Uricase variants

Abstract

Provided herein are uricase enzyme variants for use in degrading uric acid and in treating gout. More specifically, provided herein are uricase enzyme variants that exhibit increased thermal stability as measured by residual uric acid degrading activity following thermal challenge.

Description

Attorney Docket No. : 123818-0502URICASE VARIANTS TECHNICAL FIELD

[0001] This disclosure relates to enzyme variants for use in treating disease. More particularly, this disclosure relates to uric acid degrading enzyme variants that may be used in the treatment of gout, along with pharmaceutical compositions and methods of treatment using said variants.BACKGROUND

[0002] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0003] Gout is the most common form of chronic inflammatory arthritis, which can cause sudden, severe attacks of pain, swelling, redness, and tenderness in one or more joints caused by hyperuricemia, which is due to the deposition of needle-like monosodium urate (MSU) crystals, also known as uric acid (UA) or urate. In addition to causing extreme pain, MSU deposition in the tissues can lead to tophi, chronic synovitis, bony erosions, cartilage damage, kidney stones, and heart and kidney disease, which can progress to organ failure in severe cases. All humans, some higher primates, and certain New World monkeys have no detectible uricase activity.

[0004] The annual incidence of gout is approximately 1 in 400, affecting an estimated 9.2 million adults in the United States. It is estimated that approximately 40% of men and approximately 20% of women have hyperuricemia, with approximately 4-6% of men and approximately 1-2% of women with hyperuricemia developing gout. Additional factors, such as, genetics, age, lifestyle, body mass index, heart / kidney disease, increasing the risk of developing gout. Typical treatments for gout focus on urate-lowering therapeutics; however, use of current therapies come with drawbacks, such as severe allergic reactions, increased risk of cardiovascular death, anaphylaxis, and unwanted immune responses, such as development of antidrug antibodies. Thus, there is a need for developing alternative treatments approaches.SUMMARY

[0005] The present disclosure relates to uric acid degrading enzyme variants, compositions of the enzyme variants and methods of use of the variants. As shown herein, administration ofAttorney Docket No. : 123818-0502the enzyme variant can improve uric acid degradation capabilities in a subject that may have gout. The present disclosure shows that the enzyme variants may exhibit increased thermal stability and increased residual activity following application of physicochemical stress. It is to be understood that the disclosed embodiments are merely exemplary, and accordingly, the invention may be embodied in various and alternative forms. The specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the embodiments described herein.

[0006] In one aspect, the present disclosure provides a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wildtype uricase enzyme set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.

[0007] In some embodiments, at least one substitution increases the thermostability of the variant relative to the wild-type uricase enzyme. In some embodiments, the increased thermostability of the variant relative the wild-type uricase is an increase of at least 5% of residual uricase activity following a thermal challenge. In some embodiments, the increase is at least 10% of residual uricase activity following a thermal challenge. In some embodiments, the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C. In some embodiments, residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

[0008] In some embodiments, the residual uricase activity for at least one variant is retained following nebulization. In some embodiments, the residual uricase activity following nebulization is about 50% or more.

[0009] In some embodiments, the variant sequence comprises at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T,Attorney Docket No. : 123818-0502A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, SI 87V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

[0010] In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92. In some embodiments, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297. In some embodiments, the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

[0011] In some embodiments, the variant sequence comprises at least two substitutions at two or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at last two substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 93, 101, 105, 106, 108, 111, 112, 114, 116, and 117.

[0012] In some embodiments, the variant sequence comprises at least three substitutions at three or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least three substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 94, 98, 99, 102, 104, 107 109, 113, and 115.

[0013] In some embodiments, the variant sequence comprises at least four substitutions at four or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least four substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 95, 97, 100, 103, and 110.

[0014] In some embodiments, the variant sequence comprises at least five substitutions at five or more amino acid positions selected from 18, 29, 56, 63, 187, and 297, of SEQ ID NO: 1. In some embodiment, the variant sequence comprises at least five substitutions selectedAttorney Docket No. : 123818-0502from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence of SEQ ID NO: 96.

[0015] In some embodiments, the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

[0016] In another aspect, the present disclosure provides enzyme variants for use in treating gout (e.g., an enzyme variant of any one of the forgoing aspect or embodiments).

[0017] In another aspect, the present disclosure provides uses of an enzyme variant disclosed herein (e.g., an enzyme variant of any one of the forgoing aspects or embodiments) in the manufacture of a medicament for treating gout.

[0018] In another aspect, the present disclosure provides pharmaceutical compositions, comprising: (a) a uric acid degrading enzyme variant disclosed herein (e.g., an enzyme variant of any one of the forgoing aspects or embodiments), and (b) a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for aerosol administration. In some embodiments, the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration. In some embodiments, the pharmaceutical composition is formulated such that the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0019] In another aspect, the present disclosure provides pharmaceutical compositions, comprising: (a) a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization; and (b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated such that the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0020] In some embodiments, wherein the increased thermostability of the variant relative the wild-type uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge. In some embodiments, the increase is at least 10% of residualAttorney Docket No. : 123818-0502uricase activity following a thermal challenge. In some embodiments, the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C. In some embodiments, the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge. In some embodiments, the residual uricase activity following nebulization is about 50% or more.

[0021] In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92. In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297. In some embodiments, the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

[0022] In some embodiments, the variant sequence comprises at least two substitutions at two or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at last two substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 93, 101, 105, 106, 108, 111, 112, 114, 116, and 117.

[0023] In some embodiments, the variant sequence comprises at least three substitutions at three or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least three substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 94, 98, 99, 102, 104, 107, 109, 113, and 115.

[0024] In some embodiments, the variant sequence comprises at least four substitutions at four or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least four substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequenceAttorney Docket No. : 123818-0502comprises an amino acid sequence selected from any one of SEQ ID NOs: 95, 97, 100, 103, and 110.

[0025] In some embodiments, the variant sequence comprises at least five substitutions at five or more amino acid positions selected from 18, 29, 56, 63, 187, and 297, of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least five substitutions selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence of SEQ ID NO: 96.

[0026] In some embodiments, the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

[0027] In some embodiments, the pharmaceutical composition is formulated for aerosol administration. In some embodiments, the aerosol administration is selected from liquid aerosol administration or solid (e.g., dry powder) aerosol administration. In some embodiments, the pharmaceutical composition is formulated such that the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0028] In another aspect, the present disclosure provides the disclosed pharmaceutical compositions (e.g., any of the foregoing aspect of embodiments) for use in treating gout.

[0029] In another aspect, the present disclosure provides uses of the disclosed pharmaceutical compositions (e.g., any of the foregoing aspect of embodiments) in the manufacture of a medicament for treating gout.

[0030] In another aspect, the present disclosure provides methods of treating a subject with gout, the method comprising administering to the subject a variant disclosed herein or a pharmaceutical composition disclosed here (e.g., any of the foregoing aspects or embodiments). In some embodiments, the variant or the pharmaceutical composition is administered as a liquid aerosol. In some embodiments, the variant or the pharmaceutical composition is administered as a solid (e.g., dry powder) aerosol. In some embodiments, the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0031] In another aspect, the present disclosure provides methods of treating a subject with gout, the method comprising administering to the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of theAttorney Docket No. : 123818-0502wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization. In some embodiments, the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0032] In some embodiments, the increased thermostability of the variant relative the wildtype uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge. In some embodiments, the increase is at least 10% of residual uricase activity following a thermal challenge. In some embodiments, the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C. In some embodiments, the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

[0033] In some embodiments, the residual uricase activity following nebulization is about 50% or more.

[0034] In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least one substitution selected from THE, T1 IM, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92. In some embodiments, the variant sequence comprises at least one substitution at one or more of the positionsAttorney Docket No. : 123818-0502selected from 18, 22, 56, 63, 187, and 297. In some embodiments, the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

[0035] In some embodiments, the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

[0036] In some embodiments, the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises 2, 3, 4, or 5 substitutions.

[0037] In some embodiments, the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus

[0038] In some embodiments, the enzyme variant is formulated for aerosol administration. In some embodiments, the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

[0039] In another aspect, the present disclosure provides methods of degrading uric acid in a subject, the method comprising administering to a variant disclosed herein or a pharmaceutical composition disclosed here (e.g., any of the foregoing aspects or embodiments). In some embodiments, the variant or the pharmaceutical composition is administered as a liquid aerosol. In some embodiments, the variant or the pharmaceutical composition is administered as a dry powder aerosol. In some embodiments, the subject has gout. In some embodiments, the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0040] In another aspect, the present disclosure provides method of degrading uric acid in a subject, the method comprising administering to the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization. In some embodiments, the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.Attorney Docket No. : 123818-0502

[0041] In some embodiments, the increased thermostability of the variant relative the wildtype uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge, wherein the increase is at least 10% of residual uricase activity following a thermal challenge, wherein the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C, wherein the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

[0042] In some embodiments, the residual uricase activity following nebulization is about 50% or more.

[0043] In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least one substitution selected from THE, T1 IM, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92. In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297. The method of any one of claims 81-90, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, andN297P.

[0044] In some embodiments, the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. In some embodiments, the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, and N297P. In some embodiments, the variant sequence comprises 2, 3, 4, or 5 substitutions.Attorney Docket No. : 123818-0502

[0045] In some embodiments, the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

[0046] In some embodiments, the enzyme variant is formulated for aerosol administration. In some embodiments, the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration. In some embodiments, the subject has gout. In some embodiments, the uric acid degrading enzyme variant is formulated to introduce the variant to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0047] In another aspect, the present disclosure provides a method of treating a subject with gout, the method comprising administering to the lung of the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1.

[0048] In some embodiments, the variant (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization.

[0049] In some embodiments, the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1. In some embodiments, the variant sequence comprises at least one substitution selected from THE, T1 IM, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92. In some embodiments,Attorney Docket No. : 123818-0502the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297. In some embodiments, the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

[0050] In some embodiments, the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

[0051] In some embodiments, the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, andN297P.

[0052] In some embodiments, the variant sequence comprises 2, 3, 4, or 5 substitutions.

[0053] In some embodiments, the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

[0054] In some embodiments, the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

[0055] The foregoing general description and following detailed description are examples and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

[0056] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 depicts the wild-type A. globiformis uricase enzymatic activity at 60°C, 62°C, 64°C, 66°C, 68°C, and 70°C, as measured by the amount of uric acid production remaining.

[0058] FIGS. 2A, 2B, and 2C depict the activity of the uricase variants generated in the first round of variant creation as a measure of uric acid breakdown. Uricase activity was measured average activity. FIG. 2A shows average activity for the variants without thermal challenge.FIG. 2B shows average activity for the variants following thermal challenge at 66°C. FIG.2C shows average activity for the variants following thermal challenge at 68°C.Attorney Docket No. : 123818-0502

[0059] FIG. 3 depicts the activity of the uricase variants generated in the second round of variant creation as a measure of uric acid breakdown with average activity presented on the left Y-axis and % residual activity on the right Y-axis. Each enzyme has three data points. The blue bars are enzyme activity determined without thermal challenge. Orange bars are enzyme activity determined following thermal challenge. The grey dots are enzyme residual activity.

