” refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0149] “Operably linked” or “operatively linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where appropriate the encoded polypeptide of interest.

[0150] “Promoter sequence” refers to a nucleic acid sequence that defines and / or initiates expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0151] “Heterologous” refers to the relationship between two or more nucleic acid or protein sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0152] “Vector” refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide of interest and where appropriate the encoded polypeptide.Docket No. CX10-257WO3

[0153] "Expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0154] “Produces” refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0155] “Culturing” refers to the growing of a population of microbial cells under any suitable conditions (e.g., using a liquid, gel or solid medium).

[0156] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., the polynucleotides encoding the adenosine kinase variants). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0157] “pH stable” refers to an adenosine kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hr) compared to the untreated enzyme.

[0158] “Thermostable” refers to an adenosine kinase polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the same elevated temperature.

[0159] “Solvent stable” refers to an adenosine kinase polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl adenosine, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the same concentration of the same solvent.

[0160] “Thermo- and solvent stable” refers to an adenosine kinase polypeptide that is both thermostable and solvent stable.

[0161] “Stereoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively reported in the art (typically as a percentage) as the enantiomeric excess (“e.e.”)Docket No. CX10-257WO3 calculated therefrom according to the formula [major enantiomer − minor enantiomer] / [major enantiomer + minor enantiomer]. Where the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity, the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly alternatively reported as the diastereomeric excess (“d.e.”). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.

[0162] “Regioselectivity” and “regioselective reaction” refer to a reaction in which one direction of bond making or breaking occurs preferentially over all other possible directions. Reactions can completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%), or partially regioselective (x%, wherein the percentage is set dependent upon the reaction of interest), if the product of reaction at one site predominates over the product of reaction at other sites.

[0163] “Chemoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one product over another.

[0164] “Suitable reaction conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which an adenosine kinase polypeptide of the present invention is capable of converting a substrate to the desired product compound. Some exemplary “suitable reaction conditions” are provided herein.

[0165] “Loading,” such as in “compound loading” or “enzyme loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0166] “Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the engineered enzymes provided herein (e.g., engineered adenosine kinase polypeptides).

[0167] “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of an enzymatic polypeptide on a substrate.

[0168] “Increasing” yield of a product (e.g., a nucleoside triphosphate or analogue) from a reaction occurs when a particular component present during the reaction (e.g., an adenosine kinase enzyme) causes more product to be produced, compared with a reaction conducted under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

[0169] “Substantially free” of a particular enzyme if the amount of that enzyme compared with other enzymes that participate in catalyzing the reaction is less than about 2%, about 1%, or about 0.1% (wt / wt).

[0170] “Fractionating” a liquid (e.g., a culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or a combination of suchDocket No. CX10-257WO3 processes to provide a solution in which a desired protein comprises a greater percentage of total protein in the solution than in the initial liquid product.

[0171] “Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (C1-C4)alkyl refers to an alkyl of 1 to 4 carbon atoms).

[0172] “Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0173] “Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0174] “Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR’-, -PH-, - S(O)-, -S(O)2-, -S(O) NR’-, -S(O)2NR’-, and the like, including combinations thereof, where each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0175] “Alkoxy” refers to the group –OR’ wherein R’ is an alkyl group is as defined above including optionally substituted alkyl groups as also defined herein.

[0176] “Amino” refers to the group -NHI. Substituted amino refers to the group –NHR’, NR’R’, and NR’R’R’, where each R’ is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

[0177] “Oxo” refers to =O.

[0178] “Oxy” refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0179] “Carboxy” refers to -COOH.

[0180] “Carbonyl” refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0181] “Alkyloxycarbonyl” refers to -C(O)OR’, where R’ is an alkyl group as defined herein, which can be optionally substituted.Docket No. CX10-257WO3

[0182] As used herein, “aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to – C(O)NR’R’, where the amino group NR’R’ is as defined herein.

[0183] “Halogen” and “halo” refer to fluoro (F), chloro (C), bromo (B), and iodo (I).

[0184] “Hydroxy” refers to -OH.

[0185] “Cyano” refers to -CN.

[0186] “Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 12 carbon atoms inclusively having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

[0187] “Heterocycle,” “heterocyclic,” and interchangeably “heterocycloalkyl,” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, from 2 to 10 carbon ring atoms inclusively and from 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.

[0188] “Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusively selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0189] “Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl (i.e., heteroaryl-alkyl- groups), preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0190] “Heteroarylalkenyl” refers to an alkenyl substituted with a heteroaryl (i.e., heteroaryl-alkenyl- groups), preferably having from 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety.

[0191] “Heteroarylalkynyl” refers to an alkynyl substituted with a heteroaryl (i.e., heteroaryl- alkynyl- groups), preferably having from 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety.Docket No. CX10-257WO3

[0192] “Membered ring” is meant to embrace any cyclic structure. The number preceding the term “membered” denotes the number of skeletal atoms that constitute the ring. Thus, for example, cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0193] “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein.

[0194] “Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0195] “Thiophosphate” refers to an instance where a non-bridging oxygen in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP is replaced with a sulfur. In some embodiments, nucleoside 5’-thiomonophosphate is referred to as NMP-S. In some embodiments, nucleoside-5’-1- thio(diphosphate) and nucleoside-5’-1-thio(triphosphate) are referred to as NDP ^S and NTP ^S, respectively. In some embodiments, nucleoside-5’-2-thio(diphosphate) and nucleoside-5’-2- thio(triphosphate) are referred to as NDP ^S and NTP ^S, respectively.

[0196] “Dithiophosphate” refers to an instance where two non-bridging oxygens in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP are replaced with two sulfurs.

[0197] Unless otherwise specified, positions occupied by hydrogen in the foregoing groups can be further substituted with substituents exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that where open valences exist on these substituents they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further understood that the above substitutionsDocket No. CX10-257WO3 can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable.

[0198] “Optional” and “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. One of ordinary skill in the art would understand that with respect to any molecule described as containing one or more optional substituents, only sterically practical and / or synthetically feasible compounds are meant to be included.

[0199] “Optionally substituted” refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl, the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted. Engineered Adenosine Kinase Polypeptides

[0200] In one aspect, the present disclosure provides adenosine kinase enzymes engineered to have improved properties compared to the parent adenosine kinase enzyme. In some embodiments, the engineered adenosine kinase enzymes exhibit increased activity on nucleoside substrates, increased thermostability, and / or increased activity on modified nucleoside substrates. The engineered adenosine kinases are useful in the conversion of nucleosides to their corresponding nucleoside monophosphate products, particularly in enzyme cascade systems.

[0201] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2- 1142, 1194-1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0202] In some embodiments, an engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to a reference sequence corresponding toDocket No. CX10-257WO3 SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942,wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0203] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0204] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0205] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4- 1142, 1194-1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0206] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168,Docket No. CX10-257WO3 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0207] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q, 43E / K / R / S / V, 46I, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93A, 94M / N, 100G, 101F / W, 102I, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 190I, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M, 294L, 295A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0208] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution S13A / E / P, A14S / T / V, L20F / V, A21G, D23N, T24I / N / V, M26F, V27A / C / G / K / P / R / S / W, F28L / M, P29G / R, D30G / Y, Q31F / H / L / R / W / Y, K33A / P / Q / S, N34C / G / K / R, H35D / E / S / V / Y, P38C / R / V, K40H / M / R / W, V41L, H42D / G / N / Q, I43E / K / R / S / V, V46I, L49M, V50C, P51F / G / S, R52C / D / F / G / S / T, M53L / R, R54V, R55H, E56F / G / S / Y, I64M / T, N67G, G73A / C / E / M, A74F / I / K / R / T / V, P77V, G79A, T80M / S / V, Q83F / L / S / T / V, G86L / Q, P87K, R89G / K / L / T,Docket No. CX10-257WO3 E90L / R / S / T / V, H91Y, F92M, E93A, T94M / N, S100G, R101F / W, V102I, K103L / Q / R, I104A / C / D / G / L / N / P / R / S / T / W / Y, D106E / G / L / M / P / R / T, D107A / G / H / S / T, L108A / C / E, F109E / G / L / P / R / S / T, P111A / G / L, Q112I / L, A113G / S, T116M, H119C / G / M / Q / R / S / T / V, D120C, N121F / H / L / M / Q, A126L / S, H128F / V / W, P129A / G / S, G130A, M133A / L, R134H, Y136F / H, R141S, P144Q / S, V146C / I, T147A / N / S, V151L, G152A / C, G155A / I / N / T / V / W, R156Y, E157P, I160L, N162F, A163G, E164A / G / K / Q / S / W, A167F / T / V / W, A168R / V, G169A / C / D / I / L / M / S, G170D / H / K / M / R / S / V, I171C / V, P172S, P177V, Q179N, A180Q, M181A / C / L / V, P182G, L183M / S / V, N185S / T, P187H / Q / S / V, E188T, R190I, E191A / R / S / W, F192L, E194A / R, Q195G / M / T / V, A196S, D197R, N202F, S206A / H, N207A / L / M / R / T / V, L208V, Q210E / L, E211A / C / F / H / I / K / L / M / R / T / W, R212A, W215L, N216V, E217A / V, Q218D / L / N / R / V, V221A, S222A / G / M / Q, Q225E / G, T229C / H / V, Q231L / M / V, G232S, P233A / S / V, K234A / M / N / R / V / Y, A236C / S / V, L237I / K / L / M / Q / V, V238L, H239A / F / G / H / I / M / Q / R / T / W, T240L / V, P241R, E242D / T, K243F / G / R / Y, T244R / W, Y245F / Q / W, D246A / R / S / T / V, I247L, P248E / G / K / L / R / V, P249A / G / I / L / M / R / S, A250H / I / M / T, H251K / L / T / V, E252A / C / G / K / T / V, R253D / E / G / H / K / L / P / S / T / V, R254I / L / M / P / Q / T, V255A / C / E / G / I / K / S / W, V256C / H / M, P258R / S / V, F265Y, Y271W, N276D / E / G / S, Q274R, H275S, E276G, N287A / S / V / Y, L288V, A291G, L292F / I / K / V, K293M, V294L, E295A, H296N, T299G / S, R303D / F / H / M / S / V / W, D305S / T, A307F / G, E308D / F / L / V, N310A / C, D311E / F / G / L / M / Q / R / S / T, Q312R, K314Q / S / V, Q315S, Q316A / D / E / I / K / S / Y, F317M / W / Y, or A320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0209] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0210] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 24I / N / V, 27A / C / G / K / P / R / S / W, 31F / H / L / R / W / Y, 40H / M / R / W, 43E / K / R / S / V, 51F / G / S, 52C / D / F / G / S / T, 74F / I / K / R / T / V, 77V, 79A, 109E / G / L / P / R / S / T, 113G / S, 119C / G / M / Q / R / S / T / V, 121F / H / L / M / Q, 126L / S, 129A / G / S, 169A / C / D / I / L / M / S, 181A / C / L / V, 185S / T, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / T / W, 233A / S / V, 234A / M / N / R / V / Y, 237K / I / L / M / Q / V, 239A / F / G / H / I / M / Q / R / T / W, 244R / W, 246A / R / S / T / V, 249A / G / I / L / M / R / S, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 276D / E / G / S, 299G / S, 305S / T, 312R, or 316A / D / E / I / K / S / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.Docket No. CX10-257WO3

[0211] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 24N, 27W, 31Y, 40R, 43R, 51F, 52D, 74R, 77V, 79A, 109S, 113G, 119M, 121H / Q, 126L, 129G / S, 169M, 181L / V, 185S, 207R, 211A / T, 233S, 234A, 237V, 239M, 244R, 246V, 249A, 251K, 252A / T, 253S, 276E, 299G, 305T, 312R, or 316D / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0212] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution T24N, V27W, Q31Y, K40R, I43R, P51F, R52D, A74R, P77V, G79A, F109S, A113G, H119M, N121H / Q, A126L, P129G / S, G169M, M181L / V, N185S, N207R, E211A / T, P233S, K234A, L237V, H239M, T244R, D246V, P249A, H251K, E252T, R253S, N276E, T299G, D305T, Q312R, or Q316D / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0213] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 40, 43, 109, 181, 233, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 40R, 43R, 109S, 181L, 233S, or 316D, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution K40R, I43R, F109S, M181L, P233S, or Q316D, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0214] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 40 / 43 / 109 / 181 / 233 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 40R / 43R / 109S / 181L / 233S / 316D, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least aDocket No. CX10-257WO3 substitution set K40R / I43R / F109S / M181L / P233S / Q316D, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0215] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 77, 79, 211, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution 77V, 79A, 211T, or 316Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution P77V, G79A, E211T, or D316Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0216] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 77 / 79 / 211 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 77V / 79A / 211T / 316Y, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set P77V / G79A / E211T / D316Y, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0217] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 119, 129, 234, or 252, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution 119M, 129G, 234A, or 252T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution H119M, P129G, K234A, or E252T, or combinations thereof, wherein the amino acid positions are relative toDocket No. CX10-257WO3 the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0218] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 119 / 129 / 234 / 252, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 119M / 129G / 234A / 252T, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set H119M / P129G / K234A / E252T, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0219] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 181, 211, or 251, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution 181V, 211A, or 251K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution L181V, T211A, or H251K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0220] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 181 / 211 / 251, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set 181V / 211A / 251K, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set L181V / T211A / H251K, wherein the amino acidDocket No. CX10-257WO3 positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0221] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 52 or 169, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution 52D or 169M, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution R52D or G169M, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0222] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 52 / 169, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 52D / 169M, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set R52D / G169M, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0223] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 121, 129, 207, 237, or 249, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 121Q, 129S, 207R, 237V, or 249A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution H121Q, G129S, N207R, L237V, or P249A, or combinations thereof, wherein theDocket No. CX10-257WO3 amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0224] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 121 / 129 / 207 / 237 / 249, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set 121Q / 129S / 207R / 237V / 249A, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set H121Q / G129S / N207R / L237V / P249A, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0225] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 185 or 276, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 185S or 276E, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution N185S or N276E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0226] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 185 / 276, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 185S / 276E, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set N185S / N276E, wherein the amino acid positionsDocket No. CX10-257WO3 are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0227] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 31, 74, 239, 244, or 246, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 31Y, 74R, 239M, 244R, or 246V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution Q31Y, A74R, H239M, T244R, or D246V, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0228] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 31 / 74 / 239 / 244 / 246, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set 31Y / 74R / 239M / 244R / 246V, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set Q31Y / A74R / H239M / T244R / D246V, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0229] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 27, 305, or 312, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 27W, 305T, or 312R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution V27W, D305T, or Q312R, wherein the amino acid positions are relative to the referenceDocket No. CX10-257WO3 sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0230] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 27 / 305 / 312, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set 27W / 305T / 312R, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set V27W / D305T / Q312R, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0231] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 126, or 299, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 126L, or 299G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution A126L, or T299G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0232] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set at amino acid positions 126 / 299, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set 126L / 299G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set A126L / T299G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.Docket No. CX10-257WO3

[0233] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 109 / 111, 104 / 111, 38, 40, 111, 55 / 316, 258, 106 / 111, 103 / 111, 121, 207, 316, 43, 254, 233, 130, 249, 107 / 111, 253, 210, 136 / 249, 208, 34, 211, 308, 181, 248, 56, 89, 67, 111 / 121 / 233 / 316, 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316 / 320, 53 / 86 / 111 / 121 / 169 / 181 / 211 / 233 / 248 / 308 / 316 / 320, or 53 / 77 / 79 / 86 / 111 / 121 / 169 / 170 / 181 / 211 / 233 / 234 / 248 / 308 / 316 / 320, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0234] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set or amino acid residue(s) 109R / 111A, 104G / 111A, 38C, 104A / 111A, 104Y / 111A, 40R, 111G, 109S / 111A, 55H / 316S, 109G / 111A, 258V, 106E / 111A, 104N / 111A, 109T / 111A, 109P / 111A, 103Q / 111A, 104D / 111A, 104R / 111A, 121F, 104S / 111A, 104T / 111A, 207R, 111A, 109E / 111A, 103R / 111A, 109L / 111A, 104L / 111A, 106P / 111A, 316E, 43R, 103L / 111A, 316S, 254P, 233S, 130A, 316D, 249G, 104P / 111A, 104W / 111A, 249S, 107T / 111A, 106G / 111A, 253P, 121H, 210E, 136H / 249G, 208V, 34K, 107S / 111A, 107G / 111A, 211T, 308F, 181L, 233V, 107A / 111A, 34R, 38V, 248R, 56S, 316K, 248V, 248K, 89T, 308V, 106R / 111A, 67G, 111A / 121H / 233S / 316S, 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S, 53L / 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S, 53L / 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S / 320S, 53L / 86Q / 111A / 121H / 169A / 181L / 211T / 233S / 248R / 308L / 316S / 320S, or 53L / 77V / 79A / 86Q / 111A / 121H / 169A / 170D / 181L / 211T / 233S / 234R / 248R / 308L / 316S / 320S, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0235] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set F109R / P111A, I104G / P111A, P38C, I104A / P111A, I104Y / P111A, K40R, P111G, F109S / P111A, R55H / Q316S, F109G / P111A, P258V, D106E / P111A, I104N / P111A, F109T / P111A, F109P / P111A, K103Q / P111A, I104D / P111A, I104R / P111A, N121F, I104S / P111A, I104T / P111A, N207R, P111A, F109E / P111A, K103R / P111A, F109L / P111A, I104L / P111A, D106P / P111A, Q316E, I43R, K103L / P111A, Q316S, R254P, P233S, G130A, Q316D, P249G, I104P / P111A, I104W / P111A, P249S, D107T / P111A, D106G / P111A, R253P, N121H, Q210E, Y136H / P249G, L208V, N34K, D107S / P111A, D107G / P111A, E211T, E308F, M181L, P233V, D107A / P111A, N34R, P38V, P248R, E56S, Q316K, P248V, P248K, R89T, E308V, D106R / P111A, N67G, P111A / N121H / P233S / Q316S, P111A / N121H / M181L / E211T / P233S / P248R / E308L / Q316S, M53L / P111A / N121H / M181L / E211T / P233S / P248R / E308L / Q316S, M53L / P111A / N121H / M181L / E211T / P233S / P248R / E308L / Q316S / A320S,Docket No. CX10-257WO3 M53L / G86Q / P111A / N121H / G169A / M181L / E211T / P233S / P248R / E308L / Q316S / A320S, or M53L / P77V / G79A / G86Q / P111A / N121H / G169A / G170D / M181L / E211T / P233S / K234R / P248R / E308 L / Q316S / A320S, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0236] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 34, 112, 126, 128, 155, 179, 183, 229, 236, 248, 253, 254, 292, 293, or 299, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0237] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 34C, 112I, 126L, 128V, 155T, 179N, 183S, 183V, 229C, 229V, 236C, 248E, 253D, 253E, 253G, 253H, 253K, 253L, 253S, 253T, 253V, 254M, 254T, 292I, 292V, 293M, or 299G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0238] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 14, 38, 46, 108, 146, 211, 243, 275, 303, 311, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0239] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 14S, 14T, 38R, 46I, 108A, 146C, 211R, 243F, 275S, 303D, 303S, 311E, 320G, or 320R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0240] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 94, 106, 108, 194, 274, 303, 307, or 315, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0241] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 94M, 106L, 108E, 194R, 274R, 303V, 307G, or 315S, or combinations thereof, wherein the amino acid positions are relative to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0242] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 112 / 183 / 292 / 299, 112 / 292, 126 / 299, 155, 155 / 183 / 299, 155 / 299, 183 / 255 / 299, 183 / 299, 229 / 299, 255 / 299, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0243] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 112I / 183V / 292I / 299G, 112I / 292I, 126L / 299G, 155T, 155T / 183V / 299G, 155T / 299G, 183V / 255I / 299G, 183V / 299G, 229H / 299G, 255I / 299G, or 292I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0244] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 26, 27, 28, 29, 31, 33, 35, 42, 136, 212, 218, 237, 239, 245, 246, 252, 276, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0245] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 26F, 27C, 27G, 27R, 27S, 28L, 28M, 29G, 31H, 33A, 33P, 35D, 35E, 42G, 42N, 42Q, 136F, 212A, 218R, 237K, 237L, 237Q, 239A, 239G, 239H, 239I, 239T, 239W, 245F, 245Q, 246A, 246T, 252A, 252G, 276G, 316A, or 316I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0246] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 26 / 27, 26 / 27 / 28, 26 / 27 / 28 / 31, 26 / 27 / 28 / 245, 26 / 28 / 31, 27, 27 / 28, 27 / 28 / 29 / 126, 27 / 29, 27 / 29 / 126, 27 / 29 / 237, 27 / 29 / 245, 27 / 31 / 126 / 245, 27 / 31 / 155, 27 / 126, 27 / 126 / 155, 27 / 126 / 237, 27 / 126 / 237 / 245, 27 / 237, 27 / 245, 28, 28 / 31, 28 / 31 / 239, 28 / 42 / 126 / 245, 28 / 42 / 245, 28 / 239, 29 / 31, 29 / 31 / 126 / 239, 30 / 31 / 33 / 35 / 126 / 218, 30 / 31 / 33 / 126 / 276 / 316, 30 / 33 / 35, 31 / 33 / 218 / 276, 31 / 126, 31 / 126 / 155, 33, 33 / 126 / 239, 42, 126, 126 / 155, 126 / 155 / 237, 126 / 155 / 239 / 276, 126 / 155 / 239 / 316, 126 / 239, 126 / 239 / 316, 126 / 245, 126 / 316, 155 / 276 / 316, 155 / 316, 218, 218 / 239 / 316, 239, 245, 252 / 276, or 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.Docket No. CX10-257WO3

