Fitness maturation of a blood-brain barrier penetrant AAV capsid

Abstract

This application relates to engineering AAV capsids, for example, STAC-BBB and variants of STAC-BBB. In some embodiments, the engineered AAV capsids mediate delivery to the central nervous system.

Description

Attorney Docket No.: | 91355.12316 P.0288.WO1FITNESS MATURATION OF A BLOOD-BRAIN BARRIER PENETRANT AAV CAPSIDFIELD

[0001] This application relates to engineering AAV Capsids.BACKGROUND

[0002] The clinical translation of genomic medicines to treat disorders of the central nervous system (CNS) has been limited by inefficient gene delivery. Previously, the blood-brain barrier penetrant capsid STAC-BBB (CNSRCV300 in PCT Publication No. WO2024238684) was identified as a novel adeno-associated virus (AAV) capsid variant that exhibits enhanced CNS delivery after intravenous administration. STAC-BBB is derived from the parental serotype AAV9, and was discovered using the functional selection platform SIFTER (Selecting In vivo for Transduction and Expression of RNA, see, PCT Publication No. WO2020219988). Fitness maturation of engineered capsids may further improve upon their key desirable properties. There remains a need to perform fitness maturation of capsid STAC-BBB to identify variants of STAC-BBB that mediate further improvement in CNS delivery.SUMMARY

[0003] STAC-BBB is an engineered AAV capsid that has shown enhanced biodistribution across the central nervous system (CNS) compared to wild-type AAV9. Described herein are modifications to the STAC-BBB capsid. In some embodiments, these modifications result in additional enhanced biodistribution across the CNS relative to the unmodified STAC-BBB capsid. In some embodiments, the modifications comprise substituting specific amino acids within the STAC-BBB capsid. In some embodiments, the modified STAC-BBB capsid comprises a CNS-targeting molecule that may also be used to enhance biodistribution across the CNS of non-AAV compositions.

[0004] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 1-8.

[0005] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 9-17.

[0006] In an embodiment, an engineered AAV capsid protein is provided comprising a diversified region as in STAC-BBB variants SEQ ID NO: 1-17.14899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0007] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 1, wherein the diversified region comprises: at position 2, any one of the amino acids Y, S, I, K, N, F, V, T, Q, R, H, L, P, M, W, or E; and / or, at position 10, any one of the amino acids D, L, H, N, E, Q, I, S, A, F, T, Y, V, P, R, K, or W.SEQ ID NO: 1Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S Y Y V N I M D D D D Q AAt position 2: Any of Y, S, I, K, N, F, V, T, Q, R, H, L, P, M, W, or EAt position 10: Any of D, L, H, N, E, Q, I, S, A, F, T, Y, V, P, R, K, or W

[0008] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 2, wherein the diversified region comprises: at position 6, any one of the amino acids V, F, S, T, D, L, A, or M; and / or, at position 10, any one of the amino acids L, D, F, N, P, E, Y, G, H, V, K, S, R, or A.SEQ ID NO: 2Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y V N V M D D L D Q AAt position 6: Any of V, F, S, T, D, L, A, or MAt position 10: Any of L, D, F, N, P, E, Y, G, H, V, K, S, R, or A

[0009] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 3, wherein the diversified region comprises: at position 6, any one of the amino acids V or D.SEQ ID NO: 3Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y V N V M D D M D Q AAt position 6: Any of V, or D

[0010] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 4, wherein the diversified region comprises: at position 6, any one of the amino acids V, D, S, K, L, or Q; and / or, at position 13, any one of the amino acids P, S, E, L, V, W, N, T, Q, H, Y, F, G, M, I, or K.SEQ ID NO: 4Position 1 2 3 4 5 6 7 8 9 10 11 12 1324899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1Amino Acid S A Y V N V M D D M D Q PAt position 6: Any of V, D, S, K, L, or QAt position 13 : Any of P, S, E, L, V, W, N, T, Q, H, Y, F, G, M, I, or K

[0011] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 5, wherein the diversified region comprises: at position 6, any one of the amino acids V, L, D, S, P, or H; and / or, at position 12, any one of the amino acids L, V, F, G, S, H, M, W, Y, P, A, E, N, K, T, D, I, or R.SEQ ID NO: 5Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y V N L M D D M D W AAt position 6: Any of V, L, D, S, P, or HAt position 12: Any of L, V, F, G, S, H, M, W, Y, P, A, E, N, K, T, D, I, or R

[0012] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 6, wherein the diversified region comprises: at position 1, any one of the amino acids T, H, M, V, N, G, W, D, F, E, R, I, L, or K; and / or, at position 5, any one of the amino acids F, Q, I, S, W, Y, V, A, M, H, or T.SEQ ID NO: 6Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino AcidI A Y V I I M D D M D Q AAt position 1 : Any of T, H, M, V, N, G, W, D, F, E, R, I, L, or KAt position 5: Any of F, Q, I, S, W, Y, V, A, M, H, or T

[0013] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 7, wherein the diversified region comprises: at position 4, any one of the amino acids M, Y, I, L, or F.SEQ ID NO: 7Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y R N I M D D M D Q AAt position 4: Any of M, Y, I, L, or F

[0014] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 8, wherein the diversified region comprises: at position 11, any one of the amino acids N, W, K, I, H, G, F, S, Q, V, P, M, R, T,34899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1E, L, A, or Y; and / or, at position 13, any one of the amino acids H, V, P, R, W, E, S, F, L, Y, G, N, Q, I, K, D, T, or M.SEQ ID NO: 8Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y V N I M D D M K Q KAt position 11 : Any of N, W, K, I, H, G, F, S, Q, V, P, M, R, T, E, L, A, or YAt position 13 : Any of H, V, P, R, W, E, S, F, L, Y, G, N, Q, I, K, D, T, or M

[0015] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 9, wherein the diversified region comprises: at position 2, any one of the amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y; and / or, at position 4, any one of the amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E.SEQ ID NO: 9Position 1 2 3 4 5 6 7Amino Acid G V G N N Q QAt position 2: Any of V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or YAt position 4: Any of N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E

[0016] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 10, wherein the diversified region comprises: at position 2, any one of the amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y; and / or, at position 4, any one of the amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E.SEQ ID NO: 10Position 1 2 3 4 5 6 7Amino Acid G A G L N Q QAt position 2: Any of V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or YAt position 4: Any of N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E

[0017] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 11, wherein the diversified region comprises: at position 2, any one of the amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y; and / or, at position 4, any one of the amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E.44899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1SEQ ID NO: 11Position 1 2 3 4 5 6 7Amino Acid G V G T N Q QAt position 2: Any of V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or YAt position 4: Any of N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E

[0018] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 12, wherein the diversified region comprises: at position 1, any one of the amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F; and / or, at position 4, any one of the amino acids S, N, T, A, H, V, L, Y, P, W, G, D, R, or I.SEQ ID NO: 12Position 1 2 3 4 5 6 7Amino Acid N S G N N Q QAt position 1 : Any of S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or FAt position 4: Any of S, N, T, A, H, V, L, Y, P, W, G, D, R, or I

[0019] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 13, wherein the diversified region comprises: at position 4, any one of the amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K; and / or, at position 6, any one of the amino acids K, A, H, S, T, R, P, M, V, W, N, Y, G, or I.SEQ ID NO: 13Position 1 2 3 4 5 6 7Amino Acid G S G L N K QAt position 4: Any of L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or KAt position 6: Any of K, A, H, S, T, R, P, M, V, W, N, Y, G, or I

[0020] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 14, wherein the diversified region comprises: at position 4, any one of the amino acids W, K, T, H, A, R, V, S, L, G, N, M, I, or P; and / or, at position 7, any one of the amino acids E, N, F, I, H, G, R, K, P, A, M, L, or V.SEQ ID NO: 14Position 1 2 3 4 5 6 7Amino Acid G S G K N Q N54899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1At position 4: Any of W, K, T, H, A, R, V, S, L, G, N, M, I, or PAt position 7: Any of E, N, F, I, H, G, R, K, P, A, M, L, or V

[0021] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 15, wherein the diversified region comprises: at position 1, any one of the amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F; and / or, at position 4, any one of the amino acids S, N, T, A, H, V, L, Y, P, W, G, D, R, or I.SEQ ID NO: 15Position 1 2 3 4 5 6 7Amino Acid S S G S N Q QAt position 1 : Any of S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or FAt position 4: Any of S, N, T, A, H, V, L, Y, P, W, G, D, R, or I

[0022] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 16, wherein the diversified region comprises: at position 4, any one of the amino acids L, S, T, I, A, G, N, F, K, V, M, R, Y, E, D, W, H, or P; and / or, at position 5, any one of the amino acids K, H, A, S, T, R, V, M, W, Q, L, F, G, D, or E.SEQ ID NO: 16Position 1 2 3 4 5 6 7Amino Acid G S G L K Q QAt position 4: Any of L, S, T, I, A, G, N, F, K, V, M, R, Y, E, D, W, H, or PAt position 5: Any of K, H, A, S, T, R, V, M, W, Q, L, F, G, D, or E

[0023] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 17, wherein the diversified region comprises: at position 4, any one of the amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K; and / or, at position 6, any one of the amino acids K, A, H, S, T, R, P, M, V, W, N, Y, G, or I.SEQ ID NO: 17Position 1 2 3 4 5 6 7Amino Acid G S G S N K QAt position 4: Any of L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or KAt position 6: Any of K, A, H, S, T, R, P, M, V, W, N, Y, G, or I64899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0024] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant of SEQ ID NO: 35, wherein the diversified region comprises any one of the amino acids T, N, W, V, or L at position 1; Q, L, N, S, K, Y, M, T, R, I, or V at position 2; I, F, V, D, or L at position 3; M, Y, I, L, or F at position 4; V or D at position 6; I at position 7; G at position 8; E or N at position 9; L, D, E, N, K, Y, F, S, or A at position 10; I, H, S, E, A, or W at position 11; H, L, M, S, V, F, G, N, K, or D at position 12; P, S, L, V, N, T, H, Q, E, G, F, orK at position 13.SEQ ID NO: 35Position 1 2 3 4 5 6 7 8 9 10 11 12 13Amino Acid S A Y V N I M D D M D Q AAt position 1 : Any of T, N, W, V, or LAt position 2: Any of Q, L, N, S, K, Y, M, T, R, I, or VAt position 3 : Any of I, F, V, D, or LAt position 4: Any of M, Y, I, L, or FAt position 6: Any of V or DAt position 7: IAt position 8: GAt position 9: Any of E or NAt position 10: Any of L, D, E, N, K, Y, F, S, or AAt position 11 : Any of I, H, S, E, A, or WAt position 12: Any of H, L, M, S, V, F, G, N, K, or DAt position 13: Any of P, S, L, V, N, T, H, Q, E, G, F, or K

[0025] In an embodiment, an engineered AAV (e.g., STAC-BBB) sequence is provided comprising a variant diversified region SEQ ID NO: 5443, wherein the diversified region comprises any one of the amino acids N, V, A, P, L, H, F, Y, or T at position 1; A, N, G, Q, K, or V at position 2; S or T at position 3; T, A, V, S, L, I, H, or G at position 4; R, K, L, or D at position 5; A at position 6; F, K, M, or H at position 7.SEQ ID NO: 5443Position 1 2 3 4 5 6 7Amino Acid G S G Q N Q QAt position 1 : Any of N, V, A, P, L, H, F, Y, or TAt position 2: Any of A, N, G, Q, K, or VAt position 3 : Any of S or TAt position 4: Any of T, A, V, S, L, I, H, or GAt position 5: Any of R, K, L, or DAt position 6: AAt position 7: Any of F, K, M, or H74899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1. SIFTER library design utilized for screening of the STAC-BBB capsid variant libraries. Each capsid is linked to at least three unique barcodes using a bioinformatic look-up table. Hundreds to thousands of unique molecular identifiers (UMIs) are cloned per barcode to facilitate interpretation of distinct AAV transduction events. Capsid performance is evaluated based on barcoded mRNA expression from the neuron specific human synapsin 1 promoter or the ubiquitous cytomegalovirus (CMV) promoter.

[0027] FIG. 2. Neuronal mRNA expression in cynomolgus macaque brain for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598. Six coronal brain slices spanning the rostral-caudal axis were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 1-8 are annotated, along with STAC-BBB and AAV9.

[0028] FIG. 3. Ubiquitous mRNA expression in cynomolgus macaque brain for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598. Six coronal brain slices spanning the rostral-caudal axis were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 1-8 are annotated, along with STAC-BBB and AAV9.

[0029] FIG. 4. Neuronal mRNA expression in cynomolgus macaque spinal cord for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598. Cervical, thoracic, and lumbar spinal cord segments were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 1-8 are annotated, along with STAC-BBB and AAV9.

[0030] FIG. 5. Ubiquitous mRNA expression in cynomolgus macaque spinal cord for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598. Cervical, thoracic, and lumbar spinal cord segments were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 1-8 are annotated, along with STAC-BBB and AAV9.84899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0031] FIG. 6. Single amino acid substitution heatmap for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598. The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the original amino acid at each position in the STAC-BBB amino acid sequence. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0032] FIG. 7. Neuronal mRNA expression in cynomolgus macaque brain for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459. Six coronal brain slices spanning the rostral-caudal axis were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 9-17 are annotated, along with STAC-BBB.

[0033] FIG. 8. Ubiquitous mRNA expression in cynomolgus macaque brain for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459. Six coronal brain slices spanning the rostral-caudal axis were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 9-17 are annotated, along with STAC-BBB.

[0034] FIG. 9. Neuronal mRNA expression in cynomolgus macaque spinal cord for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459. Cervical, thoracic, and lumbar spinal cord segments were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 9-17 are annotated, along with STAC-BBB.

[0035] FIG. 10. Ubiquitous mRNA expression in cynomolgus macaque spinal cord for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459. Cervical, thoracic, and lumbar spinal cord segments were analyzed and performance metrics were calculated for each capsid variant within the library. Capsid variants with the diversified regions corresponding to sequence ID numbers 9-17 are annotated, along with STAC-BBB.

[0036] FIG. 11. Single amino acid substitution heatmap for the STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459. The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the original amino acid at each position in the STAC-BBB amino acid 94899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1sequence. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0037] FIG. 12. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 2 and 10 in the diversified region of amino acids 586 through 598.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 1. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0038] FIG. 13. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 6 and 10 in the diversified region of amino acids 586 through 598.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 2. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0039] FIG. 14. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 6 and 13 in the diversified region of amino acids 586 through 598.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 4. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0040] FIG. 15. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 6 and 12 in the diversified region of amino acids 586 through 598.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 5. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0041] FIG. 16. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 1 and 5 in the diversified region of amino acids 586 through 598.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 6. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0042] FIG. 17. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 11 and 13 in the diversified region of amino acids 586 through 598.104899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 8. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0043] FIG. 18. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 2 and 4 in the diversified region of amino acids 453 through 459.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 9, 10, or 11. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0044] FIG. 19. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 1 and 4 in the diversified region of amino acids 453 through 459.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 12 or 15. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0045] FIG. 20. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 4 and 6 in the diversified region of amino acids 453 through 459.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 13 or 17. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0046] FIG. 21. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 4 and 7 in the diversified region of amino acids 453 through 459.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 14. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.