[0060] FIG. 4 depicts the activity of the uricase variant H56Y and H56Y S187N as a measure of uric acid breakdown following no thermal challenge (null) and thermal challenge for 6 minutes, 18 minutes, and 30 minutes, at 72.4°C, 73.3°C, 74.6°C, 76.1°C, 77.7°C, 79.3°C, 80.9°C, 82.4°C, 83.7°C, 84.6°C, and 85°C.

[0061] FIG. 5 depicts the activity of the uricase variant wild type (WT), H56Y and H56Y S187N as a measure of uric acid breakdown following no thermal challenge (0 minutes) and thermal challenge for up to 30 minutes in 2 minute increments at 65.7°C, 67.4°C, 70.0°C, 73.0°C, 75.9°C, 79.1°C, 82.0°C, 95.0°C, 87.6°C, 89.3°C, and 90.0°C.

[0062] FIG. 6 shows an exemplary SDS-PAGE gel of uricase variants purified from shake flask culture of enzyme-expressing Escherichia coli. The shake flask protocol utilized 100 mL of medium in a 500 mL flask. Supe is the supernatant of the culture broth, FT is the flow through, Fractions is collections from the affinity column. The box represents the purified globiformis uricase.

[0063] FIG. 7 shows the exemplary SDS-PAGE gel of the H56Y HIS tagged protein purification steps from Pichia. FT : flow through od protein not binding the resin, W 1 : wash 1, W2: wash 2 fractions, E: elution of the HIS tag protein fraction. The H56Y protein is the major band.

[0064] FIG. 8A depicts a graph illustrating serum uric acid concentration (pM) in serum samples obtained from Cynomolgus non-human primates (NHP) administered a variant uricase enzyme (H56Y) as a function of time following administration. At Cmax, UA serum concentration was 74 pM ± 24.5 SD (UA Control), 59 pM ± 12 SD (Low-dose uricase (H56Y) + UA), and 46 pM ± 20 SD (High-dose uricase (H56Y) + UA).

[0065] FIG. 8B depicts a graph illustrating the Area Under the Curve (AUC) of time course data described in FIG. 8B. Serum uric acid was decreased 36.6% (Low-dose uricase; H56Y) and 56.8% (High-dose uricase; H56Y, p<0.01 ) compared to UA Control, as determined byAttorney Docket No. : 123818-0502area under the curve analysis of time course data (60 - 515 minutes). Data are shown as mean ± SD, One-way ANOVA vs Uric Acid Control. **p<0.01, ***p<0.001.

[0066] FIG. 9A depicts a graph illustrating serum uric acid concentration (pM) in serum samples obtained from mice administered a wildtype uricase or a variant uricase enzyme (H56Y) as a function of time following administration. Healthy female mice were provided with either 2 mg / mouse of A. flavus uricase or engineered variant uricase (H56Y), or vehicle (black, n=3). Time course data of serum uric acid (UA) post-dose is shown. Statistics: data shown as mean ± SD; multiple t-test comparing Control to Treatment.

[0067] FIG. 9B is a graph illustrating the Area Under the Curve (AUC) of time course data described in FIG. 9B. Serum uric acid was decreased 29.8% (WT uricase) and 34.4% (variant uricase (H56Y), p<0.01) compared to UA Control, as determined by area under the curve analysis of time course data (0-240 minutes). Statistics: data shown as mean ± SD; One-way ANOVA comparing Control to Treatment groups (*). Directional black arrows indicate percent decrease compared to Control. Unpaired t-test comparing Treatment groups (A).DETAILED DESCRIPTION

[0068] Gout is a form of chronic inflammatory arthritis that is caused by hyperuricemia where uric acid is persistently elevated in the blood.

[0069] A handful of treatment options have been developed. Two such therapies is uricases administered via IV-infusion: One such therapy is a non- PEGylated uricase from Aspergillus flavus (A. flavus) (Elitek®, rasburicase), which is only approved for hyperuricemia in malignancy and not currently approved for adults, and a PEGylated chimeric porcine / baboon uricase (Krystexxa®, pegloticase), which can have risks of anaphylaxis. Additionally, both come with risks of the patient developing antidrug antibodies that decrease efficacy.

[0070] Provided herein are uric acid degrading enzyme variants ( / .< ., uricase variants) that may be used to treat gout, along with a pharmaceutical composition and treatments utilizing the same. The disclosed variants are modified forms of the wild-type uricase enzyme, wherein the variant exhibits increased thermostability relative to the wild-type uricase enzyme and increased residual uricase activity. The variants also maintain at least 50% of residual uricase activity following nebulization. The variants can be delivered to a subject through a liquid aerosol administration or a dry powder aerosol administration. In some embodiments, the variants with increased thermostability and residual uricase activity are suited for this administration method, these methods subject the enzyme variants to physicalAttorney Docket No. : 123818-0502stressors that are not presented in other methods of administration. Such a method of treatment may facilitate increased compliance with the treatment regimen as it is not invasive, painful, or unduly limiting for the patient. However, enzyme variants with increased thermostability and residual uricase activity may be administered via other methods of administration in addition to aerosols (e.g., oral administration, injection, etc.).

[0071] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein for the purpose of describing particular embodiments only and is not intended to be limiting.I. Definitions

[0072] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0073] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). Additionally, when “about” is stated with respect to a particular value, it should be understood as the stated number and up to 10% plus or minus that number. For example, the phrase “about 10” should be understood as disclosed both 10 and a range of 9-11.

[0074] As used herein, the phrases “therapeutically effective amount” and “therapeutic level” mean a uricase enzyme variant dosage or plasma concentration in a subject that provides the specific pharmacological effect for which the uricase enzyme variant is administered to a subject in need of such treatment, i.e. to facilitate degradation of uric acid and to mitigateAttorney Docket No. : 123818-0502symptoms of gout. It is emphasized that a therapeutically effective amount or therapeutic level of the uricase enzyme variant will not always be effective in treating the gout symptoms of a given subject, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary amounts are provided below.

[0075] Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject and / or the subject’s condition, including the amount of uric acid in the subject’s bloodstream at the time of treatment, and / or the degree of gout disease manifestation in the subject.

[0076] The terms of “treatment” or “treating” as used herein with reference to gout refer to one or more of: reducing, ameliorating or eliminating one or more symptoms or effects of uric acid accumulation; reducing the subject’s blood levels of uric acid; and / or reducing the amount of uric acid localized in specific tissues of the subject.

[0077] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammal subject, e.g., bovine, canine, feline, equine, or human.II. Gout

[0078] Gout is a common form of chronic inflammatory arthritis that occurs due to the accumulation of uric acid in the blood ( / .< ., hyperuricemia). Gout develops, and is characterized, when hyperuricemia is at persistently elevated levels of about >6.8 mg / dL of UA in the blood.

[0079] Gout can cause sudden, severe attacks of pain, swelling, redness, and tenderness in one or more joints due to the deposition of uric acid. Uricase is an enzyme that can break down uric acid. All humans have no detectible uricase activity.III. Uricase Enzyme

[0080] The enzyme uricase (urate oxidase or factor-independent urate hydroxylase), catalyzes the oxidation of uric acid to 5-hydroxyisourate then to allantoin: Uric acid + O2 + H2O — 5-hydroxyisourate + H2O — allantoin + CO2.

[0081] Uricase is a homotetrameric enzyme containing four identical active sites situated at the interfaces between its four subunits. Enzyme variants of the bacteria ArthrobacterAttorney Docket No. : 123818-0502globiformis (A. globiformis) uricase, known to breakdown uric acid into allantoin and CO2, were engineered towards lung delivery as a potential therapy for gout.IV. Uricase Enzyme Variants

[0082] Disclosed herein are variants of a uricase enzyme for use in treating gout and in degrading uric acid in the bloodstream of a subject. The variants may be selected based on their thermostability and residual enzyme activity following physicochemical and / or thermal stress, (e.g., nebulization and heat treatment). The uricase enzyme variants may be variants of the wild-type uricase enzyme from A. globiformis.

[0083] At least one mutation that increases the thermostability of the uricase enzyme may be introduced into the wild-type uricase enzyme, thereby creating a uricase variant, allowing the variant to remain active and degrade uric acid at higher temperatures or following thermal challenge as compared to the wild-type uricase enzyme. The increase in thermostability may be measured by comparing the amount of uric acid degraded by the variant compared to wildtype following thermal challenge. In some embodiments, the thermal challenge may be for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes incubation period. In some embodiments, the thermal challenge may be for about 6 minutes, about 18 minutes, or about 30 minutes incubation period. In some embodiments, the thermal challenge is for about a 10 minute incubation period. The thermal challenge may be at about 66°C to about 68°C. In some embodiments, the thermal challenge may be at about 56°C. In some embodiments, the thermal challenge may be at about 60°C to about 70°C. In some embodiments, the thermal challenge may be at about 72°C to about 85°C. In some embodiments, the thermal challenge may be at about 65°C to about 90°C. In some embodiments, the thermal challenge is at 66°C. In some embodiments, the thermal challenge is at 68°C. In some embodiments, the thermal challenge is greater than or equal to 80°C.

[0084] The amount of uricase activity may be detected by measuring the amount of uric acid measured via fluorescence following a 30-minute incubation period of about Img / L to about 2mg / L of uric acid. A variant may be selected for use to degrade uric acid or treat gout if it exhibits an increase of at least about 5% to about 10% residual uricase activity following thermal challenge. A variant may be selected for use to degrade uric acid or to treat gout if it shows an increase of at least 5% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 100% or more) residual activity followingAttorney Docket No. : 123818-0502thermal challenge relative to wild-type uricase enzyme. The residual uricase activity may be calculated by first measuring the uricase activity of the enzyme prior to thermal challenge and second measuring the uricase activity of the enzyme following thermal challenge and dividing the latter by the former.

[0085] At least one mutation that increases the residual uricase activity following physicochemical stress may be introduced into the variant, allow the variant to remain active and oxidize of uric acid to allantoin following application of the physicochemical stress as compared to the wild-type uricase enzyme. One such physicochemical stress may be nebulization, wherein the enzyme in a liquid sample is converted into a mist. The nebulization challenge assay may include loading 3 mL of a liquid enzyme solution into a nebulizer, pulling air into the nebulizer via vacuum to convert the liquid enzyme solution into a mist, capturing the mist from the nebulizer in an ice trap, and recovering a portion of the liquid enzyme sample. A variant may be selected for use to degrade uric acid or to treat gout if it shows an increase of at least 10% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 100% or more) residual activity relative to wild-type uricase enzyme. The residual uricase activity may be calculated by first measuring the uricase activity of the enzyme prior to nebulization and second measuring the uricase activity of the enzyme following nebulization challenge and dividing the latter by the former.

[0086] At least one mutation that increases the catalytic activity of the enzyme may be introduced into the variant, allowing the variant to more rapidly and / or more efficiently break-down uric acid. Such a mutation may improve various measures of enzymatic performance, including but not limited to, increasing kcat, lowering KM, increasing kcat / K , and / or increasing Vmax.