[0247] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 26F / 27G / 28M, 26F / 27R, 26F / 27R / 28L / 31H, 26F / 27R / 28M / 245F, 26F / 28M / 31H, 27G, 27G / 28M, 27G / 29G, 27G / 29G / 237K, 27G / 29G / 237Q, 27G / 29G / 245F, 27G / 31H / 126A / 245F, 27G / 31H / 155T, 27G / 126A, 27G / 126A / 155T, 27G / 126A / 237K, 27G / 126A / 237Q / 245F, 27G / 237K, 27G / 245F, 27R, 27R / 28M / 29G / 126A, 27R / 29G / 126A, 27R / 245F, 28L / 31H / 239T, 28L / 239T, 28M, 28M / 31H, 28M / 42N / 126A / 245F, 28M / 42N / 245F, 29G / 31H, 29G / 31H / 126A / 239T, 30G / 31H / 33A / 35D / 126A / 218N, 30G / 31H / 33A / 126A / 276G / 316I, 30G / 33A / 35D, 31H / 33A / 218N / 276G, 31H / 126A, 31H / 126A / 155T, 33A, 33A / 126A / 239H, 42N, 126A, 126A / 155T, 126A / 155T / 237K, 126A / 155T / 239H / 316I, 126A / 155T / 239T / 276G, 126A / 239H, 126A / 239H / 316I, 126A / 245F, 126A / 316I, 155T / 276G / 316I, 155T / 316I, 218N, 218N / 239H / 316I, 239H, 245F, 252T / 276G, or 316I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0248] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at an amino acid position set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0249] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution as set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0250] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0251] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2,Docket No. CX10-257WO3 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0252] In some embodiments, the adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0253] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0254] In some embodiments, the engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808, and 1818-2124.

[0255] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.Docket No. CX10-257WO3

[0256] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0257] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 27, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 276, 287, 288, 291, 292, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0258] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D, 43E / K / R / S / V, 42G / N / Q, 46I, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93A, 94M / N, 100G, 101F / W, 102I, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126A / L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / M / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 190I, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V, 208V,Docket No. CX10-257WO3 210E / L, 211A / C / F / H / I / K / L / M / T / W, 211R, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / T / D / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251H / K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M, 294L, 295A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0259] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0260] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 24I / N / V, 27A / C / G / K / P / R / S / W, 31F / H / L / R / W / Y, 40H / M / R / W, 43E / K / R / S / V, 51F / G / S, 52C / D / F / G / S / T, 74F / I / K / R / T / V, 77V, 79A, 109E / G / L / P / R / S / T, 113G / S, 119C / G / M / Q / R / S / T / V, 121F / H / L / M / Q, 126A / L / S, 129A / G / S, 169A / C / D / I / L / M / S, 181A / C / L / M / V, 185S / T, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / T / W, 233A / S / V, 234A / M / N / R / V / Y, 237K / I / L / M / Q / V, 239A / F / G / H / I / M / Q / R / T / W, 244R / W, 246A / D / R / S / T / V, 249A / G / I / L / M / R / S, 251H / K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 276D / E / G / S, 299G / S, 305S / T, 312R, or 316A / D / E / I / K / S / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0261] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 24N, 27W, 31Y, 40R, 43R, 51F, 52D, 74R, 77V, 79A, 109S, 113G, 119M, 121H / Q, 126L, 129G / S, 169M, 181L / V, 185S, 207R, 211A / T, 233S, 234A,Docket No. CX10-257WO3 237V, 239M, 244R, 246V, 249A, 251K, 252A / T, 253S, 276E, 299G, 305T, 312R, or 316D / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0262] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or to the reference sequence corresponding to SEQ ID NO: 90, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0263] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 148-358, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0264] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 111 / 233 / 316, 43 / 111 / 233 / 316, 109 / 181 / 316, 109 / 316, 111 / 233, 109 / 233 / 316, 38 / 208 / 211 / 308, 111 / 181 / 233 / 316, 109 / 111 / 233 / 316, 111 / 181 / 233 / 239 / 316, 111 / 233 / 239 / 316, 109 / 111 / 181 / 233 / 316, 111 / 181 / 316, 43 / 233, 109 / 111 / 316, 181 / 233, 34 / 211 / 248 / 249, 34 / 38 / 248 / 249, 34 / 308, 233 / 316, 43 / 233 / 316, 181 / 316, 34 / 249, 43 / 181 / 233 / 316, 34 / 38 / 103 / 116 / 253, 34, 40 / 43 / 109 / 181 / 233 / 316, 34 / 38 / 208 / 253, 103 / 249 / 253 / 308, 34 / 38 / 116 / 208, 38 / 249, 34 / 38 / 116 / 211 / 248 / 253 / 308, 43 / 109 / 316, 116 / 253, 43 / 111 / 181 / 316, 34 / 38 / 308, 109 / 111 / 181, 111 / 130 / 233, 233, 34 / 38 / 116 / 308, 109 / 233, 40 / 43 / 109 / 111 / 181 / 233 / 316, 109 / 130 / 181 / 233, 34 / 38 / 253, 253, 34 / 38 / 103 / 208 / 249, 103 / 208 / 210 / 253, 130 / 181 / 233 / 316, 109 / 111 / 130 / 181 / 233, 34 / 38 / 210 / 211 / 253 / 303, 103 / 116 / 208, 34 / 116 / 248 / 252 / 253, 34 / 38 / 103 / 210 / 211 / 249, 40 / 233 / 316, 34 / 38, 109 / 130 / 233, 111 / 130 / 181 / 233 / 316, 34 / 38 / 249 / 253, 34 / 38 / 208 / 248, 40 / 109 / 111 / 181 / 233 / 316, 130 / 316, 40 / 316, 38 / 116, 34 / 38 / 116, 38 / 116 / 208 / 210 / 249 / 253, 40 / 130 / 181 / 233 / 316, 103 / 248 / 249, or 103 / 248, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.Docket No. CX10-257WO3