[0047] FIG. 22. Double amino acid substitution heatmap for the STAC-BBB capsid with mutations at positions 4 and 5 in the diversified region of amino acids 453 through 459.The heat map indicates the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in SEQ ID No. 16. White squares without a value represent capsid sequences with a log2 fold change that is less than 1.114899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0048] FIG. 23 shows Table 1.

[0049] FIG. 24 shows Table 2.

[0050] FIG. 25 shows Table 3.DETAILED DESCRIPTION

[0051] In some embodiments, the present invention relates to engineered capsids identified through fitness maturation of the STAC-BBB capsid. In some embodiments, an engineered capsid comprises a diversified region sequence as shown in Tables 1, 2, 3, 4, 7, or 8. In some embodiments, the engineered STAC-BBB variant sequences enable enhanced delivery to the central nervous system (CNS).

[0052] In an embodiment, an engineered AAV capsid protein is provided. In embodiments, the engineered AAV capsid protein comprises at least 3, 4, 5, 6 or 7 contiguous amino acids from any of the diversified region sequences according to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 described herein (see, e g., Table 4). In some embodiments, the engineered AAV capsid proteins comprise modifications of SEQ ID NO: 1-17, such as at least one or two substitutions to any amino acids within any of SEQ ID NO: 1-17.

[0053] In an embodiment, an engineered AAV capsid protein is provided. In embodiments, the engineered AAV capsid protein comprises at least 3, 4, 5, 6 or 7 contiguous amino acids from any of the diversified region sequences according to any one of SEQ ID NOs: 37-79 described herein (see, e.g., Table 7). In some embodiments, the engineered AAV capsid proteins comprise modifications of SEQ ID NO: 37-79, such as at least one or two substitutions to any amino acids within any of SEQ ID NO: 37-79.

[0054] In another embodiment, an engineered STAC-BBB AAV capsid protein is provided. In embodiments, the engineered STAC-BBB capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises the sequence outside the diversified region in any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34 described herein. In some embodiments, the engineered STAC-BBB capsid protein is124899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1at least 80%, 85%, 90%, 95%, or 99% identical to or comprises any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 120, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34 described herein. In some embodiments, the engineered STAC-BBB capsid protein comprises modifications within any one of a VP1 sequence as described herein, for example, any one of SEQ ID NOs: 18-34, such as at least one or two substitutions to any amino acids within any one of a VP1 sequence as described herein, for example, any one of SEQ ID NOs: 18-34. In some embodiments, the at least one or two substitutions within any one of a VP1 sequence as described herein, for example, any one of SEQ ID NOs: 18-34 comprise substitutions of conserved amino acids, wherein the original and substituted amino acids have identical or similar biochemical properties. In some embodiments, the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in any one of SEQ ID NOs: 18-25 or 81-122.Engineered AAV Capsid Proteins

[0055] In an embodiment, described herein are compositions comprising engineered AAV capsid proteins and methods of making and using the same. In some embodiments, the AAV capsid protein has enhanced tropism for a cell or tissue, e.g., for the delivery of genetic material to a specific cell or tissue, for example a CNS tissue or a CNS cell. In some embodiments, a parent capsid protein is engineered for enhanced function. In a preferred embodiment, the parent capsid is the STAC-BBB capsid.

[0056] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids within a diversified region of a parent capsid protein. In some embodiments the diversified region is within or near a surface-exposed loop of the capsid, for example, VR8. In some embodiments the diversified region is within or near amino acids 586 through 598 corresponding to the sequence of AAV9. In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 1-8 or SEQ ID NOs: 38-79. In some embodiments, an engineered AAV capsid protein sequence is provided wherein the diversified region comprises a sequence wherein the first, second, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and / or134899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1thirteenth amino acid position is modified in any one of SEQ ID NOs: 1-8 or SEQ ID NOs: 38-79.

[0057] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, or 7 contiguous amino acids within a diversified region of a parent capsid protein. In some embodiments the diversified region is within or near is within or near a surface-exposed loop of the capsid, for example, VR4. In some embodiments the diversified region is within or near amino acids 453 through 459 corresponding to the sequence of AAV9. In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 9-17 or SEQ ID NO: 37. In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, or 7 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 9-17. In some embodiments, an engineered AAV capsid protein sequence is provided wherein the diversified region comprises a sequence wherein the first, second, fourth, fifth, sixth, and / or seventh amino acid position is modified in any one of SEQ ID NOs: 9-17 or SEQ ID NO: 37.

[0058] In some embodiments, an engineered AAV parent capsid is modified by combinations of diversified regions. In some embodiments, an engineered AAV parent capsid is modified by combining any one of SEQ ID NOs: 1-8 or SEQ ID NOs: 38-79 with any one of SEQ ID NOs: 9-17 or SEQ ID NO: 37. In some embodiments, an engineered AAV parent capsid comprises any one of SEQ ID NOs: 1-8 or SEQ ID NOs: 38-79 and any one of SEQ ID NOs: 9-17 or SEQ ID NO: 37.

[0059] In some embodiments, the engineered AAV capsid proteins have advantages over wildtype AAV capsid proteins. In some embodiments, these advantages including (i) enhanced cell or tissue tropism as compared to the natural / wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural / wild-type AAV serotype (ii) increased penetrance through the blood brain barrier following administration to a subject, (iii) wider distribution throughout the multiple brain regions, for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus, (iv) elevated expression of genetic material in multiple brain regions. In some embodiments, the engineered AAV capsids enhance the delivery of genetic material to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus,144899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1caudate, and / or hippocampus, (v) is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0060] In embodiments, the engineered AAV capsid proteins and genetic material described herein may be delivered to one ormore (such as 1-10) target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins have enhanced tropism for a specific target cell type, tissue or organ. As a non-limiting example, the engineered AAV capsid protein has enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, engineered AAV capsid proteins are produced recombinantly and are an adeno-associated virus (AAV) serotype such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74, or a combination thereof. In some embodiments, engineered AAV capsid proteins are produced recombinantly and are based on any one or more (such as 1-15) AAV serotypes known in the art.

[0061] Fitness Maturation of Modified STAC-BBB Parent Capsids

[0062] In some embodiments, the engineered AAV capsid protein comprises a modified STAC-BBB parent capsid. In some embodiments, the engineered AAV capsid protein comprises a modified STAC-BBB parent capsid sequence, wherein the parent sequence comprises SEQ ID NO: 36. In some embodiments, the engineered STAC-BBB capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or essentially comprises the sequence of SEQ ID NO: 36. In some embodiments, the engineered AAV capsid protein comprises a modified STAC-BBB parent capsid comprising at least one or two amino acid modifications to SEQ ID NO: 36. In some embodiment, the at least one or two amino acid modifications to SEQ ID NO: 36 are made in a surface-exposed loop of the sequence upon its formation of a capsid protein, for example, within or near VR8 and / or VR4. In some embodiments, the at least one or two amino acid modifications to SEQ ID NO: 36 are made in a diversified region of the sequence, for example, within or near amino acids 586 through 598 or amino acids 453 through 459 corresponding to the sequence of AAV9.

[0063] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGY KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGK SGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN154899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS QAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQY LYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDAD KVMITNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPG MVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTA FNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTE GVYSEPRPIGTRYLTRNL (SEQ ID NO: 36)

[0064] In some embodiments, the diversified region of STAC-BBB comprises at least one or two substitutions in SEQ ID NO: 35. In some embodiments, the at least one or two substitutions of SEQ ID NO: 36 occurs at one or two of any of the following the amino acids 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and 598 (amino acids shown in bold in SEQ ID NO: 36). In some embodiments, amino acids 586-598 of the STAC-BBB capsid comprise the diversified region of the STAC-BBB parent capsid. In some embodiments, the diversified region of the STAC-BBB parent capsid comprises the sequence: SAYVNIMDDMDQA (SEQ ID NO: 35).

[0065] Modifications to amino acids in SEQ ID NO: 36 (STAC-BBB parent capsid)

[0066] In some embodiments, the STAC-BBB parent capsid is modified, z.e., comprises a substitution, insertion, deletion or combinations thereof. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 586. In some embodiments, the modification at position 586 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 587. In some embodiments, the modification at position 587 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 588. In some embodiments, the modification at position 588 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 589. In some embodiments, the modification at position 589 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 590. In some embodiments, the modification at position 590 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 591. In some embodiments, the modification at position 591 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 592. In some embodiments, the modification at position 592 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 593. In some embodiments, the modification at position 593 comprises a164899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 594. In some embodiments, the modification at position 594 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 595. In some embodiments, the modification at position 595 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 596. In some embodiments, the modification at position 596 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 596. In some embodiments, the modification at position 596 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 597. In some embodiments, the modification at position 597 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 598. In some embodiments, the modification at position 598 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and / or 598. In some embodiments, the STAC-BBB parent capsid is modified in at least two different positions selected from amino acid position 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and / or 598. In some embodiments, the modification at position 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and / or 598 comprises a substitution, insertion, deletion or combinations thereof.

[0067] In some embodiments, the STAC-BBB parent capsid is modified, z.e., comprises a substitution, insertion, deletion or combinations thereof. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 453. In some embodiments, the modification at position 453 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 454. In some embodiments, the modification at position 454 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 455. In some embodiments, the modification at position 455 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 456. In some embodiments, the modification at position 456 comprises a substitution. In some embodiments, the diversified region of the STAC-BBB parent capsid is modified at amino acid position 457. In some embodiments, the modification at position 457 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 458. In some embodiments, the modification at position 458 comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 459. In some embodiments, the modification at position 459174899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1comprises a substitution. In some embodiments, the STAC-BBB parent capsid is modified at amino acid position 453, 545, 455, 456, 457, 458, and / or 459. In some embodiments, the STAC-BBB parent capsid is modified in at least two different positions selected from amino acid position 453, 545, 455, 456, 457, 458, and / or 459. In some embodiments, the modification at position 453, 545, 455, 456, 457, 458, and / or 459 comprises a substitution, insertion, deletion or combinations thereof.

[0068] Disclosed are combinations of at least two positions that can be modified in the diversified region of the STAC-BBB parent capsid, which are described in this section with respect to the STAC-BBB parent capsid sequence (i.e., SEQ ID NO: 36).

[0069] In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 595. In some embodiments, the modifications at positions 587 and 595, comprise one substitution at each of positions 587 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 595. In some embodiments, the modifications at positions 591 and 595 comprise one substitution at each of positions 591 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 598. In some embodiments, the modifications at positions 591 and 598, comprise one substitution at each of positions 591 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 597. In some embodiments, the modifications at positions 591 and 597, comprise one substitution at each of positions 591 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 590. In some embodiments, the modifications at positions 586 and 590 comprise one substitution at each of positions 586 and 590. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 596 and 598. In some embodiments, the modifications at positions 596 and 598 comprise one substitution at each of positions 596 and 598.