[0087] As noted above, the uricase enzyme variants may exhibit increased uricase activity, increased thermostability, increased residual enzyme activity following nebulization, and / or decreased immunogenicity relative to the wild-type uricase enzyme. The variants may comprise one or more mutations to the amino acid sequence of wild-type uricase encoding gene as disclosed in SEQ ID NO: 1, including one or more deletions, additions, or substitutions. (SEQ ID NO:1 -MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAA HTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWA AQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPAttorney Docket No. : 123818-0502RDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQ QTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIE ATIQREGSRADHPIWSNIAGFC.

[0088] The uricase enzyme variants may include one or more mutations, such as a mutation at one or more positions selected from any one of amino acid residues 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297 of SEQ ID NO:1. For the purposes of the present disclosure, the N-terminal or the C-terminal of SEQ ID NO: 1 or the uricase enzyme variant including one or more mutations may further comprise a polyhistidine-tag (His-tag).

[0089] The variant may include one, two, three, four, five, six, seven, eight, nine, ten, or more mutations or substitutions. The variant may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more substitutions at positions selected 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297 of SEQ ID NO: 1.

[0090] The disclosed uricase enzyme variants can comprise at least one substitution at one or more of positions selected from 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297 of SEQ ID NO:1. The uricase enzyme variant sequence may comprise at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297 of SEQ ID NO: 1. The uricase enzyme variant sequence may include at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E,Attorney Docket No. : 123818-0502and N297P. The uricase enzyme variant sequence may be any one of SEQ ID NOs: 2-92. The uricase enzyme variant sequence may include at least one substitution selected from N18H, H56Y, H63F, S187N, andN297P.

[0091] The uricase enzyme variant sequence may include at least two substitutions at two or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. The uricase enzyme variant sequence may include at least two substitutions selected from N18H, H56Y, H63F, S187N, and N297P. The uricase enzyme variant sequence may include at least two substitutions selected from: a) H56Y, H63F; b) H56Y, N18H; c) H56Y, N297P; d) H56Y, S187N; e) H63F, N18H; f) H63F, N297P; g) H63F, S187N; h) N18H, N297P; i) N18H, S187N; and j) S187N_N297P. The uricase enzyme variant may be any one of SEQ ID NOs: 93, 101, 105, 106, 108, 111, 112, 114, 116, and 117.

[0092] The uricase enzyme variant sequence may include at least three substitutions at three or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. The uricase enzyme variant sequence may include at least three substitutions selected from N18H, H56Y, H63F, S187N, and N297P. The uricase enzyme variant sequence may include at least three substitutions, wherein the combination of at least three substitutions is selected from: a) H56Y, H63F, N18H; b) H56Y, H63F, N297P; c) H56Y, H63F, S187N; d) H56Y, N18H, N297; e) H56Y, N18H, S187N; f) H56Y, S187N, N297P; g) H63F, N18H, N297P; h) H63F, S187N, N297P; and i) N18H, N297P, S187N. The uricase enzyme variant sequence may be any one of SEQ ID NOs: 94, 98, 99, 102, 104, 107, 109, 113, and 115.

[0093] The uricase enzyme variant sequence may include at least four substitutions at four or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. The uricase enzyme variant may include at least four substitutions selected N18H, H56Y, H63F, S187N, and N297P. The uricase enzyme variant sequence may include at least four substitutions, wherein the combination of at least four substitutions is selected from: a) H56Y H63F N18H N297P; b) H56Y H63F N18H S187N; c) H56Y_H63F_S187N_N297P d) H56Y_N18H_N297P_S187N; and e) H63F_N18H_N297P_S187N. The uricase enzyme variant sequence may be any one of SEQ ID NOs: 95, 97, 100, 103, and 110.

[0094] The uricase enzyme variant sequence may include at least five substitutions at five or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1. The uricase enzyme variant may include at least five substitutions selected from N18H, H56Y,Attorney Docket No. : 123818-0502H63F, S187N, and N297P. The uricase enzyme variant sequence may include at least five substitutions, wherein the combination of at least five substitutions is H56Y, H63F, N18H, N297P, S187N. The uricase enzyme variant sequence may be SEQ ID NO: 96.

[0095] The amino acid sequence of exemplary variants of uricase are set forth in Table 1 below. These sequences are merely examples of variants within the scope of the disclosure.Table 1: Examples of Uricase VariantsSEQ IDEnzyme Mutations SequenceNO.agURIC Ol THE 2 MTATAETSTGEKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_02 T11M 3 MTATAETSTGMKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_03 Q17D 4 MTATAETSTGTKVVLGDNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_04 Q17E 5 MTATAETSTGTKVVLGENQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_05 Q17K 6 MTATAETSTGTKVVLGKNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_06 Q17S 7 MTATAETSTGTKVVLGSNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_07 N18H 8 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_08 Y20F 9 MTATAETSTGTKVVLGQNQFGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_09 K22I 10 MTATAETSTGTKVVLGQNQYGIAEVRLVKVTRN TARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVA TDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGF DWVTGGRWAAQQFFWDRINDHDHAFSRNKSEV RTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFP RDKYTTLQETTDRILATDVSARWRYNTVEVDFD AVYASVRGLLLKAFAETHSLALQQTMYEMGRA VIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC lO K22M 11 MTATAETSTGTKVVLGQNQYGMAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC ll K22W 12 MTATAETSTGTKVVLGQNQYGVAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_12 K22V 13 MTATAETSTGTKVVLGQNQYGWAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_13 K29D 14 MTATAETSTGTKVVLGQNQYGKAEVRLVDVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_14 T31D 15 MTATAETSTGTKVVLGQNQYGKAEVRLVKVDR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_15 N33E 16 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR ETARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_16 N33T 17 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR TTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_17 N33V 18 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR VTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_18 I39L 19 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHELQDLNVTSQLRGDFEAAHTAGDNAHV VATDTQKNTVYAFARDGFATTEEFLLRLGKHFT EGFDWVTGGRWAAQQFFWDRINDHDHAFSRNK SEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFH GFPRDKYTTLQETTDRILATDVSARWRYNTVEV DFDAVYASVRGLLLKAFAETHSLALQQTMYEM GRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQD NPNEVFYAADRPYGLIEATIQREGSRADHPIWSNI AGFCagURIC_19 S46T 20 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTTQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_20 G50A 21 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRADFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_21 E53H 22 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFFAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_22 E53L 23 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFHAAHTAGDNAHV VATDTQKNTVYAFARDGFATTEEFLLRLGKHFT EGFDWVTGGRWAAQQFFWDRINDHDHAFSRNK SEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFH GFPRDKYTTLQETTDRILATDVSARWRYNTVEV DFDAVYASVRGLLLKAFAETHSLALQQTMYEM GRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQD NPNEVFYAADRPYGLIEATIQREGSRADHPIWSNI AGFCagURIC_23 E53F 24 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFLAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_24 H56Y 25 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_25 H63N 26 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_26 H63F 27 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNANVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_27 V64I 28 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHIV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_28 T67F 29 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV AFDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_29 V74I 30 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTIYAFARDGFATTEEFLLRLGKHFTEG FDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGF PRDKYTTLQETTDRILATDVSARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_30 A83K 31 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFKTTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_31 H95F 32 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKFFTEG FDWVTGGRWAAQQFFWDRINDHDHAFSRNKSE VRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGF PRDKYTTLQETTDRILATDVSARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_32 H95Y 33 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKYFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_33 T97V 34 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFVE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_34 F100Y 35 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GYDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_35 A109S 36 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWSAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_36 Q112E 37 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQEFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_37 F113A 38 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQAFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_38 D116N 39 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWERINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_39 D116E 40 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWKRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_40 D116K 41 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWNRINDHDHAFSRNKSAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_41 V136T 42 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTATLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_42 S142D 43 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGDEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_43 QI 441 44 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEIAIVAGIEGLTVLKSTGSEFHGF PRDKYTTLQETTDRILATDVSARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_44 QI 44 V 45 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEVAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_45 A145C 46 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQCIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_46 A145S 47 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQSIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_47 A145T 48 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQTIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_48 A148S 49 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVSGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_49 A148T 50 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVTGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_50 T159D 51 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSDGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_51 Q175E 52 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLEETTDRILATDVSARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_52 T177F 53 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQEFTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_53 D185T 54 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATTVSARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_54 D185V 55 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATWSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_55 V186I 56 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDISARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_56 V186L 57 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDL S ARWRYNTVEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_57 S187D 58 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVDARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_58 S187N 59 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_59 S187T 60 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVTARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_60 SI 87V 61 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVVARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_61 V195T 62 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTTEVDF DAVYASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_62 E196N 63 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVDVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_63 E196D 64 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVNVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_64 Y203P 65 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVPASVRGLLLKAFAETHSLALQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_65 S205A 66 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYAAVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_66 H218D 67 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETDSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_67 S219D 68 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHDLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_68 L220P 69 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSPALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_69 A221N 70 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLNLQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_70 A221S 71 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLSLQQTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_71 Q224H 72 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.FDAVYASVRGLLLKAFAETHSLALQFTMYEMGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_72 Q224F 73 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQHTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_73 Q224Y 74 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQYTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_74 M229A 75 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEAGR AVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPN EVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCagURIC_75 K244R 76 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIRMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_76 M245A 77 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKASLPNKHHFLVDLQPFGQDNPAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_77 N249D 78 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPDKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_78 N249L 79 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPLKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_79 H251E 80 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKEHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_80 H251P 81 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKPHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_81 F253Y 82 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHYLVDLQPFGQDN PNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC_82 Q258R 83 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLKPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_83 Q258K 84 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLRPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_84 Q258S 85 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLSPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_85 F269Y 86 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVYYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_86 I279V 87 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLVEATIQREGSRADHPIWSNIA GFCagURIC_87 Q284R 88 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIKREGSRADHPIWSNIA GFCagURIC_88 Q284K 89 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIRREGSRADHPIWSNIA GFCagURIC_89 S296D 90 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWDNIA GFCagURIC_90 N297E 91 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSEIAG FCagURIC_91 N297P 92 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 93 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVVH56Y H63FATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_2 94 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH56Y H63F N18HFPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_3 95 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y H63F N18H EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG _N297P FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_4 96 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y H63F N18H EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG _N297P_S187N FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_5 97 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y H63F N18H EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG _S187N FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_6 98 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVVH56Y H63F N297 ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE P GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_7 99 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y H63F S187 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG N FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_8 100 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y H63F S187 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG N_N297P FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_9 101 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH56Y N18HFPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC st l 102 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 0 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH56Y_N18H_N297FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 103 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 1 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEH56Y_N18H_N297 GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS P_S187N EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 104 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 2 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y N18H S187 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG N FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC st l 105 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 3 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH56Y N297PFPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 106 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 4 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH56Y_S187NFPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC st l 107 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 5 NTARHEIQDLNVTSQLRGDFEAAYTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH56Y_S187N_N29 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG 7P FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 108 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 6 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH63F N18HFPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFCAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.agURIC st l 109 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 7 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH63F_N18H_N297 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG P FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 110 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 8 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH63F_N18H_N297 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG P_S187N FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC st l 111 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 9 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH63F N297PFPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_2 112 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 0 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGH63F_S187NFPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_2 113 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 1 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAFVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSH63F_S187N_N297 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG P FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_2 114 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTRN18H_N297P2 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVAttorney Docket No. : 123818-0502SEQ IDEnzyme Mutations SequenceNO.ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG FPRDKYTTLQETTDRILATDVSARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_2 115 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 3 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSN18H_N297P_S187 EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHG N FPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAG FCagURIC_st_2 116 MTATAETSTGTKVVLGQHQYGKAEVRLVKVTR 4 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGN18H_S187NFPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSNIA GFCagURIC_st_2 117 MTATAETSTGTKVVLGQNQYGKAEVRLVKVTR 5 NTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVV ATDTQKNTVYAFARDGFATTEEFLLRLGKHFTE GFDWVTGGRWAAQQFFWDRINDHDHAFSRNKS EVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGS187N_N297PFPRDKYTTLQETTDRILATDVNARWRYNTVEVD FDAVYASVRGLLLKAFAETHSLALQQTMYEMG RAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNP NEVFYAADRPYGLIEATIQREGSRADHPIWSPIAGFC

[0096] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 2. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 2.Attorney Docket No. : 123818-0502

[0097] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 3. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 3.