[0265] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 111A / 233S / 316S, 111A / 233S / 316E, 43R / 111A / 233S / 316S, 109S / 181L / 316S, 109S / 316E, 111A / 233S, 109S / 233S / 316E, 38C / 208V / 211T / 308F, 111A / 181L / 233S / 316S, 109S / 111A / 233S / 316S, 111A / 181L / 233S / 239Q / 316S, 111A / 233S / 239Q / 316S, 109S / 111A / 181L / 233S / 316S, 111A / 181L / 316E, 43R / 233S, 109S / 111A / 316S, 181L / 233S, 34K / 211T / 248R / 249G, 34K / 38C / 248R / 249G, 34K / 308F, 233S / 316E, 43R / 233S / 316D, 181L / 316S, 34R / 249G, 43R / 233S / 316S, 43R / 181L / 233S / 316S, 34R / 38C / 103R / 116M / 253P, 34K, 40R / 43R / 109S / 181L / 233S / 316D, 34R / 38C / 208V / 253P, 103R / 249G / 253P / 308F, 34R / 38C / 116M / 208V, 38C / 249G, 34K / 38C / 116M / 211T / 248R / 253P / 308L, 43R / 109S / 316S, 116M / 253P, 109S / 111A / 233S / 316D, 43R / 111A / 181L / 316E, 34R / 38C / 308F, 109S / 111A / 181L, 111A / 130A / 233S, 233S, 34R / 38C / 116M / 308F, 109S / 233S, 40R / 43R / 109S / 111A / 181L / 233S / 316S, 109S / 130A / 181L / 233S, 34K / 38C / 253P, 253P, 34R / 38C / 103R / 208V / 249S, 103R / 208V / 210E / 253P, 130A / 181L / 233S / 316D, 109S / 111A / 130A / 181L / 233S, 34R / 38C / 210E / 211T / 253P / 303H, 103R / 116M / 208V, 34R / 116M / 248R / 252K / 253P, 34R / 38C / 103R / 210E / 211T / 249G, 40R / 233S / 316S, 34R / 38C, 109S / 130A / 233S, 111A / 130A / 181L / 233S / 316S, 34K / 38C / 249G / 253P, 34K / 38C / 208V / 248R, 40R / 109S / 111A / 181L / 233S / 316S, 130A / 316S, 40R / 316S, 38C / 116M, 34K / 38C / 116M, 38C / 116M / 208V / 210E / 249G / 253P, 40R / 130A / 181L / 233S / 316D, 34R / 38C / 116M, 34R / 38C / 253P, 130A / 316D, 103R / 248R / 249G, or 103R / 248R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0266] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set P111A / P233S / Q316S, P111A / P233S / Q316E, I43R / P111A / P233S / Q316S, F109S / M181L / Q316S, F109S / Q316E, P111A / P233S, F109S / P233S / Q316E, P38C / L208V / E211T / E308F, P111A / M181L / P233S / Q316S, F109S / P111A / P233S / Q316S, P111A / M181L / P233S / H239Q / Q316S, P111A / P233S / H239Q / Q316S, F109S / P111A / M181L / P233S / Q316S, P111A / M181L / Q316E, I43R / P233S, F109S / P111A / Q316S, M181L / P233S, N34K / E211T / P248R / P249G, N34K / P38C / P248R / P249G, N34K / E308F, P233S / Q316E, I43R / P233S / Q316D, M181L / Q316S, N34R / P249G, I43R / P233S / Q316S, I43R / M181L / P233S / Q316S, N34R / P38C / K103R / T116M / R253P, N34K, K40R / I43R / F109S / M181L / P233S / Q316D, N34R / P38C / L208V / R253P, K103R / P249G / R253P / E308F, N34R / P38C / T116M / L208V, P38C / P249G, N34K / P38C / T116M / E211T / P248R / R253P / E308L, I43R / F109S / Q316S, T116M / R253P, F109S / P111A / P233S / Q316D, I43R / P111A / M181L / Q316E, N34R / P38C / E308F, F109S / P111A / M181L, P111A / G130A / P233S, P233S, N34R / P38C / T116M / E308F, F109S / P233S, K40R / I43R / F109S / P111A / M181L / P233S / Q316S, F109S / G130A / M181L / P233S, N34K / P38C / R253P, R253P, N34R / P38C / K103R / L208V / P249S, K103R / L208V / Q210E / R253P,Docket No. CX10-257WO3 G130A / M181L / P233S / Q316D, F109S / P111A / G130A / M181L / P233S, N34R / P38C / Q210E / E211T / R253P / R303H, K103R / T116M / L208V, N34R / T116M / P248R / E252K / R253P, N34R / P38C / K103R / Q210E / E211T / P249G, K40R / P233S / Q316S, N34R / P38C, F109S / G130A / P233S, P111A / G130A / M181L / P233S / Q316S, N34K / P38C / P249G / R253P, N34K / P38C / L208V / P248R, K40R / F109S / P111A / M181L / P233S / Q316S, G130A / Q316S, K40R / Q316S, P38C / T116M, N34K / P38C / T116M, P38C / T116M / L208V / Q210E / P249G / R253P, K40R / G130A / M181L / P233S / Q316D, N34R / P38C / T116M, N34R / P38C / R253P, G130A / Q316D, K103R / P248R / P249G, K103R / P248R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0267] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or to the reference sequence corresponding to SEQ ID NO: 254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0268] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0269] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 77 / 111 / 170 / 211 / 236, 77 / 111 / 211, 77 / 211, 53 / 77 / 170 / 211 / 320, 53 / 77 / 211 / 234 / 320, 77 / 79 / 211 / 316, 77 / 111 / 234 / 236 / 308, 111 / 170 / 211 / 234 / 236 / 248, 111 / 169 / 170 / 234 / 236 / 248, 77 / 211 / 248 / 320, 53 / 169 / 170 / 234 / 320, 111 / 211 / 234 / 236 / 248, 77, 53 / 77 / 111 / 170 / 248, 211 / 234 / 248, 77 / 86 / 170 / 211 / 234 / 308 / 320, 77 / 86, 53 / 111 / 170, 77 / 79 / 111, 53 / 77 / 111 / 169 / 236, 77 / 211 / 234, 211, 53 / 77, 86 / 169 / 234, 53 / 211 / 234 / 248, 111 / 169 / 170 / 236 / 248, 77 / 79 / 211, 77 / 79 / 169 / 170, 77 / 79 / 86 / 169 / 236, 79 / 211 / 248, 79 / 211, 53 / 86 / 211 / 234 / 236, 79 / 170 / 211, 308, 53 / 211, 169 / 234 / 236, 77 / 79 / 169 / 234 / 236, 53 / 77 / 79, 77 / 111, 53 / 234 / 236, 169 / 170, 53 / 77 / 79 / 169, 53 / 86 / 234 / 236, 53 / 169 / 234 / 236, 53 / 234, 77 / 79 / 86 / 170 / 211 / 234 / 236 / 308 / 320, 53 / 170, 77 / 248, 53 / 86, 252, 180, 129, 121, 255, 119, 256, 237, 249, 20, 234, 254, 56, 287, 317, 299, 229, 250, 295, 144 / 255, 291, 181, 233, 248, 231, 251, 128, 156,Docket No. CX10-257WO3 247, 288, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0270] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 77V / 111A / 170D / 211T / 236V, 77V / 111A / 211T, 77V / 211T, 53L / 77V / 170D / 211T / 320S, 53L / 77V / 211T / 234R / 320S, 77V / 79A / 211T / 316Y, 77V / 111A / 234R / 236V / 308L, 111A / 170D / 211T / 234R / 236V / 248R, 111A / 169A / 170D / 234R / 236V / 248R, 77V / 211T / 248R / 320S, 53L / 169A / 170D / 234R / 320S, 111A / 211T / 234R / 236V / 248R, 77V, 53L / 77V / 111A / 170D / 248R, 211T / 234R / 248R, 77V / 86Q / 170D / 211T / 234R / 308L / 320S, 77V / 86Q, 53L / 111A / 170D, 77V / 79A / 111A, 53L / 77V / 111A / 169A / 236V, 77V / 211T / 234R, 211T, 53L / 77V, 86Q / 169A / 234R, 53L / 211T / 234R / 248R, 111A / 169A / 170D / 236V / 248R, 77V / 79A / 211T, 77V / 79A / 169A / 170D, 77V / 79A / 86Q / 169A / 236V, 79A / 211T / 248R, 79A / 211T, 53L / 86Q / 211T / 234R / 236V, 79A / 170D / 211T, 308L, 53L / 211T, 169A / 234R / 236V, 77V / 79A / 169A / 234R / 236V, 53L / 77V / 79A, 77V / 111A, 53L / 234R / 236V, 169A / 170D, 53L / 77V / 79A / 169A, 53L / 86Q / 234R / 236V, 53L / 169A / 234R / 236V, 53L / 234R, 77V / 79A / 86Q / 170D / 211T / 234R / 236V / 308L / 320S, 53L / 170D, 77V / 248R, 53L / 86Q, 252T, 180Q, 129G, 121M, 255A, 255S, 121L, 255E, 119M, 256C, 255G, 237I, 249L, 20F, 255K, 237V, 234A, 254Q, 256M, 56Y, 56F, 249M, 119T, 287V, 121F, 119S, 252K, 56G, 317W, 119V, 299S, 229C, 119C, 250I, 254L, 234M, 229V, 234Y, 287Y, 295A, 144Q / 255W, 119Q, 291G, 250T, 181M, 287S, 119R, 317Y, 119G, 250M, 249G, 249I, 20V, 237M, 233A, 248G, 231V, 251L, 128F, 287A, 181V, 156Y, 247L, 288V, 292K, 181A, 249R, or 250H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0271] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set P77V / P111A / G170D / E211T / A236V, P77V / P111A / E211T, P77V / E211T, M53L / P77V / G170D / E211T / A320S, M53L / P77V / E211T / K234R / A320S, P77V / G79A / E211T / D316Y, P77V / P111A / K234R / A236V / E308L, P111A / G170D / E211T / K234R / A236V / P248R, P111A / G169A / G170D / K234R / A236V / P248R, P77V / E211T / P248R / A320S, M53L / G169A / G170D / K234R / A320S, P111A / E211T / K234R / A236V / P248R, P77V, M53L / P77V / P111A / G170D / P248R, E211T / K234R / P248R, P77V / G86Q / G170D / E211T / K234R / E308L / A320S, P77V / G86Q, M53L / P111A / G170D, P77V / G79A / P111A, M53L / P77V / P111A / G169A / A236V, P77V / E211T / K234R, E211T, M53L / P77V, G86Q / G169A / K234R, M53L / E211T / K234R / P248R, P111A / G169A / G170D / A236V / P248R, P77V / G79A / E211T, P77V / G79A / G169A / G170D, P77V / G79A / G86Q / G169A / A236V, G79A / E211T / P248R, G79A / E211T, M53L / G86Q / E211T / K234R / A236V, G79A / G170D / E211T, E308L, M53L / E211T, G169A / K234R / A236V, P77V / G79A / G169A / K234R / A236V, M53L / P77V / G79A, P77V / P111A,Docket No. CX10-257WO3 M53L / K234R / A236V, G169A / G170D, M53L / P77V / G79A / G169A, M53L / G86Q / K234R / A236V, M53L / G169A / K234R / A236V, M53L / K234R, P77V / G79A / G86Q / G170D / E211T / K234R / A236V / E308L / A320S, M53L / G170D, P77V / P248R, M53L / G86Q, E252T, A180Q, P129G, H121M, V255A, V255S, H121L, V255E, H119M, V256C, V255G, L237I, P249L, L20F, V255K, L237V, K234A, R254Q, V256M, E56Y, E56F, P249M, H119T, N287V, H121F, H119S, E252K, E56G, F317W, H119V, T299S, T229C, H119C, A250I, R254L, K234M, T229V, K234Y, N287Y, E295A, P144Q / V255W, H119Q, A291G, A250T, L181M, N287S, H119R, F317Y, H119G, A250M, P249G, P249I, L20V, L237M, S233A, P248G, Q231V, H251L, H128F, N287A, L181V, R156Y, I247L, L288V, L292K, L181A, P249R, or A250H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0272] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or to the reference sequence corresponding to SEQ ID NO: 370, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0273] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0274] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 299, 21, 113, 152, 169 / 255, 251, 294, 255, 202 / 211, 31, 129, 252, 27 / 64, 128, 248, 234, 288, 20, 111, 112, 181, 119, 64, 155, 256, 254, 292, 265, 177, 232, 120, 183, 234 / 252, 181 / 250 / 252 / 299, 119 / 181 / 234 / 250 / 299, 129 / 234 / 252, 119 / 129 / 234, 119 / 181 / 234 / 250 / 256 / 299, 119 / 181 / 250 / 252, 119 / 181 / 234 / 299, 234 / 250 / 252, 119 / 181 / 252 / 299, 129 / 181 / 234 / 299, 56 / 121 / 229, 181 / 255 / 299, 119 / 299, 119 / 250 / 255 / 299, 80 / 119 / 252, 119 / 129, 119 / 129 / 250, 181 / 234 / 252 / 299, 181 / 250 / 299, 252 / 299, 119 / 181 / 299, 119 / 129 / 299, 119 / 129 / 234 / 252, 119 / 129 / 234 / 250 / 299, 119 / 181 / 234 / 252, 129 / 234 / 255 / 299, 181 / 299, 250 / 252, 250 / 255 / 299, 119 / 234 / 299, 119 / 181 / 250, 56 / 237, 250 / 299, 119 / 234 / 255 / 256 / 299, 56 / 229 / 254, 119 / 129 / 181, 56 / 121 / 295 / 317, 119 / 181 / 250 / 255 / 256, 229, 250, 119 / 234, 20 / 229 / 237 / 317,Docket No. CX10-257WO3 181 / 250, 119 / 255, 121 / 156 / 229, 56 / 121 / 237, 237 / 317, 56 / 295, 56 / 121 / 229 / 236 / 254 / 295, 56 / 254, 119 / 250 / 299, 229 / 237 / 295, 20 / 229 / 317, 229 / 295, 156 / 229, 56 / 121 / 229 / 237 / 295, 237 / 254, 20 / 56, 56 / 156, 56 / 156 / 254 / 295, 20 / 56 / 229, 119 / 129 / 181 / 234, 237, 56 / 156 / 229 / 237, 20 / 56 / 102 / 229, 119 / 181 / 234, 20 / 229, 56 / 121 / 156 / 237, 56 / 156 / 229, 20 / 56 / 156 / 229 / 237 / 295, 20 / 56 / 121 / 237 / 317, 156, 156 / 237 / 295, 20 / 56 / 121, 119 / 181, 20 / 56 / 229 / 254, 56, 121 / 254, 20 / 295, 255 / 256, 56 / 121 / 229 / 254, or 119 / 129 / 234 / 255 / 256, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0275] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 299G, 21G, 299S, 113S, 152C, 169D / 255C, 251K, 294L, 255S, 202F / 211A, 31L, 129A, 252V, 27A / 64M, 128W, 248L, 251V, 234V, 288V, 20V, 111L, 112L, 181C, 119C, 64T, 251T, 155W, 256H, 128F, 254I, 248G, 292F, 252C, 234N, 181A, 31W, 265Y, 251L, 152A, 234M, 177V, 232S, 120C, 31F, 183M, 234A / 252T, 181V / 250I / 252T / 299S, 119R / 181V / 234A / 250I / 299S, 129G / 234A / 252T, 119M / 129G / 234A, 119M / 181V / 234A / 250I / 256M / 299S, 119R / 181V / 250I / 252T, 119M / 181V / 234A / 299S, 234A / 250I / 252T, 119R / 181V / 252T / 299S, 129G / 181V / 234A / 299S, 56G / 121L / 229V, 181V / 255A / 299S, 119M / 299S, 119R / 250I / 255K / 299S, 119R / 181V / 234A / 299S, 252T, 80M / 119M / 252T, 119M / 129G, 119M / 129G / 250I, 181V / 234A / 252T / 299S, 181V / 250I / 299S, 252T / 299S, 119M / 181V / 299S, 119R / 129G / 299S, 119M / 129G / 234A / 252T, 119M / 129G / 234A / 250I / 299S, 119M / 181V / 234A / 252T, 129G / 234A / 255K / 299S, 181V / 299S, 250I / 252T, 250I / 255K / 299S, 119R / 181V / 299S, 119M / 234A / 299S, 119R / 299S, 119M / 181V / 250I, 129G, 56G / 237V, 250I / 299S, 119M / 234A / 255K / 256M / 299S, 56G / 229V / 254L, 119R / 129G / 181V, 56G / 121M / 295A / 317Y, 119R / 181V / 250I / 255A / 256M, 229V, 250I, 119M, 119M / 234A, 20F / 229V / 237V / 317Y, 181V / 250I, 119M / 255K, 121M / 156Y / 229V, 56G / 121M / 237V, 237V / 317Y, 56G / 295A, 56G / 121M / 229V / 236S / 254L / 295A, 56G / 254L, 119R / 250I / 299S, 229V / 237V / 295A, 20F / 229V / 317Y, 234A, 119R / 234A, 254L, 229V / 295A, 156Y / 229V, 56F / 121M / 229V / 237V / 295A, 56F / 237V, 237V / 254L, 299S, 20F / 56G, 56G / 156Y, 56G / 156Y / 254L / 295A, 20F / 56G / 229V, 119M / 129G / 181V / 234A, 237V, 20F, 56F / 156Y / 229V / 237V, 56F / 254L, 20F / 56G / 102I / 229V, 20F / 56F, 256M, 119M / 181V / 234A, 20F / 229V, 56F / 121M / 156Y / 237V, 56F / 156Y / 229V, 20F / 56G / 156Y / 229V / 237V / 295A, 56F / 229V / 254L, 20F / 56F / 121M / 237V / 317Y, 20F / 56F / 229V, 119R / 181V / 234A, 119R, 156Y, 156Y / 237V / 295A, 20F / 56F / 121M, 119M / 181V, 20F / 56G / 229V / 254L, 56F, 121M / 254L, 20F / 295A, 255K / 256M, 56F / 121M / 229V / 254L, 119M / 129G / 234A / 255A / 256M, or 119R / 181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0276] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set T299G, A21G, T299S, A113S, G152C, G169D / V255C,Docket No. CX10-257WO3 H251K, V294L, V255S, N202F / T211A, Q31L, P129A, E252V, V27A / I64M, H128W, P248L, H251V, K234V, L288V, L20V, P111L, Q112L, L181C, H119C, I64T, H251T, G155W, V256H, H128F, R254I, P248G, L292F, E252C, K234N, L181A, Q31W, F265Y, H251L, G152A, K234M, P177V, G232S, D120C, Q31F, L183M, K234A / E252T, L181V / A250I / E252T / T299S, H119R / L181V / K234A / A250I / T299S, P129G / K234A / E252T, H119M / P129G / K234A, H119M / L181V / K234A / A250I / V256M / T299S, H119R / L181V / A250I / E252T, H119M / L181V / K234A / T299S, K234A / A250I / E252T, H119R / L181V / E252T / T299S, P129G / L181V / K234A / T299S, E56G / H121L / T229V, L181V / V255A / T299S, H119M / T299S, H119R / A250I / V255K / T299S, H119R / L181V / K234A / T299S, E252T, T80M / H119M / E252T, H119M / P129G, H119M / P129G / A250I, L181V / K234A / E252T / T299S, L181V / A250I / T299S, E252T / T299S, H119M / L181V / T299S, H119R / P129G / T299S, H119M / P129G / K234A / E252T, H119M / P129G / K234A / A250I / T299S, H119M / L181V / K234A / E252T, P129G / K234A / V255K / T299S, L181V / T299S, A250I / E252T, A250I / V255K / T299S, H119R / L181V / T299S, H119M / K234A / T299S, H119R / T299S, H119M / L181V / A250I, P129G, E56G / L237V, A250I / T299S, H119M / K234A / V255K / V256M / T299S, E56G / T229V / R254L, H119R / P129G / L181V, E56G / H121M / E295A / F317Y, H119R / L181V / A250I / V255A / V256M, T229V, A250I, H119M, H119M / K234A, L20F / T229V / L237V / F317Y, L181V / A250I, H119M / V255K, H121M / R156Y / T229V, E56G / H121M / L237V, L237V / F317Y, E56G / E295A, E56G / H121M / T229V / A236S / R254L / E295A, E56G / R254L, H119R / A250I / T299S, T229V / L237V / E295A, L20F / T229V / F317Y, K234A, H119R / K234A, R254L, T229V / E295A, R156Y / T229V, E56F / H121M / T229V / L237V / E295A, E56F / L237V, L237V / R254L, T299S, L20F / E56G, E56G / R156Y, E56G / R156Y / R254L / E295A, L20F / E56G / T229V, H119M / P129G / L181V / K234A, L237V, L20F, E56F / R156Y / T229V / L237V, E56F / R254L, L20F / E56G / V102I / T229V, L20F / E56F, V256M, H119M / L181V / K234A, L20F / T229V, E56F / H121M / R156Y / L237V, E56F / R156Y / T229V, L20F / E56G / R156Y / T229V / L237V / E295A, E56F / T229V / R254L, L20F / E56F / H121M / L237V / F317Y, L20F / E56F / T229V, H119R / L181V / K234A, H119R, R156Y, R156Y / L237V / E295A, L20F / E56F / H121M, H119M / L181V, L20F / E56G / T229V / R254L, E56F, H121M / R254L, L20F / E295A, V255K / V256M, E56F / H121M / T229V / R254L, H119M / P129G / K234A / V255A / V256M, or H119R / L181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0277] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO: 734, wherein the amino acid sequence comprises one or more substitutions relative toDocket No. CX10-257WO3 the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0278] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0279] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 56 / 229, 21 / 56, 181, 181 / 211 / 251, 181 / 211, 21 / 121 / 299, 229, 56, 56 / 169, 311, 52, 170, 31 / 181 / 211, 167, 303, 222, 43, 83, 113 / 181 / 211, 251, 187, 218, 181 / 251, 195, 121 / 229, 107, 141, 31 / 181, 307, 100, 169, 86, 90, 164, 310, 276, 35, 56 / 134, 211, 50, 41, 121 / 169 / 229, 74, 225, 106, 121, 31 / 113 / 181, 191, 190, 171, 168, 31 / 181 / 211 / 294, 73, 31 / 251, 31, 196, 89, 197, 242, 51, 103, 101, 147, 40, 146, 243, 56 / 121 / 229, or 31 / 211, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0280] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 56G / 229V, 21G / 56G, 181V, 181V / 211A / 251K, 181V / 211A, 21G / 121L / 299S, 229V, 56G, 56G / 169D, 311Q, 52D, 170R, 31L / 181V / 211A, 167W, 170H, 303M, 222G, 43V, 83V, 113S / 181V / 211A, 251K, 187V, 218V, 181V / 251K, 195T, 121L / 229V, 107H, 141S, 31L / 181V, 307F, 100G, 169L, 169M, 52G, 86L, 90L, 311G, 164W, 310C, 167F, 276E, 35V, 56G / 134H, 211M, 211M, 170V, 50C, 41L, 311S, 121L / 169D / 229V, 74I, 225G, 106G, 121L, 31L / 113S / 181V, 191A, 191S, 211I, 35S, 190I, 164K, 171C, 211F, 211L, 225E, 195G, 311R, 167V, 311L, 168V, 168R, 211C, 31L / 181V / 211A / 294L, 73M, 276S, 74V, 164S, 31L / 251K, 169S, 31L, 169D, 196S, 196S, 52C, 218L, 169C, 89G, 90R, 73E, 73E, 167T, 197R, 242D, 51S, 103L, 191R, 101F, 83T, 52F, 164G, 195M, 43K, 147S, 187Q, 311M, 40W, 169I, 242T, 101W, 83L, 222M, 170S, 311F, 146I, 83S, 243R, 74T, 311T, 218N, 74F, 303V, 83F, 90V, 74K, 303F, 310A, 243G, 222A, 43S, 303W, 56G / 121L / 229V, or 31L / 211A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0281] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set E56G / T229V, A21G / E56G, L181V, L181V / T211A / H251K, L181V / T211A, A21G / H121L / T299S, T229V, E56G, E56G / G169D, D311Q, R52D, G170R, Q31L / L181V / T211A, A167W, G170H, R303M, S222G, R43V, Q83V, A113S / L181V / T211A,Docket No. CX10-257WO3 H251K, P187V, Q218V, L181V / H251K, Q195T, H121L / T229V, D107H, R141S, Q31L / L181V, A307F, S100G, G169L, G169M, R52G, G86L, E90L, D311G, E164W, N310C, A167F, N276E, H35V, E56G / R134H, T211M, T211M, G170V, V50C, V41L, D311S, H121L / G169D / T229V, A74I, Q225G, D106G, H121L, Q31L / A113S / L181V, E191A, E191S, T211I, H35S, R190I, E164K, I171C, T211F, T211L, Q225E, Q195G, D311R, A167V, D311L, A168V, A168R, T211C, Q31L / L181V / T211A / V294L, G73M, N276S, A74V, E164S, Q31L / H251K, G169S, Q31L, G169D, A196S, A196S, R52C, Q218L, G169C, R89G, E90R, G73E, G73E, A167T, D197R, E242D, P51S, K103L, E191R, R101F, Q83T, R52F, E164G, Q195M, R43K, T147S, P187Q, D311M, R40W, G169I, E242T, R101W, Q83L, S222M, G170S, D311F, V146I, Q83S, K243R, A74T, D311T, Q218N, A74F, R303V, Q83F, E90V, A74K, R303F, N310A, K243G, S222A, R43S, R303W, E56G / H121L / T229V, or Q31L / T211A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0282] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or to the reference sequence corresponding to SEQ ID NO: 896, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0283] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1196-1344, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1196- 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0284] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 52 / 169, 52 / 141 / 169, 52, 41, 169, 51 / 52 / 211, 52 / 141 / 187, 51 / 52, 141, 41 / 103, 311, 167, 41 / 311, 133, 207, 14, 34, 241, 33, 27, 206, 217, 239, 13, 246, 162, 108, 160, 245, 92, 163, 104, 157, 215, 80, 54, 240, 210, 23, 53, 87, 188, 151, 29, 258, 238, 244, 192, 91, or 30, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.Docket No. CX10-257WO3

[0285] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 52D / 169M, 52D / 141S / 169M, 52G, 52G / 169M, 52D, 41L, 52G / 141S / 169M, 169M, 51S / 52G / 211M, 52G / 141S / 187V, 51S / 52D, 141S, 41L / 103L, 311Q, 167F, 41L / 311Q, 51S / 52G, 133L, 207R, 14V, 34R, 241R, 33Q, 27S, 206A, 217V, 239R, 207T, 13A, 246T, 13P, 207M, 162F, 207L, 27P, 108C, 160L, 33S, 245W, 239F, 92M, 163G, 239T, 104C, 157P, 215L, 80V, 54V, 239A, 13E, 207V, 240V, 133A, 210L, 207A, 23N, 53R, 87K, 188T, 151L, 29R, 258S, 34G, 238L, 27R, 14S, 244W, 192L, 91Y, 206H, 30Y, 80S, 258R, 240L, or 104L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0286] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set R52D / G169M, R52D / R141S / G169M, R52G, R52G / G169M, R52D, V41L, R52G / R141S / G169M, , G169M, P51S / R52G / A211M, R52G / R141S / P187V, P51S / R52D, R141S, V41L / K103L, D311Q, A167F, V41L / D311Q, P51S / R52G, , M133L, N207R, A14V, N34R, P241R, K33Q, , V27S, S206A, E217V, H239R, N207T, S13A, D246T, S13P, N207M, N162F, N207L, V27P, L108C, I160L, K33S, , Y245W, H239F, F92M, A163G, H239T, , I104C, E157P, W215L, T80V, R54V, H239A, S13E, N207V, T240V, M133A, Q210L, N207A, D23N, M53R, P87K, E188T, V151L, P29R, P258S, N34G, V238L, V27R, A14S, T244W, F192L, H91Y, S206H, D30Y, T80S, P258R, T240L, or I104L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO: 896.