[0070] In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 587. In some embodiments, the modifications at positions 586 and 587, comprise one substitution at each of positions 586 and 587. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 588. In some embodiments, the modifications at positions 586 and 588, comprise one substitution at each of positions 586 and 588. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 589. In some embodiments, the modifications at positions 586 and 589, comprise one 184899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1substitution at each of positions 586 and 589. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 590. In some embodiments, the modifications at positions 586 and 590, comprise one substitution at each of positions 586 and 590, In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 591. In some embodiments, the modifications at positions 586 and 591, comprise one substitution at each of positions 586 and 591. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 592. In some embodiments, the modifications at positions 586 and 592, comprise one substitution at each of positions 586 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 593. In some embodiments, the modifications at positions 586 and 593, comprise one substitution at each of positions 586 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 594. In some embodiments, the modifications at positions 586 and 594, comprise one substitution at each of positions 586 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 595. In some embodiments, the modifications at positions 586 and 595, comprise one substitution at each of positions 586 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 596. In some embodiments, the modifications at positions 586 and 596, comprise one substitution at each of positions 586 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 597. In some embodiments, the modifications at positions 586 and 597, comprise one substitution at each of positions 586 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 586 and 598. In some embodiments, the modifications at positions 586 and 598, comprise one substitution at each of positions 586 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 588. In some embodiments, the modifications at positions 587 and 588, comprise one substitution at each of positions 587 and 588. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 589. In some embodiments, the modifications at positions 587 and 589, comprise one substitution at each of positions 587 and 589. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 590. In some embodiments, the modifications at positions 587 and 590, comprise one substitution at each of positions 587 and 590. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 591. In some embodiments, the modifications at positions 587 and 591, comprise one substitution at each of positions 587 and 591. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 194899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1587 and 592. In some embodiments, the modifications at positions 587 and 592, comprise one substitution at each of positions 587 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 593. In some embodiments, the modifications at positions 587 and 593, comprise one substitution at each of positions 587 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 594. In some embodiments, the modifications at positions 587 and 594, comprise one substitution at each of positions 587 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 595. In some embodiments, the modifications at positions 587 and 595, comprise one substitution at each of positions 587 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 596. In some embodiments, the modifications at positions 587 and 596, comprise one substitution at each of positions 587 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 597. In some embodiments, the modifications at positions 587 and 597, comprise one substitution at each of positions 587 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 587 and 598. In some embodiments, the modifications at positions 587 and 598, comprise one substitution at each of positions 587 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 589. In some embodiments, the modifications at positions 588 and 589, comprise one substitution at each of positions 588 and 589. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 590. In some embodiments, the modifications at positions 588 and 590, comprise one substitution at each of positions 588 and 590. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 591. In some embodiments, the modifications at positions 588 and 591, comprise one substitution at each of positions 588 and 591. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 592. In some embodiments, the modifications at positions 588 and 592, comprise one substitution at each of positions 588 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 593. In some embodiments, the modifications at positions 588 and 593, comprise one substitution at each of positions 588 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 594. In some embodiments, the modifications at positions 588 and 594, comprise one substitution at each of positions 588 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 595. In some embodiments, the modifications at positions 588 and 595, comprise one substitution at each of positions 588 and 204899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 596. In some embodiments, the modifications at positions 588 and 596, comprise one substitution at each of positions 588 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 597. In some embodiments, the modifications at positions 588 and 597, comprise one substitution at each of positions 588 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 588 and 598. In some embodiments, the modifications at positions 588 and 598, comprise one substitution at each of positions 588 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 590. In some embodiments, the modifications at positions 589 and 590, comprise one substitution at each of positions 589 and 590. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 591. In some embodiments, the modifications at positions 589 and 591, comprise one substitution at each of positions 589 and 591. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 592. In some embodiments, the modifications at positions 589 and 592, comprise one substitution at each of positions 589 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 593. In some embodiments, the modifications at positions 589 and 593, comprise one substitution at each of positions 589 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 594. In some embodiments, the modifications at positions 589 and 594, comprise one substitution at each of positions 589 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 595. In some embodiments, the modifications at positions 589 and 595, comprise one substitution at each of positions 589 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 596. In some embodiments, the modifications at positions 589 and 596, comprise one substitution at each of positions 589 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 597. In some embodiments, the modifications at positions 589 and 597, comprise one substitution at each of positions 589 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 589 and 598. In some embodiments, the modifications at positions 589 and 598, comprise one substitution at each of positions 589 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 591. In some embodiments, the modifications at positions 590 and 591, comprise one substitution at each of positions 590 and 591. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 592. In some embodiments, the 214899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1modifications at positions 590 and 592, comprise one substitution at each of positions 590 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 593. In some embodiments, the modifications at positions 590 and 593, comprise one substitution at each of positions 590 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 594. In some embodiments, the modifications at positions 590 and 594, comprise one substitution at each of positions 590 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 595. In some embodiments, the modifications at positions 590 and 595, comprise one substitution at each of positions 590 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 596. In some embodiments, the modifications at positions 590 and 596, comprise one substitution at each of positions 590 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 597. In some embodiments, the modifications at positions 590 and 597, comprise one substitution at each of positions 590 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 590 and 598. In some embodiments, the modifications at positions 590 and 598, comprise one substitution at each of positions 590 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 592. In some embodiments, the modifications at positions 591 and 592, comprise one substitution at each of positions 591 and 592. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 593. In some embodiments, the modifications at positions 591 and 593, comprise one substitution at each of positions 591 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 594. In some embodiments, the modifications at positions 591 and 594, comprise one substitution at each of positions 591 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 595. In some embodiments, the modifications at positions 591 and 595, comprise one substitution at each of positions 591 and 595In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 596. In some embodiments, the modifications at positions 590 and 595, comprise one substitution at each of positions 591 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 597. In some embodiments, the modifications at positions 591 and 597, comprise one substitution at each of positions 591 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 591 and 598. In some embodiments, the modifications at positions 591 and 598, comprise one substitution at each of positions 591 and 598. In some embodiments, the STAC-BBB parent 224899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1capsid is modified at amino acid positions 592 and 593. In some embodiments, the modifications at positions 592 and 593, comprise one substitution at each of positions 592 and 593. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 592 and 594. In some embodiments, the modifications at positions 592 and 594, comprise one substitution at each of positions 592 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 592 and 595. In some embodiments, the modifications at positions 592 and 595, comprise one substitution at each of positions 592 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 592 and 596. In some embodiments, the modifications at positions 592 and 596, comprise one substitution at each of positions 592 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 592 and 597. In some embodiments, the modifications at positions 592 and 597, comprise one substitution at each of positions 592 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 592 and 598. In some embodiments, the modifications at positions 592 and 598, comprise one substitution at each of positions 592 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 593 and 594. In some embodiments, the modifications at positions 593 and 594, comprise one substitution at each of positions 593 and 594. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 593 and 595. In some embodiments, the modifications at positions 593 and 595, comprise one substitution at each of positions 593 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 593 and 596. In some embodiments, the modifications at positions 593 and 596, comprise one substitution at each of positions 593 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 593 and 597. In some embodiments, the modifications at positions 593 and 597, comprise one substitution at each of positions 593 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 593 and 598. In some embodiments, the modifications at positions 593 and 598, comprise one substitution at each of positions 593 and 598In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 594 and 595. In some embodiments, the modifications at positions 594 and 595, comprise one substitution at each of positions 594 and 595. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 594 and 596. In some embodiments, the modifications at positions 594 and 596, comprise one substitution at each of positions 594 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 594 and 597. In some embodiments, the modifications at positions 594 and 597, comprise one 234899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1substitution at each of positions 594 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 594 and 598. In some embodiments, the modifications at positions 594 and 598, comprise one substitution at each of positions 594 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 595 and 596. In some embodiments, the modifications at positions 595 and 596, comprise one substitution at each of positions 595 and 596. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 595 and 597. In some embodiments, the modifications at positions 595 and 597, comprise one substitution at each of positions 595 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 595 and 598. In some embodiments, the modifications at positions 595 and 598, comprise one substitution at each of positions 595 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 596 and 597. In some embodiments, the modifications at positions 596 and 597, comprise one substitution at each of positions 596 and 597. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 596 and 598. In some embodiments, the modifications at positions 596 and 598, comprise one substitution at each of positions 596 and 598. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 597 and 598. In some embodiments, the modifications at positions 597 and 598, comprise one substitution at each of positions 597 and 598.

[0071] In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 456. In some embodiments, the modifications at positions 454 and 456, comprise one substitution at each of positions 454 and 456. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 456. In some embodiments, the modifications at positions 453 and 456, comprise one substitution at each of positions 453 and 456. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 458. In some embodiments, the modifications at positions 456 and 458, comprise one substi6ution at each of positions 456 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 459. In some embodiments, the modifications at positions 456 and 459, comprise one substi6ution at each of positions 456 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 457. In some embodiments, the modifications at positions 456 and 457, comprise one substi6ution at each of positions 456 and 457.244899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0072] In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 454. In some embodiments, the modifications at positions 453 and 454, comprise one substitution at each of positions 453 and 454. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 455. In some embodiments, the modifications at positions 453 and 455, comprise one substitution at each of positions 453 and 455. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 456. In some embodiments, the modifications at positions 453 and 456, comprise one substitution at each of positions 453 and 456. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 457. In some embodiments, the modifications at positions 453 and 457, comprise one substitution at each of positions 453 and 457. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 458. In some embodiments, the modifications at positions 453 and 458, comprise one substitution at each of positions 453 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 453 and 459. In some embodiments, the modifications at positions 453 and 459, comprise one substitution at each of positions 453 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 455. In some embodiments, the modifications at positions 454 and 455, comprise one substitution at each of positions 454 and 455. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 456. In some embodiments, the modifications at positions 454 and 456, comprise one substitution at each of positions 454 and 456. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 457. In some embodiments, the modifications at positions 454 and 457, comprise one substitution at each of positions 454 and 457. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 458. In some embodiments, the modifications at positions 454 and 458, comprise one substitution at each of positions 454 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 454 and 459. In some embodiments, the modifications at positions 454 and 459, comprise one substitution at each of positions 454 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 455 and 456. In some embodiments, the modifications at positions 455 and 456, comprise one substitution at each of positions 455 and 456. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 455 and 457. In some embodiments, the modifications at positions 455 and 457, comprise one substitution at each of positions 455 and 457. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 455 and 458. In some embodiments, the 254899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1modifications at positions 455 and 458, comprise one substitution at each of positions 455 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 455 and 459. In some embodiments, the modifications at positions 455 and 459, comprise one substitution at each of positions 455 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 457. In some embodiments, the modifications at positions 456 and 457, comprise one substitution at each of positions 456 and 457. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 458. In some embodiments, the modifications at positions 456 and 458, comprise one substitution at each of positions 456 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 456 and 459. In some embodiments, the modifications at positions 456 and 459, comprise one substitution at each of positions 456 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 457 and 458. In some embodiments, the modifications at positions 457 and 458, comprise one substitution at each of positions 457 and 458. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 457 and 459. In some embodiments, the modifications at positions 457 and 459, comprise one substitution at each of positions 457 and 459. In some embodiments, the STAC-BBB parent capsid is modified at amino acid positions 458 and 459. In some embodiments, the modifications at positions 458 and 459, comprise one substitution at each of positions 458 and 459.

[0073] In some embodiments, the STAC-BBB parent capsid is modified by combinations of diversified regions. In some embodiments, an engineered STAC-BBB parent capsid is made by modifying the STAC-BBB parent capsid, such that the engineered STAC-BBB capsid comprises at least two diversified sequences, and wherein each of the at least two sequences comprises a different diversified region sequence. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 with SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID264899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 1 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 2 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 3 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 4 and SEQ ID NO 17. In some embodiments, an engineered STAC- 274899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 9. In some embodiments, an engineered STAC -BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 10. In some embodiments, an engineered STAC -BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 11. In some embodiments, an engineered STAC -BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 5 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 16 In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 6 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 7 and SEQ ID NO 17. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 9. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID 284899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1NO 10. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 11. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 12. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 13. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 14. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 15. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 16. In some embodiments, an engineered STAC-BBB parent capsid comprises SEQ ID NO 8 and SEQ ID NO 17.

[0074] In some embodiments, an engineered STAC-BBB parent capsid comprises any sequence variant of any one of SEQ ID NOs: 1-8 or SEQ ID NOs: 37-79 and any sequence variant of any one of SEQ ID NO 9-17 or SEQ ID NO: 37, for example, any one of the sequence variants as described herein.

[0075] In some embodiments, an engineered STAC-BBB parent capsid comprises any one of SEQ ID NOs: 1-8 or SEQ ID NOs: 37-79 and any one of SEQ ID NOs: 9-17 or SEQ ID NO: 37. In some embodiments, an engineered STAC-BBB parent capsid comprises any one of a sequence variant of SEQ ID NOs: 1-8 or SEQ ID NOs: 37-79 as described herein and any one of a sequence variant of SEQ ID NOs: 9-17 or SEQ ID NO: 37 as described herein.

[0076] In some embodiments, an engineered variant STAC-BBB parent capsid comprises a diversified region of amino acids 453 through 459 and a diversified region of amino acids 586 through 598, wherein each diversified region is selected from a sequences shown in Tables 4 or 7 and / or comprises one or more mutations as shown in Table 5 or Table 6.

[0077] In some embodiments, an engineered STAC-BBB parent capsid comprises a combination of a diversified region of amino acids 453 through 459 with another diversified region of amino acids 586 through 598, wherein the combination of diversified regions is as shown in a single row of Table 8.

[0078] Engineered AAV Capsid Library Generation

[0079] In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins, wherein members of the library encode engineered AAV capsid proteins having different sequences, and wherein some members of the library encode an AAV capsid protein having a desired characteristic compared to a natural / wild-type AAV serotype. In one embodiment, disclosed herein is the development of libraries encoding 294899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1engineered AAV capsid proteins with a desired characteristic compared to the parent capsid STAC-BBB. Thus, described herein are libraries of AAV capsid proteins with a desired characteristic compared to the parent capsid STAC-BBB. In some embodiments, the desired characteristic is enhanced cell or tissue tropism as compared to the parent capsid STAC-BBB, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the parent capsid STAC-BBB. In some embodiments, the desired characteristic is increased penetrance through the blood brain barrier following administration to a subject. In some embodiments, the desired characteristic is wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the desired characteristic is elevated genetic material expression in multiple brain regions. In some embodiments, the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0080] In some embodiments, each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising an engineered STAC-BBB variant sequence; b) a nucleic acid sequence encoding barcode; c) nucleic acid sequence(s) encoding a promoter(s); d) a nucleic acid sequence encoding a unique molecular identifier (UMI); and combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to and expressed in a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence.

[0081] In some embodiments, each engineered AAV capsid protein was synthesized as an oligo pool. In some embodiments, each member of a library comprises one or more (such as 1-10) of a nucleic acid sequence encoding an AAV capsid protein comprising a) nucleic acid sequences encoding one or more (such as 1-10) barcodes: b) nucleic acid sequences encoding one or more (such as 1-10) promoters; c) nucleic acid sequences encoding one or more (such as 1-10,000) unique molecular identifiers (UMIs); or combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence. In some embodiments, each of the one or more (such as 1-10) barcodes is linked to the identity of a single engineered AAV capsid protein. In some embodiments, each of the barcodes is linked to one or more (such as 1-10,000) UMIs.304899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0082] In some embodiments, a nucleic acid comprising a barcode is added to the genome of each AAV capsid in a library. In some embodiments, a unique barcode is bioinformatically linked to each different variant sequence that is represented within the library, for example, each different variant STAC-BBB sequence. In some embodiments, the DNA sequences encoding a STAC-BBB variant sequence are synthesized to further comprise a random or specified barcode. The barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides. In some embodiments, each targeting STAC-BBB variant sequence is linked to at least 2 distinct barcodes. In some embodiments, each barcode is linked to one or more (such as 1-10,000) UMIs.

[0083] In some embodiments, each member of the library comprises a nucleic acid comprising more than one barcode sequences (such as 1-10). In some embodiments, each member of the library comprises two or more nucleic acids (such as 1-10) each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter. In some embodiments, each capsid is linked to at least one unique barcode. In some embodiments, each capsid is linked to at least two unique barcodes using a bioinformatic look-up table. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific human Synapsin 1 promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the ubiquitous CMV promoter.

[0084] In some embodiments, libraries are created encoding engineered AAV capsid proteins that comprise at least one mutation relative to a parent capsid, for example, the parent capsid STAC-BBB. In some embodiments, the engineered AAV capsid proteins contains two mutations relative to the parent capsid STAC-BBB. In some embodiments, each mutated position of an engineered AAV capsid protein is changed to all possible amino acids except cysteine and the original amino acid in STAC-BBB.314899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0085] In some embodiments, the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, El A and E1B) are supplied in trans. In some embodiments, the AAV rep function comprises rep78, rep 68, rep 52, and rep40 genes. In some embodiments, the rep genes are supplied in trans. In some embodiments, the start codon of the rep78 and / or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield. In some embodiments, the cap genes are supplied as genetic material that is packaged into the manufactured AAVs. In some embodiments, the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.

[0086] Performance of Engineered AAV Capsid Proteins

[0087] In some embodiments, high performing capsids have the following features in the bubble plots: (1) High log2 fold enrichment (these data are normalized by input abundance (y-axis)); (2) Low coefficient of variation (x-axis); (3) High fraction of sequenced samples in which a capsid is found (large bubble size); (4) Robust unique molecular identifier recovery (green color); (5) and combinations thereof.