[0098] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 4. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 4.

[0099] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of, SEQ ID NO: 5. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 5.

[0100] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 6. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 6.

[0101] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 7. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 7.

[0102] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 8. In some implementations, a uricase enzyme variant asAttorney Docket No. : 123818-0502described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 8.

[0103] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 9. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 9.

[0104] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 10. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 10.

[0105] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 11. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 11.

[0106] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 12. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 12.

[0107] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 13. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 13.

[0108] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 14. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 14.

[0109] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 15. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 15.

[0110] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 16. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 16.

[0111] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 17. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 17.

[0112] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 18. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 18.Attorney Docket No. : 123818-0502

[0113] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 19. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 19.

[0114] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 20. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 20.

[0115] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 21. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 21.

[0116] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 22. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 22.

[0117] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 23. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 23.

[0118] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 24. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 24.

[0119] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 25. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 25.

[0120] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 26. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 26.

[0121] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 27. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 27.

[0122] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 28. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 28.

[0123] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 29. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 29.

[0124] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 30. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 30.

[0125] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 31. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 31.

[0126] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 32. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 32.

[0127] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 33. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 33.

[0128] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 34. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 34.Attorney Docket No. : 123818-0502

[0129] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 35. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 35.

[0130] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 36. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 36.

[0131] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 37. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 37.

[0132] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 38. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 38.

[0133] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 39. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 39.

[0134] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 40. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 40.

[0135] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 41. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 41.

[0136] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 42. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 42.

[0137] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 43. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 43.

[0138] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 44. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 44.

[0139] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 45. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 45.

[0140] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 46. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 46.

[0141] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 47. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 47.

[0142] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 48. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 48.

[0143] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 49. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 49.

[0144] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 50. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 50.Attorney Docket No. : 123818-0502

[0145] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 51. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 51.

[0146] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 52. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 52.

[0147] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 53. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 53.

[0148] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 54. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 54.

[0149] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 55. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 55.

[0150] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 56. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 56.

[0151] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 57. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 57.

[0152] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 58. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 58.

[0153] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 59. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 59.

[0154] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 60. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 60.

[0155] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 61. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 61.

[0156] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 62. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 62.

[0157] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 63. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 63.

[0158] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 64. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 64.

[0159] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 65. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 65.

[0160] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 66. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 66.Attorney Docket No. : 123818-0502

[0161] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 67. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 67.

[0162] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 68. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 68.

[0163] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 69. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 69.

[0164] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 70. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 70.

[0165] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 71. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 71.

[0166] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 72. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 72.

[0167] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 73. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 73.

[0168] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 74. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 74.

[0169] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 75. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 75.

[0170] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 76. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 76.

[0171] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 77. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 77.

[0172] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 78. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 78.

[0173] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 79. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 79.

[0174] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 80. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 80.

[0175] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 81. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 81.

[0176] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 82. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 82.Attorney Docket No. : 123818-0502

[0177] in some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 83. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 83.

[0178] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 84. In some implementations, a URICASE enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 84.

[0179] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 85. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 85.

[0180] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 86. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 86.

[0181] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 87. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 87.

[0182] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 88. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 88.

[0183] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 89. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 89.

[0184] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 90. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 90.

[0185] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 91. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 91.

[0186] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 92. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 92.

[0187] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 93. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 93.

[0188] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 94. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 94.

[0189] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 95. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 95.

[0190] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 96. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 96.

[0191] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 97. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 97.

[0192] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 98. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 98.Attorney Docket No. : 123818-0502

[0193] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 99. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 99.

[0194] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 100. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 100.

[0195] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 101. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 101.

[0196] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 102. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 102.

[0197] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 103. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 103.

[0198] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 104. In some implementations, a uricase enzyme variantAttorney Docket No. : 123818-0502as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 104.

[0199] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 105. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 105.

[0200] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 106. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 106.

[0201] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 107. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 107.

[0202] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 108. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 108.

[0203] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 109. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, aboutAttorney Docket No. : 123818-050292%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 109.

[0204] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 110. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 110.

[0205] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 111. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 111.

[0206] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 112. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 112.

[0207] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 113. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 113.

[0208] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 114. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 114.Attorney Docket No. : 123818-0502

[0209] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 115. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 115.

[0210] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 116. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 116.

[0211] In some implementations, a uricase enzyme variant as described herein may comprise, or consist of SEQ ID NO: 117. In some implementations, a uricase enzyme variant as described herein has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the variant sequence of SEQ ID NO: 117.

[0212] For the purposes of the present disclosure, it is expected that the disclosed engineered uricase enzymes comprise at least one substitution mutation, deletion, and / or insertion that increases stability and / or enzymatic activity relative to a non-engineered enzyme comprising the same amino acid sequence but without the modification. In some implementations, a variant as described herein exhibits increased activity relative to wild-type uricase (SEQ ID NO: 1), such that its activity is at least about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1100%, about 1200%, about 1300%, about 1400%, about 1500%, about 1600%, about 1700%, about 1800%, about 1900%, about 2000%, about 2250%, about 2500%, about 2750%, aboutAttorney Docket No. : 123818-05023000%, about 3250%, about 3500%, about 3750%, about 4000%, about 4250%, about 4500%, about 4750%, about 5000%, about 10000%, about 100000%, about 1000000%, about 10000000%, or about 20000000% or more than that of the wild-type uricase, as determined by a method disclosed herein. Similarly, in some implementations, a variant as described herein exhibits increased activity relative to wild-type uricase (SEQ ID NO: 1), such that its activity is at least about 2-fold, at least about 4-fold, about 5-fold, about 10-fold, about 18-fold, about 20-fold, about 50-fold, about 100-fold, about 200-fold, about 1000-fold, about 5000-fold, about 10000-fold, about 20000-fold, about 50000-fold, about 100000-fold, about 200000-fold, about 500000-fold, or about 1000000-fold, or more, than that of the wild-type uricase, as determined by a method disclosed herein.V. Pharmaceutical Compositions

[0213] The uricase enzyme variants disclosed herein can be formulated into pharmaceutical compositions suitable for administration to the target subject ( / .< ., a human or other mammal) via a predetermined route of administration, as discussed in more detail below.

[0214] Pharmaceutical compositions may include one or more variants as described herein and a pharmaceutically acceptable carrier or diluent.

[0215] The compositions may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, oral, nasal, pulmonary, ocular, vaginal, or rectal administration. The composition may be formulated for administration by injection or infusion. The composition may be formulated for oral administration. The compositions may be formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as in a solution, suspension, emulsion, liposome formulation, etc. The pharmaceutical compositions can be formulated to be an immediate-release composition, sustained-release composition, delay ed-release composition, etc., using techniques known in the art.

[0216] Preferably, the composition is formulated for liquid aerosol administration or dry powder aerosol administration. Inhaled protein therapeutics meet a growing interest for treatment of various disease. However, in aerosols, proteins and enzymes face stresses that may generate instabilities, such as physicochemical denaturation, aggregation, and loss of activity. Thus, it is important that the protein or enzyme used in liquid aerosol administration or dry powder aerosol administration is designed to better withstand said stresses. When administered to the lung of a subject, the disclosed enzyme variants may be formulated suchAttorney Docket No. : 123818-0502that the enzymes are delivered systemically throughout the subject’s body or locally (i.e., to the lung only) without systemic distribution. In other words, the disclosed enzyme variants can be formulated under conditions that inhibit or do not support systemic delivery of the variants or exclude pulmonary transmucosal delivery of the variant, which leads to systemic delivery in the subject.

[0217] Formulation for administration to pulmonary tissue of a subject may be carried out for any suitable route but under conditions that do not support a significant or substantial permeation of the agent beyond pulmonary tissue (i.e., minimization of systemic introduction). For example, such a pharmaceutical composition may be formulated for administration by inhalation or via the trachea (i.e., intratracheal administration). However, to an extent significant or substantial systemic introduction is to be avoided, inhalation of an atomized solution of an agent is preferably avoided. Avoiding or minimization of systemic introduction means that a majority of the agent introduced continues to reside in pulmonary tissue. In certain embodiments of this disclosure, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the agent introduced permeates mucosal tissue (i.e., introduction is not transmucosal) into a subject’s blood or serum. A pharmaceutical composition may be formulated for administration intratracheally, the enzyme variants introduced eventually finding their way into pulmonary tissue of a subject.

[0218] Pharmaceutically acceptable carriers for various dosage forms are known in the art. For example, excipients, lubricants, binders, and disintegrants for solid preparations are known; solvent, solubilizing agents, suspending agents, isotonicity agents, buffers, and soothing agents for liquid preparations are known. The pharmaceutical compositions may include one or more additional components, such as one or more preservatives, antioxidants, colorants, sweetening / flavoring agents, absorbing agents, wetting agents, and the like.

[0219] In some embodiments, the uricase enzyme variant may be a long-acting variant that has been modified in order to extend its half-life in vivo (after administration). Various techniques are known in the art for extending the circulating half-life of peptides. For example, the variant may be conjugated to polyethylene glycol (PEG) or a similar polymer that prolongs half-life. Alternatively, the variant may be fused to an albumin-binding peptide, an albumin-binding protein domain, human serum albumin, or an inert polypeptide.Exemplary inert polypeptides that have been used to increase the circulating half-life of peptides include, but are not limited to, XTEN® (also known as recombinant PEG orAttorney Docket No. : 123818-0502“rPEG”), a homo-amino acid polymer (HAP; HAPylation), a proline-alanine serine polymer (PAS; PASylation), or an elastin-like peptide (ELP; ELPylation). As used herein, “fused to” includes genetic fusion, directly or through a linker, resulting in a single polypeptide containing multiple domains, unless otherwise specified.VI. Methods and Treatments

[0220] As noted above, the variants described herein are useful in methods of degrading uric acid in a mammalian subject in need thereof. The subject may be a human subject. The human subject may have gout. Thus, the variants described herein may be useful in methods of treating gout in a mammalian subject in need thereof.