[0287] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or to the reference sequence corresponding to SEQ ID NO: 1196, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0288] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1346-1364, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1346- 1364, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.Docket No. CX10-257WO3

[0289] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 24, 234 / 236, 251 / 252, 119, 234, or 231 / 234, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0290] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 24V, 24I, 234K / 236C, 24N, 251H / 252A, 119S, 119T, 234K, 234K / 236S, or 231L / 234K, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0291] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set T24V, T24I, A234K / A236C, T24N, K251H / T252A, M119S, M119T, A234K, A234K / A236S, or Q231L / A234K, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

[0292] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or to the reference sequence corresponding to SEQ ID NO: 1366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0293] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1366-1410, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1366- 1410, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0294] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 182, 121, 249, 129, 152, 237, 155, 121 / 129 / 207 / 237 / 249, 121 / 129 / 207 / 249, 27 / 121 / 207, 27 / 121 / 207 / 249, 121 / 207, 121 / 237, 121 / 249, 121 / 207 / 252, 27 / 121 / 129 / 207, 27 / 121 / 129 / 237, 207, 182 / 207, 121 / 129, or 121 / 237 / 252, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.Docket No. CX10-257WO3

[0295] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 182G, 121Q, 249A, 129S, 152A, 237V, 155A, 121Q / 129S / 207R / 237V / 249A, 121Q / 129S / 207R / 249A, 27P / 121Q / 207R, 27P / 121Q / 207R / 249A, 121Q / 207R, 121Q / 237V, 121Q / 249A, 121Q / 207R / 252V, 27P / 121Q / 129S / 207R, 27P / 121Q / 129S / 237V, 207R, 182G / 207R, 121Q / 129S, 121Q / 237V / 252V, or 155I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0296] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set P182G, H121Q, P249A, G129S, G152A, L237V, G155A, H121Q / G129S / N207R / L237V / P249A, H121Q / G129S / N207R / P249A, V27P / H121Q / N207R, V27P / H121Q / N207R / P249A, H121Q / N207R, H121Q / L237V, H121Q / P249A, H121Q / N207R / T252V, V27P / H121Q / G129S / N207R, V27P / H121Q / G129S / L237V, N207R, P182G / N207R, H121Q / G129S, H121Q / L237V / T252V, or G155I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

[0297] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or to the reference sequence corresponding to SEQ ID NO: 1382, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0298] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1412-1564, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1412- 1564, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0299] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 314, 221, 170, 40, 49, 73, 194, 195, 147, 218, 216, 90, 89, 42, 211, 106, 171, 93, 296, 100, 243, 172, 51, 271, 90 / 171 / 191, 90 / 271, 90 / 191, 90 / 147 / 171 / 271, 191, 51 / 90, 187 / 211, 171 / 191, 170 / 211, 49 / 51, 51 / 171 / 191 / 271, 40 / 211, 106 / 187 / 211, 211 / 221, 40 / 170 / 211, 49 / 51 / 90 / 147 / 171, 51 / 90 / 171, 51 / 171, 49 / 51 / 191, 49 / 51 / 90 / 147,Docket No. CX10-257WO3 49 / 51 / 171 / 271, 106 / 211 / 221, 187 / 221, 49 / 171 / 191 / 314, 51 / 191 / 271, 51 / 90 / 171 / 191 / 271, 52 / 221, 90 / 171 / 191 / 271, 191 / 271, 170 / 221, , 50 / 52, 50 / 52 / 106 / 211, 50 / 211 / 308, 50 / 52 / 106, 40 / 50 / 187 / 211 / 308, 50 / 52 / 106 / 170 / 187 / 308, 40 / 52 / 170, 50 / 106, 50 / 221 / 308, 49 / 51 / 90 / 271, 40 / 52 / 170 / 211, 187, 50 / 211 / 221 / 308, or 40 / 50 / 52 / 221, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0300] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 314S, 221A, 170K, 40H, 314V, 49M, 73C, 314Q, 194A, 195V, 147N, 218L, 216V, 90S, 89L, 42D, 211H, 90T, 106M, 171C, 93A, 296N, 100G, 243Y, 172S, 73A, , 51F, 271W, 90S / 171V / 191W, 90S / 271W, 90S / 191W, 90S / 147A / 171V / 271W, 191W, 51F / 90S, 187H / 211W, 171C / 191W, 170M / 211K, 49M / 51G, 51G / 171C / 191W / 271W, 40M / 211K, 106T / 187H / 211K, 211W / 221A, 40M / 170M / 211K, 211W, 49M / 51G / 90S / 147A / 171V, 51G / 90S / 171V, 51G / 171V, 49M / 51F / 191W, 49M / 51G / 90S / 147A, 51G, 49M / 51F / 171V / 271W, 49M / 51F / 171C / 271W, 106T / 211K / 221A, 211K, 187H / 221A, 49M / 171C / 191W / 314S, 51G / 191W / 271W, 51G / 90S / 171C / 191W / 271W, 52T / 221A, 90S / 171C / 191W / 271W, 191W / 271W, 170M / 221A, , 50C / 52T, 50C / 52T / 106T / 211W, 50C / 211K / 308D, 50C / 52S / 106T, 40M, 40M / 50C / 187H / 211K / 308D, 50C / 52T / 106T / 170M / 187H / 308D, 40M / 52T / 170M, 50C / 106T, 50C / 221A / 308D, 49M / 51F / 90S / 271W, 40M / 52S / 170M / 211W, 187H, 50C / 211K / 221A / 308D, or 40M / 50C / 52S / 221A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0301] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set K314S, V221A, G170K, R40H, K314V, L49M, G73C, K314Q, E194A, Q195V, T147N, Q218L, N216V, E90S, R89L, H42D, A211H, E90T, D106M, I171C, E93A, H296N, S100G, K243Y, P172S, G73A, , P51F, Y271W, E90S / I171V / E191W, E90S / Y271W, E90S / E191W, E90S / T147A / I171V / Y271W, E191W, P51F / E90S, P187H / A211W, I171C / E191W, G170M / A211K, L49M / P51G, P51G / I171C / E191W / Y271W, R40M / A211K, D106T / P187H / A211K, A211W / V221A, R40M / G170M / A211K, A211W, L49M / P51G / E90S / T147A / I171V, P51G / E90S / I171V, P51G / I171V, L49M / P51F / E191W, L49M / P51G / E90S / T147A, P51G, L49M / P51F / I171V / Y271W, L49M / P51F / I171C / Y271W, D106T / A211K / V221A, A211K, P187H / V221A, L49M / I171C / E191W / K314S, P51G / E191W / Y271W, P51G / E90S / I171C / E191W / Y271W, D52T / V221A, E90S / I171C / E191W / Y271W, E191W / Y271W, G170M / V221A, , V50C / D52T, V50C / D52T / D106T / A211W, V50C / A211K / E308D, V50C / D52S / D106T, R40M, R40M / V50C / P187H / A211K / E308D, V50C / D52T / D106T / G170M / P187H / E308D, R40M / D52T / G170M, V50C / D106T, V50C / V221A / E308D, L49M / P51F / E90S / Y271W, R40M / D52S / G170M / A211W, P187H, V50C / A211K / V221A / E308D, or R40M / V50C / D52S / V221A,Docket No. CX10-257WO3 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

[0302] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or to the reference sequence corresponding to SEQ ID NO: 1464, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0303] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1566-1578, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1566- 1578, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0304] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 317, 113, 21, 155, 126, 155, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0305] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 317M, 113G, 21G, 155V, 126S, 155N, or 231M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0306] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution F317M, A113G, A21G, G155V, A126S, G155N, or Q231M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

[0307] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of SEQ ID NO: 1568, or to the reference sequence corresponding to SEQ ID NO: 1568, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0308] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1580-1648, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1580- 1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0309] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 246, 74, 185, 244, 164, 276, 43, 163, 217, 31, 43 / 276, 185 / 187 / 276, 43 / 187 / 222 / 276, 185 / 187 / 222 / 276, 187 / 276, 185 / 276, 164 / 222, 164 / 187 / 222 / 276, 43 / 222 / 276, 164 / 187 / 222, 222, 43 / 164 / 185 / 187 / 222, 222 / 276, 239, or 53, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0310] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 246V, 74R, 185T, 244R, 246R, 164S, 276G, 43E, 246S, 163G, 217A, 31H, 43E / 276E, 185S / 187S / 276G, 43E / 187S / 222Q / 276D, 185S / 187S / 222Q / 276D, 187S / 276E, 187S / 276D, 185S / 276E, 164S / 222Q, 164S / 187S / 222Q / 276G, 43E / 222Q / 276G, 164S / 187S / 222Q, 222Q, 43E / 164S / 185S / 187S / 222Q, 222Q / 276G, 276E, 276D, 31L, 31Y, 164Q, 239M, 53L, 185S, or 164A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

[0311] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set D246V, A74R, N185T, T244R, D246R, E164S, N276G, R43E, D246S, A163G, E217A, Q31H, R43E / N276E, N185S / P187S / N276G, R43E / P187S / S222Q / N276D, N185S / P187S / S222Q / N276D, P187S / N276E, P187S / N276D, N185S / N276E, E164S / S222Q, E164S / P187S / S222Q / N276G, R43E / S222Q / N276G, E164S / P187S / S222Q, S222Q, R43E / E164S / N185S / P187S / S222Q, S222Q / N276G, , N276E, N276D, , Q31L, Q31Y, E164Q, H239M, M53L, N185S, or E164A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.Docket No. CX10-257WO3

[0312] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or to the reference sequence corresponding to SEQ ID NO: 1616, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0313] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1650-1742, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1650- 1742, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0314] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 74 / 239 / 244 / 246, 31 / 74 / 207 / 239 / 244 / 246, 31 / 246, 74 / 244, 31 / 74, 53 / 74 / 239 / 244 / 246, 31 / 74 / 207 / 244 / 246, 31 / 74 / 163 / 239 / 244, 31 / 74 / 239 / 244 / 246, 31 / 244 / 246, 31 / 53 / 164, 31 / 239 / 244, 239 / 246, 163 / 164 / 207 / 239 / 244 / 246, 31 / 74 / 163 / 246, 244, 31 / 239 / 244 / 246, 31 / 53 / 239 / 244 / 246, 31 / 207 / 244 / 246, 31, 31 / 53 / 74 / 239 / 246, 31 / 74 / 207, 31 / 163 / 207 / 244, 31 / 53 / 244, 31 / 163, 31 / 53 / 239 / 246, 74 / 207 / 239 / 244, 31 / 163 / 164 / 207 / 239 / 244 / 246, 74 / 207, 31 / 163 / 207 / 239 / 244, 31 / 74 / 163 / 164 / 207 / 246, 31 / 53 / 207 / 239, 207, 53 / 163 / 207 / 246, 31 / 53 / 74 / 163 / 207 / 246, 31 / 163 / 239 / 246 / 308, 31 / 74 / 207 / 239 / 308, or 163 / 207 / 246, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0315] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 74T / 239M / 244R / 246V, 31Y / 74R / 207M / 239M / 244R / 246V, 31Y / 246S, 74R / 244R, 31R / 74R, 53L / 74T / 239M / 244R / 246S, 53L / 74R / 239M / 244R / 246V, 31R / 74T / 207M / 244R / 246S, 31Y / 74R / 163G / 239M / 244R, 31Y / 74R / 239M / 244R / 246V, 31L / 244R / 246S, 31Y / 244R / 246S, 31R / 53L / 164Q, 31R / 239M / 244R, 239M / 246V, 163G / 164Q / 207M / 239M / 244R / 246V, 31Y / 74R / 163G / 246V, 244R, 31L / 239M / 244R / 246S, 31Y / 74T, 31L / 53L / 239M / 244R / 246V, 31Y / 74R, 31Y / 207M / 244R / 246V, 31Y / 53L / 239M / 244R / 246V, 31Y, 31L / 53L / 74T / 239M / 246S, 31Y / 74R / 207M, 31Y / 163G / 207M / 244R, 31R / 207M / 244R / 246V, 31Y / 53L / 244R, 31Y / 163G, 31R, 31R / 53L / 239M / 246S, 74T / 207M / 239M / 244R, 31L / 163G / 164Q / 207M / 239M / 244R / 246V, 74T / 207M,Docket No. CX10-257WO3 31Y / 163G / 207M / 239M / 244R, 31L / 74R / 163G / 164Q / 207M / 246V, 31Y / 53L / 207M / 239M, 31Y / 207M / 244R / 246S, 207M, 53L / 163G / 207M / 246S, 31R / 53L / 74T / 163G / 207M / 246S, 31Y / 163G / 239M / 246V / 308K, 31R / 207M / 244R / 246S, 31Y / 74R / 207M / 239M / 308K, or 163G / 207M / 246V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0316] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set A74T / H239M / T244R / D246V, Q31Y / A74R / R207M / H239M / T244R / D246V, Q31Y / D246S, A74R / T244R, Q31R / A74R, M53L / A74T / H239M / T244R / D246S, M53L / A74R / H239M / T244R / D246V, Q31R / A74T / R207M / T244R / D246S, Q31Y / A74R / A163G / H239M / T244R, Q31Y / A74R / H239M / T244R / D246V, Q31L / T244R / D246S, Q31Y / T244R / D246S, Q31R / M53L / E164Q, Q31R / H239M / T244R, H239M / D246V, A163G / E164Q / R207M / H239M / T244R / D246V, Q31Y / A74R / A163G / D246V, T244R, Q31L / H239M / T244R / D246S, Q31Y / A74T, Q31L / M53L / H239M / T244R / D246V, Q31Y / A74R, Q31Y / R207M / T244R / D246V, Q31Y / M53L / H239M / T244R / D246V, Q31Y, Q31L / M53L / A74T / H239M / D246S, Q31Y / A74R / R207M, Q31Y / A163G / R207M / T244R, Q31R / R207M / T244R / D246V, Q31Y / M53L / T244R, Q31Y / A163G, Q31R, Q31R / M53L / H239M / D246S, A74T / R207M / H239M / T244R, Q31L / A163G / E164Q / R207M / H239M / T244R / D246V, A74T / R207M, Q31Y / A163G / R207M / H239M / T244R, Q31L / A74R / A163G / E164Q / R207M / D246V, Q31Y / M53L / R207M / H239M, Q31Y / R207M / T244R / D246S, R207M, M53L / A163G / R207M / D246S, Q31R / M53L / A74T / A163G / R207M / D246S, Q31Y / A163G / H239M / D246V / E308K, Q31R / R207M / T244R / D246S, Q31Y / A74R / R207M / H239M / E308K, or A163G / R207M / D246V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

[0317] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or to the reference sequence corresponding to SEQ ID NO: 1668, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0318] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1744-1808, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1744- 1808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0319] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 312, 305, 246, 89, 33, 218, 147, 27 / 218 / 305 / 312, 27 / 35 / 106 / 218 / 312, 27 / 218 / 312, 27 / 312, 27 / 305 / 312, 27 / 106 / 218 / 312, 27 / 218, 27 / 106 / 312, 35 / 305, 27 / 144 / 312, 27 / 218 / 305, 35 / 312, 27 / 106 / 218 / 305 / 312, 27 / 35 / 106 / 312, 106 / 218 / 305, 106 / 218 / 312, 27 / 106 / 305, 27 / 35 / 94 / 106 / 305 / 312, 35 / 218 / 312, 218 / 312, 27 / 106 / 218, 35 / 106 / 312, or 27 / 106, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0320] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 312R, 305T, 246D, 89K, 33A, 218D, 147N, 27W / 218D / 305T / 312R, 27K / 35Y / 106P / 218D / 312R, 27W / 218D / 312R, 27W / 312R, 27W / 305T / 312R, 27W / 106P / 218D / 312R, 27W / 218D, 27W / 106P / 312R, 35Y / 305T, 27W / 144S / 312R, 27W / 218D / 305S, 35Y / 312R, 27W / 106P / 218D / 305S / 312R, 27W / 35Y / 106P / 312R, 106P / 218D / 305T, 27W / 106P / 218D / 305T / 312R, 106P / 218D / 312R, 27W / 106P / 305T, 27W / 35Y / 94N / 106P / 305S / 312R, 35Y / 218D / 312R, 218D / 312R, 27W / 106P / 218D, 35Y / 106P / 312R, 27W / 106P, or 27W / 106P / 305S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0321] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set Q312R, D305T, V246D, R89K, K33A, Q218D, T147N, V27W / Q218D / D305T / Q312R, V27K / H35Y / D106P / Q218D / Q312R, V27W / Q218D / Q312R, V27W / Q312R, V27W / D305T / Q312R, V27W / D106P / Q218D / Q312R, V27W / Q218D, V27W / D106P / Q312R, H35Y / D305T, V27W / P144S / Q312R, V27W / Q218D / D305S, H35Y / Q312R, V27W / D106P / Q218D / D305S / Q312R, V27W / H35Y / D106P / Q312R, D106P / Q218D / D305T, V27W / D106P / Q218D / D305T / Q312R, D106P / Q218D / Q312R, V27W / D106P / D305T, V27W / H35Y / T94N / D106P / D305S / Q312R, H35Y / Q218D / Q312R, Q218D / Q312R, V27W / D106P / Q218D, H35Y / D106P / Q312R, V27W / D106P, or V27W / D106P / D305S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

[0322] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of SEQ ID NO: 1766, or to the reference sequence corresponding to SEQ ID NO: 1766, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0323] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1818-1870, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1818- 1870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0324] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 34, 112, 126, 128, 155, 179, 183, 229, 236, 248, 253, 254, 292, 293, or 299, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0325] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 34C, 112I, 126L, 128V, 155T, 179N, 183S, 183V, 229C, 229V, 236C, 248E, 253D, 253E, 253G, 253H, 253K, 253L, 253S, 253T, 253V, 254M, 254T, 292I, 292V, 293M, or 299G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0326] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution N34C, Q112I, A126L, H128V, G155T, Q179N, L183S, L183V, T229C, T229V, A236C, P248E, R253D, R253E, R253G, R253H, R253K, R253L, R253S, R253T, R253V, R254M, R254T, L292I, L292V, K293M, or T299G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

[0327] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or to the reference sequence corresponding to SEQ ID NO: 1826, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.Docket No. CX10-257WO3

[0328] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1872-1936, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1872- 1936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0329] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 14, 38, 46, 108, 146, 211, 243, 275, 303, 311, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0330] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 14S, 14T, 38R, 46I, 108A, 146C, 211R, 243F, 275S, 303D, 303S, 311E, 320G, or 320R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0331] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution A14S, A14T, P38R, V46I, L108A, V146C, A211R, K243F, H275S, R303D, R303S, D311E, A320G, or A320R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0332] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 94, 106, 108, 194, 274, 303, 307, or 315, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0333] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 94M, 106L, 108E, 194R, 274R, 303V, 307G, or 315S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0334] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution T94M, D106L, L108E, E194R, Q274R, R303V, A307G, or Q315S, orDocket No. CX10-257WO3 combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0335] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 112 / 183 / 292 / 299, 112 / 292, 126 / 299, 155, 155 / 183 / 299, 155 / 299, 183 / 255 / 299, 183 / 299, 229 / 299, 255 / 299, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0336] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 112I / 183V / 292I / 299G, 112I / 292I, 126L / 299G, 155T, 155T / 183V / 299G, 155T / 299G, 183V / 255I / 299G, 183V / 299G, 229H / 299G, 255I / 299G, or 292I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0337] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set Q112I / L183V / L292I / T299G, Q112I / L292I, A126L / T299G, G155T, G155T / L183V / T299G, G155T / T299G, L183V / V255I / T299G, L183V / T299G, T229H / T299G, V255I / T299G, or L292I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

[0338] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or to the reference sequence corresponding to SEQ ID NO: 1918, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0339] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1938-2010, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1938- 2010, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.Docket No. CX10-257WO3

[0340] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 26, 27, 28, 29, 31, 33, 35, 42, 136, 212, 218, 237, 239, 245, 246, 252, 276, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0341] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 26F, 27C, 27G, 27R, 27S, 28L, 28M, 29G, 31H, 33A, 33P, 35D, 35E, 42G, 42N, 42Q, 136F, 212A, 218R, 237K, 237L, 237Q, 239A, 239G, 239H, 239I, 239T, 239W, 245F, 245Q, 246A, 246T, 252A, 252G, 276G, 316A, or 316I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0342] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution M26F, W27C, W27G, W27R, W27S, F28L, F28M, P29G, Y31H, K33A, K33P, H35D, H35E, H42G, H42N, H42Q, Y136F, R212A, Q218R, V237K, V237L, V237Q, M239A, M239G, M239H, M239I, M239T, M239W, Y245F, Y245Q, V246A, V246T, T252A, T252G, E276G, Y316A, or Y316I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