[0088] In some embodiments, STAC-BBB variants (i.e. capsids having substitutions relative to the parent capsid STAC-BBB) exhibit an increase in mRNA expression relative to the parent STAC-BBB. In some embodiments, coronal slices from the forebrain, midbrain, and / or hindbrain were analyzed for mRNA expression of modified STAC-BBB parent capsid relative to the parent capsid STAC-BBB. In some embodiments, STAC-BBB variants exhibit an increase in mRNA expression in the brain relative to the parent capsid STAC-BBB. In some embodiments, STAC-BBB variants exhibit an increase in mRNA expression in the cervical, thoracic, and lumbar spinal cord regions relative to the parent capsid STAC-BBB. Some STAC-BBB variants exhibit enhanced mRNA expression relative to STAC-BBB in CNS tissues, while others exhibit performance that is lower than STAC-BBB but still significantly higher than wild type AAV9.Adeno-associated virus (AAV)

[0089] AAV are capable of infecting a wide range of cells including quiescent cells and dividing cells. In some embodiments, AAV can be modified so that it contains the components necessary for the assembly of a functional recombinant virus or viral particle. In some324899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1embodiments, AAV is engineered to target a specific tissue and / or cell. In some embodiments, the AAV is engineered to deliver a specific genetic material to a tissue and / or cell.

[0090] Modified AAV Serotypes

[0091] In some embodiments, an engineered AAV may be based on any natural or recombinant AAV serotype. Different AAV serotypes have different characteristics such as different packaging, tropism, and transduction profiles. In some embodiments, the engineered AAV capsid proteins are based on a wild-type AAV serotype. In some embodiments, the AAV serotype comprises AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the AAV serotype comprises less well-characterized AAV serotypes such as AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74. In some embodiments, the engineered AAV capsid protein is derived from multiple wild-type AAV serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes. In some embodiments, AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined to create chimeric capsids. In some embodiments, combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.

[0092] In some embodiments, different AAV serotypes are different in their ability to direct or modulate an AAV particle to a particular cell or tissue. In some embodiments, the AAV serotype can be modified to increase the tropism of the AAV particle to cells or tissues of the central nervous system (CNS). In some embodiments, the AAV serotype can be modified to increase tropism of the AAV particle to cells or tissues of the peripheral nervous system (PNS).

[0093] In some embodiments, the modified AAV serotype has a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, the modified AAV serotype allows for increased penetration of the blood brain barrier following administration to a subject. In some embodiments, the modified AAV serotype causes increased biodistribution to a brain region. In some embodiments, the brain region comprises the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the hippocampus, the thalamus, or the putamen. In some embodiments, the brain comprises any brain region known in the art. In some embodiments, the modified AAV serotype causes increased biodistribution to more than one brain regions, for example, 2 brain regions, 3 brain regions, 4 brain regions, 5 brain regions, 6 brain regions, 7 brain regions, 8 brain regions, 9 brain regions, or 10 brain regions. In some334899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1embodiments, the modified AAV serotype causes increased biodistribution to 1- 10 brain regions. In some embodiments, the modified AAV serotype are useful in elevating genetic material expression in multiple brain regions. In some embodiments, the modified AAV serotype are used to deliver genetic material of interest to a desired tissue, cell, or organelle.

[0094] In some embodiments, the modified AAV serotype causes increased biodistribution to regions of the spinal cord. In some embodiments, the region of the spinal cord comprises any of the thoracic spinal cord region, the lumbar spinal cord region, and / or the cervical spinal cord region. In some embodiments, the region of the spinal cord includes any region of the spinal cord known in the art. In some embodiments, the modified AAV serotype cases increased biodistribution to more than one region of the spinal cord, for example, 2 spinal cord regions, 3 spinal cord regions, 4 spinal cord regions, 5 spinal cord regions, 6 spinal cord regions, 7 spinal cord regions, 8 spinal cord regions, 9 spinal cord regions, or 10 spinal cord regions. In some embodiments, the modified AAV serotype cases increased biodistribution to 1- 10 spinal cord regions.

[0095] In some embodiments, the modified AAV serotype is modified with a peptide that comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the modified AAV serotype is modified with a peptide set forth in any one of SEQ ID NOs: 1-17, or any variant of any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79, for example, any variant as described herein. In some embodiments, the modified AAV serotype is modified with any of the peptides described herein.

[0096] Structure of AAV

[0097] In some embodiments, the genome of the AAV comprises a single-strand DNA (ssDNA) molecule that is approximately between about 2.5 kb and about 5.0 kb in length. In some embodiments, the genome of the AAV comprises a self-complementary DNA (scDNA) molecule that is approximately between about 0.5 kb and about 2.5 kb in length. In some embodiments, the AAV genome contains inverted terminal repeats (ITRs) that flank the 5’ and 3’ ends of the AAV molecule. In some embodiments, the ITRs contain origins of replication for the viral genome. In some embodiments, the length of the ITRs is about 145 bp in length, for example, between about 130 bp in length and 160 bp in length.344899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0098] In some embodiments, the AAV genome comprises the rep and cap genes. In some embodiments, the AAV genome nucleotide includes nucleotide sequences that encode four non- structural Rep proteins (Rep 78, Rep68, Rep52, Rep40, encoded by Rep genes). In some embodiments, the AAV viral genome includes nucleotide sequences that encode the three capsid, or structural, proteins (i.e., VP1, VP2, VP3, encoded by the cap gene). In some embodiments, the rep proteins are used for replication and packaging. In some embodiments, the capsid proteins are assembled to create the protein shell of the AAV.

[0099] AAV particles

[0100] In some embodiments, the engineered AAV capsid proteins assemble to form AAV particles. In some embodiments, the AAV particles that have enhanced tropism for a target tissue (e.g., CNS and PNS) are provided. In some embodiments, the AAV particles include engineered STAC-BBB variant sequences that alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. In some embodiments, the AAV particles are capable of penetrating the blood brain barrier.

[0101] Delivery of AAV particles

[0102] The AAV particles may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively), or cells and tissues of a muscle. The AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.

[0103] In some embodiments, the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating. In some embodiments, the AAV particles are used to deliver therapeutic cargoes.

[0104] AAV viral genomes

[0105] In some embodiments, the AAV particles are used to deliver a viral genome (i.e., a genetic payload) to a tissue or cells such as CNS or PNS cell or tissue.

[0106] The delivered viral genome may include genetic material of interest, such as, for example, genetic material that encodes an engineered DNA recombinase protein, a fusion354899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1protein comprising a DNA-binding domain (e.g., a zinc finger or a TALE protein) fused to a functional domain (e.g., to modulate DNA function or to cleave DNA), an antibody, an enzyme, regulatory RNA, a CRISPR protein, or a cDNA, amongst others. In some embodiments, the viral genome includes 2 ITR sequences. In some embodiments, the ITR sequences flank the genetic material of interest. In some embodiments, the ITR sequences are complementary to each other. In some embodiments, the ITR sequences are not complementary to each other. In some embodiments, one ITR sequence is a self-complementary ITR. In some embodiments, the ITR regions are derived from the same serotype as the capsid protein. In some embodiments, the ITR regions are derived from AAV2 serotype. In some embodiments, the ITR regions are derived from a serotype known to the art. ITR regions may be between 100 and 150 nucleotides in length.AAV Diversified Region Sequences

[0107] In embodiments, sequences of AAV diversified regions disclosed herein. In some embodiments, the sequences can function as a general CNS-targeting molecule or sequence.

[0108] In embodiments, the sequences function as a general CNS-targeting molecule sequence. In some embodiments, the CNS-targeting sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein. In some embodiments, any of SEQ ID NO: 1-17 or 37-79 is fused or conjugated to a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein. In some embodiments, CNS-targeting sequences may be utilized to enable a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.

[0109] In some embodiments, the sequences are part of an engineered AAV capsid protein. In some embodiments, the engineered AAV capsid protein is any engineered AAV capsid protein disclosed herein.

[0110] In some embodiments, the sequences may increase tropism of an AAV capsid protein to a cell or tissue of the CNS. In some embodiments, the cell of the CNS is a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial cells (e.g., microglia, astrocytes, oligodendrocytes) and / or a supporting cells 364899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1of the brain such as immune cells (e.g., T cells). In some embodiments, the CNS tissue is the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, entorhinal cortex, cerebellum, or spinal cord.[OHl] In some embodiments, the sequences increase tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).

[0112] In some embodiments, the sequences decrease tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).

[0113] In some embodiments, the sequence comprises at least 4 contiguous amino acids of a sequence set forth in any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the sequence comprises the sequence set forth in any one of SEQ ID NO: 1-17 or SEQ ID NOs: 37-79.

[0114] In some embodiments, the sequence comprises variants of the amino acid sequences set forth in any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the variant comprises 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the variant comprises 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the variant comprises 1, 2, 3, or 4 additions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the variant comprises any combination of the substitutions, deletions, or additions described above. In some embodiments, the variant amino acid sequences comprise conservative replacement of one or more amino acid(s) within the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79.

[0115] In some embodiments, a variant refers to a variant in the nucleotide sequence that encodes any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, the variant in the nucleotide sequence results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid 374899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 additions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In embodiments, the variant in the nucleotide sequence encodes any combination of the substitutions, deletions, or additions described above.

[0116] In some embodiments the capsid protein comprises any of the serotypes of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9.

[0117] In some embodiments, the capsid protein comprises a variant of the STAC-BBB parent capsid, for example, a variant as described herein. In some embodiments, the capsid protein comprises a variant of the sequence set forth in SEQ ID NO: 36. In embodiments, a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids in the amino acid sequences set forth in SEQ ID NO: 36. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions of any of the amino acids of the amino acid sequence of SEQ ID NO: 36. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 deletions of any of the amino acids of the amino acid sequence set forth of SEQ ID NO: 36. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions of any of the amino acids of the amino acid sequence set forth SEQ ID NO: 36.

[0118] In some embodiments, a variant refers to a variant in the nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 36, for example, a variant as described herein. In some embodiments, the variant in the nucleotide sequence results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions of any of the amino acids of the amino acid sequence of SEQ ID NO: 36. In some embodiments,384899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 deletions of any of the amino acids of the amino acid sequence of SEQ ID NO: 36. In some some embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 additions of any of the amino acids of the amino acid sequence of SEQ ID NO: 36.

[0119] Administering AAVs Having Diversified Region Sequences to Subjects

[0120] In some embodiments, when administered to subjects, capsid proteins containing the engineered diversified region sequences described herein are capable of higher expression in neurons relative to capsid proteins that lack the engineered diversified region sequences. In some embodiments, the capsid protein administered to subjects comprises an engineered STAC-BBB sequence described herein.

[0121] Length and Structure of Diversified Regions (Sequences modified within Engineered AAV)

[0122] The diversified region within an engineered AAV (e.g., STAC-BBB) variant sequence, may vary in length. In some embodiments, the diversified region is about 3 to about 20 amino acids in length. In some examples, the diversified region is about 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18 amino acids in length. In some embodiments, the diversified region is between about 8-10, 10-12, 12-14, 14-16, or 16-18 amino acids in length. In some embodiments, the diversified region is about 13 amino acids in length. In some embodiments, the diversified region is about 7 amino acids in length.

[0123] In some embodiments, a diversified region may comprise an amino acid sequence of any one of SEQ ID NOs: 1-17, as described in Table 4, or any of SEQ ID NOs: 37-79, as described in Table 7. In some embodiments, a diversified region may comprise at least 3 contiguous amino acid sequences of any one of SEQ ID NO: 1-17, as described in Table 4, or any one of SEQ ID NOs: 37-79, as described in Table 7. In some embodiments, a diversified region may comprise an amino acid sequence of any one of SEQ ID NOs: 1-17, as described in Table 4, or any of SEQ ID NOs: 37-79, as described in Table 7, wherein one of more underlined position(s) in the diversified regions is / are modified by any other amino acid,394899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1preferably an amino acid having similar biochemical properties as the underlined amino acid(s) shown in any one of SEQ ID NOs: 1-17 or any of SEQ ID NOs: 37-79.

[0124] In some embodiments, a diversified region comprises a modified version of any one of SEQ IDs NOs: 1, 2, 4-6, or 8-17, wherein one or two positions are modified in each sequence, and wherein the modified first position and the modified second position are as shown in Table 5 or 6. In some embodiments, a diversified region comprises a modified version of SEQ ID No. 35 or 5443, wherein at least one position is modified as shown in Table 5 or 6.

[0125] In some embodiments, the diversified region comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 3 contiguous amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 4 contiguous amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 5 contiguous amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 6 contiguous amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 7 contiguous amino acids of any sequence set forth in SEQ ID NO: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the diversified region comprises at least 8 contiguous amino acids of any sequence set forth in SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79.

[0126] In some embodiments, the diversified region comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92% percent, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 37-79. In some embodiments, the peptide comprises any one of SEQ ID NOs: 1-17.

[0127] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the diversified region comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 1.404899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 1.

[0128] In some embodiments, SEQ ID NO: 1 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position N2 in SEQ ID NO: 1 is modified. In some embodiments, the modification at position N2 comprises a substitution. In some embodiments, position N10 in SEQ ID NO: 1 is modified. In some embodiments, the modification at position N10 comprises a substitution. In some embodiments, both positions N2 and N10 are modified in SEQ ID NO: 1. In some embodiments, the modifications at positions N2 andNIO, comprise a substitution at both N2 and N10 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 1.

[0129] In some embodiments, position N2 of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 comprises any one of the amino acid Y, S, I, K, N, F, V, T, Q, R, H, L, P, M, W, or E. In some embodiments, position N10 comprises any one of the amino acid D, L, H, N, E, Q, I, S, A, F, T, Y, V, P, R, K, or W. In some embodiments, position N2 and position N10 comprise any combination of the foregoing.

[0130] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the diversified region comprises an amino acid sequence comprising at least one, two, or three, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 2.

[0131] In some embodiments, SEQ ID NO: 2 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, Nl 1, N12, and N13. In some embodiments, position N6 in SEQ ID NO: 2 is modified. In some embodiments, the modification at position N6 comprises a substitution. In some embodiments, position N10 in SEQ ID NO: 2 is modified. In some embodiments, the modification at position N10 comprises a substitution. In some embodiments, both positions N6 and N10 are modified in SEQ ID NO: 2. In some embodiments, the modifications at positions N6 and N10 , comprise a substitution at both N6 and N10 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 2.414899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0132] In some embodiments, position N6 of SEQ ID NO: 2 or a variant of SEQ ID NO: 2 comprises any one of the amino acid V, F, S, T, D, L, A, or M. In some embodiments, position N10 comprises any one of the amino acid L, D, F, N, P, E, Y, G, H, V, K, S, R, or A. In some embodiments, position N6 and position N10 comprise any combination of the foregoing.

[0133] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the diversified region comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 3.

[0134] In some embodiments, SEQ ID NO: 3 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position N6 in SEQ ID NO: 3 is modified. In some embodiments, the modification at position N6 comprises a substitution. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 3.