[0221] The methods generally involve administering a therapeutically effective amount of a uricase enzyme variant as described herein (or a pharmaceutical composition comprising the same) to the subject. A therapeutically effective amount would be an amount that degrades uric acid to improve gout or to negate the effects of uric acid accumulation in the subject. The method may include administering the enzyme variant itself to the subject. However, the method may include administering a nucleic acid encoding the uricase enzyme variant in a construct that expresses the variant in vivo. For example, the nucleic acid can be provided in a suitable vector, such as an adeno-associated virus (AAV) gene transfer vector. Other exemplary vectors that are suitable for use in such methods are known in the art.Exemplary vectors may include one or more enhancers (e.g., a cytomegalovirus (CMV) enhancer), promoters (e.g., chicken P-actin promoter), and / or other elements enhancing the properties of the expression cassette. Methods of making suitable vectors and general methods of using expression vectors in vivo are known in the art.

[0222] A subject in need of treatment for gout is a human subject who has increased levels of uric acid in their bloodstream. Such a subject may or may not suffer sudden, severe attacks of pain, swelling, redness, tenderness in one or more joints, tophi, chronic synovitis, bony erosions, cartilage damage, kidney stones, and heart and kidney disease.

[0223] The specific amount of a uricase enzyme that is administered may depend on one or more of the age and / or weight of the subject, the amount of uric acid the subject can degrade without treatment, and / or the specific mutation causing gout. A variant may be administered at a dose of from about 0.01 to about 20 mg / kg, about 0.1 mg / kg to about 18 mg / kg, about 1 mg / kg to about 16 mg / kg, about 2 mg / kg to about 14 mg / kg, or about 5 mg / kg to about 10 mg / kg. In some embodiments, a variant is administered at a dose of about 0.01Attorney Docket No. : 123818-0502mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8 / 5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about lOmg / kg, about 10.5 mg / kg, about 12 mg / kg, about 12.5 mg / kg, about 13 mg / kg, about 13.5 mg / kg, about 14 mg / kg, about 14.5 mg / kg, about 15 mg / kg, about 15.5 mg / kg, about 16 mg / kg, aboutl6.5 mg / kg, about 17 mg / kg, about 17.5 mg / kg, about 18 mg / kg, about 18.5 mg / kg, about 19mg / kg, about 19.5 mg / kg, or about 20 mg / kg. A variant may be administered at a dose of about 0.5 mg, about 1 mg, about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about20 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about550 mg, about 600, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, or about 2500 mg. When more than one variant is administered, the total amount of variants administered may be in accordance with the foregoing guidance.

[0224] The methods may be administered via a single dose of a uricase enzyme variant(s) (or composition comprising the same). The method may be administered via repeated doses, such as for a predetermined period of time until the symptoms or effects of uricase are reduced, ameliorated, or eliminated. Treatment may be repeated with additional doses of the variant(s) if signs / symptoms / effects persist. Treatment may be repeated for the duration of a subject’s life. In some embodiments, the variant is administered to the lung or lungs of the subject. In some embodiments, the variant is administered to the trachea of the subject.

[0225] Administration or introduction to pulmonary tissue of a subject may be carried out by any suitable route but under conditions that do not support a significant or substantialAttorney Docket No. : 123818-0502permeation of the agent beyond pulmonary tissue (i.e., minimization of systemic introduction). Administration or introduction can be carried out by inhalation. However, to an extent significant or substantial systemic introduction is to be avoided, inhalation of an atomized solution of an agent is preferably avoided. Avoiding or minimization of systemic introduction means that a majority of the agent introduced continues to reside in pulmonary tissue. In certain embodiments of this disclosure, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the agent introduced permeates mucosal tissue (i.e., introduction is not transmucosal) into a subject’s blood or serum. Administration or introduction can be carried out intratracheally, the agent(s) introduced eventually finding their way into pulmonary tissue of a subject.

[0226] The methods may include administering a uricase enzyme variant(s) (or composition including the same) three or more times a day, twice a day, or once a day. The methods may include administering a uricase enzyme variant(s) (or composition comprising the same) once every other day, three times a week, twice a week, once a week, once every other week, once every three weeks, once a month, or less frequently. The uricase enzyme variant may be a long-acting uricase enzyme variant as described above.

[0227] Treatment may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more weeks; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; or 1, 2, 3 or more years or until the subject no longer exhibits symptoms of gout.

[0228] The method may be a method of treating gout in a subject with the disease. The method may be a method of degrading uric acid in a subject.

[0229] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the disclosure. The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not limited to the specific conditions or details of these examples.EXAMPLES

[0230] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: Enzyme Activity and Selection.Attorney Docket No. : 123818-0502

[0231] Proteins tend to misfold and aggregate during delivery to and occupancy in the lungs. Literature and experts believe this misfolding and aggregation is caused by mechanical stress induced during aerosolization and protein migration at the air-liquid interfaces present in both aerosol droplets and mucus within the lungs. Ultimately, protein drugs delivered to the lungs must stay properly folded during delivery and after deposition. Thus, development of hyper-folded protein variants could enable a competitive advantage for both goals.

[0232] Metabolites diffuse between the lungs and the bloodstream. This interaction may be exploited to treat gout by depositing enzymes in the lungs and depleting specific metabolites in the blood. Presumably, it would be possible to deposit an enzyme acting on specific blood metabolites in the lungs and then metabolize target molecules that freely diffuse between the lungs and blood. This hypothesis does not require the enzyme to translocate across the lung membrane and into the bloodstream. It, instead, depends upon the metabolites translocating from the bloodstream and into the lungs and upon the enzymes remaining active in the lungs.

[0233] Uricase from Arthrobacter globiformis (A. globiformis) was selected as the enzyme used to generate variants. The enzyme was synthesis by cloning in an E. coli expression vector and a His-tag was at the amino terminal was added for ease of purification at scale.

[0234] To test the stability of the wild type E. coli produced uricase, a thermostability challenge assay was performed. The E. coli produced uricase was heat treated for 10 minutes under a gradient of fixed of temperature, from 60°C, 62°C, 64°C, 66°C, 68°C, and 70°C, in duplicate at a dilution of 32-fold and 64-fold, and compared to non-challenged uricase. Uric acid added to the reaction was measured using a uricase detection assay commercial kit (#A22181; ThermoFisher®). In assay, uricase catalyzes the conversion of uric acid to allantoin, hydrogen peroxide, and carbon dioxide. In the presence of horseradish peroxidase (HRP), hydrogen peroxide reacts stoichiometrically with the Amplex™ Red reagent to generate the red-fluorescent oxidation product resorufin. The reaction is performed at 37°C and the readout is fluorometric. A significant decrease in uricase activity was observed after heat treatment (FIG. 1). Wild type protein retained between 5-7% of its residual activity at 66°C and 68°C and these temperatures were selected for further validation and screening studies.Attorney Docket No. : 123818-0502

[0235] A sequential process was applied to identify and qualify uricase mutants. Two rounds of selection were used to identify candidate variants. The first round focused on single amino acid changes, while the second round focused on stacking the best single variants to generate variants with more than one amino acid change.Example 2: Library Design and Screening.

[0236] 91 alleles of A. globiformis uricase were selected (Table 2) utilized the protein crystal structure (PDB: 2YZB, 2YZC, 2YZD, and 2YZE) of uricase from A. globiformis. A convolutional neural network (CNN) ensemble network consisting of 24 models was used to identify suboptimal residues at specific positions and predict mutations that would enhance the overall structural and functional properties of the protein, including folding, stability, activity, and expression levels. These models provided fitness probabilities for both ground truth amino acids (Prob GT) and the remaining 19 amino acids as mutations (Prob Mut) at each position. Our selection process for the top 40 variants followed these four sequential filtering criteria:!) Selection of wildtype residues with probabilities < 0.1, indicating lower fitness; 2) Selection of mutations with high probabilities (> 0.1) at these low-fitness residue positions; 3) Elimination of any conserved residues located in the enzyme's active site; and 4) Final selection based on Prob Diff (Prob Mut - Prob GT) and Prob Ratio (Mut / GT) parameters derived from Prob Mut and Prob GT.Table 2: List of Uricase Mutants Generated in the First Round of Identification.SEQ ID NOName MutationagURIC 00 WT URICASE SEQ ID NO 1agURIC Ol THE SEQ ID NO 2agURIC_02 T11M SEQ ID NO 3agURIC_03 Q17D SEQ ID NO 4agURIC_04 Q17E SEQ ID NO 5agURIC_05 Q17K SEQ ID NO 6agURIC_06 Q17S SEQ ID NO 7agURIC_07 N18H SEQ ID NO 8agURIC_08 Y20F SEQ ID NO 9agURIC_09 K22I SEQ ID NO 10agURIC lO K22M SEQ ID NO 11agURIC ll K22V SEQ ID NO 12agURIC_12 K22W SEQ ID NO 13agURIC_13 K29D SEQ ID NO 14agURIC_14 T31D SEQ ID NO 15agURIC_15 N33E SEQ ID NO 16Attorney Docket No. : 123818-0502agURIC_16 N33T SEQ ID NO 17 agURIC_17 N33V SEQ ID NO 18 agURIC_18 139L SEQ ID NO 19 agURIC_19 S46T SEQ ID NO 20 agURIC_20 G50A SEQ ID NO 21 agURIC_21 E53H SEQ ID NO 22 agURIC_22 E53L SEQ ID NO 23 agURIC_23 E53F SEQ ID NO 24 agURIC_24 H56Y SEQ ID NO 25 agURIC_25 H63N SEQ ID NO 26 agURIC_26 H63F SEQ ID NO 27 agURIC_27 V64I SEQ ID NO 28 agURIC_28 T67F SEQ ID NO 29 agURIC_29 V74I SEQ ID NO 30 agURIC_30 A83K SEQ ID NO 31 agURIC_31 H95F SEQ ID NO 32 agURIC_32 H95Y SEQ ID NO 33 agURIC_33 T97V SEQ ID NO 34 agURIC_34 F100Y SEQ ID NO 35 agURIC_35 A109S SEQ ID NO 36 agURIC_36 Q112E SEQ ID NO 37 agURIC_37 F113A SEQ ID NO 38 agURIC_38 D116N SEQ ID NO 39 agURIC_39 D116E SEQ ID NO 40 agURIC_40 D116K SEQ ID NO 41 agURIC_41 V136T SEQ ID NO 42 agURIC_42 S142D SEQ ID NO 43 agURIC_43 QI 441 SEQ ID NO 44 agURIC_44 QI 44 V SEQ ID NO 45 agURIC_45 A145C SEQ ID NO 46 agURIC_46 A145S SEQ ID NO 47 agURIC_47 A145T SEQ ID NO 48 agURIC_48 A148S SEQ ID NO 49 agURIC_49 A148T SEQ ID NO 50 agURIC_50 T159D SEQ ID NO 51 agURIC_51 Q175E SEQ ID NO 52 agURIC_52 T177F SEQ ID NO 53 agURIC_53 D185T SEQ ID NO 54 agURIC_54 D185V SEQ ID NO 55 agURIC_55 V186I SEQ ID NO 56 agURIC_56 V186L SEQ ID NO 57 agURIC_57 S187D SEQ ID NO 58 agURIC_58 S187N SEQ ID NO 59 agURIC_59 S187T SEQ ID NO 60Attorney Docket No. : 123818-0502agURIC_60 SI 87V SEQ ID NO 61agURIC_61 V195T SEQ ID NO 62agURIC_62 E196N SEQ ID NO 63agURIC_63 E196D SEQ ID NO 64agURIC_64 Y203P SEQ ID NO 65agURIC_65 S205A SEQ ID NO 66agURIC_66 H218D SEQ ID NO 67agURIC_67 S219D SEQ ID NO 68agURIC_68 L220P SEQ ID NO 69agURIC_69 A221N SEQ ID NO 70agURIC_70 A221S SEQ ID NO 71agURIC_71 Q224H SEQ ID NO 72agURIC_72 Q224F SEQ ID NO 73agURIC_73 Q224Y SEQ ID NO 74agURIC_74 M229A SEQ ID NO 75agURIC_75 K244R SEQ ID NO 76agURIC_76 M245A SEQ ID NO 77agURIC_77 N249D SEQ ID NO 78agURIC_78 N249L SEQ ID NO 79agURIC_79 H251E SEQ ID NO 80agURIC_80 H251P SEQ ID NO 81agURIC_81 F253Y SEQ ID NO 82agURIC_82 Q258R SEQ ID NO 83agURIC_83 Q258K SEQ ID NO 84agURIC_84 Q258S SEQ ID NO 85agURIC_85 F269Y SEQ ID NO 86agURIC_86 I279V SEQ ID NO 87agURIC_87 Q284R SEQ ID NO 88agURIC_88 Q284K SEQ ID NO 89agURIC_89 S296D SEQ ID NO 90agURIC_90 N297E SEQ ID NO 91agURIC_91 N297P SEQ ID NO 92