[0343] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or to the reference sequence corresponding to SEQ ID NO: 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0344] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2012-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2012- 2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0345] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 26 / 27, 26 / 27 / 28, 26 / 27 / 28 / 31, 26 / 27 / 28 / 245, 26 / 28 / 31, 27, 27 / 28, 27 / 28 / 29 / 126, 27 / 29, 27 / 29 / 126, 27 / 29 / 237, 27 / 29 / 245,Docket No. CX10-257WO3 27 / 31 / 126 / 245, 27 / 31 / 155, 27 / 126, 27 / 126 / 155, 27 / 126 / 237, 27 / 126 / 237 / 245, 27 / 237, 27 / 245, 28, 28 / 31, 28 / 31 / 239, 28 / 42 / 126 / 245, 28 / 42 / 245, 28 / 239, 29 / 31, 29 / 31 / 126 / 239, 30 / 31 / 33 / 35 / 126 / 218, 30 / 31 / 33 / 126 / 276 / 316, 30 / 33 / 35, 31 / 33 / 218 / 276, 31 / 126, 31 / 126 / 155, 33, 33 / 126 / 239, 42, 126, 126 / 155, 126 / 155 / 237, 126 / 155 / 239 / 276, 126 / 155 / 239 / 316, 126 / 239, 126 / 239 / 316, 126 / 245, 126 / 316, 155 / 276 / 316, 155 / 316, 218, 218 / 239 / 316, 239, 245, 252 / 276, or 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0346] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 26F / 27G / 28M, 26F / 27R, 26F / 27R / 28L / 31H, 26F / 27R / 28M / 245F, 26F / 28M / 31H, 27G, 27G / 28M, 27G / 29G, 27G / 29G / 237K, 27G / 29G / 237Q, 27G / 29G / 245F, 27G / 31H / 126A / 245F, 27G / 31H / 155T, 27G / 126A, 27G / 126A / 155T, 27G / 126A / 237K, 27G / 126A / 237Q / 245F, 27G / 237K, 27G / 245F, 27R, 27R / 28M / 29G / 126A, 27R / 29G / 126A, 27R / 245F, 28L / 31H / 239T, 28L / 239T, 28M, 28M / 31H, 28M / 42N / 126A / 245F, 28M / 42N / 245F, 29G / 31H, 29G / 31H / 126A / 239T, 30G / 31H / 33A / 35D / 126A / 218N, 30G / 31H / 33A / 126A / 276G / 316I, 30G / 33A / 35D, 31H / 33A / 218N / 276G, 31H / 126A, 31H / 126A / 155T, 33A, 33A / 126A / 239H, 42N, 126A, 126A / 155T, 126A / 155T / 237K, 126A / 155T / 239H / 316I, 126A / 155T / 239T / 276G, 126A / 239H, 126A / 239H / 316I, 126A / 245F, 126A / 316I, 155T / 276G / 316I, 155T / 316I, 218N, 218N / 239H / 316I, 239H, 245F, 252T / 276G, or 316I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

[0347] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set M26F / W27G / F28M, M26F / W27R, M26F / W27R / F28L / Y31H, M26F / W27R / F28M / Y245F, M26F / F28M / Y31H, W27G, W27G / F28M, W27G / P29G, W27G / P29G / V237K, W27G / P29G / V237Q, W27G / P29G / Y245F, W27G / Y31H / L126A / Y245F, W27G / Y31H / G155T, W27G / L126A, W27G / L126A / G155T, W27G / L126A / V237K, W27G / L126A / V237Q / Y245F, W27G / V237K, W27G / Y245F, W27R, W27R / F28M / P29G / L126A, W27R / P29G / L126A, W27R / Y245F, F28L / Y31H / M239T, F28L / M239T, F28M, F28M / Y31H, F28M / H42N / L126A / Y245F, F28M / H42N / Y245F, P29G / Y31H, P29G / Y31H / L126A / M239T, D30G / Y31H / K33A / H35D / L126A / Q218N, D30G / Y31H / K33A / L126A / E276G / Y316I, D30G / K33A / H35D, Y31H / K33A / Q218N / E276G, Y31H / L126A, Y31H / L126A / G155T, K33A, K33A / L126A / M239H, H42N, L126A, L126A / G155T, L126A / G155T / V237K, L126A / G155T / M239H / Y316I, L126A / G155T / M239T / E276G, L126A / M239H, L126A / M239H / Y316I, L126A / Y245F, L126A / Y316I, G155T / E276G / Y316I, G155T / Y316I, Q218N, Q218N / M239H / Y316I, M239H, Y245F, A252T / E276G, or Y316I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.Docket No. CX10-257WO3

[0348] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at an amino acid position set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0349] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0350] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0351] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0352] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,Docket No. CX10-257WO3 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0353] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally included 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine kinase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine optionally includes 1, 2, 3, 4, or 5 substitutions.

[0354] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence comprising residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or comprises SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine kinase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered adenosine kinase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenosine optionally includes 1, 2, 3, 4, or 5 substitutions.

[0355] In some embodiments, the engineered adenosine kinase has adenosine kinase activity and at least one improved property as compared to a reference adenosine kinase.

[0356] In some embodiments, the engineered adenosine kinase has increased activity on a nucleoside substrate as compared to the reference adenosine kinase. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, 15, or 20 fold or more activity compared to the reference adenosine kinase. In some embodiments, the increased activity is onDocket No. CX10-257WO3 guanosine, adenosine, cytidine, uridine, or thymidine. Examples of increased activity on nucleosides is presented in the Examples.

[0357] In some embodiments, the engineered adenosine kinase has increased activity (e.g., product formation) on cytidine or guanosine. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 fold or more activity on cytidine and / or guanosine compared to the reference adenosine kinase.

[0358] In some embodiments, the engineered adenosine kinase is capable of converting nucleoside substrate to NMP product at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate adenosine, guanosine, cytidine, uridine, or thymidine to product AMP, GMP, CMP, UMP, or TMP, respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0359] In some embodiments, the engineered kinase has increased activity (e.g., product formation) on 2’-fluoro modified nucleosides. In some embodiments, the 2’-F modified nucleosides are 2’-F-2’- deoxyadenosine, 2’-F-2’-deoxycytidine, 2’-F-2’-deoxyguanosine, and / or 2’-F-2’-deoxyuridine. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 fold or more activity on 2’-F modified nucleosides as compared to the reference adenosine kinase.

[0360] In some embodiments, the engineered kinase has increased activity (e.g., product formation) on 2’-O-CH3modified nucleosides. In some embodiments, the 2’-O-CH3modified nucleosides are 2’-O-CH3adenosine, 2’-O-CH3cytidine, 2’-O-CH3guanosine, and / or 2’-O-CH3uridine. In some embodiments, the engineered adenosine kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 fold or more activity on 2’-O-CH3modified nucleosides as compared to the reference adenosine kinase.

[0361] In some embodiments, the engineered adenosine kinase is capable of converting substrate 2’- fluoro-nucleoside to product 2’-fluoro-NMP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’- fluoro-cytidine, 2’-fluoro-uridine, or 2’-fluoro-thymidine to product 2’-fluoro-AMP, 2’-fluoro-GMP, 2’-fluoro-CMP, 2’-fluoro-UMP, or 2’-fluoro-TMP, respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0362] In some embodiments, the engineered adenosine kinase is capable of converting 2’-O-methyl- nucleoside substrate to product 2’-O-methyl-NMP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenosine kinase is capable of converting substrate 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine to product 2’-O- methyl-AMP, 2’-O-methyl-GMP, 2’-O-methyl-CMP, 2’-O-methyl-UMP, or 2’-O-methyl-TMP,Docket No. CX10-257WO3 respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0363] Exemplary conditions for the conversion of a nucleoside to the corresponding NMP, including the conversion of 2’-fluoro-nucleoside to 2’-fluoro NMP and the conversion of 2’-O-methyl nucleoside to 2’-O-methyl NMP are provided in the Examples.

[0364] In some embodiments, the engineered adenosine kinase has increased thermostability as compared to the reference adenosine kinase. In some embodiments, increased thermal stability is at condition of 45 °C for 60 min.

[0365] In some embodiments, the engineered adenosine kinase has an improved property selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine and / or guanosine, iv) increased activity on 2’-F modified nucleosides, and v) increased activity on 2’-O-CH3modified nucleosides, or any combinations of i), ii), iii), iv), and v), compared to a reference adenosine kinase.

[0366] In some embodiments, for comparing the improved property, the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or an amino acid sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942. In some embodiments, the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 2, or an amino acid sequence corresponding to SEQ ID NO: 2.

[0367]

[0368] In some embodiments, the present disclosure further provides an adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to (a) a sequence corresponding to residues 12 to 339 of SEQ ID NO: 1818, a sequence corresponding to residues 12 to 339 of SEQ ID NO: 1820, or (b) a sequence corresponding to SEQ ID NO: 2; a sequence corresponding to SEQ ID NO: 1818; or a sequence corresponding to SEQ ID NO: 1820.

[0369] In some embodiments, the present disclosure provides an adenosine kinase comprising an amino acid sequence comprising: (a) residues 12 to 339 of SEQ ID NO: 1818, or residues 12 to 339 of SEQ ID NO: 1820, (b) SEQ ID NO: 2;Docket No. CX10-257WO3 SEQ ID NO: 1818; or SEQ ID NO: 1820.

[0370] In some embodiments, the engineered adenosine kinase is provided in the form of a fusion polypeptide. In some embodiments, the engineered adenosine kinase is fused to variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification. In some embodiments, the fusion protein or polypeptide of the engineered adenosine kinase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion protein or polypeptide of the engineered adenosine kinase comprises a polylysine, for example, 2-12 lysine units, such as for conjugation to a support medium. In some embodiments, the fusion protein of the engineered adenosine kinase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered adenosine kinase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) terminus of engineered adenosine kinase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the adenosine kinase polypeptide.

[0371] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of engineered adenosine kinase polypeptides described herein. Thus, for each and every embodiment herein of an engineered adenosine kinase, a functional fragment or biologically active fragment of the engineered adenosine kinase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of an engineered adenosine kinase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the adenosine kinase polypeptide from which it was derived (i.e., the parent adenosine kinase). In some embodiments, functional fragments or biologically active fragments comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the adenosine kinase. In some embodiments the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0372] In some embodiments, a functional fragment of an engineered adenosine kinase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered adenosine kinase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

[0373] In some embodiments, the functional fragments or biologically active fragments of the engineered adenosine kinase polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of an engineered adenosine kinase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered adenosineDocket No. CX10-257WO3 kinase displays the enhanced or improved property associated with the mutation or mutation set in the parent adenosine kinase.

[0374] In some embodiments, the engineered adenosine kinase is purified, as described herein. In some embodiments, the purified preparation has the engineered adenosine kinase at least 60%, 70%, 80%, 85%, 90%, or 95% greater of the protein content of the preparation.

[0375] In some embodiments, an engineered adenosine kinase described herein is provided immobilized on a substrate or support medium, such as a solid substrate, a porous substrate, a membrane, or particles. The polypeptide can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, and collagen. In some embodiments, the solid matrices, includes, among others, activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0376] In some embodiments, the engineered adenosine kinase is immobilized on the surface of a support material. In some embodiments, the polypeptide is adsorbed on the support material. In some embodiments, the polypeptide is immobilized on the support material by covalent attachment. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polymethacrylate, polyacrylate, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.

[0377] As further described below, solid supports useful for immobilizing the engineered polypeptides of the present disclosure include, but are not limited to, beads or resins comprising polymethacrylate or polyacrylate with epoxide functional groups, polymethacrylate or polyacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate or polyacrylate with octadecyl functional groups. In some embodiments, exemplary solid supports useful for immobilizing the engineered polypeptides of the present invention include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120). Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells

[0378] In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered adenosine kinases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the engineered adenosine kinase. In some embodiments, an expression construct containing at least oneDocket No. CX10-257WO3 heterologous polynucleotide encoding the engineered adenosine kinase polypeptide(s) is introduced into appropriate host cells to express the corresponding adenosine kinase polypeptide(s).

[0379] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered adenosine kinase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the engineered adenosine kinase polypeptides described herein by selecting combinations based on the possible codon choices, and all such polynucleotide variants are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2) and in the Sequence Listing.

[0380] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding an engineered adenosine kinase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0381] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure comprises a polynucleotide sequence encoding an engineered adenosine kinase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide has preferred codons for expression (e.g., codon optimized). In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized for expression in eukaryotic or prokaryotic cells, as further discussed below.

[0382] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808 and 1818- 2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2- 1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or moreDocket No. CX10-257WO3 substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0383] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to a reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0384] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0385] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0386] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Docket No. CX10-257WO3 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818- 2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4- 1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0387] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0388] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0389] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 109 / 111, 104 / 111, 38, 40, 111, 55 / 316, 258, 106 / 111, 103 / 111, 121, 207, 316, 43, 254, 233, 130, 249, 107 / 111, 253, 210, 136 / 249, 208, 34, 211, 308, 181, 248, 56, 89, 67, 111 / 121 / 233 / 316, 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316 / 320, 53 / 86 / 111 / 121 / 169 / 181 / 211 / 233 / 248 / 308 / 316 / 320, or 53 / 77 / 79 / 86 / 111 / 121 / 169 / 170 / 181 / 211 / 233 / 234 / 248 / 308 / 316 / 320, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.Docket No. CX10-257WO3

[0390] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least substitution at an amino acid position set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0391] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0392] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0393] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0394] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2,Docket No. CX10-257WO3 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0395] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0396] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124.

[0397] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0398] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutionsDocket No. CX10-257WO3 relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0399] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 27, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 276, 287, 288, 291, 292, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0400] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

[0401] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or to the reference sequence corresponding to SEQ ID NO: 90, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.Docket No. CX10-257WO3

[0402] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0403] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 111 / 233 / 316, 43 / 111 / 233 / 316, 109 / 181 / 316, 109 / 316, 111 / 233, 109 / 233 / 316, 38 / 208 / 211 / 308, 111 / 181 / 233 / 316, 109 / 111 / 233 / 316, 111 / 181 / 233 / 239 / 316, 111 / 233 / 239 / 316, 109 / 111 / 181 / 233 / 316, 111 / 181 / 316, 43 / 233, 109 / 111 / 316, 181 / 233, 34 / 211 / 248 / 249, 34 / 38 / 248 / 249, 34 / 308, 233 / 316, 43 / 233 / 316, 181 / 316, 34 / 249, 43 / 181 / 233 / 316, 34 / 38 / 103 / 116 / 253, 34, 40 / 43 / 109 / 181 / 233 / 316, 34 / 38 / 208 / 253, 103 / 249 / 253 / 308, 34 / 38 / 116 / 208, 38 / 249, 34 / 38 / 116 / 211 / 248 / 253 / 308, 43 / 109 / 316, 116 / 253, 43 / 111 / 181 / 316, 34 / 38 / 308, 109 / 111 / 181, 111 / 130 / 233, 233, 34 / 38 / 116 / 308, 109 / 233, 40 / 43 / 109 / 111 / 181 / 233 / 316, 109 / 130 / 181 / 233, 34 / 38 / 253, 253, 34 / 38 / 103 / 208 / 249, 103 / 208 / 210 / 253, 130 / 181 / 233 / 316, 109 / 111 / 130 / 181 / 233, 34 / 38 / 210 / 211 / 253 / 303, 103 / 116 / 208, 34 / 116 / 248 / 252 / 253, 34 / 38 / 103 / 210 / 211 / 249, 40 / 233 / 316, 34 / 38, 109 / 130 / 233, 111 / 130 / 181 / 233 / 316, 34 / 38 / 249 / 253, 34 / 38 / 208 / 248, 40 / 109 / 111 / 181 / 233 / 316, 130 / 316, 40 / 316, 38 / 116, 34 / 38 / 116, 38 / 116 / 208 / 210 / 249 / 253, 40 / 130 / 181 / 233 / 316, 103 / 248 / 249, or 103 / 248, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO: 90.

[0404] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or to the reference sequence corresponding to SEQ ID NO: 254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0405] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Docket No. CX10-257WO3 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0406] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 77 / 111 / 170 / 211 / 236, 77 / 111 / 211, 77 / 211, 53 / 77 / 170 / 211 / 320, 53 / 77 / 211 / 234 / 320, 77 / 79 / 211 / 316, 77 / 111 / 234 / 236 / 308, 111 / 170 / 211 / 234 / 236 / 248, 111 / 169 / 170 / 234 / 236 / 248, 77 / 211 / 248 / 320, 53 / 169 / 170 / 234 / 320, 111 / 211 / 234 / 236 / 248, 77, 53 / 77 / 111 / 170 / 248, 211 / 234 / 248, 77 / 86 / 170 / 211 / 234 / 308 / 320, 77 / 86, 53 / 111 / 170, 77 / 79 / 111, 53 / 77 / 111 / 169 / 236, 77 / 211 / 234, 211, 53 / 77, 86 / 169 / 234, 53 / 211 / 234 / 248, 111 / 169 / 170 / 236 / 248, 77 / 79 / 211, 77 / 79 / 169 / 170, 77 / 79 / 86 / 169 / 236, 79 / 211 / 248, 79 / 211, 53 / 86 / 211 / 234 / 236, 79 / 170 / 211, 308, 53 / 211, 169 / 234 / 236, 77 / 79 / 169 / 234 / 236, 53 / 77 / 79, 77 / 111, 53 / 234 / 236, 169 / 170, 53 / 77 / 79 / 169, 53 / 86 / 234 / 236, 53 / 169 / 234 / 236, 53 / 234, 77 / 79 / 86 / 170 / 211 / 234 / 236 / 308 / 320, 53 / 170, 77 / 248, 53 / 86, 252, 180, 129, 121, 255, 119, 256, 237, 249, 20, 234, 254, 56, 287, 317, 299, 229, 250, 295, 144 / 255, 291, 181, 233, 248, 231, 251, 128, 156, 247, 288, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO: 254.

[0407] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or to the reference sequence corresponding to SEQ ID NO: 370, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0408] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding toDocket No. CX10-257WO3 residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0409] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 299, 21, 113, 152, 169 / 255, 251, 294, 255, 202 / 211, 31, 129, 252, 27 / 64, 128, 248, 234, 288, 20, 111, 112, 181, 119, 64, 155, 256, 254, 292, 265, 177, 232, 120, 183, 234 / 252, 181 / 250 / 252 / 299, 119 / 181 / 234 / 250 / 299, 129 / 234 / 252, 119 / 129 / 234, 119 / 181 / 234 / 250 / 256 / 299, 119 / 181 / 250 / 252, 119 / 181 / 234 / 299, 234 / 250 / 252, 119 / 181 / 252 / 299, 129 / 181 / 234 / 299, 56 / 121 / 229, 181 / 255 / 299, 119 / 299, 119 / 250 / 255 / 299, 80 / 119 / 252, 119 / 129, 119 / 129 / 250, 181 / 234 / 252 / 299, 181 / 250 / 299, 252 / 299, 119 / 181 / 299, 119 / 129 / 299, 119 / 129 / 234 / 252, 119 / 129 / 234 / 250 / 299, 119 / 181 / 234 / 252, 129 / 234 / 255 / 299, 181 / 299, 250 / 252, 250 / 255 / 299, 119 / 234 / 299, 119 / 181 / 250, 56 / 237, 250 / 299, 119 / 234 / 255 / 256 / 299, 56 / 229 / 254, 119 / 129 / 181, 56 / 121 / 295 / 317, 119 / 181 / 250 / 255 / 256, 229, 250, 119 / 234, 20 / 229 / 237 / 317, 181 / 250, 119 / 255, 121 / 156 / 229, 56 / 121 / 237, 237 / 317, 56 / 295, 56 / 121 / 229 / 236 / 254 / 295, 56 / 254, 119 / 250 / 299, 229 / 237 / 295, 20 / 229 / 317, 229 / 295, 156 / 229, 56 / 121 / 229 / 237 / 295, 237 / 254, 20 / 56, 56 / 156, 56 / 156 / 254 / 295, 20 / 56 / 229, 119 / 129 / 181 / 234, 237, 56 / 156 / 229 / 237, 20 / 56 / 102 / 229, 119 / 181 / 234, 20 / 229, 56 / 121 / 156 / 237, 56 / 156 / 229, 20 / 56 / 156 / 229 / 237 / 295, 20 / 56 / 121 / 237 / 317, 156, 156 / 237 / 295, 20 / 56 / 121, 119 / 181, 20 / 56 / 229 / 254, 56, 121 / 254, 20 / 295, 255 / 256, 56 / 121 / 229 / 254, or 119 / 129 / 234 / 255 / 256, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO: 370.