[0135] In some embodiments, position N6 of SEQ ID NO: 3 or a variant of SEQ ID NO: 3 comprises the amino acid V. In some embodiments, position N6 comprises the amino acid D.

[0136] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the diversified region comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 4.

[0137] In some embodiments, SEQ ID NO: 4 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, Nl 1, N12, and N13. In some embodiments, position N6 in SEQ ID NO: 4 is modified. In some embodiments, the modification at position N6 comprises a substitution. In some embodiments, position N13 in SEQ ID NO: 4 is modified. In some embodiments, the modification at position N13 comprises a substitution. In some embodiments, both positions N6 and N13 are modified in SEQ ID NO: 4. In some embodiments, the modifications at positions N6 and N13, comprise424899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1a substitution at both N6 and N13 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 4.

[0138] In some embodiments, position N6 of SEQ ID NO: 4 or a variant of SEQ ID NO: 4 comprises any one of the amino acid V, D, S, K, L, or Q. In some embodiments, position N13 comprises any one of the amino acid P, S, E, L, V, W, N, T, Q, H, Y, F, G, M, I, or K. In some embodiments, position N6 and position N13 comprise any combination of the foregoing.

[0139] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence comprises at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 5.

[0140] In some embodiments, SEQ ID NO: 5 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position N6 in SEQ ID NO: 5 is modified. In some embodiments, the modification at position N6 comprises a substitution. In some embodiments, position N 12 in SEQ ID NO: 5 is modified. In some embodiments, the modification at position N12 comprises a substitution. In some embodiments, both positions N6 and N12 are modified in SEQ ID NO: 5. In some embodiments, the modifications at positions N6 and N12, comprise a substitution at both N6 and N12 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 5.

[0141] In some embodiments, position N6 of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 comprises any one of the amino acid V, L, D, S, P, or H. In some embodiments, position N12 comprises any one of the amino acid L, V, F, G, S, H, M, W, Y, P, A, E, N, K, T, D, I, or R. In some embodiments, position N6 and position N12 comprise any combination of the foregoing.

[0142] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 6.434899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0143] In some embodiments, SEQ ID NO: 6 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position Nl in SEQ ID NO: 6 is modified. In some embodiments, the modification at position Nl comprises a substitution. In some embodiments, position N5 in SEQ ID NO: 6 is modified. In some embodiments, the modification at position N5 comprises a substitution. In some embodiments, both positions Nl and N5 are modified in SEQ ID NO: 6. In some embodiments, the modifications at positions Nl and N5, comprise a substitution at both Nl and N5 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 6.

[0144] In some embodiments, positionNl of SEQ ID NO: 6 or a variant of SEQ ID NO: 6 comprises any one of the amino acid T, H, M, V, N, G, W, D, F, E, R, I, L, or K. In some embodiments, position N5 comprises any one of the amino acid F, Q, I, S, W, Y, V, A, M, H, or T. In some embodiments, position Nl and position N5 comprise any combination of the foregoing.

[0145] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 7, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 7.

[0146] In some embodiments, SEQ ID NO: 7 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, Nil, N12, and N13. In some embodiments, position N4 in SEQ ID NO: 7 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 7.

[0147] In some embodiments, position N4 of SEQ ID NO: 7 or a variant of SEQ ID NO: 7 comprises the amino acid M, Y, I, L, or F.

[0148] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 8, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence 444899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ IDNO: 8.

[0149] In some embodiments, SEQ ID NO: 8 comprises 13 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position Nil in SEQ ID NO: 8 is modified. In some embodiments, the modification at position Nil comprises a substitution. In some embodiments, position N13 in SEQ ID NO: 8 is modified. In some embodiments, the modification at position N13 comprises a substitution. In some embodiments, both positions Nil and N13 are modified in SEQ ID NO: 8. In some embodiments, the modifications at positions Nil and N13, comprise a substitution at both Nil and N13 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 8.

[0150] In some embodiments, position Nl 1 of SEQ ID NO: 8 or a variant of SEQ ID NO: 8 comprises any one of the amino acid N, W, K, I, H, G, F, S, Q, V, P, M, R, T, E, L, A, or Y. In some embodiments, position N13 comprises any one of the amino acid H, V, P, R, W, E, S, F, L, Y, G, N, Q, I, K, D, T, or M. In some embodiments, position Nl 1 and position Nl 3 comprise any combination of the foregoing.

[0151] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 9, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the modifications at these positions are as shown in SEQ IDNO: 9.

[0152] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 10, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the modifications at these positions are as shown in SEQ IDNO: 10.454899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0153] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 11, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 11.

[0154] In some embodiments, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 comprises 7 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, and N7. In some embodiments, position N2 in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 is modified. In some embodiments, the modification at position N2 comprises a substitution. In some embodiments, position N4 in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, both positions N2 and N4 are modified in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the modifications at positions N2 and N4, comprise a substitution at both N2 and N4 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 11.

[0155] In some embodiments, position N2 of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or variants of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 comprises any one of the amino acid V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y. In some embodiments, position N4 comprises any one of the amino acid N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E. In some embodiments, position N2 and position N4 comprise any combination of the foregoing.

[0156] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 12, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 12.

[0157] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 15, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the464899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 15.

[0158] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 15, each comprising 7 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, and N7. In some embodiments, position Nl in SEQ ID NO: 12 or SEQ ID NO: 15 is modified. In some embodiments, the modification at position Nl comprises a substitution. In some embodiments, position N4 in SEQ ID NO: 12 or SEQ ID NO: 15 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, both positions N 1 and N4 are modified in SEQ ID NO: 12 or SEQ ID NO: 15. In some embodiments, the modifications at positions Nl and N4, comprise a substitution at both Nl and N4 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 12 or SEQ ID NO: 15.

[0159] In some embodiments, the diversified region comprises the amino acid sequence of , wherein position Nl of SEQ ID NO: 12 or SEQ ID NO: 15, or variants of SEQ ID NO: 12 and SEQ ID NO: 15, comprises any one of the amino acid S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F. In some embodiments, position N4 comprises any one of the amino acid S, N, T, A, H, V, L, Y, P, W, G, D, R, or I. In some embodiments, position Nl and / or position N4 comprise any combination of the foregoing.

[0160] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 13, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 13.

[0161] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 17, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the amino acid sequence474899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ IDNO: 17.

[0162] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 13, or SEQ ID NO: 17, each comprising 7 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, and N7. In some embodiments, position N4 in SEQ ID NO: 13 or SEQ ID NO: 17 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, position N6 in SEQ ID NO: 13, or SEQ ID NO: 17 is modified. In some embodiments, the modification at position N6 comprises a substitution. In some embodiments, both positions N4 and N6 are modified in SEQ ID NO: 13, or SEQ ID NO: 17. In some embodiments, the modifications at positions N4 and N6, comprise a substitution at both N4 and / or N6 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 13 or SEQ IDNO: 17.

[0163] In some embodiments, position N2 of SEQ ID NO: 13, or SEQ ID NO: 17 or variants of SEQ ID NO: 13, and SEQ ID NO: 17 comprises any one of the amino acid L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K. In some embodiments, position N6 comprises any one of the amino acid K, A, H, S, T, R, P, M, V, W, N, Y, G, or I. In some embodiments, position N4 and position N6 comprise any combination of the foregoing.

[0164] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 14, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ IDNO: 14.

[0165] In some embodiments, SEQ ID NO: 14 comprises 7 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, and N7. In some embodiments, position N4 in SEQ ID NO: 14 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, position N7 in SEQ ID NO: 14 is modified. In some embodiments, the modification at position N7 comprises a substitution. In some embodiments, both positions N4 and N7 are modified in SEQ ID NO: 14. In some embodiments, the modifications at positions N4 and N7, comprise a substitution484899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1at both N4 and N7 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 14.

[0166] In some embodiments, position N4 of SEQ ID NO: 14 or variants of SEQ ID NO: 14 comprises any one of the amino acid W, K, T, H, A, R, V, S, L, G, N, M, I, or P. In some embodiments, position N7 comprises any one of the amino acid E, N, F, I, H, G, R, K, P, A, M, L, or V. In some embodiments, position N4 and position N7 comprise any combination of the foregoing.

[0167] In some embodiments, the diversified region comprises the amino acid sequence of SEQ ID NO: 16, an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the amino acid sequence comprises at least one, two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 16.

[0168] In some embodiments, SEQ ID NO: 16 comprises 7 amino acids with each amino acid comprising consecutive amino acid positions, Nl, N2, N3, N4, N5, N6, and N7. In some embodiments, position N4 in SEQ ID NO: 16 is modified. In some embodiments, the modification at position N4 comprises a substitution. In some embodiments, position N5 in SEQ ID NO: 16 is modified. In some embodiments, the modification at position N5 comprises a substitution. In some embodiments, both positions N4 and N5 are modified in SEQ ID NO: 16. In some embodiments, the modifications at positions N4 and N5, comprise a substitution at both N4 and N5 positions. In some embodiments, the modifications at these positions are as shown in SEQ ID NO: 16.

[0169] In some embodiments, position N4 of SEQ ID NO: 16 or variants of SEQ ID NO: 16 comprises any one of the amino acid L, S, T, I, A, G, N, F, K, V, M, R, Y, E, D, W, H, or P. In some embodiments, position N5 comprises any one of the amino acid K, H, A, S, T, R, V, M, W, Q, L, F, G, D, or E. In some embodiments, position N4 and position N5 comprise any combination of the foregoing.Functional Properties of Engineered AAV Capsid Sequences

[0170] In some embodiments, an engineered AAV capsid sequence comprising a diversified region sequence as described herein (e.g., STAC-BBB) is used for enhanced or improved494899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1transduction of a target cell or tissue (e.g., cells or tissues of the central nervous system (CNS) or peripheral nervous system (PNS)). In some embodiments, the diversified region is used to facilitate the AAV capsid protein across the blood brain barrier following administration to a subject. In some embodiments, the diversified region is used for enhanced or improved distribution of the genetic material throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the diversified region is used for enhanced or improved genetic material expression in multiple brain regions. In some embodiments, the diversified region is used for enhanced or improved delivery genetic material of interest to a desired tissue, cell, or organelle.

[0171] In some embodiments, the diversified region is included in an engineered AAV capsid protein and increases tropism of the AAV capsid protein to a cell, region, or tissue of the CNS. Examples of CNS cells include but are not limited to neurons (e.g., excitatory neurons, inhibitory neurons, and motor neurons) and glial cells (e.g., ependymal cells, astrocytes, oligodendrocytes). Examples of CNS tissue include but are not limited to the cortex (e.g., frontal cortex, parietal cortex, occipital cortex, temporal cortex), thalamus, hypothalamus, striatum, hippocampus, entorhinal cortex, and basal ganglia.

[0172] In some embodiments, the AAV capsid protein containing a diversified region described herein is capable of mediating increased delivery and transgene expression in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a targeting diversified region. In some embodiments, the AAV capsid protein containing a diversified region is capable of increased expression between 1 and 1000-fold relative to an AAV capsid protein that lacks a diversified region, for example, between 1-fold and 10-fold, between 10-fold and 100-fold, or between 100-fold and 1000-fold. In some embodiments, the AAV capsid protein containing a diversified region is capable of increased expression of greater than 1000-fold in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a diversified region.Genetic Material

[0173] In some embodiments, the engineered AAV capsid proteins described herein encapsulate genetic material to be delivered to a cell of interest. As such, in embodiments the engineered AAV capsid proteins described herein enable delivery of genetic material to a cell of interest. In embodiments, the genetic material encodes a zinc finger protein, a TALE protein, and / or a CRISPR protein, or fragments thereof. In embodiments, the genetic material encodes 504899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1one or more antibodies or an antibody fragments. In some embodiments the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNAs.

[0174] In some embodiments, the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are noncoding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.

[0175] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.

[0176] In some embodiments, the genetic material encodes a gene therapy product. A gene therapy product can include a peptide, a polypeptide, or an RNA molecule that when expressed carries out a desired therapeutic effect. In some embodiments, the therapeutic effect is treating any one or more diseases or disorders described herein.

[0177] In some embodiments, a promoter is operably linked to the genetic material to be delivered to the cell. In some embodiments, the promoter comprises a tissue and / or cell specific promoter. In some embodiments, the one more promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others. In some embodiments, the promoter comprises a cell type and / or tissue specific type promoter. Exemplary cell type and / or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes. Other non-limiting cell type and / or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific, Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), MHCK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes). In embodiments, tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS. In embodiments, tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and / or Schwann cells.514899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0178] In some embodiments, the promoters are naturally occurring promoters. In some embodiments, the promoter is synthetic. In some embodiments, the promoter is derived from mammals, humans, viruses, or plants. In some embodiments, the promoters are truncated. In some embodiments, the promoter is mutated.Active Agents

[0179] In some embodiments, engineered AAV sequences described herein (e.g., variants of STAC-BBB) are fused or coupled to an active agent. In some embodiments, a sequence is fused or coupled to an active agent through conjugation. In some embodiments, the active agent comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region). In some embodiments, the sequence is fused to a Fc region of an antibody. In some embodiments, the sequence is fused to the C-terminus of the Fc region. In some embodiments, the sequence is fused to the N-terminus of the Fc region. In some embodiments, the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the sequence is fused or coupled directly to at least on strand of the RNAi. In some embodiments, the sequence is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the sequence is fused or coupled to the sense strand of RNAi. In some embodiments, the sequence is fused or coupled to the antisense strand of RNAi.

[0180] In some embodiments the active agent comprises a diagnostic agent. In some embodiments, the diagnostic agent comprises a detectable moiety such as a fluorophore. In some embodiments, the active agent is a small molecule.Pharmaceutical Compositions and Dosage Forms

[0181] Compositions herein e.g., engineered STAC-BBB variant sequences, AAV particles, and engineered AAV capsid proteins) can be included in pharmaceutical compositions. In some embodiments, the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material; (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and / or (7) allow for regulatable expression of the genetic material.524899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0182] The pharmaceutical compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period. For example, pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, or every three years.

[0183] In some embodiments, the compositions described herein (e.g., engineered STAC-BBB variant sequences, engineered AAV capsid proteins, and modified STAC-BBB parent capsids) can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical. Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients. The compositions described herein can also be formulated for injection, insufflation, infusion, or intradermal exposure. For instance, an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity. The compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.