[0237] The next step was to test the 91 enzyme variants for stability and uric activity in vitro. Each single mutation was introduced into the parent backbone by site directed mutagenesis using oligonucleotide targeting the specific residue to change. BugBuster treated ceil extracts were tested for uricase activity. The protein extracts were diluted 800-fold and subjected to a thermal challenge assay as described in Example. 1. Specifically, the uricase variants were tested for stability and uric acid activity before (untreated) and after heat treatment for 10 minutes at 66°C and 68°C. The average uricase activity was determined (FIG. 2A-C). Six mutations were observed as having retained uricase activity, at positionsAttorney Docket No. : 123818-050218, 22, 56, 63,187 and 297. However, the mutation at position 22, though having good activity at high temperature, showed weaker activity prior to the temperature challenge, and was therefore not further pursued. (Table 3).Table 3: Results of Residual Activity Before and After Thermo-challenge of Top 5 Mutations.Description SEQ ID NO: Residual activity (%) Residual activity (%)- 66°C - 68°CWild Type SEQ ID NO 1 1.18 1.91H56Y SEQ ID NO 25 45 74.5H63F SEQ ID NO 27 11.85 6.11N18H SEQ ID NO 8 4.44 11.82N297P SEQ ID NO 92 6.41 4.93S187N SEQ ID NO 58 7.24 4.97

[0238] The five variants from the single variant library (e.g., H56Y, H63F, N18H, N297P, and S187N) were used to create a stacked library of 26 variants, with 2, 3, 4, and 5 mutations per construct (Table 4). To construct the library, each mutated protein sequence was back translated using the appropriate codon optimization, and the resulting coding sequence was synthetized by TWIST Bioscience and cloned into an expression vector.Table 4: List of Uricase Stacked Mutants Generated in the Second Round of Identification.Name Mutation SEQ ID NO agURIC st O H56Y SEQ ID NO 25 agURIC st l H56Y H63F SEQ ID NO 93 agURIC_st_2 H56Y H63F N18H SEQ ID NO 94 agURIC_st_3 H56Y H63F N 18H N297P SEQ ID NO 95 agURIC_st_4 H56Y H63F N 18H_N297P_S 187N SEQ ID NO 96 agURIC_st_5 H56Y H63F N 18H_S 187N SEQ ID NO 97 agURIC_st_6 H56Y H63F N297P SEQ ID NO 98 agURIC_st_7 H56Y H63F S187N SEQ ID NO 99 agURIC_st_8 H56Y H63F S 187N_N297P SEQ ID NO 100 agURIC_st_9 H56Y N18H SEQ ID NO 101 agURIC st lO H56Y_N18H_N297 SEQ ID NO 102 agURIC st ll H56Y_N 18H_N297P_S 187N SEQ ID NO 103 agURIC_st_12 H56Y_N18H_S187N SEQ ID NO 104 agURIC_st_13 H56Y N297P SEQ ID NO 105 agURIC_st_14 H56Y_S187N SEQ ID NO 106 agURIC_st_15 H56Y_S187N_N297P SEQ ID NO 107agURIC_st_16 H63F N18H SEQ ID NO 108Attorney Docket No. : 123818-0502agURIC_st_17 H63F N18H N297P SEQ ID NO 109 agURIC_st_18 H63F N 18H_N297P_S 187N SEQ ID NO 110 agURIC_st_19 H63F N297P SEQ ID NO 111 agURIC_st_20 H63F_S187N SEQ ID NO 112 agURIC_st_21 H63F_S187N_N297P SEQ ID NO 113 agURIC_st_22 N18H_N297P SEQ ID NO 114 agURIC_st_23 N18H_N297P_S187N SEQ ID NO 115 agURIC_st_24 N18H_S187N SEQ ID NO 116 agURIC_st_25 S187N_N297P SEQ ID NO 117agURIC_st_26 WT SEQ ID NO 118

[0239] The protein extracts of the stack variants were diluted 100-fold and thermostability was assessed using the thermal challenge assay as described in Example 1. Uric acid residual activity was determined in each stack variant, in duplication, before and after heat treatment. As the effects of heat treatment is protein concentration dependent, and higher protein concentration can lead to higher heat tolerance and higher residual activity, the heat treatment was performed at 56°C for 10 minutes (FIG.3). Two double mutants, agURIC_st_14 (H56Y S187N; SEQ ID NO: 106) and agURIC_st_13 (H56Y N297P; SEQ ID NO: 105), retrain the highest level of residual activity and highest stability as compared to wild type, as well as superior stability and residual activity compared to the best single variant of agURIC st O (H56Y; SEQ ID NO: 25) (Table 5).Table 5: Residual Activity Before and After Thermo-challenge of Top Variants from the Single and Stacked Libraries.Description SEQ ID NO: Residual Activity (%) WT SEQ ID NO 1 1.03H56Y SEQ ID NO 25 10.15H56Y N297P SEQ ID NO 105 19.6H56Y S187N SEQ ID NO 106 62.3

[0240] The protein extracts of the stack variant agURIC_st_14 (H56Y S187N; SEQ ID NO: 106) were tested for stability at higher heat temperatures and compared to the single variant of agURIC st O (H56Y; SEQ ID NO: 25). Thermostability was assessed without heat treatment and with heat treatment at 72.4°C, 73.3°C, 74.6°C, 76.1°C, 77.7°C, 79.3°C, 80.9°C, 82.4°C, 83.7°C, 84.6°C, and 85°C. The heat treatment was for 10 minutes. The protein was diluted to Img / L. Uric acid was measured. The double mutant, agURIC_st_14 (H56Y S187N; SEQ ID NO: 106) retained a higher level of stability and higher level of residual activity compared to the single variant of agURIC st O (H56Y; SEQ ID NO: 25) atAttorney Docket No. : 123818-0502> 80°C (FIG. 4). Additionally, the protein extracts of the stack variant agURIC_st_14 (H56Y S187N; SEQ ID NO: 106) were tested for stability at higher heat temperatures and compared to the single variant of agURIC st O (H56Y; SEQ ID NO: 25) and wild type (SEQ ID NO: 1). Thermostability was assessed without and with heat treatment of 65.7°C, 67.4°C, 70.0°C, 73.0°C, 75.9°C, 79.1°C, 82.0°C, 95.0°C, 87.6°C, 89.3°C, and 90.0°C. The heat treatment was for 10 minutes. The protein was diluted to 2mg / L. Uric acid was measured several times over the course of 30 minutes. The double mutant, agURIC_st_14(H56Y S187N; SEQ ID NO: 106) retained a higher level of stability and higher level of residual activity compared to the single variant of agURIC st O (H56Y; SEQ ID NO: 25) and wild-type (SEQ ID NO: 1) (FIG. 5).Example 3: Nebulization Challenge and Post-nebulization Challenge Activity Assay.

[0241] Uricase variant nebulization was accomplished using the PARI Vios nebulizer (model #31080003). 3 ml of the sample was loaded into the chamber and nebulization was started. The fog was precipitated and captured by an ice trap. Protein concentration and activity of the captured liquid was determined. The recovered protein was then tested for the ability to metabolize uric acid, and the data revealed the protein retained over 50% its activity after nebulization.Example 4: Protein Scale up for in vivo studies

[0242] The H56Y (SEQ ID NO: 25) variant from A. globiformis was expressed in E. coli and H56Y __S187N variant from A. globiformis was expressed in P. pastoris to get sufficient material for mice and non-human primate studies. The same design was applied to express the H56Y S187N A. globiformis variant in P. pastoris with the coding sequence was optimized for this host for expression (start and stop codon with bold; without purification tag):ATGACCGCTACAGCCGAAACTTCTACTGGTACTAAGGTTGTCTTGGGTCAAAACC AAIACGGlAAGGCl GAAGlCAGATTGGrCAAAGrCAClAGAAACACCGClAGAC ACGAAATCCAAGACTTGAACGTTACCTCTCAATTAAGAGGTGATTTTGAAGCCGC TTATACCGCTGGTGATAACGCTCACGTTGTCGCTACTGACACCCAAAAGAACACT GTTTACGCCTTCGCTAGAGACGGCTTCGCTACCACTGAGGAATTTCTATTGAGAT TGGGTAAGCATTTTACTGAAGGTTTCGACTGGGTCACCGGCGGTCGTTGGGCCGC TCAACAATTTTTCTGGGATAGAATTAACGATCACGATCATGCCTTCTCTAGAAAC AAATCTGAAGTCAGAACTGCTGTTTTAGAAATTTCTGGTTCCGAACAAGCTATCGAttorney Docket No. : 123818-0502TTGCTGGTATCGAAGGTTTGACCGTTTTGAAGTCCACCGGTTCCGAGTTCCATGG TTTCCCTAGAGACAAGTACACCACTTTGCAAGAAACTACCGACCGTATTTTAGCC ACTGATGTTAACGCTAGATGGCGTTACAACACTGTTGAAGTCGATTTTGACGCTG TCTACGCTTCCGTCAGAGGTTTATTGTTAAAGGCTTTCGCTGAAACCCACTCTCTA GCCTTACAACAAACTATGTACGAAATGGGTAGAGCCGTCATCGAAACTCACCCA GAAATTGACGAGATTAAGATGTCCTTACCTAACAAGCATCACTTCTTGGTTGATC TACAACCATTCGGCCAAGAIAACCCAAATGAAGTCTTTTACGCCGCTGACAGACC ATACGGTTTGATCGAAGCCACCATCCAAAGAGAAGGTTCCCGTGCCGATCACCC AATCTGGTCTAACATTGCTGGTTTCTGTTGA (SEQ ID NO: 119).