[0410] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO: 734, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0411] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192, and 1194, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192, and 1194, wherein the amino acid sequence comprises one or more substitutions relative to theDocket No. CX10-257WO3 reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO: 734.

[0412] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenosine kinase comprising an amino acid sequence comp...

Claims

Docket No. CX10-257WO3 CLAIMS What is claimed is:

1. An engineered adenosine kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808, and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1142, 1194-1808, and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942 or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

2. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942 or to a reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942 or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

3. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

4. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366,Docket No. CX10-257WO3 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4- 1142, 1194-1808, and 1818-2124 or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808, and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 46, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 179, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 212, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 274, 275, 276, 287, 288, 291, 292, 293, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 315, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered adenosine kinase of any one of claims 1-6, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D / G / N / Q, 43E / K / R / S / V, 46I, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93A, 94M / N, 100G, 101F / W, 102I, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T,Docket No. CX10-257WO3 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 190I, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / R / T / W, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M, 294L, 295A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

8. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

9. The engineered adenosine kinase of any one of claims 1-5 and 8, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 24I / N / V, 27A / C / G / K / P / R / S / W, 31F / H / L / R / W / Y, 40H / M / R / W, 43E / K / R / S / V, 51F / G / S, 52C / D / F / G / S / T, 74F / I / K / R / T / V, 77V, 79A, 109E / G / L / P / R / S / T, 113G / S, 119C / G / M / Q / R / S / T / V, 121F / H / L / M / Q, 126L / S, 129A / G / S, 169A / C / D / I / L / M / S, 181A / C / L / V, 185S / T, 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / T / W, 233A / S / V, 234A / M / N / R / V / Y, 237K / I / L / M / Q / V, 239A / F / G / H / I / M / Q / R / T / W, 244R / W, 246A / R / S / T / V, 249A / G / I / L / M / R / S, 251K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 276D / E / G / S, 299G / S, 305S / T, 312R, or 316A / D / E / I / K / S / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

10. The engineered adenosine kinase of any one of claims 1-5 and 8, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acidDocket No. CX10-257WO3 residue 24N, 27W, 31Y, 40R, 43R, 51F, 52D, 74R, 77V, 79A, 109S, 113G, 119M, 121H / Q, 126L, 129G / S, 169M, 181L / V, 185S, 207R, 211A / T, 233S, 234A, 237V, 239M, 244R, 246V, 249A, 251K, 252A / T, 253S, 276E, 299G, 305T, 312R, or 316D / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

11. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 109 / 111, 104 / 111, 38, 40, 111, 55 / 316, 258, 106 / 111, 103 / 111, 121, 207, 316, 43, 254, 233, 130, 249, 107 / 111, 253, 210, 136 / 249, 208, 34, 211, 308, 181, 248, 56, 89, 67, 111 / 121 / 233 / 316, 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316, 53 / 111 / 121 / 181 / 211 / 233 / 248 / 308 / 316 / 320, 53 / 86 / 111 / 121 / 169 / 181 / 211 / 233 / 248 / 308 / 316 / 320, or 53 / 77 / 79 / 86 / 111 / 121 / 169 / 170 / 181 / 211 / 233 / 234 / 248 / 308 / 316 / 320, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

12. The engineered adenosine kinase of any one of claims 1-5 and 11, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set 109R / 111A, 104G / 111A, 38C, 104A / 111A, 104Y / 111A, 40R, 111G, 109S / 111A, 55H / 316S, 109G / 111A, 258V, 106E / 111A, 104N / 111A, 109T / 111A, 109P / 111A, 103Q / 111A, 104D / 111A, 104R / 111A, 121F, 104S / 111A, 104T / 111A, 207R, 111A, 109E / 111A, 103R / 111A, 109L / 111A, 104L / 111A, 106P / 111A, 316E, 43R, 103L / 111A, 316S, 254P, 233S, 130A, 316D, 249G, 104P / 111A, 104W / 111A, 249S, 107T / 111A, 106G / 111A, 253P, 121H, 210E, 136H / 249G, 208V, 34K, 107S / 111A, 107G / 111A, 211T, 308F, 181L, 233V, 107A / 111A, 34R, 38V, 248R, 56S, 316K, 248V, 248K, 89T, 308V, 106R / 111A, 67G, 111A / 121H / 233S / 316S, 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S, 53L / 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S, 53L / 111A / 121H / 181L / 211T / 233S / 248R / 308L / 316S / 320S, 53L / 86Q / 111A / 121H / 169A / 181L / 211T / 233S / 248R / 308L / 316S / 320S, or 53L / 77V / 79A / 86Q / 111A / 121H / 169A / 170D / 181L / 211T / 233S / 234R / 248R / 308L / 316S / 320S, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

13. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the referenceDocket No. CX10-257WO3 sequence corresponding to SEQ ID NO:

2.

14. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

15. The engineered adenosine kinase of any one of claims 1-5, wherein the adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

16. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

17. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124.

18. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464,Docket No. CX10-257WO3 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

19. The engineered adenosine kinase of claim 1 or 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194- 1808 and 1818-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

20. The engineered adenosine kinase of claim 18 or 19, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 13, 14, 20, 21, 23, 24, 27, 29, 30, 31, 33, 34, 35, 38, 40, 41, 42, 43, 49, 50, 51, 52, 53, 54, 55, 56, 64, 67, 73, 74, 77, 79, 80, 83, 86, 87, 89, 90, 91, 92, 93, 94, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 112, 113, 116, 119, 120, 121, 126, 128, 129, 130, 133, 134, 136, 141, 144, 146, 147, 151, 152, 155, 156, 157, 160, 162, 163, 164, 167, 168, 169, 170, 171, 172, 177, 180, 181, 182, 183, 185, 187, 188, 190, 191, 192, 194, 195, 196, 197, 202, 206, 207, 208, 210, 211, 215, 216, 217, 218, 221, 222, 225, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 258, 265, 271, 276, 287, 288, 291, 292, 294, 295, 296, 299, 303, 305, 307, 308, 310, 311, 312, 314, 316, 317, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

21. The engineered adenosine kinase of any one of claims 18-20, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 13A / E / P, 14S / T / V, 20F / V, 21G, 23N, 24I / N / V, 26F, 27A / C / G / K / P / R / S / W, 28L / M, 29G / R, 30G / Y, 31F / H / L / R / W / Y, 33A / P / Q / S, 34C / G / K / R, 35D / E / S / V / Y, 38C / R / V, 40H / M / R / W, 41L, 42D,Docket No. CX10-257WO3 43E / K / R / S / V, 42G / N / Q, 46I, 49M, 50C, 51F / G / S, 52C / D / F / G / S / T, 53L / R, 54V, 55H, 56F / G / S / Y, 64M / T, 67G, 73A / C / E / M, 74F / I / K / R / T / V, 77V, 79A, 80M / S / V, 83F / L / S / T / V, 86L / Q, 87K, 89G / K / L / T, 90L / R / S / T / V, 91Y, 92M, 93A, 94M / N, 100G, 101F / W, 102I, 103L / Q / R, 104A / C / D / G / L / N / P / R / S / T / W / Y, 106E / G / L / M / P / R / T, 107A / G / H / S / T, 108A / C / E, 109E / G / L / P / R / S / T, 111A / G / L, 112I / L, 113G / S, 116M, 119C / G / M / Q / R / S / T / V, 120C, 121F / H / L / M / Q, 126A / L / S, 128F / V / W, 129A / G / S, 130A, 133A / L, 134H, 136F / H, 141S, 144Q / S, 146C / I, 147A / N / S, 151L, 152A / C, 155A / I / N / T / V / W, 156Y, 157P, 160L, 162F, 163G, 164A / G / K / Q / S / W, 167F / T / V / W, 168R / V, 169A / C / D / I / L / M / S, 170D / H / K / M / R / S / V, 171C / V, 172S, 177V, 179N, 180Q, 181A / C / L / M / V, 182G, 183M / S / V, 185S / T, 187H / Q / S / V, 188T, 190I, 191A / R / S / W, 192L, 194A / R, 195G / M / T / V, 196S, 197R, 202F, 206A / H, 207A / L / M / R / T / V, 208V, 210E / L, 211A / C / F / H / I / K / L / M / T / W, 211R, 212A, 215L, 216V, 217A / V, 218D / L / N / R / V, 221A, 222A / G / M / Q, 225E / G, 229C / H / V, 231L / M / V, 232S, 233A / S / V, 234A / M / N / R / V / Y, 236C / S / V, 237K / I / L / M / Q / V, 238L, 239A / F / G / H / I / M / Q / R / T / W, 240L / V, 241R, 242D / T, 243F / G / R / Y, 244R / W, 245F / Q / W, 246A / T / D / R / S / T / V, 247L, 248E / G / K / L / R / V, 249A / G / I / L / M / R / S, 250H / I / M / T, 251H / K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 254I / L / M / P / Q / T, 255A / C / E / G / I / K / S / W, 256C / H / M, 258R / S / V, 265Y, 271W, 274R, 275S, 276D / E / G / S, 287A / S / V / Y, 288V, 291G, 292F / I / K / V, 293M, 294L, 295A, 296N, 299G / S, 303D / F / H / M / S / V / W, 305S / T, 307F / G, 308D / F / L / V, 310A / C, 311E / F / G / L / M / Q / R / S / T, 312R, 314Q / S / V, 315S, 316A / D / E / I / K / S / Y, 317M / W / Y, or 320G / R / S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

22. The engineered adenosine kinase of any one of claims 18-20, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 24, 27, 31, 40, 43, 51, 52, 74, 77, 79, 109, 113, 119, 121, 126, 129, 169, 181, 185, 207, 211, 233, 234, 237, 239, 244, 246, 249, 251, 252, 253, 276, 299, 305, 312, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

23. The engineered adenosine kinase of any one of claims 18-20, and 22, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 24I / N / V, 27A / C / G / K / P / R / S / W, 31F / H / L / R / W / Y, 40H / M / R / W, 43E / K / R / S / V, 51F / G / S, 52C / D / F / G / S / T, 74F / I / K / R / T / V, 77V, 79A, 109E / G / L / P / R / S / T, 113G / S, 119C / G / M / Q / R / S / T / V, 121F / H / L / M / Q, 126A / L / S, 129A / G / S, 169A / C / D / I / L / M / S, 181A / C / L / M / V, 185S / T,Docket No. CX10-257WO3 207A / L / M / R / T / V, 211A / C / F / H / I / K / L / M / T / W, 233A / S / V, 234A / M / N / R / V / Y, 237K / I / L / M / Q / V, 239A / F / G / H / I / M / Q / R / T / W, 244R / W, 246A / D / R / S / T / V, 249A / G / I / L / M / R / S, 251H / K / L / T / V, 252A / C / G / K / T / V, 253D / E / G / H / K / L / P / S / T / V, 276D / E / G / S, 299G / S, 305S / T, 312R, or 316A / D / E / I / K / S / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

24. The engineered adenosine kinase of any one of claims 18-20, and 22, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 24N, 27W, 31Y, 40R, 43R, 51F, 52D, 74R, 77V, 79A, 109S, 113G, 119M, 121H / Q, 126L, 129G / S, 169M, 181L / V, 185S, 207R, 211A / T, 233S, 234A, 237V, 239M, 244R, 246V, 249A, 251K, 252A / T, 253S, 276E, 299G, 305T, 312R, or 316D / Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

25. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or to the reference sequence corresponding to SEQ ID NO: 90, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO:

90.

26. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 148 -358, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO:

90.

27. The engineered adenosine kinase of claim 25 or 26 wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 111 / 233 / 316, 43 / 111 / 233 / 316, 109 / 181 / 316, 109 / 316, 111 / 233, 109 / 233 / 316,Docket No. CX10-257WO3 38 / 208 / 211 / 308, 111 / 181 / 233 / 316, 109 / 111 / 233 / 316, 111 / 181 / 233 / 239 / 316, 111 / 233 / 239 / 316, 109 / 111 / 181 / 233 / 316, 111 / 181 / 316, 43 / 233, 109 / 111 / 316, 181 / 233, 34 / 211 / 248 / 249, 34 / 38 / 248 / 249, 34 / 308, 233 / 316, 43 / 233 / 316, 181 / 316, 34 / 249, 43 / 181 / 233 / 316, 34 / 38 / 103 / 116 / 253, 34, 40 / 43 / 109 / 181 / 233 / 316, 34 / 38 / 208 / 253, 103 / 249 / 253 / 308, 34 / 38 / 116 / 208, 38 / 249, 34 / 38 / 116 / 211 / 248 / 253 / 308, 43 / 109 / 316, 116 / 253, 43 / 111 / 181 / 316, 34 / 38 / 308, 109 / 111 / 181, 111 / 130 / 233, 233, 34 / 38 / 116 / 308, 109 / 233, 40 / 43 / 109 / 111 / 181 / 233 / 316, 109 / 130 / 181 / 233, 34 / 38 / 253, 253, 34 / 38 / 103 / 208 / 249, 103 / 208 / 210 / 253, 130 / 181 / 233 / 316, 109 / 111 / 130 / 181 / 233, 34 / 38 / 210 / 211 / 253 / 303, 103 / 116 / 208, 34 / 116 / 248 / 252 / 253, 34 / 38 / 103 / 210 / 211 / 249, 40 / 233 / 316, 34 / 38, 109 / 130 / 233, 111 / 130 / 181 / 233 / 316, 34 / 38 / 249 / 253, 34 / 38 / 208 / 248, 40 / 109 / 111 / 181 / 233 / 316, 130 / 316, 40 / 316, 38 / 116, 34 / 38 / 116, 38 / 116 / 208 / 210 / 249 / 253, 40 / 130 / 181 / 233 / 316, 103 / 248 / 249, or 103 / 248, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO:

90.

28. The engineered adenosine kinase of any one of claims 25-27, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 111A / 233S / 316S, 111A / 233S / 316E, 43R / 111A / 233S / 316S, 109S / 181L / 316S, 109S / 316E, 111A / 233S, 109S / 233S / 316E, 38C / 208V / 211T / 308F, 111A / 181L / 233S / 316S, 109S / 111A / 233S / 316S, 111A / 181L / 233S / 239Q / 316S, 111A / 233S / 239Q / 316S, 109S / 111A / 181L / 233S / 316S, 111A / 181L / 316E, 43R / 233S, 109S / 111A / 316S, 181L / 233S, 34K / 211T / 248R / 249G, 34K / 38C / 248R / 249G, 34K / 308F, 233S / 316E, 43R / 233S / 316D, 181L / 316S, 34R / 249G, 43R / 233S / 316S, 43R / 181L / 233S / 316S, 34R / 38C / 103R / 116M / 253P, 34K, 40R / 43R / 109S / 181L / 233S / 316D, 34R / 38C / 208V / 253P, 103R / 249G / 253P / 308F, 34R / 38C / 116M / 208V, 38C / 249G, 34K / 38C / 116M / 211T / 248R / 253P / 308L, 43R / 109S / 316S, 116M / 253P, 109S / 111A / 233S / 316D, 43R / 111A / 181L / 316E, 34R / 38C / 308F, 109S / 111A / 181L, 111A / 130A / 233S, 233S, 34R / 38C / 116M / 308F, 109S / 233S, 40R / 43R / 109S / 111A / 181L / 233S / 316S, 109S / 130A / 181L / 233S, 34K / 38C / 253P, 253P, 34R / 38C / 103R / 208V / 249S, 103R / 208V / 210E / 253P, 130A / 181L / 233S / 316D, 109S / 111A / 130A / 181L / 233S, 34R / 38C / 210E / 211T / 253P / 303H, 103R / 116M / 208V, 34R / 116M / 248R / 252K / 253P, 34R / 38C / 103R / 210E / 211T / 249G, 40R / 233S / 316S, 34R / 38C, 109S / 130A / 233S, 111A / 130A / 181L / 233S / 316S, 34K / 38C / 249G / 253P, 34K / 38C / 208V / 248R, 40R / 109S / 111A / 181L / 233S / 316S, 130A / 316S, 40R / 316S, 38C / 116M, 34K / 38C / 116M, 38C / 116M / 208V / 210E / 249G / 253P, 40R / 130A / 181L / 233S / 316D, 34R / 38C / 116M, 34R / 38C / 253P, 130A / 316D, 103R / 248R / 249G, or 103R / 248R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 90, or relative to the reference sequence corresponding to SEQ ID NO:

90.

29. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,Docket No. CX10-257WO3 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or to the reference sequence corresponding to SEQ ID NO: 254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO:

254.

30. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 360-592, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO:

254.

31. The engineered adenosine kinase of claim 29 or 30, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 77 / 111 / 170 / 211 / 236, 77 / 111 / 211, 77 / 211, 53 / 77 / 170 / 211 / 320, 53 / 77 / 211 / 234 / 320, 77 / 79 / 211 / 316, 77 / 111 / 234 / 236 / 308, 111 / 170 / 211 / 234 / 236 / 248, 111 / 169 / 170 / 234 / 236 / 248, 77 / 211 / 248 / 320, 53 / 169 / 170 / 234 / 320, 111 / 211 / 234 / 236 / 248, 77, 53 / 77 / 111 / 170 / 248, 211 / 234 / 248, 77 / 86 / 170 / 211 / 234 / 308 / 320, 77 / 86, 53 / 111 / 170, 77 / 79 / 111, 53 / 77 / 111 / 169 / 236, 77 / 211 / 234, 211, 53 / 77, 86 / 169 / 234, 53 / 211 / 234 / 248, 111 / 169 / 170 / 236 / 248, 77 / 79 / 211, 77 / 79 / 169 / 170, 77 / 79 / 86 / 169 / 236, 79 / 211 / 248, 79 / 211, 53 / 86 / 211 / 234 / 236, 79 / 170 / 211, 308, 53 / 211, 169 / 234 / 236, 77 / 79 / 169 / 234 / 236, 53 / 77 / 79, 77 / 111, 53 / 234 / 236, 169 / 170, 53 / 77 / 79 / 169, 53 / 86 / 234 / 236, 53 / 169 / 234 / 236, 53 / 234, 77 / 79 / 86 / 170 / 211 / 234 / 236 / 308 / 320, 53 / 170, 77 / 248, 53 / 86, 252, 180, 129, 121, 255, 119, 256, 237, 249, 20, 234, 254, 56, 287, 317, 299, 229, 250, 295, 144 / 255, 291, 181, 233, 248, 231, 251, 128, 156, 247, 288, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO:

254.