[0184] In some embodiments, the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and / or alter the release profile.Gene Editing System

[0185] In some embodiments, the genetic material of interest comprises a gene editing system or portions of a gene editing system. In some embodiments, the gene editing system is capable of inducing single or double-stranded breaks into nucleic acid sequences. In some embodiments, the gene editing system is capable of inserting, substituting, or deleting a base or a sequences of bases into nucleic acid sequences. In some embodiments, the gene editing system includes a CRISPR-Cas system. In some embodiments, the gene editing system includes a TALEN. In some embodiments, the gene editing system includes a zinc finger nuclease.Engineered AAV Capsid Proteins Delivered to a Target Cell534899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0186] The engineered AAV capsid proteins may be delivered to one or more of target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins may have enhanced tropism for cells and tissues of the central or peripheral nervous systems, or cells and tissues of a muscle. The engineered AAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins that are desired to have tropism for CNS cells may have enhanced tropism for neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, Schwann cells, and reduced tropism for liver and dorsal root ganglion.Methods of Detecting Engineered AAV Capsid Proteins

[0187] In some embodiments, a method of identifying an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype is provided comprising: (i) contacting a cell, cell line, or tissue in vitro or in vivo with any one of the libraries of engineered AAV capsid proteins, (ii) allowing the engineered AAV capsid proteins in said library to transduce the cell, cell line, or tissue; (iii) recovering from the cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.

[0188] In another embodiment, disclosed herein are methods for directed evolution of engineered AAV capsid proteins and identification of an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype comprise (i) modifying the natural / wild-type AAV serotype to create variant capsids; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where variants are isolated from a target tissue or cell line; and (viii) full evaluation of enriched variants. In some embodiments, the desired characteristic includes enhanced tissue tropism as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes544899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism of the central nervous system as compared to the natural / wild-type AAV serotype. In some embodiments, modification of the AAV capsid protein results in an AAV capsid protein comprising any of the STAC-BBB variant sequences described herein.Methods of Delivery and Treatment

[0189] In some embodiments, methods for introducing the compositions described herein (e.g. , engineered STAC-BBB variant sequences, AAV particles, and engineered AAV capsid proteins) into cells and / or tissues are provided. In some embodiments, the methods comprise introducing into cells and / or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and / or protein in the cells and / or tissues.

[0190] In some embodiments, the compositions described herein are delivered via a localized delivery route. In some embodiments, the localized delivery route includes any one or more of intramuscular administration, intraparenchymal administration, and intracerebral administration, amongst others. In some embodiments, the compositions described herein are administered via a localized delivery route through a bolus infusion.

[0191] In some embodiments, the compositions described herein are administered through systemic administration. In some embodiments, systemic administration includes intravenous administration.

[0192] In some embodiments, the compositions described herein are administered to the central nervous system of via intraventricular administration and / or intrathecal administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the compositions described herein are administered to the central nervous system via administration into the cerebrospinal fluid.

[0193] In some embodiments the compositions can be delivered to target cells or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell554899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1or tissue includes, but is not limited to CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell or target tissue is a CNS cell or tissue. In some embodiments, the target cell or tissue is liver cell or tissue.

[0194] In some embodiments, the target cell includes, but is not limited to, neurons, glial cells, astrocytes, oligodendroglia, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat storing cells, Kupffer cells, liver endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.

[0195] In some embodiments, the compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery. In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via injection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.

[0196] In some embodiments, the compositions described herein are administered as part of a composition that allows for extended release. In some embodiments, the compositions comprises a formulation that includes a depot.

[0197] Disclosed herein are methods of treatment using any of the compositions described herein (engineered STAC-BBB variant sequences, AAV particles, and engineered AAV capsid proteins). In embodiments, the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases / disorders, and neurodegenerative disorders, amongst others. In embodiments, the disclosed compositions can be used to treat any one or more of Alzheimer's disease, Huntington's disease; autism; Parkinson's disease; Spinal muscular atrophy, Friedreich's ataxia. In embodiments, the disclosed compositions are used in treatments through any of the methods of delivery described herein.564899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0198] In some embodiments, disclosed are methods for treating, or ameliorating a disease or condition associated with abnormal gene and / or protein in a subject in need of treatment, the methods comprising administering to the subject any effective amount of at least one of the compositions described herein (e.g., engineered STAC-BBB variant sequences, AAV particles, and engineered AAV capsid proteins), delivering the compositions described herein into targeted cells, inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject.Equivalents and Scope

[0199] The disclosure includes many equivalents to the specific embodiments described herein. A person of skill in the art will be able to ascertain equivalents to the specific embodiments, through routine experimentation.

[0200] It is assumed that words of this disclosure are for the purpose of description and not limitation. Changes to words in the claims can be made, while still retaining the scope of the disclosure in its broad embodiment. Specific embodiments of the disclosure have been described herein. However, these embodiments are not intended to be limiting of the broad scope of this disclosure.

[0201] The disclosure is further illustrated by the following examples, which are intended to be purely illustrative and not limiting of the disclosure herein.EXAMPLESExample 1. Methods

[0202] Fitness maturation of AAV capsid STAC-BBB. Fitness maturation was performed by creating libraries of AAV capsids with 1 or 2 amino acid mutations introduced relative to the parental capsid STAC-BBB. In one fitness maturation library, the amino acids 586-598 in STAC-BBB were each changed to all possible amino acids except cysteine and the original amino acid in STAC-BBB. In a separate fitness maturation library, the amino acids 453-459 in STAC-BBB were each changed to all possible amino acids except cysteine and the original amino acid in STAC-BBB.

[0203] AAV capsid library generation. Capsid variants for library screening were synthesized as an oligo pool. Each diversified region was synthesized with three unique nucleotide sequences encoding the diversified region, and each diversified region was linked to a distinct barcode. The oligo pool was cloned into a linearized intermediate plasmid,574899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1followed by cloning of a constant donor sequences to separate the barcode and diversified region and generate the full AAV vector construct. The resulting constructs included either a neuron specific human Synapsin 1 promoter or a ubiquitous CMV promoter driving expression of the barcodes, wherein each barcode is linked to the identity of a single capsid. This enables an assessment of which capsids drive the most functional mRNA expression in the central nervous system. Moreover, each barcode is linked to a unique molecular identifier (UMI). Based on the overall size of the cloned library each barcode is appended to hundreds to thousands of UMIs. The utility of the UMI is to additionally approximate how many distinct AAV transduction events give rise to the NGS read counts that are measured.

[0204] These AAV plasmid libraries were manufactured in HEK293 cells. Briefly, libraries were produced by transient transfection including supplementation of Rep in trans, capsids were purified by cesium density centrifugation, and buffer exchanged into PBS by Amicon filtration. DNase-resistant viral genomic titers were measured by quantitative real time PCR.

[0205] Administration of AAV libraries to cynomolgus macaques. The AAV capsid libraries were each administered to cynomolgus macaques as an intravenous bolus injection into the saphenous vein.

[0206] Tissue collection and preservation. Three weeks post-administration, animals were euthanized and a necropsy was performed. The brain was removed and placed in a coronal brain matrix in an ice-cold nuclease free PBS bath for approximately 10 minutes. The brain was then sliced using a brain matrix at 4 mm coronal slice thickness. Each brain slice was immersed in RNAlater (ratio 1:10) and refrigerated at 4°C for 24 hrs. Following removal from RNAlater, slices were rinsed briefly in cold RNAse-free phosphate-buffered saline and 2 millimeter punches were collected from different brain regions. Punches and remaining tissue slices were frozen at < -65°C. The same workflow was used to process spinal cord and dorsal root ganglia tissues.

[0207] RNA isolation from entire coronal brain slices and spinal cord. Hemisected brain slices or spinal cord samples were weighed and placed into 50 mL Bigprep Lysing Matrix D tubes or 2 mL Lysing Matrix D tubes. The tubes were filled with TRIZOL Reagent at 10 mL per gram of tissue. The tissue samples were homogenized at 1800 RPM for 1.5 minute with a 1 minute rest. The tissue homogenization was repeated 4-6 times. The lysate was centrifuged at 4,000 xg for 5 minutes at 4°C. The clarified lysate was removed and a second round of584899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1centrifugation and removal of clarified lysate to a fresh tube was conducted. Aqueous phase was separated by adding 0.2 mL molecular grade chloroform per mL of Trizol. The samples were mixed by inversion and vortexing for 30 seconds then centrifuged at 4,000g for 20 minutes at 4°C. The aqueous phase, approximately 50 % of the lysate volume, was carefully removed to a new tube. 0.5 mL per mL Trizol of molecular grade isopropanol was added to the tubes and mixed by inversion. The samples were incubated for 10 minutes on ice followed by centrifugation at 10,000 xg for 10 minutes at 4°C. The RNA pellet was resuspended in 75% ethanol at 1 mL per mL Trizol, vortexed, and centrifuged at 10,000 xg for 5 minutes at 4°C. The RNA pellet was air dried for 5 minutes and dissolved in 0.2 mL per mL Trizol with RNAse free DEPC treated water. The yield and purity of the RNA was determined using a spectrophotometer.

[0208] Reverse transcription and next generation sequencing. Isolated RNA was reverse transcribed using a gene-specific primer with the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit. The synthesized complementary DNA (cDNA) was then carried forward for next-generation sequencing (NGS).

[0209] Primers were designed to amplify the region of interest on the cDNA, and these primers also included sequences that bind the adapters for NGS. Using the Roche Kapa HiFi DNA polymerase, a PCR was set up to amplify the region of interest of the cDNA or vector genomic DNA and a second PCR was set up to attach the adapters necessary for NGS sample barcoding. These amplicons were carried forward for NGS and analyzed using a custom bioinformatics pipeline.

[0210] Example 2. Evaluation of STAC-BBB variant performance for delivery to cynomolgus macaque CNS tissues. The performance of each AAV capsid variant in the library was assessed by next-generation sequencing to quantify enrichment in barcoded mRNA transcripts expressed in macaque CNS tissues.

[0211] Next-generation sequencing reads containing barcodes derived from the neuronspecific human Synapsin 1 promoter or ubiquitous CMV promoter were demultiplexed. A log2 fold change for neuronal or ubiquitous expression was then computed for each capsid in the library by totaling the number of barcoded NGS reads recovered from brain or spinal cord mRNA and normalizing by the input abundance of each capsid in the administered library. The log2 fold change for neuronal and ubiquitous mRNA expression in the brain and spinal cord594899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1are shown in Tables 1, 2, and 3. Empty cells indicate that the capsid was not detected in that assay, potentially due to limited sample recovery or sequencing depth.

[0212] Table 1. Performance of STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598 and log2 fold change in neuronal expression in the brain that is greater than zero.

[0213] Table 1 is shown in Figure 23.

[0214] Table 2. Performance of STAC-BBB capsid variant library with mutations in the diversified region of amino acids 586 through 598, log2 fold change in neuronal expression in the brain that is less than zero, and ubiquitous expression in the brain greater than 1.

[0215] Table 2 is shown in Figure 24.

[0216] Table 3. Performance of STAC-BBB capsid variant library with mutations in the diversified region of amino acids 453 through 459 and log2 fold change in neuronal expression in the brain that is greater than zero.

[0217] Table 3 is shown in Figure 25.Tables 1 through 3 highlight variant sequences that exhibit enrichment in the CNS relative to the input abundance in the library. The performance of the entire library is depicted in the bubble plots in FIGs. 2-5 and 7-10.

[0218] Capsids with especially exemplary performance have the following features in the bubble plots:1) High log2 fold enrichment, these data are normalized by input abundance (y-axis)2) Low coefficient of variation (x-axis)3) High fraction of sequenced samples in which a capsid is found (large bubble size)4) Robust UMI recovery (green color)

[0219] STAC-BBB and a non-limiting subset of exemplary STAC-BBB variants are annotated.

[0220] Conclusions of in vivo assessment in cynomolgus macaques. Thousands of STAC-BBB variants exhibit enhanced CNS delivery relative to AAV9. Among these, some variants also exhibit enhanced CNS delivery relative to STAC-BBB. Some non-limiting examples include the STAC-BBB capsid variants with diversified regions corresponding to sequence ID numbers 1-4. These capsids exhibit a higher degree of neuronal mRNA expression throughout 604899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1the CNS compared to STAC-BBB. The STAC-BBB variants with diversified regions corresponding to sequence ID numbers 5-8 exhibit high ubiquitous mRNA expression and low neuronal mRNA expression, suggestive of a shift in cellular tropism relative to STAC-BBB. STAC-BBB variants with diversified regions corresponding to sequence ID numbers 9-17 exhibit similar or enhanced neuronal mRNA expression compared to the STAC-BBB sequence. STAC-BBB variants corresponding to SEQ ID NO. 1-17 are summarized in Table 4. The unmodified sequences for each of the diversified regions in STAC-BBB are provided in SEQ ID NO. 35 and SEQ ID NO. 5443.

[0221] Table 4.SEQ ID Sequence of diversified region (underlinedDiversified regionNO: positions are mutations relative to STAC-BBB)1 SYYVNIMDDDDQA Amino acids 586 through 598 2 SAYVNVMDDLDQA Amino acids 586 through 598 3 SAYVNVMDDMDQA Amino acids 586 through 598 4 SAYVNVMDDMDQP Amino acids 586 through 598 5 SAYVNLMDDMDWA Amino acids 586 through 598 6 IAYVIIMDDMDQA Amino acids 586 through 598 7 SAYRNIMDDMDQA Amino acids 586 through 598 8 SAYVNIMDDMKQK Amino acids 586 through 598 9 GVGNNQQ Amino acids 453 through 459 10 GAGLNQQ Amino acids 453 through 459 11 GVGTNQQ Amino acids 453 through 459 12 NSGNNQQ Amino acids 453 through 459 13 GSGLNKQ Amino acids 453 through 459 14 GSGKNQN Amino acids 453 through 459 15 SSGSNQQ Amino acids 453 through 459 16 GSGLKQQ Amino acids 453 through 459 17 GSGSNKQ Amino acids 453 through 459 35 SAYVNIMDDMDQA Amino acids 586 through 598 5443 GSGQNQQ Amino acids 453 through 459614899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1

[0222] Analysis of mutation landscape for STAC-BBB variants. Saturation mutagenesis enables a comprehensive understanding of the impact of amino acid mutations on the CNS delivery mediated by STAC-BBB. The impact of single amino acid changes is summarized in FIG. 6 (diversified region corresponding to amino acids 586 through 598) and FIG. 11 (diversified region corresponding to amino acids 453 through 459) respectively. In these figures the heat map shows the log2 fold change in neuronal mRNA expression in cynomolgus macaque brain. (*) indicates the original amino acid at each position in the STAC-BBB amino acid sequence.