[0243] Histidine residues were added to the amino end of the proteins, thereby increasing the affinity of the protein for metal ion is increased allowing the increased affinity to be exploited to selectively isolate the protein of interest. When a protein with a His-tag is brought into contact with a carrier on which a metal ion is immobilized, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier or bind only very weakly, they can be removed by washing the carrier with an appropriate buffer. The poly-histidine tagged protein can then be recovered by eluting it off the resin. Example 5: Production of Uricase Variant: E. coli Expression - Bacteria

[0244] His-tagged H56Y uricase (SEQ ID NO: 25) was made in milligram quantities for animal studies in a 5L fermenter in standard lactose-containing autoinduction media (ZYP-5052). Fermentation was conducted at 24°C. After 16 hours, when CO2 production dropped, the media was collected and centrifuged at 7460 G for 20 minutes to pellet the cells. The cells were then resuspended in 20 mM Tris buffer (pH 7.4) and lysed by passing the cells through a cell homogenizer twice. The cell lysate was clarified by centrifugation (14,000 G for 1 hour), and the supernatant was applied to a His-tag affinity column. After binding, the column was washed with Buffer A (20mM Na-phosphate, 500mM NaCl pH 7.4) and then with 12 % Buffer B (20mM Na-phosphate, 500mM NaCl, 500mM imidazole pH 7.4) and eluted in Buffer B and diluted back in diluted immediately with 7x volume of 20mM Na-P, 200mM NaCl, pH 7.4. The elute was buffer exchanged to 20mM Na-phosphate, 200mM NaCl, pH 7.0, concentrated to 70 g / L. The samples were run on an SDS-PAGE gel. (FIG. 6)

[0245] To calculate the concentration of the uricase protein, the Thermo Fisher Nanodrop One ® was used. The Nanodrop unit calculated from the absorbance measured at 280 nM (A280), the molecular extinction coefficient (40.91), molecular weight (34.71 kDa),Attorney Docket No. : 123818-0502and the protein concentration in grams per liter. The molar extinction coefficient was a calculated value from the protein sequence.Example 6: Production of Uricase Variant: Pichia Expression - Yeast

[0246] The coding DNA sequence to express H56Y carrying the HIS-tag sequence, for ease of purification of the protein, at the 5 ’end was cloned into an integrative expression system under the control of the A0X1 methanol induced promoter. After DNA sequencing confirmation of the construct integrity, the plasmid was transformed in K. pastoris and colonies grown in the selective medium were picked and grown in liquid flaks; cell lysates were prepared, and the level of H56Y protein abundance were assessed using SDS-PAGE.

[0247] A top expressing candidate was selected for further studies and was grown into stirred tank, cell pellet was disrupted by homogenization and apply to the resin to purify the protein as a discreet band as seen on SDS-PAGE.

[0248] His-tagged H56Y uricase was made in grams quantities for animal studies in a 22L fermenter in Invitrogen Pichia process. Fermentation was conducted at 30°C.Fermentation was started with glycerol fed batch, then switched to MetOH fed batch. MetOH feeding was stopped at the age of 120 h. The media was collected and centrifuged 7460 G for 20 minutes to pellet the cells. The cells were then resuspended in 20 mM Tris buffer (pH 7.4) and lysed by passing the cells through a cell homogenizer twice. The cell lysate was clarified by centrifugation (14,000 G for 1 hour), and the supernatant was applied to a His-tag affinity column. After binding, the column was washed with Buffer A (20mM Na-phosphate, 500mM NaCl pH 7.4) and then with 12 % Buffer B (20mM Na-phosphate, 500mM NaCl, 500mM imidazole pH 7.4) and eluted in Buffer B and diluted back in diluted immediately with 7x volume of 20mM Na-P, 200mM NaCl, pH 7.4. The elute was buffer exchanged to 20mM Na-phosphate, 200mM NaCl, pH 7.0, concentrated to 70 g / L. The samples were run on an SDS-PAGE gel showing purification of the H56Y protein (FIG. 7).Example 7: In vivo reduction of circulating uric acid levels by variant uricase deposition in mouse and monkey lung

[0249] The present Example describes the effect of a variant uricase enzyme deposition in the lung tissue of mice and monkeys on circulating uric acid levels.

[0250] An induced model of hyperuricemia was produced in Cynomolgus non-human primates (NHP) by IV injection of uric acid (UA; 8.15 mg / kg) to assess the activity of anAttorney Docket No. : 123818-0502inhaled engineered variant uricase (H56Y (SEQ ID NO: 25) variant from A. globiformis) on UA in blood serum.

[0251] Briefly, on the morning of the experiment, 12 male non-naive NHPs, 4-5 kg, were randomized into four groups of three animals: 1) Neg. Control: no treatment, no UA; 2) UA Control: no treatment, + UA control; 3) Low dose uricase (H56Y) + UA; 4) High dose uricase (H56Y) + UA. Animals had been fasted overnight. A baseline blood collection was obtained at time 0, and inhalation treatment was then administered via the Aeroneb® Solo vibrating mesh nebulizer system with spacer and oronasal mask within 15 minutes of initial blood draw. Nebulized treatment was administered for 20 minutes (uricase buffer, 60 mg / mL uricase, or 20 mg / mL uricase) with an inhaled target uricase dose of 25 mg / kg and 8.3 mg / kg in treatment groups. At 75 minutes, an IV injection of uric acid was provided (to induce hyperuricemia) or 0.9% saline (Control).

[0252] Blood samples were collected at 60- and 65-minutes (post-inhalation and pre-UA), and at 95-, 155-, 275-, and 515-minutes (post-inhalation and UA injection) corresponding to 1-, 2-, 4-, and 8-hours following completion of nebulized treatment. Food was provided to the animals after the final blood collection. A crossover design was used to maximize the number of NHPs per group (n = 6 total), which incorporated a 7-day washout between each dosing day. Serum was isolated from blood samples using standard protocols and frozen at -80°C until analysis by tandem mass spectrometry to quantify uric acid.

[0253] Healthy baseline UA, prior to inhalation treatment and UA injection, was 4 pM ± 3.4 SD, and Group 1 (Neg. Control) maintained this concentration throughout the time course. UA injection resulted in high UA in the serum (Cmax = 75 minutes) and exhibited a subsequent gradual decrease returning to baseline by -275 minutes for uricase-treated animals, while UA Control uric acid was still elevated at the end of the experiment.

[0254] Variant uricase treatment targeted to the lungs resulted in a dose responsive reduction in circulating uric acid observable at all time points. For example, at Cmax, UA serum concentration was 74 pM ± 24.5 SD (UA Control), 59 pM ± 12 SD (Low-dose uricase + UA), and 46 pM ± 20 SD (High-dose uricase + UA) (FIG. 8A). Serum uric acid was decreased 36.6% (Low-dose uricase (H56Y)) and 56.8% (High-dose uricase (H56Y), p<0.01) compared to UA Control, as determined by area under the curve analysis of time course data (60 - 515 minutes). (Fig. 8B). No adverse effects of any kind were observed throughout the study.Attorney Docket No. : 123818-0502

[0255] In a second experiment, the ability of the variant uricase to decrease uric acid levels in vivo following lung deposition in vivo was assessed by directly comparing its activity to that of a WT uricase in mice. Briefly, mice (3 per group) were administered either 2 mg / mouse of uricase (A. flavus WT uricase enzyme or engineered A. globiformis variant uricase (H56Y) or vehicle (uricase buffer) by intratracheal (IT) administration. The mice were maintained on a commercial diet (LabDiet 5K52) and water purified by reverse osmosis, which was available ad libitum. Uricase was prepared on the day of dosing. Animals received 2 mg / mouse in a 50 pL dosing volume, by direct deposition into the lungs (IT).

[0256] Following uricase enzyme administration, blood samples were obtained from the mice 30, 60, 120, 180, and 240 minutes after delivery. Blood samples were also obtained immediately before uricase enzyme delivery (time 0). Blood samples were analyzed by fluorometric or colorimetric assay using a Uric Acid Assay Kit (AbCam #ab65344) for quantification of blood uric acid levels.

[0257] A head-to-head comparison of the commercial A. flavus WT uricase and an engineered globiformis variant uricase (H56Y) was performed, in which 2 mg of enzyme was delivered to the lungs of healthy female mice. Serum uric acid levels were then measured to determine efficacy compared to untreated controls.

[0258] Circulating uric acid levels were significantly reduced in all treated animals (FIG. 9). The observed decrease in UA levels was similar independent of the enzyme in the time course analysis (FIG. 9A). WT delivery decreased UA by 29.8% while variant uricase delivery decreased it by 34.4% compared to untreated controls (AUC), which was a statistically significant improvement over WT (p<0.01) (Fig. 9B).

[0259] These data further validate the potential benefits of lung delivery of therapeutic enzymes, which can be enhanced using protein engineering techniques to improve enzyme stability in the lung, which may have broad opportunities towards treatment of gout and other metabolic diseases.EQUIVALENTS

[0260] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses withinAttorney Docket No. : 123818-0502the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0261] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent that are not inconsistent with the explicit teachings of this specification.

Claims

Attorney Docket No. : 123818-0502CLAIMSWhat is claimed is:

1. A uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO:

1. wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.

2. The variant of claim 1, wherein the at least one substitution increases the thermostability of the variant relative to the wild-type uricase enzyme.

3. The variant of claim 2, wherein the increased thermostability of the variant relative the wild-type uricase is an increase of at least 5% of residual uricase activity following a thermal challenge.

4. The variant of claim 3, wherein the increase is at least 10% of residual uricase activity following a thermal challenge.

5. The variant of claim 3 or 4, wherein the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C.

6. The variant of any one of claims 3-5, wherein residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

7. The variant of any one of claims 1-6, wherein the residual uricase activity for at least one variant is retained following nebulization, wherein the residual uricase activity following nebulization is about 50% or more.

8. The variant of any one of claims 1-7, wherein the variant sequence comprises at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S,Attorney Docket No. : 123818-0502A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

9. The variant of any one of claims 1-8, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92.

10. The variant of any one of claims 1-9, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297.

11. The variant of any one of claims 1-10, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

12. The variant of any one of claims 1-11, wherein the variant sequence comprises at least two substitutions at two or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

13. The variant of claim 12, wherein the variant sequence comprises at last two substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

14. The variant of claim 12 or 13, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 93, 101, 105, 106, 108, 111, 112, 114, 116, and 117.