32. The engineered adenosine kinase of any one of claims 29-31, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set 77V / 111A / 170D / 211T / 236V, 77V / 111A / 211T, 77V / 211T, 53L / 77V / 170D / 211T / 320S, 53L / 77V / 211T / 234R / 320S, 77V / 79A / 211T / 316Y, 77V / 111A / 234R / 236V / 308L, 111A / 170D / 211T / 234R / 236V / 248R, 111A / 169A / 170D / 234R / 236V / 248R, 77V / 211T / 248R / 320S, 53L / 169A / 170D / 234R / 320S, 111A / 211T / 234R / 236V / 248R, 77V, 53L / 77V / 111A / 170D / 248R, 211T / 234R / 248R, 77V / 86Q / 170D / 211T / 234R / 308L / 320S, 77V / 86Q, 53L / 111A / 170D, 77V / 79A / 111A, 53L / 77V / 111A / 169A / 236V, 77V / 211T / 234R, 211T, 53L / 77V, 86Q / 169A / 234R,Docket No. CX10-257WO3 53L / 211T / 234R / 248R, 111A / 169A / 170D / 236V / 248R, 77V / 79A / 211T, 77V / 79A / 169A / 170D, 77V / 79A / 86Q / 169A / 236V, 79A / 211T / 248R, 79A / 211T, 53L / 86Q / 211T / 234R / 236V, 79A / 170D / 211T, 308L, 53L / 211T, 169A / 234R / 236V, 77V / 79A / 169A / 234R / 236V, 53L / 77V / 79A, 77V / 111A, 53L / 234R / 236V, 169A / 170D, 53L / 77V / 79A / 169A, 53L / 86Q / 234R / 236V, 53L / 169A / 234R / 236V, 53L / 234R, 77V / 79A / 86Q / 170D / 211T / 234R / 236V / 308L / 320S, 53L / 170D, 77V / 248R, 53L / 86Q, 252T, 180Q, 129G, 121M, 255A, 255S, 121L, 255E, 119M, 256C, 255G, 237I, 249L, 20F, 255K, 237V, 234A, 254Q, 256M, 56Y, 56F, 249M, 119T, 287V, 121F, 119S, 252K, 56G, 317W, 119V, 299S, 229C, 119C, 250I, 254L, 234M, 229V, 234Y, 287Y, 295A, 144Q / 255W, 119Q, 291G, 250T, 181M, 287S, 119R, 317Y, 119G, 250M, 249G, 249I, 20V, 237M, 233A, 248G, 231V, 251L, 128F, 287A, 181V, 156Y, 247L, 288V, 292K, 181A, 249R, or 250H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 254, or relative to the reference sequence corresponding to SEQ ID NO:

254.

33. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or to the reference sequence corresponding to SEQ ID NO: 370, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO:

370.

34. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 594-888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO:

370.

35. The engineered adenosine kinase of claim 33 or 34, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 299, 21, 113, 152, 169 / 255, 251, 294, 255, 202 / 211, 31, 129, 252, 27 / 64, 128, 248, 234, 288, 20, 111, 112, 181, 119, 64, 155, 256, 254, 292, 265, 177, 232, 120, 183, 234 / 252, 181 / 250 / 252 / 299, 119 / 181 / 234 / 250 / 299, 129 / 234 / 252, 119 / 129 / 234, 119 / 181 / 234 / 250 / 256 / 299, 119 / 181 / 250 / 252, 119 / 181 / 234 / 299, 234 / 250 / 252, 119 / 181 / 252 / 299, 129 / 181 / 234 / 299, 56 / 121 / 229, 181 / 255 / 299, 119 / 299, 119 / 250 / 255 / 299, 80 / 119 / 252, 119 / 129, 119 / 129 / 250, 181 / 234 / 252 / 299, 181 / 250 / 299, 252 / 299, 119 / 181 / 299, 119 / 129 / 299, 119 / 129 / 234 / 252, 119 / 129 / 234 / 250 / 299,Docket No. CX10-257WO3 119 / 181 / 234 / 252, 129 / 234 / 255 / 299, 181 / 299, 250 / 252, 250 / 255 / 299, 119 / 234 / 299, 119 / 181 / 250, 56 / 237, 250 / 299, 119 / 234 / 255 / 256 / 299, 56 / 229 / 254, 119 / 129 / 181, 56 / 121 / 295 / 317, 119 / 181 / 250 / 255 / 256, 229, 250, 119 / 234, 20 / 229 / 237 / 317, 181 / 250, 119 / 255, 121 / 156 / 229, 56 / 121 / 237, 237 / 317, 56 / 295, 56 / 121 / 229 / 236 / 254 / 295, 56 / 254, 119 / 250 / 299, 229 / 237 / 295, 20 / 229 / 317, 229 / 295, 156 / 229, 56 / 121 / 229 / 237 / 295, 237 / 254, 20 / 56, 56 / 156, 56 / 156 / 254 / 295, 20 / 56 / 229, 119 / 129 / 181 / 234, 237, 56 / 156 / 229 / 237, 20 / 56 / 102 / 229, 119 / 181 / 234, 20 / 229, 56 / 121 / 156 / 237, 56 / 156 / 229, 20 / 56 / 156 / 229 / 237 / 295, 20 / 56 / 121 / 237 / 317, 156, 156 / 237 / 295, 20 / 56 / 121, 119 / 181, 20 / 56 / 229 / 254, 56, 121 / 254, 20 / 295, 255 / 256, 56 / 121 / 229 / 254, or 119 / 129 / 234 / 255 / 256, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the reference sequence corresponding to SEQ ID NO:

370.

36. The engineered adenosine kinase of any one of claims 33-35, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set 299G, 21G, 299S, 113S, 152C, 169D / 255C, 251K, 294L, 255S, 202F / 211A, 31L, 129A, 252V, 27A / 64M, 128W, 248L, 251V, 234V, 288V, 20V, 111L, 112L, 181C, 119C, 64T, 251T, 155W, 256H, 128F, 254I, 248G, 292F, 252C, 234N, 181A, 31W, 265Y, 251L, 152A, 234M, 177V, 232S, 120C, 31F, 183M, 234A / 252T, 181V / 250I / 252T / 299S, 119R / 181V / 234A / 250I / 299S, 129G / 234A / 252T, 119M / 129G / 234A, 119M / 181V / 234A / 250I / 256M / 299S, 119R / 181V / 250I / 252T, 119M / 181V / 234A / 299S, 234A / 250I / 252T, 119R / 181V / 252T / 299S, 129G / 181V / 234A / 299S, 56G / 121L / 229V, 181V / 255A / 299S, 119M / 299S, 119R / 250I / 255K / 299S, 119R / 181V / 234A / 299S, 252T, 80M / 119M / 252T, 119M / 129G, 119M / 129G / 250I, 181V / 234A / 252T / 299S, 181V / 250I / 299S, 252T / 299S, 119M / 181V / 299S, 119R / 129G / 299S, 119M / 129G / 234A / 252T, 119M / 129G / 234A / 250I / 299S, 119M / 181V / 234A / 252T, 129G / 234A / 255K / 299S, 181V / 299S, 250I / 252T, 250I / 255K / 299S, 119R / 181V / 299S, 119M / 234A / 299S, 119R / 299S, 119M / 181V / 250I, 129G, 56G / 237V, 250I / 299S, 119M / 234A / 255K / 256M / 299S, 56G / 229V / 254L, 119R / 129G / 181V, 56G / 121M / 295A / 317Y, 119R / 181V / 250I / 255A / 256M, 229V, 250I, 119M, 119M / 234A, 20F / 229V / 237V / 317Y, 181V / 250I, 119M / 255K, 121M / 156Y / 229V, 56G / 121M / 237V, 237V / 317Y, 56G / 295A, 56G / 121M / 229V / 236S / 254L / 295A, 56G / 254L, 119R / 250I / 299S, 229V / 237V / 295A, 20F / 229V / 317Y, 234A, 119R / 234A, 254L, 229V / 295A, 156Y / 229V, 56F / 121M / 229V / 237V / 295A, 56F / 237V, 237V / 254L, 299S, 20F / 56G, 56G / 156Y, 56G / 156Y / 254L / 295A, 20F / 56G / 229V, 119M / 129G / 181V / 234A, 237V, 20F, 56F / 156Y / 229V / 237V, 56F / 254L, 20F / 56G / 102I / 229V, 20F / 56F, 256M, 119M / 181V / 234A, 20F / 229V, 56F / 121M / 156Y / 237V, 56F / 156Y / 229V, 20F / 56G / 156Y / 229V / 237V / 295A, 56F / 229V / 254L, 20F / 56F / 121M / 237V / 317Y, 20F / 56F / 229V, 119R / 181V / 234A, 119R, 156Y, 156Y / 237V / 295A, 20F / 56F / 121M, 119M / 181V, 20F / 56G / 229V / 254L, 56F, 121M / 254L, 20F / 295A, 255K / 256M, 56F / 121M / 229V / 254L, 119M / 129G / 234A / 255A / 256M, or 119R / 181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 370, or relative to the referenceDocket No. CX10-257WO3 sequence corresponding to SEQ ID NO:

370.

37. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO: 734, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO:

734.

38. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 890-1142, 1192 and 1194, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO:

734.

39. The engineered adenosine kinase of claim 37 or 38, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 56 / 229, 21 / 56, 181, 181 / 211 / 251, 181 / 211, 21 / 121 / 299, 229, 56, 56 / 169, 311, 52, 170, 31 / 181 / 211, 167, 303, 222, 43, 83, 113 / 181 / 211, 251, 187, 218, 181 / 251, 195, 121 / 229, 107, 141, 31 / 181, 307, 100, 169, 86, 90, 164, 310, 276, 35, 56 / 134, 211, 50, 41, 121 / 169 / 229, 74, 225, 106, 121, 31 / 113 / 181, 191, 190, 171, 168, 31 / 181 / 211 / 294, 73, 31 / 251, 31, 196, 89, 197, 242, 51, 103, 101, 147, 40, 146, 243, 56 / 121 / 229, or 31 / 211, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO:

734.

40. The engineered adenosine kinase of any one of claims 37-39, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 56G / 229V, 21G / 56G, 181V, 181V / 211A / 251K, 181V / 211A, 21G / 121L / 299S, 229V, 56G, 56G / 169D, 311Q, 52D, 170R, 31L / 181V / 211A, 167W, 170H, 303M, 222G, 43V, 83V, 113S / 181V / 211A, 251K, 187V, 218V, 181V / 251K, 195T, 121L / 229V, 107H, 141S, 31L / 181V, 307F, 100G, 169L, 169M, 52G, 86L, 90L, 311G, 164W, 310C, 167F, 276E, 35V, 56G / 134H, 211M, 211M, 170V, 50C, 41L, 311S, 121L / 169D / 229V, 74I, 225G, 106G, 121L, 31L / 113S / 181V, 191A, 191S, 211I, 35S, 190I, 164K, 171C, 211F, 211L, 225E, 195G, 311R, 167V, 311L, 168V, 168R, 211C, 31L / 181V / 211A / 294L, 73M, 276S, 74V, 164S, 31L / 251K, 169S, 31L, 169D, 196S, 196S, 52C, 218L, 169C, 89G, 90R, 73E, 73E, 167T, 197R, 242D, 51S, 103L, 191R, 101F, 83T, 52F, 164G, 195M, 43K,Docket No. CX10-257WO3 147S, 187Q, 311M, 40W, 169I, 242T, 101W, 83L, 222M, 170S, 311F, 146I, 83S, 243R, 74T, 311T, 218N, 74F, 303V, 83F, 90V, 74K, 303F, 310A, 243G, 222A, 43S, 303W, 56G / 121L / 229V, or 31L / 211A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 734, or relative to the reference sequence corresponding to SEQ ID NO:

734.

41. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or to the reference sequence corresponding to SEQ ID NO: 896, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO:

896.

42. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1196-1344, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1196- 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO:

896.

43. The engineered adenosine kinase of claim 41 or 42, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 52 / 169, 52 / 141 / 169, 52, 41, 169, 51 / 52 / 211, 52 / 141 / 187, 51 / 52, 141, 41 / 103, 311, 167, 41 / 311, 133, 207, 14, 34, 241, 33, 27, 206, 217, 239, 13, 246, 162, 108, 160, 245, 92, 163, 104, 157, 215, 80, 54, 240, 210, 23, 53, 87, 188, 151, 29, 258, 238, 244, 192, 91, or 30, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO:

896.

44. The engineered adenosine kinase of any one of claims 41-43, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 52D / 169M, 52D / 141S / 169M, 52G, 52G / 169M, 52D, 41L, 52G / 141S / 169M, 169M, 51S / 52G / 211M, 52G / 141S / 187V, 51S / 52D, 141S, 41L / 103L, 311Q, 167F, 41L / 311Q, 51S / 52G, 133L, 207R, 14V, 34R, 241R, 33Q, 27S, 206A, 217V, 239R, 207T, 13A, 246T, 13P, 207M, 162F, 207L, 27P, 108C, 160L, 33S, 245W, 239F, 92M, 163G, 239T, 104C, 157P, 215L, 80V, 54V, 239A, 13E, 207V, 240V, 133A, 210L, 207A, 23N, 53R, 87K, 188T, 151L, 29R, 258S, 34G, 238L, 27R, 14S, 244W, 192L, 91Y, 206H, 30Y, 80S, 258R, 240L, or 104L, wherein the amino acidDocket No. CX10-257WO3 positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 896, or relative to the reference sequence corresponding to SEQ ID NO:

896.

45. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or to the reference sequence corresponding to SEQ ID NO: 1196, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

46. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1346-1364, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1346- 1364, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

47. The engineered adenosine kinase of claim 45 or 46, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 24, 234 / 236, 251 / 252, 119, 234, or 231 / 234, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

48. The engineered adenosine kinase of any one of claims 45-47, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 24V, 24I, 234K / 236C, 24N, 251H / 252A, 119S, 119T, 234K, 234K / 236S, or 231L / 234K, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1196, or relative to the reference sequence corresponding to SEQ ID NO: 1196.

49. The engineered adenosine kinase of claim 18 comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or to the reference sequence corresponding to SEQ ID NO: 1366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to theDocket No. CX10-257WO3 reference sequence corresponding to SEQ ID NO: 1366.

50. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1366-1410, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1366- 1410, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

51. The engineered adenosine kinase of claim 49 or 50, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 182, 121, 249, 129, 152, 237, 155, 121 / 129 / 207 / 237 / 249, 121 / 129 / 207 / 249, 27 / 121 / 207, 27 / 121 / 207 / 249, 121 / 207, 121 / 237, 121 / 249, 121 / 207 / 252, 27 / 121 / 129 / 207, 27 / 121 / 129 / 237, 207, 182 / 207, 121 / 129, or 121 / 237 / 252, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

52. The engineered adenosine kinase of any one of claims 49-51, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 182G, 121Q, 249A, 129S, 152A, 237V, 155A, 121Q / 129S / 207R / 237V / 249A, 121Q / 129S / 207R / 249A, 27P / 121Q / 207R, 27P / 121Q / 207R / 249A, 121Q / 207R, 121Q / 237V, 121Q / 249A, 121Q / 207R / 252V, 27P / 121Q / 129S / 207R, 27P / 121Q / 129S / 237V, 207R, 182G / 207R, 121Q / 129S, 121Q / 237V / 252V, or 155I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1366, or relative to the reference sequence corresponding to SEQ ID NO: 1366.

53. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or to the reference sequence corresponding to SEQ ID NO: 1382, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

54. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1412-1564, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1412- 1564, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

55. The engineered adenosine kinase of claim 53 or 54, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 314, 221, 170, 40, 49, 73, 194, 195, 147, 218, 216, 90, 89, 42, 211, 106, 171, 93, 296, 100, 243, 172, 51, 271, 90 / 171 / 191, 90 / 271, 90 / 191, 90 / 147 / 171 / 271, 191, 51 / 90, 187 / 211, 171 / 191, 170 / 211, 49 / 51, 51 / 171 / 191 / 271, 40 / 211, 106 / 187 / 211, 211 / 221, 40 / 170 / 211, 49 / 51 / 90 / 147 / 171, 51 / 90 / 171, 51 / 171, 49 / 51 / 191, 49 / 51 / 90 / 147, 49 / 51 / 171 / 271, 106 / 211 / 221, 187 / 221, 49 / 171 / 191 / 314, 51 / 191 / 271, 51 / 90 / 171 / 191 / 271, 52 / 221, 90 / 171 / 191 / 271, 191 / 271, 170 / 221, , 50 / 52, 50 / 52 / 106 / 211, 50 / 211 / 308, 50 / 52 / 106, 40 / 50 / 187 / 211 / 308, 50 / 52 / 106 / 170 / 187 / 308, 40 / 52 / 170, 50 / 106, 50 / 221 / 308, 49 / 51 / 90 / 271, 40 / 52 / 170 / 211, 187, 50 / 211 / 221 / 308, or 40 / 50 / 52 / 221, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

56. The engineered adenosine kinase of any one of claims 53-55, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 314S, 221A, 170K, 40H, 314V, 49M, 73C, 314Q, 194A, 195V, 147N, 218L, 216V, 90S, 89L, 42D, 211H, 90T, 106M, 171C, 93A, 296N, 100G, 243Y, 172S, 73A, , 51F, 271W, 90S / 171V / 191W, 90S / 271W, 90S / 191W, 90S / 147A / 171V / 271W, 191W, 51F / 90S, 187H / 211W, 171C / 191W, 170M / 211K, 49M / 51G, 51G / 171C / 191W / 271W, 40M / 211K, 106T / 187H / 211K, 211W / 221A, 40M / 170M / 211K, 211W, 49M / 51G / 90S / 147A / 171V, 51G / 90S / 171V, 51G / 171V, 49M / 51F / 191W, 49M / 51G / 90S / 147A, 51G, 49M / 51F / 171V / 271W, 49M / 51F / 171C / 271W, 106T / 211K / 221A, 211K, 187H / 221A, 49M / 171C / 191W / 314S, 51G / 191W / 271W, 51G / 90S / 171C / 191W / 271W, 52T / 221A, 90S / 171C / 191W / 271W, 191W / 271W, 170M / 221A, , 50C / 52T, 50C / 52T / 106T / 211W, 50C / 211K / 308D, 50C / 52S / 106T, 40M, 40M / 50C / 187H / 211K / 308D, 50C / 52T / 106T / 170M / 187H / 308D, 40M / 52T / 170M, 50C / 106T, 50C / 221A / 308D, 49M / 51F / 90S / 271W, 40M / 52S / 170M / 211W, 187H, 50C / 211K / 221A / 308D, or 40M / 50C / 52S / 221A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1382, or relative to the reference sequence corresponding to SEQ ID NO: 1382.

57. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or to the reference sequence corresponding toDocket No. CX10-257WO3 SEQ ID NO: 1464, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

58. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1566-1578, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1566- 1578, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

59. The engineered adenosine kinase of claim 57 or 58, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or amino acid residue 317M, 113G, 21G, 155V, 126S, 155N, or 231M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1464, or relative to the reference sequence corresponding to SEQ ID NO: 1464.

60. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or to the reference sequence corresponding to SEQ ID NO: 1568, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

61. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1580-1648, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1580- 1648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

62. The engineered adenosine kinase of claim 60 or 61, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 246, 74, 185, 244, 164, 276, 43, 163, 217, 31, 43 / 276, 185 / 187 / 276, 43 / 187 / 222 / 276,Docket No. CX10-257WO3 185 / 187 / 222 / 276, 187 / 276, 185 / 276, 164 / 222, 164 / 187 / 222 / 276, 43 / 222 / 276, 164 / 187 / 222, 222, 43 / 164 / 185 / 187 / 222, 222 / 276, 239, or 53, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

63. The engineered adenosine kinase of any one of claims 60-62, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 246V, 74R, 185T, 244R, 246R, 164S, 276G, 43E, 246S, 163G, 217A, 31H, 43E / 276E, 185S / 187S / 276G, 43E / 187S / 222Q / 276D, 185S / 187S / 222Q / 276D, 187S / 276E, 187S / 276D, 185S / 276E, 164S / 222Q, 164S / 187S / 222Q / 276G, 43E / 222Q / 276G, 164S / 187S / 222Q, 222Q, 43E / 164S / 185S / 187S / 222Q, 222Q / 276G, 276E, 276D, 31L, 31Y, 164Q, 239M, 53L, 185S, or 164A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12- 321 of SEQ ID NO: 1568, or relative to the reference sequence corresponding to SEQ ID NO: 1568.

64. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or to the reference sequence corresponding to SEQ ID NO: 1616, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

65. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1650-1742, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1650- 1742, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

66. The engineered adenosine kinase of claim 64 or 65, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 74 / 239 / 244 / 246, 31 / 74 / 207 / 239 / 244 / 246, 31 / 246, 74 / 244, 31 / 74, 53 / 74 / 239 / 244 / 246, 31 / 74 / 207 / 244 / 246, 31 / 74 / 163 / 239 / 244, 31 / 74 / 239 / 244 / 246, 31 / 244 / 246, 31 / 53 / 164, 31 / 239 / 244, 239 / 246, 163 / 164 / 207 / 239 / 244 / 246, 31 / 74 / 163 / 246, 244, 31 / 239 / 244 / 246, 31 / 53 / 239 / 244 / 246, 31 / 207 / 244 / 246, 31, 31 / 53 / 74 / 239 / 246, 31 / 74 / 207, 31 / 163 / 207 / 244, 31 / 53 / 244, 31 / 163, 31 / 53 / 239 / 246, 74 / 207 / 239 / 244, 31 / 163 / 164 / 207 / 239 / 244 / 246, 74 / 207, 31 / 163 / 207 / 239 / 244, 31 / 74 / 163 / 164 / 207 / 246, 31 / 53 / 207 / 239, 207, 53 / 163 / 207 / 246, 31 / 53 / 74 / 163 / 207 / 246,Docket No. CX10-257WO3 31 / 163 / 239 / 246 / 308, 31 / 74 / 207 / 239 / 308, or 163 / 207 / 246, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

67. The engineered adenosine kinase of any one of claims 64-66, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 74T / 239M / 244R / 246V, 31Y / 74R / 207M / 239M / 244R / 246V, 31Y / 246S, 74R / 244R, 31R / 74R, 53L / 74T / 239M / 244R / 246S, 53L / 74R / 239M / 244R / 246V, 31R / 74T / 207M / 244R / 246S, 31Y / 74R / 163G / 239M / 244R, 31Y / 74R / 239M / 244R / 246V, 31L / 244R / 246S, 31Y / 244R / 246S, 31R / 53L / 164Q, 31R / 239M / 244R, 239M / 246V, 163G / 164Q / 207M / 239M / 244R / 246V, 31Y / 74R / 163G / 246V, 244R, 31L / 239M / 244R / 246S, 31Y / 74T, 31L / 53L / 239M / 244R / 246V, 31Y / 74R, 31Y / 207M / 244R / 246V, 31Y / 53L / 239M / 244R / 246V, 31Y, 31L / 53L / 74T / 239M / 246S, 31Y / 74R / 207M, 31Y / 163G / 207M / 244R, 31R / 207M / 244R / 246V, 31Y / 53L / 244R, 31Y / 163G, 31R, 31R / 53L / 239M / 246S, 74T / 207M / 239M / 244R, 31L / 163G / 164Q / 207M / 239M / 244R / 246V, 74T / 207M, 31Y / 163G / 207M / 239M / 244R, 31L / 74R / 163G / 164Q / 207M / 246V, 31Y / 53L / 207M / 239M, 31Y / 207M / 244R / 246S, 207M, 53L / 163G / 207M / 246S, 31R / 53L / 74T / 163G / 207M / 246S, 31Y / 163G / 239M / 246V / 308K, 31R / 207M / 244R / 246S, 31Y / 74R / 207M / 239M / 308K, or 163G / 207M / 246V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1616, or relative to the reference sequence corresponding to SEQ ID NO: 1616.

68. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or to the reference sequence corresponding to SEQ ID NO: 1668, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

69. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1744-1808, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1744- 1808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

70. The engineered adenosine kinase of claim 68 or 69, wherein the amino acid sequenceDocket No. CX10-257WO3 of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 312, 305, 246, 89, 33, 218, 147, 27 / 218 / 305 / 312, 27 / 35 / 106 / 218 / 312, 27 / 218 / 312, 27 / 312, 27 / 305 / 312, 27 / 106 / 218 / 312, 27 / 218, 27 / 106 / 312, 35 / 305, 27 / 144 / 312, 27 / 218 / 305, 35 / 312, 27 / 106 / 218 / 305 / 312, 27 / 35 / 106 / 312, 106 / 218 / 305, 106 / 218 / 312, 27 / 106 / 305, 27 / 35 / 94 / 106 / 305 / 312, 35 / 218 / 312, 218 / 312, 27 / 106 / 218, 35 / 106 / 312, or 27 / 106, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

71. The engineered adenosine kinase of any one of claims 68-70, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set, or amino acid residue(s) 312R, 305T, 246D, 89K, 33A, 218D, 147N, 27W / 218D / 305T / 312R, 27K / 35Y / 106P / 218D / 312R, 27W / 218D / 312R, 27W / 312R, 27W / 305T / 312R, 27W / 106P / 218D / 312R, 27W / 218D, 27W / 106P / 312R, 35Y / 305T, 27W / 144S / 312R, 27W / 218D / 305S, 35Y / 312R, 27W / 106P / 218D / 305S / 312R, 27W / 35Y / 106P / 312R, 106P / 218D / 305T, 27W / 106P / 218D / 305T / 312R, 106P / 218D / 312R, 27W / 106P / 305T, 27W / 35Y / 94N / 106P / 305S / 312R, 35Y / 218D / 312R, 218D / 312R, 27W / 106P / 218D, 35Y / 106P / 312R, 27W / 106P, or 27W / 106P / 305S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1668, or relative to the reference sequence corresponding to SEQ ID NO: 1668.

72. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or to the reference sequence corresponding to SEQ ID NO: 1766, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

73. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1818-1870, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1818- 1870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

74. The engineered adenosine kinase of claim 72 or 73, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 34, 112, 126, 128, 155, 179, 183, 229, 236, 248, 253, 254, 292, 293, or 299, or combinations thereof, whereinDocket No. CX10-257WO3 the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

75. The engineered adenosine kinase of any one of claims 72-74, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 34C, 112I, 126L, 128V, 155T, 179N, 183S, 183V, 229C, 229V, 236C, 248E, 253D, 253E, 253G, 253H, 253K, 253L, 253S, 253T, 253V, 254M, 254T, 292I, 292V, 293M, or 299G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1766, or relative to the reference sequence corresponding to SEQ ID NO: 1766.

76. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or to the reference sequence corresponding to SEQ ID NO: 1826, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

77. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1872-1936, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1872- 1936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

78. The engineered adenosine kinase of claim 76 or 77, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 14, 38, 46, 108, 146, 211, 243, 275, 303, 311, or 320, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

79. The engineered adenosine kinase of any one of claims 76-78, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 14S, 14T, 38R, 46I, 108A, 146C, 211R, 243F, 275S, 303D, 303S, 311E, 320G, or 320R, or combinations thereof, wherein the amino acid positions are relative to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

80. The engineered adenosine kinase of claim 76 or 77, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 94, 106, 108, 194, 274, 303, 307, or 315, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

81. The engineered adenosine kinase of any one of claims 76, 77, and 80, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 94M, 106L, 108E, 194R, 274R, 303V, 307G, or 315S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

82. The engineered adenosine kinase of claim 76 or 77, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 112 / 183 / 292 / 299, 112 / 292, 126 / 299, 155, 155 / 183 / 299, 155 / 299, 183 / 255 / 299, 183 / 299, 229 / 299, 255 / 299, or 292, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

83. The engineered adenosine kinase of any one of claims 76, 77, and 82 wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set, or amino acid residue(s) 112I / 183V / 292I / 299G, 112I / 292I, 126L / 299G, 155T, 155T / 183V / 299G, 155T / 299G, 183V / 255I / 299G, 183V / 299G, 229H / 299G, 255I / 299G, and 292I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1826, or relative to the reference sequence corresponding to SEQ ID NO: 1826.

84. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or to the reference sequence corresponding to SEQ ID NO: 1918, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

85. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequenceDocket No. CX10-257WO3 corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1938-2010, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1938- 2010, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

86. The engineered adenosine kinase of claim 84 or 85, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution at amino acid position 26, 27, 28, 29, 31, 33, 35, 42, 136, 212, 218, 237, 239, 245, 246, 252, 276, or 316, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

87. The engineered adenosine kinase of any one of claims 84-86, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution, or amino acid residue 26F, 27C, 27G, 27R, 27S, 28L, 28M, 29G, 31H, 33A, 33P, 35D, 35E, 42G, 42N, 42Q, 136F, 212A, 218R, 237K, 237L, 237Q, 239A, 239G, 239H, 239I, 239T, 239W, 245F, 245Q, 246A, 246T, 252A, 252G, 276G, 316A, or 316I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1918, or relative to the reference sequence corresponding to SEQ ID NO: 1918.

88. The engineered adenosine kinase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or to the reference sequence corresponding to SEQ ID NO: 1942, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

89. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2012-2124, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2012- 2124, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

90. The engineered adenosine kinase of claim 88 or 89, wherein the amino acid sequenceDocket No. CX10-257WO3 of the engineered adenosine kinase comprises at least a substitution or substitution set at amino acid position(s) 26 / 27, 26 / 27 / 28, 26 / 27 / 28 / 31, 26 / 27 / 28 / 245, 26 / 28 / 31, 27, 27 / 28, 27 / 28 / 29 / 126, 27 / 29, 27 / 29 / 126, 27 / 29 / 237, 27 / 29 / 245, 27 / 31 / 126 / 245, 27 / 31 / 155, 27 / 126, 27 / 126 / 155, 27 / 126 / 237, 27 / 126 / 237 / 245, 27 / 237, 27 / 245, 28, 28 / 31, 28 / 31 / 239, 28 / 42 / 126 / 245, 28 / 42 / 245, 28 / 239, 29 / 31, 29 / 31 / 126 / 239, 30 / 31 / 33 / 35 / 126 / 218, 30 / 31 / 33 / 126 / 276 / 316, 30 / 33 / 35, 31 / 33 / 218 / 276, 31 / 126, 31 / 126 / 155, 33, 33 / 126 / 239, 42, 126, 126 / 155, 126 / 155 / 237, 126 / 155 / 239 / 276, 126 / 155 / 239 / 316, 126 / 239, 126 / 239 / 316, 126 / 245, 126 / 316, 155 / 276 / 316, 155 / 316, 218, 218 / 239 / 316, 239, 245, 252 / 276, or 316, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

91. The engineered adenosine kinase of any one of claims 88-90, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution set or amino acid residues 26F / 27G / 28M, 26F / 27R, 26F / 27R / 28L / 31H, 26F / 27R / 28M / 245F, 26F / 28M / 31H, 27G, 27G / 28M, 27G / 29G, 27G / 29G / 237K, 27G / 29G / 237Q, 27G / 29G / 245F, 27G / 31H / 126A / 245F, 27G / 31H / 155T, 27G / 126A, 27G / 126A / 155T, 27G / 126A / 237K, 27G / 126A / 237Q / 245F, 27G / 237K, 27G / 245F, 27R, 27R / 28M / 29G / 126A, 27R / 29G / 126A, 27R / 245F, 28L / 31H / 239T, 28L / 239T, 28M, 28M / 31H, 28M / 42N / 126A / 245F, 28M / 42N / 245F, 29G / 31H, 29G / 31H / 126A / 239T, 30G / 31H / 33A / 35D / 126A / 218N, 30G / 31H / 33A / 126A / 276G / 316I, 30G / 33A / 35D, 31H / 33A / 218N / 276G, 31H / 126A, 31H / 126A / 155T, 33A, 33A / 126A / 239H, 42N, 126A, 126A / 155T, 126A / 155T / 237K, 126A / 155T / 239H / 316I, 126A / 155T / 239T / 276G, 126A / 239H, 126A / 239H / 316I, 126A / 245F, 126A / 316I, 155T / 276G / 316I, 155T / 316I, 218N, 218N / 239H / 316I, 239H, 245F, 252T / 276G, or 316I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-321 of SEQ ID NO: 1942, or relative to the reference sequence corresponding to SEQ ID NO: 1942.

92. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least one substitution set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

93. The engineered adenosine kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least a substitution or substitution set of anDocket No. CX10-257WO3 engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

94. The engineered adenosine kinase of any one of claims 1-5, wherein the adenosine kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenosine kinase variant set forth in Tables 7.1, 7.2, 8.1, 9.1, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 45.2, 46.2, 47.2, 48.2, 49.2 and 50.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or relative to the reference sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

95. The engineered adenosine kinase of claim 1, wherein the amino acid sequence of the engineered adenosine kinase comprises residues 12-321 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1142, 1194-1808 and 1818-2124, optionally wherein the engineered adenosine kinase has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

96. The engineered adenosine kinase of claim 1, wherein the amino acid sequence of the engineered adenosine kinase comprises residues 12-321 of SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or wherein the amino acid sequence comprises SEQ ID NO: 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, optionally wherein the engineered adenosine kinase has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

97. The engineered adenosine kinase of any one of claims 1-96, having adenosine kinase activity and at least one improved property as compared to a reference adenosine kinase.

98. The engineered adenosine kinase of claim 97, wherein the improved property is selected from i) increased activity on unmodified nucleoside, ii) increased thermostability, iii) increased activity on cytidine, iv) increased activity on 2-fluoro modified nucleosides, and v)Docket No. CX10-257WO3 increased activity on 2-O-methyl modified nucleosides, or any combinations of i), ii), iii), iv), and v), compared to the reference adenosine kinase.

99. The engineered adenosine kinase of claims 97 or 98, wherein the reference adenosine kinase has an amino acid sequence corresponding to residues 12-321 of SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942, or an amino acid sequence corresponding to SEQ ID NO: 2, 90, 254, 370, 734, 896, 1196, 1366, 1382, 1464, 1568, 1616, 1668, 1766, 1826, 1918, or 1942.

100. The engineered adenosine kinase of any one of claims 1-99, comprising a fusion polypeptide.

101. The engineered adenosine kinase of any one of claims 1-100, wherein the engineered adenosine kinase is purified.

102. The engineered adenosine kinase of any one of claims 1-101, wherein the engineered adenosine kinase is immobilized on a substrate or support medium.

103. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase of any one of claims 1-99.

104. The recombinant polynucleotide of claim 103, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, or 1941, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, or 1941, wherein the recombinant polynucleotide encodes an adenosine kinase.

105. The recombinant polynucleotide of claim 103, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123 or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123 wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

106. The recombinant polynucleotide of any one of claims 103-105, wherein the polynucleotide sequence has preferred codons or is codon optimized.Docket No. CX10-257WO3 107. The recombinant polynucleotide of claim 103, wherein the polynucleotide sequence comprises nucleotide residues 34-963 of SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, or 1941, or comprises SEQ ID NO: 1, 89, 253, 369, 733, 895, 1195, 1365, 1381, 1463, 1567, 1615, 1667, 1765, 1825, 1917, or 1941,.

108. The recombinant polynucleotide of claim 103, wherein the polynucleotide sequence comprises nucleotide residues 34-963 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-1141, 1193-1807, and 1817-2123.

109. An expression vector comprising a recombinant polynucleotide of any one of claims 103-108.

110. The expression vector of claim 109, wherein the recombinant polynucleotide is operably linked to a control sequence.

111. The expression vector of claim 110, wherein the control sequence comprises at least a promoter.

112. A host cell comprising an expression vector of any one of claims 109-111.

113. The host cell of claim 112 comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

114. A method of producing an engineered adenosine kinase polypeptide, comprising culturing a host cell of claim 112 or 119 under suitable culture conditions such that the engineered adenosine kinase polypeptide is produced.

115. The method of claim 114, further comprising recovering the engineered adenosine kinase polypeptide from the culture and / or host cells.

116. The method of claim 114 or 115, further comprising purifying the engineered adenosine kinase polypeptide.

117. A composition comprising an engineered adenosine kinase of any one of claims 1- 102.

118. The composition of claim 117, further comprising a nucleoside substrate.

119. The composition of claim 118, wherein the nucleoside is a modified nucleoside substrate.Docket No. CX10-257WO3 120. A method of converting a nucleoside or a modified nucleoside to corresponding nucleoside monophosphate (NMP), comprising contacting a nucleoside with an engineered adenosine kinase of any one of claims 1-102 in the presence of phosphate donor NTP under suitable reaction conditions to convert the nucleoside to the corresponding nucleoside monophosphate (NMP).

121. The method of claim 120, wherein the nucleoside is an unmodified nucleoside.

122. The method of claim 121, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine.

123. The method of any one of claims 120-122, wherein the nucleoside has at the 2’- position of the sugar moiety a H or OH.

124. The method of claim 123, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and wherein the nucleoside has at the 2’-position of the sugar moiety an OH, thereby by resulting in corresponding product rAMP, rGMP, rUMP, rCMP, or rTMP, respectively.

125. The method of claim 123, wherein the nucleoside is adenosine, guanosine, uridine, cytidine, or thymidine, and wherein the nucleoside has at the 2’-position of the sugar moiety an H, thereby by resulting in corresponding product dAMP, dGMP, dUMP, dCMP, or dTMP, respectively.

126. The method of claim 120, wherein the nucleoside is a modified nucleoside, wherein the nucleoside has a modified sugar moiety or modified nucleobase, or a combination thereof.

127. The method of claim 126, wherein the modified nucleoside comprises a modified sugar moiety, wherein the sugar moiety has a modified 2’-position or 3’-position.

128. The method of claim 127, wherein the modified sugar moiety has a modified 2’- position of the sugar moiety.

129. The method of claim 128, wherein the 2’-position is a 2’-O-alkyl or 2-halo.

130. The method of claim 129, wherein the 2’-position is a 2’-O-alkyl, and wherein the 2’- O-alkyl is 2’-O-CH3or 2’-O-CH2CH3.

131. The method of claim 129, wherein the 2’-position is 2-halo.

132. The method of claim 131, wherein the 2’-position is 2’-F or 2’-Br.

133. The method of any one of claims 127-132, wherein the modified sugar moiety is modified at the 3’-position.

134. The method of claim 133, wherein the 3’-position is modified with a blocking group,Docket No. CX10-257WO3 wherein the blocking group is formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (- ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, O-(2- nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, or triphosphate.

135. The method of any one of claims 126-134, wherein the modified nucleoside comprises a modified nucleobase, wherein the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

136. The method of any one of claims 120-135, wherein the phosphate donor NTP is rATP or dATP.

137. The method of any one of claims 120-135, wherein the phosphate donor NTP is nucleoside-5’-gamma-thiotriphosphate (NTP ^S), thereby resulting in product nucleoside 5’- thiophosphate (NMP-S).

138. The method of any one of claims 120-135, wherein the phosphate donor NTP has the same nucleoside structure of the nucleoside substrate.

139. The method of any one of claims 120-138, further comprising regenerating the NTP using a NTP regeneration system.

140. The method of claim 139, wherein the NTP regenerating system comprises creatine kinase, pyruvate kinase, polyphosphate kinase, and / or acetate-kinase.

141. The method of claim 140, wherein the NTP regenerating system is pyruvate kinase and substrate phosphoenolpyruvate.

142. The method of claim 140, wherein the NTP regenerating system is creatine kinase and substrate creatine phosphate.

143. The method of claim 140, wherein the NTP regenerating system is polyphosphate kinase and substrate polyphosphate.

144. The method of claim 140, wherein the NTP regenerating system is acetate kinase and substrate acetyl phosphate.

145. The method of claim 144, further comprising pyruvate oxidase and substrate pyruvate and inorganic phosphate.

146. The method of claim 145, further comprising including a catalase.

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