[0223] The impact of introducing two mutations in a diversified region was also evaluated. STAC-BBB variants that exceed a cutoff value for log2 fold change enrichment are highlighted in Tables 1, 2, and 3 for each respective library. In addition to these tables, heat maps highlighting the performance of all two amino acid substitutions are provided (FIGs. 12 through 22) specifically for the two positions that were modified in SEQ ID NO. 1, 2, 4-7, and 8-17. The axes of each heat map shows the different amino acids that were evaluated at each of the two positions. The intensity of each square in greyscale is proportional to the log2 fold enrichment, with darker shades representing higher enrichment and lighter shades indicating lower enrichment for the combination of amino acids at each position. White squares without a value represent capsid sequences with a log2 fold change that is less than 1. The position numbering refers to the position of each amino acid in the diversified region. (*) indicates the amino acid combination at these positions in STAC-BBB. (+) indicates the amino acid combination at these positions in one or more of the STAC-BBB variants listed in Table 4. Taken together, the heat maps provide an overview of which mutations at each mutated position in STAC-BBB enhance or impair CNS transduction. A summary of beneficial amino acid changes for the diversified region corresponding to amino acids 586 through 598 is provided in Table 5.

[0224] Table 5.Type of First Amino acids at amino acid position Amino acids at Second position the second substitution modified the first position modified positionSingle 1 T, N, W, V, or L N / A N / A624899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1Type of First Amino acids at amino acid position Amino acids at Second position the second substitution modified the first position modified positionQ, L, N, S, K, Y, M, Not applicable Not applicable Single 2 T, R, I, orVSingle 3 I, F, V, D, or L Not applicable Not applicable Single 4 M, Y, I, L, orF Not applicable Not applicable Single 5 Not applicable Not applicable Not applicable Single 6 VorD Not applicable Not applicable Single 7 I Not applicable Not applicable Single 8 G Not applicable Not applicable Single 9 E or N Not applicable Not applicable L, D, E, N, K, Y, F, Not applicable Not applicable Single 10 S, or ASingle 11 I, H, S, E, A, orW Not applicable Not applicable H, L, M, S, V, F, G, Not applicable Not applicable Single 12 N, K, orDP, S, L, V, N, T, H, Not applicable Not applicable Single 13 Q, E, G, F, or KDouble 1 T, H, M, V, N, G, 5 F, Q, I, S, W, Y, V,W, D, F, E, R, I, L, A, M, H, or T or KDouble 2 Y, S, I, K, N, F, V, 10 D, L, H, N, E, Q, I,T, Q, R, H, L, P, M, S, A, F, T, Y, V, P, W, orE R, K, orW Double 6 V, F, S, T, D, L, A, 10 L, D, F, N, P, E, Y, or M G, H, V, K, S, R, or ADouble 6 V, L, D, S, P, orH 12 L, V, F, G, S, H, M,W, Y, P, A, E, N, K, T, D, I, or R Double 6 V, D, S, K, L, or Q 13 P, S, E, L, V, W, N,T, Q, H, Y, F, G, M, I, or K634899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1Type of First Amino acids at amino acid position Amino acids at Second position the second substitution modified the first position modified position Double 11 N, W, K, I, H, G, F, 13 H, V, P, R, W, E, S,S, Q, V, P, M, R, T, F, L, Y, G, N, Q, I, E, L, A, orY K, D, T, or M

[0225] A summary of beneficial amino acid changes for the diversified region corresponding to amino acids 453 through 459 provided in Table 6.

[0226] Table 6.Type of First Amino acids at amino acid position Amino acids at Second position the second substitution modified the first position modified positionN, V, A, P, L, H, F, Not applicable Not applicable Single 1 Y, orTSingle 2 A, N, G, Q, K, orV Not applicable Not applicable Single 3 S orT Not applicable Not applicable T, A, V, S, L, I, H, Not applicable Not applicable Single 4 or GSingle 5 R, K, L, or D Not applicable Not applicable Single 6 A Not applicable Not applicable Single 7 F, K, M, orH Not applicable Not applicable Double 1 S, N, A, Q, V, H, T, 4 S, N, T, A, H, V, L,D, Y, P, R, K, M, I, Y, P, W, G, D, R, or L, E, or F IDouble 2 V, A, M, Q, T, P, I, 4 N, L, T, I, S, M, A,G, N, W, R, H, L, V, H, R, G, K, W, K, orY D, or EDouble 4 L, S, T, I, A, G, N, 5 K, H, A, S, T, R, V,F, K, V, M, R, Y, E, M, W, Q, L, F, G, D, W, H, or P D, or EDouble 4 L, T, S, R, H, W, A, 6 K, A, H, S, T, R, P,I, F, D, V, N, P, Y, M, V, W, N, Y, G, G, E, orK or I644899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1Type of First Amino acids at amino acid position Amino acids at Second position the second substitution modified the first position modified position Double 4 W, K, T, H, A, R, 7 E,N, F, I, H, G, R,V, S, L, G, N, M, I, K, P, A, M, L, orV or P

[0227] The double amino acid substitutions in Tables 5 and 6 describe exemplary sets of amino acid combinations at the two positions that were modified in SEQ IDs NO. 1, 2, 4-6, and 8-17. Table 7 highlights additional STAC-BBB variants that exhibit efficient CNS delivery and are distinct from the amino acid combinations listed in Tables 5 and 6.

[0228] Table 7.Sequence of diversified region(underlined positions aremutations relative to STAC- Diversified regionSEQ ID NO: BBB)37 GSMQNQY Amino acids 453 through 45938 SAWVNIMNDMDQA Amino acids 586 through 59839 SAYVNWMDKMDQA Amino acids 586 through 59840 SAQVNIMLDMDQA Amino acids 586 through 59841 SAYVNYMDYMDQA Amino acids 586 through 59842 SAYVRIMQDMDQA Amino acids 586 through 59843 SAYVNIMQRMDQA Amino acids 586 through 59844 SAYVNIMYAMDOA Amino acids 586 through 59845 SAWVNINDDMDQA Amino acids 586 through 59846 SAEVNIKDDMDQA Amino acids 586 through 59847 SAYVNIWDVMDQA Amino acids 586 through 59848 SDYVRIMDDMDQA Amino acids 586 through 59849 SAPKNIMDDMDQA Amino acids 586 through 59850 SGYVNIMFDMDQA Amino acids 586 through 59851 SAYVNNEDDMDQA Amino acids 586 through 59852 SAYQNIMLDMDQA Amino acids 586 through 59853 SANVNIKDDMDQA Amino acids 586 through 59854 SAYDNIMRDMDQA Amino acids 586 through 59855 SAQVNITDDMDQA Amino acids 586 through 59856 SAYPNIHDDMDQA Amino acids 586 through 59857 SAYVPIMDAMDQA Amino acids 586 through 598Amino acids 586 through 59858 SAYWNIMTDMDQA654899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO159 SAYVPIWDDMDQA Amino acids 586 through 59860 SAHQNIMDDMDQA Amino acids 586 through 59861 SAAVNIVDDMDQA Amino acids 586 through 59862 SAYVNIMIGMDQA Amino acids 586 through 59863 SAYENIADDMDQA Amino acids 586 through 59864 SAYVNIDDWMDQA Amino acids 586 through 59865 SAYRNIMFDMDQA Amino acids 586 through 59866 SAYTNIKDDMDQA Amino acids 586 through 59867 SAYVRIMDKMDQA Amino acids 586 through 59868 AAYVNIMDSMDQA Amino acids 586 through 59869 SAYVNWMDWMDQA Amino acids 586 through 59870 SAYVNIEDIMDQA Amino acids 586 through 59871 AAYVNIRDDMDQA Amino acids 586 through 59872 SAYGNIDDDMDQA Amino acids 586 through 59873 SAYANIMDVMDQA Amino acids 586 through 59874 QAYVNIMNDMDQA Amino acids 586 through 59875 SAHVGIMDDMDQA Amino acids 586 through 59876 SAYVNRWDDMDOA Amino acids 586 through 59877 SAYVNIVDQMDQA Amino acids 586 through 59878 SAYVNILDIMDQA Amino acids 586 through 598Amino acids 586 through 59879 SAYRNIMDIMDQA

[0229] The mutations introduced in each of the diversified regions can also be combined in a single VP1 capsid sequence that comprises a diversified region between amino acids 453 through 459 as well as a diversified region for amino acids 586 through 589. Non-limiting examples of this concept are provided in Table 8.

[0230] Table 8.Sequence of diversified region for amino Sequence of diversified region for amino acids 453 through 459 (underlined acids 586 through 598 (underlinedpositions are mutations relative to STAC- positions are mutations relative to STAC- BBB) BBB)GVGNNQQ SYYVNIMDDDDQA GAGLNQQ SYYVNIMDDDDQA GVGTNQQ SYYVNIMDDDDQA NSGNNQQ SYYVNIMDDDDQA GSGLNKQ SYYVNIMDDDDQA GVGNNQQ SAYVNVMDDLDQA664899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1Sequence of diversified region for amino Sequence of diversified region for amino acids 453 through 459 (underlined acids 586 through 598 (underlinedpositions are mutations relative to STAC- positions are mutations relative to STAC- BBB) BBB)GAGLNQQ SAYVNVMDDLDQA GVGTNQQ SAYVNVMDDLDQA NSGNNQQ SAYVNVMDDLDQA GSGLNKQ SAYVNVMDDLDQA GVGNNQQ SAYVNVMDDMDQP GAGLNQQ SAYVNVMDDMDQP GVGTNQQ SAYVNVMDDMDQP NSGNNQQ SAYVNVMDDMDQP GSGLNKQ SAYVNVMDDMDQP

[0231] The full VP1 capsid amino acid sequences for STAC-BBB (SEP ID NO: 36) andexemplary STAC-BBB variants (SEQ ID NO: 18-34 and SEQ ID NO: 80-122) are provided. In the sequences below, the diversified region in STAC-BBB and its variants are indicated in bold. Mutations relative to STAC-BBB are underlined.

[0232] STAC-BBB Parent Capsid (SEQ ID NO: 36) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0233] STAC-BBB Capsid Variant Amino Acid Sequences

[0234] STAC-BBB Variant SEQ ID NO: 18674899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSYYVNIMDDDDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0235] STAC-BBB Variant SEP ID NO: 19 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNVMDDLDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0236] STAC-BBB Variant SEP ID NO: 20 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNVMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0237] STAC-BBB Variant SEP ID NO: 21684899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNVMDDMDQPQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0238] STAC-BBB Variant SEP ID NO: 22 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNLMDDMDWAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0239] STAC-BBB Variant SEP ID NO: 23 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQIAYVIIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAI<IPHTDGNFHPSPLMGGFGMI<HPPPQILII<NTPVPADPPTAFNI<D KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0240] STAC-BBB Variant SEP ID NO: 24694899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYRNIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0241] STAC-BBB Variant SEP ID NO: 25 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMKQKQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0242] STAC-BBB Variant SEP ID NO: 26 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGVGNNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0243] STAC-BBB Variant SEP ID NO: 27704899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGAGLNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0244] STAC-BBB Variant SEP ID NO: 28 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGVGTNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0245] STAC-BBB Variant SEP ID NO: 29 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINNSGNNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0246] STAC-BBB Variant SEP ID NO: 30714899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGLNKQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0247] STAC-BBB Variant SEP ID NO: 31 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGKNQNTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0248] STAC-BBB Variant SEP ID NO: 32 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINSSGSNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0249] STAC-BBB Variant SEP ID NO: 33724899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGLKQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0250] STAC-BBB Variant SEP ID NO: 34 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGSNKQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0251] STAC-BBB Variant SEP ID NO: 80 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSMQNQYTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0252] STAC-BBB Variant SEP ID NO: 81734899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAWVNIMNDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0253] STAC-BBB Variant SEP ID NO: 82 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNWMDKMDQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0254] STAC-BBB Variant SEP ID NO: 83 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQVNIMLDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0255] STAC-BBB Variant SEP ID NO: 84744899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNYMDYMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0256] STAC-BBB Variant SEP ID NO: 85 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVRIMQDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0257] STAC-BBB Variant SEP ID NO: 86 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMQRMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0258] STAC-BBB Variant SEP ID NO: 87754899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMYAMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0259] STAC-BBB Variant SEP ID NO: 88 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAWVNINDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0260] STAC-BBB Variant SEP ID NO: 89 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAEVNIKDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0261] STAC-BBB Variant SEP ID NO: 90764899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIWDVMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0262] STAC-BBB Variant SEP ID NO: 91 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSDYVRIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0263] STAC-BBB Variant SEP ID NO: 92 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAPKNIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0264] STAC-BBB Variant SEP ID NO: 93774899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSGYVNIMFDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0265] STAC-BBB Variant SEP ID NO: 94 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNNEDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0266] STAC-BBB Variant SEP ID NO: 95 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYQNIMLDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0267] STAC-BBB Variant SEP ID NO: 96784899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSANVNIKDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0268] STAC-BBB Variant SEP ID NO: 97 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYDNIMRDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0269] STAC-BBB Variant SEP ID NO: 98 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQVNITDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0270] STAC-BBB Variant SEP ID NO: 99794899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYPNIHDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0271] STAC-BBB Variant SEP ID NO: 100 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVPIMDAMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0272] STAC-BBB Variant SEP ID NO: 101 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYWNIMTDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0273] STAC-BBB Variant SEP ID NO: 102804899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVPIWDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0274] STAC-BBB Variant SEP ID NO: 103 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAHQNIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0275] STAC-BBB Variant SEP ID NO: 104 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAAVNIVDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSE PRPIGTRYLTRNL

[0276] STAC-BBB Variant SEP ID NO: 105814899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMIGMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0277] STAC-BBB Variant SEP ID NO: 106 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYENIADDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0278] STAC-BBB Variant SEP ID NO: 107 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIDDWMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0279] STAC-BBB Variant SEP ID NO: 108824899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYRNIMFDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0280] STAC-BBB Variant SEP ID NO: 109 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYTNIKDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0281] STAC-BBB Variant SEP ID NO: 110 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVRIMDKMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0282] STAC-BBB Variant SEP ID NO: 111834899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQAAYVNIMDSMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0283] STAC-BBB Variant SEP ID NO: 112 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNWMDWMDQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0284] STAC-BBB Variant SEP ID NO: 113 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIEDIMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0285] STAC-BBB Variant SEP ID NO: 114844899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQAAYVNIRDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0286] STAC-BBB Variant SEP ID NO: 115 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYGNIDDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0287] STAC-BBB Variant SEP ID NO: 116 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYANIMDVMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0288] STAC-BBB Variant SEP ID NO: 117854899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQQAYVNIMNDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0289] STAC-BBB Variant SEP ID NO: 118 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAHVGIMDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0290] STAC-BBB Variant SEP ID NO: 119 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNRWDDMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0291] STAC-BBB Variant SEP ID NO: 120864899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIVDQMDQAQTGWVQNQGILPGMVW QDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVY SEPRPIGTRYLTRNL