15. The variant of any one of claims 1-11, wherein the variant sequence comprises at least three substitutions at three or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

16. The variant of claim 15, wherein the variant sequence comprises at least three substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

17. The variant of claim 15 or 16, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 94, 98, 99, 102, 104, 107, 109, 113, and 115.Attorney Docket No. : 123818-050218. The variant of any one of claims 1-11, wherein the variant sequence comprises at least four substitutions at four or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

19. The variant of claim 18, wherein the variant sequence comprises at least four substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

20. The variant of claim 18 or 19, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 95, 97, 100, 103, and 110.

21. The variant of any one of claims 1-11, wherein the variant sequence comprises at least five substitutions at five or more amino acid positions selected from 18, 29, 56, 63, 187, and 297, of SEQ IDNO: 1.

22. The variant of claim 21, wherein the variant sequence comprises at least five substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

23. The variant of claim 21 or 22, wherein the variant sequence comprises an amino acid sequence of SEQ ID NO: 96.

24. The variant of any one of claims 1-23, wherein the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

25. The variants of any one of claims 1-24, for use in treating gout.

26. Use of a variant of any one of claims 1-24 in the manufacture of a medicament for treating gout.

27. A pharmaceutical composition, comprising: (a) a uric acid degrading enzyme variant according to any one of claims 1-25, and (b) a pharmaceutically acceptable carrier or excipient.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is formulated for aerosol administration.

29. The pharmaceutical composition of claim 28, wherein the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

30. A pharmaceutical composition, comprising:Attorney Docket No. : 123818-0502(a) A uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization; and(b) a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, wherein the increased thermostability of the variant relative the wild-type uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge.

32. The pharmaceutical composition of claim 31, wherein the increase is at least 10% of residual uricase activity following a thermal challenge.

33. The pharmaceutical composition of claim 31 or 32, wherein the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C.

34. The pharmaceutical composition of any one of claims 31-33, wherein the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

35. The pharmaceutical composition of any one of claims 31-34, wherein the residual uricase activity following nebulization is about 50% or more.

36. The pharmaceutical composition of any one of claims 30-35, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.

37. The pharmaceutical composition of any one of claims 30-36, wherein the variant sequence comprises at least one substitution selected from T1 IE, T1 IM, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V,Attorney Docket No. : 123818-0502I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, QI 12E, Fl 13 A, DI 16N, DI 16E, DI 16K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

38. The pharmaceutical composition of any one of claims 30-37, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92.

39. The pharmaceutical composition of any one of claims 30-38, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297.

40. The pharmaceutical composition of any one of claims 30-39, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

41. The pharmaceutical composition of any one of claims 30-40, wherein the variant sequence comprises at least two substitutions at two or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

42. The pharmaceutical composition of claim 41, wherein the variant sequence comprises at last two substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

43. The pharmaceutical composition of claim 41 or 42, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 93, 101, 105, 106, 108, 111, 112, 114, 116, and 117.

44. The pharmaceutical composition of any one of claims 30-40, wherein the variant sequence comprises at least three substitutions at three or more positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

45. The pharmaceutical composition of claim 44, wherein the variant sequence comprises at least three substitutions selected from N18H, H56Y, H63F, S187N, and N297P.Attorney Docket No. : 123818-050246. The pharmaceutical composition of claim 44 or 45, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 94, 98, 99, 102, 104, 107, 109, 113, and 115.

47. The pharmaceutical composition of any one of claims 30-40, wherein the variant sequence comprises at least four substitutions at four or more amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

48. The pharmaceutical composition of claim 47, wherein the variant sequence comprises at least four substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

49. The pharmaceutical composition of claim 47 or 48, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 95, 97, 100, 103, and 110.

50. The pharmaceutical composition of any one of claims 30-40, wherein the variant sequence comprises at least five substitutions at five or more amino acid positions selected from 18, 29, 56, 63, 187, and 297, of SEQ ID NO: 1.

51. The pharmaceutical composition of claim 50, wherein the variant sequence comprises at least five substitutions selected from N18H, H56Y, H63F, S187N, and N297P.

52. The pharmaceutical composition of claim 50 or 51, wherein the variant sequence comprises an amino acid sequence of SEQ ID NO: 96.

53. The pharmaceutical composition of any one of claims 30-52, wherein the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

54. The pharmaceutical composition of any one of claims 30-53, wherein the pharmaceutical composition is formulated for aerosol administration.

55. The pharmaceutical composition of claim 54, wherein the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

56. The pharmaceutical composition of any one of claims 27-55 for use in treating gout.

57. Use of a pharmaceutical composition of any one of claims 27-55 in the manufacture of a medicament for treating gout.Attorney Docket No. : 123818-050258. A method of treating a subject with gout, the method comprising administering to the subject a variant according to any one of claims 1-24 or a pharmaceutical composition according to any one of claims 27-55.

59. The method of claim 58, wherein the variant or the pharmaceutical composition is administered as a liquid aerosol or a dry powder aerosol.

60. A method of treating a subject with gout, the method comprising administering to the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization.

61. The method of claim 60, wherein the increased thermostability of the variant relative the wild-type uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge.

62. The method of claim 61, wherein the increase is at least 10% of residual uricase activity following a thermal challenge.

63. The method of claim 61 or 62, wherein the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C.

64. The method of any one of claims 60-63, wherein the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.

65. The method of any one of claims 60-64, wherein the residual uricase activity following nebulization is about 50% or more.

66. The method of any one of claims 60-65, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.Attorney Docket No. : 123818-050267. The method of any one of claims 60-66, wherein the variant sequence comprises at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

68. The method of any one of claims 60-67, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92.

69. The method of any one of claims 60-68, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297.

70. The method of any one of claims 60-69, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

71. The method of any one of claims 60-70, wherein the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

72. The method of claim 71, wherein the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, andN297P.

73. The method of claim 71 or 72, wherein the variant sequence comprises 2, 3, 4, or 5 substitutions.

74. The method of any one of claims 60-73, wherein the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus75. The method of any one of claims 60-74, wherein the enzyme variant is formulated for aerosol administration.Attorney Docket No. : 123818-050276. The method of claim 75, wherein the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

77. A method of degrading uric acid in a subject, the method comprising administering to the subject a variant according to any one of claims 1-24 or a pharmaceutical composition according to any one of claims 27-55.

78. The method of claim 77, wherein the variant or the pharmaceutical composition is formulated for aerosol administration.

79. The method of claim 78, wherein the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

80. The method of any one of claims 77-79, wherein the subject has gout.

81. A method of degrading uric acid in a subject, the method comprising administering to the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1 that (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization.

82. The method of claim 81, wherein the increased thermostability of the variant relative the wild-type uricase enzyme is an increase of at least 5% of residual uricase activity following a thermal challenge.

83. The method of claim 82, wherein the increase is at least 10% of residual uricase activity following a thermal challenge.

84. The method of claim 82 or 83, wherein the thermal challenge comprises an incubation for about 10 minutes at about 66 °C to about 68 °C.

85. The method of any one of claims 81-84, wherein the residual uricase activity for each variant is calculated by dividing the amount of uricase activity following thermal challenge by the amount of uricase activity prior to thermal challenge.Attorney Docket No. : 123818-050286. The method of any one of claims 81-85, wherein the residual uricase activity following nebulization is about 50% or more.

87. The method of any one of claims 81-86, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.

88. The method of any one of claims 81-87, wherein the variant sequence comprises at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H, E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

89. The method of any one of claims 81-88, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92.

90. The method of any one of claims 81-89, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297.

91. The method of any one of claims 81-90, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

92. The method of any one of claims 81-91, wherein the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

93. The method of claim 92, wherein the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, andN297P.Attorney Docket No. : 123818-050294. The method of claim 92 or 93, wherein the variant sequence comprises 2, 3, 4, or 5 substitutions.

95. The method of any one of claims 81-94, wherein the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus96. The method of any one of claims 81-95, wherein the enzyme variant is formulated for aerosol administration.

97. The method of claim 96, wherein the aerosol administration is selected from liquid aerosol administration or dry powder aerosol administration.

98. The method of any one of claims 81-97, wherein the subject has gout.

99. The method of any one of claims 58-98, wherein the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

100. A method of treating a subject with gout, the method comprising administering to the lung of the subject a uric acid degrading enzyme variant comprising an amino acid sequence that is a variant of the amino acid sequence of the wild-type uricase enzyme set forth in SEQ ID NO: 1.

101. The method of claim 100, wherein the variant (i) increases thermostability of the enzyme variant relative to the wild-type uricase enzyme, (ii) retains residual uricase enzyme activity following nebulization relative to the wild-type uricase enzyme, or (iii) increases thermostability of the enzyme variant relative to wild-type uricase enzyme and retains residual uricase enzyme activity following nebulization.

102. The method of claim 100 or 101, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 11, 17, 18, 20, 22, 29, 31, 33, 39, 46, 50, 53, 56, 63, 64, 67, 74, 83, 95, 97, 100, 109, 112, 113, 116, 136, 142, 144, 145, 148, 159, 175, 177, 185, 186, 187, 195, 196, 203, 205, 218, 219, 220, 221, 224, 229, 244, 245, 249, 251, 253, 258, 269, 279, 284, 296, and 297, of SEQ ID NO: 1.

103. The method of any one of claims 100-102, wherein the variant sequence comprises at least one substitution selected from THE, T11M, Q17D, Q17E, Q17K, Q17S, N18H, Y20F, K22I, K22M, K22W, K22V, K29D, T31D, N33E, N33T, N33V, I39L, S46T, G50A, E53H,Attorney Docket No. : 123818-0502E53L, E53F, H56Y, H63N, H63F, V64I, T67F, V74I, A83K, H95F, H95Y, T97V, F100Y, A109S, Q112E, F113A, D116N, D116E, D116K, V136T, S142D, Q144I, Q144V, A145C, A145S, A145T, A148S, A148T, T159D, Q175E, T177F, D185T, D185V, V186I, V186L, S187N, S187D, S187T, S187V, V195T, E196N, E196D, Y203P, S205A, H218D, S219D, L220P, A221N, A221S, Q224H, Q224F, Q224Y, M229A, K244R, M245A, N249D, N249L, H251E, H251P, F253Y, Q258R, Q258K, Q258S, F269Y, I279V, Q284R, Q284K, S296D, N297E, and N297P.

104. The method of any one of claims 100-103, wherein the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-92.

105. The method of any one of claims 100-104, wherein the variant sequence comprises at least one substitution at one or more of the positions selected from 18, 22, 56, 63, 187, and 297.

106. The method of any one of claims 100-105, wherein the variant sequence comprises at least one substitution selected from N18H, H56Y, H63F, S187N, and N297P.

107. The method of any one of claims 100-105, wherein the variant sequence comprises two to five substitutions at two to five amino acid positions selected from 18, 29, 56, 63, 187, and 297 of SEQ ID NO: 1.

108. The method of claim 107, wherein the two to five variant substitutions are selected from N18H, H56Y, H63F, S187N, andN297P.

109. The method of claim 107 or 108, wherein the variant sequence comprises 2, 3, 4, or 5 substitutions.

110. The method of any one of claims 100-109, wherein the amino acid sequence further comprises a polyhistidine-tag (His-tag) at the N-terminus or C-terminus.

111. The method of any one of claims 100-110, wherein the uric acid degrading enzyme variant is introduced to the subject’s lung under conditions that inhibit or do not support systemic delivery of the uric acid degrading enzyme variant to the subject.

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