[0292] STAC-BBB Variant SEP ID NO: 121 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNILDIMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNL

[0293] STAC-BBB Variant SEP ID NO: 122 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYRNIMDIMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNLSelected Embodiments:874899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO11. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of a variant sequence of SEQ ID NO: 35.2. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids T, N, W, V, or L at amino acid position 1 of SEQ ID NO: 35.3. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids Q, L, N, S, K, Y, M, T, R, I, or V at amino acid position 2 of SEQ ID NO: 35.4. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids I, F, V, D, or L at amino acid position 3 of SEQ ID NO: 35.5. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids M, Y, I, L, or F at amino acid position 4 of SEQ ID NO: 35.6. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids V or D at amino acid position 6 of SEQ ID NO: 35.7. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises amino acid I at amino acid position 7 of SEQ ID NO: 35.8. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises amino acid G at amino acid position 8 of SEQ ID NO: 35.9. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids E or N at amino acid position 9 of SEQ ID NO: 35.10. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids L, D, E, N, K, Y, F, S, or A at amino acid position 10 of SEQ ID NO: 35.11. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids I, H, S, E, A, or W at amino acid position 11 of SEQ ID NO: 35.12. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids H, L, M, S, V, F, G, N, K, or D at amino acid position 12 of SEQ ID NO: 35.13. The engineered AAV capsid protein of embodiment 1, wherein the variant sequence comprises any of amino acids P, S, L, V, N, T, H, Q, E, G, F, or K at amino acid position 13 of SEQ ID NO: 35.884899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO114. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence of any one of SEQ ID NOs: 1-8 or 38- 79.15. The engineered capsid protein of embodiment 14, comprising a variant amino acid sequence of a sequence set forth in SEQ ID NO: 1-8 or SEQ ID NOs: 38-79.16. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 1, wherein the variant sequence comprises (i) any of amino acids Y, S, I, K, N, F, V, T, Q, R, H, L, P, M, W, or E at amino acid position 2 of SEQ ID NO: 1 and / or (ii) any of amino acids D, L, H, N, E, Q, I, S, A, F, T, Y, V, P, R, K, or W at amino acid position 10 of SEQ ID NO: 1; optionally wherein the AAV capsid protein comprises SEQ ID NO: 1.17. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 2, wherein the variant sequence comprises (i) any of amino acids V, F, S, T, D, L, A, or M at amino acid position 6 of SEQ ID NO: 2 and / or (ii) any of amino acids L, D, F, N, P, E, Y, G, H, V, K, S, R, or A at amino acid position 10 of SEQ ID NO: 2; optionally wherein the AAV capsid protein comprises SEQ ID NO: 2. 18. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 3, wherein the variant sequence comprises (i) any of amino acids V or D at amino acid position 6 of SEQ ID NO: 3.19. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 4, wherein the variant sequence comprises (i) any of amino acids V, D, S, K, L, or Q at amino acid position 6 of SEQ ID NO: 4 and / or (ii) any of amino acids P, S, E, L, V, W, N, T, Q, H, Y, F, G, M, I, or K at amino acid position 13 of SEQ ID NO: 4; optionally wherein the AAV capsid protein comprises SEQ ID NO: 4. 20. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 5, wherein the variant sequence comprises (i) any of amino acids V, L, D, S, P, or H at amino acid position 6 of SEQ ID NO: 5 and / or (ii) any of amino acids L, V, F, G, S, H, M, W, Y, P, A, E, N, K, T, D, I, or R at amino acid position 12 of SEQ ID NO: 5; optionally wherein the AAV capsid protein comprises SEQ ID NO: 5.21. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 6, wherein the variant sequence comprises (i) any of amino acids T, H, M, V, N, G, W, D, F, E, R, I, L, or K at amino acid position 1 of SEQ ID NO: 6 and / or (ii) any of amino acids F, Q, I, S, W, Y, V, A, M, H, or T at amino acid position 5 of SEQ ID NO: 6; optionally wherein the AAV capsid protein comprises SEQ ID NO: 6.894899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO122. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 7, wherein the variant sequence comprises (i) any of amino acids M, Y, I, L, or F at amino acid position 4 of SEQ ID NO: 7.23. The engineered AAV capsid protein of embodiment 14 or embodiment 15, comprising a variant sequence of SEQ ID NO: 8, wherein the variant sequence comprises (i) any of amino acids N, W, K, I, H, G, F, S, Q, V, P, M, R, T, E, L, A, or Y at amino acid position 11 of SEQ ID NO: 8 and / or (ii) any of amino acids H, V, P, R, W, E, S, F, L, Y, G, N, Q, I, K, D, T, or M at amino acid position 13 of SEQ ID NO: 8; optionally wherein the AAV capsid protein comprises SEQ ID NO: 8.24. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises a sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 18-25 or 81-122, optionally comprising a substitution of at least one or two amino acids at any one or two amino acid positions 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and / or 598 of any one of SEQ ID NOs: 18-25 or 81-122.25. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, or 7 contiguous amino acids of a variant sequence of SEQ ID NO: 5443.26. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids N, V, A, P, L, H, F, Y, or T at amino acid position 1 of SEQ ID NO: 5443.27. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids A, N, G, Q, K, or V at amino acid position 2 of SEQ ID NO: 5443.28. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids S or T at amino acid position 3 of SEQ ID NO: 5443.29. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids T, A, V, S, L, I, H, or G at amino acid position 4 of SEQ ID NO: 5443.30. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids R, K, L, or D at amino acid position 5 of SEQ ID NO: 5443.31. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises amino acid A at amino acid position 6 of SEQ ID NO: 5443.32. The engineered AAV capsid protein of embodiment 25, wherein the variant sequence comprises any of amino acids F, K, M, or H at amino acid position 7 of SEQ ID NO: 5443.904899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO133. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence of any one of SEQ ID NOs: 9-17 or 37.34. The engineered capsid protein of embodiment 33, comprising a variant amino acid sequence of a sequence set forth in SEQ ID NO: 9-17 or 37.35. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 9, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 9 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 9; optionally wherein the AAV capsid protein comprises SEQ ID NO: 9.36. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 10, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 10 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 10; optionally wherein the AAV capsid protein comprises SEQ ID NO: 10.37. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 11, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 11 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 11; optionally wherein the AAV capsid protein comprises SEQ ID NO: 11.38. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 12, wherein the variant sequence comprises (i) any of amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F at amino acid position 1 of SEQ ID NO: 12 and / or (ii) any of amino S, N, T, A, H, V, L, Y, P, W, G, D, R, or I at amino acid position 4 of SEQ ID NO: 12; optionally wherein the AAV capsid protein comprises SEQ ID NO: 12.39. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 13, wherein the variant sequence comprises (i) any of amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K at amino acid position 4 of SEQ ID NO: 13 and / or (ii) any of amino K, A, H, S, T, R, P, M, V, W, N, Y, G, or lat amino acid position 6 of SEQ ID NO: 13; optionally wherein the AAV capsid protein comprises SEQ ID NO: 13.914899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO140. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 14, wherein the variant sequence comprises (i) any of amino acids W, K, T, H, A, R, V, S, L, G, N, M, I, or P at amino acid position 4 of SEQ ID NO: 14 and / or (ii) any of amino E, N, F, I, H, G, R, K, P, A, M, L, or V at amino acid position 7 of SEQ ID NO: 14; optionally wherein the AAV capsid protein comprises SEQ ID NO: 14.41. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 15, wherein the variant sequence comprises (i) any of amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F at amino acid position 1 of SEQ ID NO: 15 and / or (ii) any of amino S, N, T, A, H, V, L, Y, P, W, G, D, R, or I at amino acid position 4 of SEQ ID NO: 15; optionally wherein the AAV capsid protein comprises SEQ ID NO: 15.42. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 16, wherein the variant sequence comprises (i) any of amino acids L, S, T, I, A, G, N, F, K, V, M, R, Y, E, D, W, H, or P at amino acid position 4 of SEQ ID NO: 16 and / or (ii) any of amino K, H, A, S, T, R, V, M, W, Q, L, F, G, D, or E at amino acid position 5 of SEQ ID NO: 16; optionally wherein the AAV capsid protein comprises SEQ ID NO: 16.43. The engineered AAV capsid protein of embodiment 33 or embodiment 34, comprising a variant sequence of SEQ ID NO: 17, wherein the variant sequence comprises (i) any of amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K at amino acid position 4 of SEQ ID NO: 17 and / or (ii) any of amino K, A, H, S, T, R, P, M, V, W, N, Y, G, or lat amino acid position 6 of SEQ ID NO: 17; optionally wherein the AAV capsid protein comprises SEQ ID NO: 17.44. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises a sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 26-34 or 80, optionally comprising a substitution of at least one or two amino acids at any one or two amino acid positions 453, 454, 455, 456, 457, 458, and / or 459 of any one of SEQ ID NOs: 26-34 or 80.45. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of embodiments 1-23); and optionally ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of embodiments 25-43).924899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO146. The engineered AAV capsid protein of embodiment 45, wherein the engineered AAV capsid protein comprises: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of embodiments 1-23); and ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of embodiments 25-43). 47. An engineered AAV capsid protein, comprising diversified region sequences as shown in a single row in Table 8.48. An engineered AAV capsid protein, comprising a diversified region sequence as shown in Table 4 or Table 7.49. The engineered AAV capsid protein of embodiment 48, wherein the diversified region sequence is modified as shown in Table 5 or Table 6.50. A central nervous system targeting molecule comprising: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of embodiments 1-23); and optionally ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of embodiments 25-43).51. The central nervous system targeting molecule of embodiment 50, comprising: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of embodiments 1-23); and ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of embodiments 25-43).52. The central nervous system targeting molecule of any one of embodiments 50-51, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.53. An engineered AAV capsid protein comprising a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of embodiments 1-23), wherein the engineered AAV capsid protein further comprises an amino acid insert immediately adjacent at the C- terminal end of the variant sequence; optionally wherein the amino acid is alanine (A). 54. The engineered AAV capsid protein of embodiment 53, wherein the engineered AAV capsid protein comprises an amino acid insert between amino acid positions 598 and 599 of a sequence set forth in any one of SEQ ID NOs: 18-25 or 81-122; optionally wherein the amino acid is alanine (A).55. A nucleic acid molecule encoding an engineered AAV capsid protein of any one of embodiments 1-50 and 53-54.56. A host cell comprising the nucleic acid molecule of embodiment 55.934899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO157. A pharmaceutical composition comprising an engineered AAV capsid protein of any one of embodiments 1-50 and 53-54, or a central nervous system targeting molecule according to any one of embodiments 50-52.58. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an engineered AAV capsid protein according to any one of embodiments 1-50 and 53-54.59. A method of delivering a payload of interest to a cell or a tissue, wherein the payload of interest is fused or conjugated to a central nervous system targeting molecule according to any one of embodiments 50-52.60. A method of treating a disease in a subject, comprising administering to the subject a composition comprising an engineered AAV capsid protein according to any one of embodiments 1-50 and 53-54, or a central nervous system targeting molecule according to any one of embodiments 50-52.61. Use of an engineered AAV capsid protein of any one of embodiments 1-50 and 53-54, a central nervous system targeting molecule of any one of embodiments 50-52, a nucleic acid molecule of embodiment 55, or a host cell of embodiment 56 for the manufacture of a medicament in the method of any one of embodiments 58-60.944899-7730-5762

Claims

Attorney Docket No.: | 91355.12316 P.0288.WO1WHAT IS CLAIMED IS:

1. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of a variant sequence of SEQ ID NO: 35.

2. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids T, N, W, V, or L at amino acid position 1 of SEQ ID NO: 35.

3. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids Q, L, N, S, K, Y, M, T, R, I, or V at amino acid position 2 of SEQ ID NO: 35.

4. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids I, F, V, D, or L at amino acid position 3 of SEQ ID NO: 35.

5. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids M, Y, I, L, or F at amino acid position 4 of SEQ ID NO: 35.

6. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids V or D at amino acid position 6 of SEQ ID NO: 35.

7. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises amino acid I at amino acid position 7 of SEQ ID NO: 35.

8. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises amino acid G at amino acid position 8 of SEQ ID NO: 35.

9. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids E or N at amino acid position 9 of SEQ ID NO: 35.

10. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids L, D, E, N, K, Y, F, S, or A at amino acid position 10 of SEQ ID NO: 35.

11. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids I, H, S, E, A, or W at amino acid position 11 of SEQ ID NO: 35.

12. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids H, L, M, S, V, F, G, N, K, or D at amino acid position 12 of SEQ ID NO: 35.

13. The engineered AAV capsid protein of claim 1, wherein the variant sequence comprises any of amino acids P, S, L, V, N, T, H, Q, E, G, F, or K at amino acid position 13 of SEQ ID NO: 35.954899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO114. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence of any one of SEQ ID NOs: 1-8 or 38- 79.

15. The engineered capsid protein of claim 14, comprising a variant amino acid sequence of a sequence set forth in SEQ ID NO: 1-8 or SEQ ID NOs: 38-79.

16. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 1, wherein the variant sequence comprises (i) any of amino acids Y, S, I, K, N, F, V, T, Q, R, H, L, P, M, W, or E at amino acid position 2 of SEQ ID NO: 1 and / or (ii) any of amino acids D, L, H, N, E, Q, I, S, A, F, T, Y, V, P, R, K, or W at amino acid position 10 of SEQ ID NO: 1; optionally wherein the AAV capsid protein comprises SEQ ID NO: 1.

17. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 2, wherein the variant sequence comprises (i) any of amino acids V, F, S, T, D, L, A, or M at amino acid position 6 of SEQ ID NO: 2 and / or (ii) any of amino acids L, D, F, N, P, E, Y, G, H, V, K, S, R, or A at amino acid position 10 of SEQ ID NO: 2; optionally wherein the AAV capsid protein comprises SEQ ID NO: 2.

18. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 3, wherein the variant sequence comprises (i) any of amino acids V or D at amino acid position 6 of SEQ ID NO: 3.

19. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 4, wherein the variant sequence comprises (i) any of amino acids V, D, S, K, L, or Q at amino acid position 6 of SEQ ID NO: 4 and / or (ii) any of amino acids P, S, E, L, V, W, N, T, Q, H, Y, F, G, M, I, or K at amino acid position 13 of SEQ ID NO: 4; optionally wherein the AAV capsid protein comprises SEQ ID NO: 4.

20. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 5, wherein the variant sequence comprises (i) any of amino acids V, L, D, S, P, or H at amino acid position 6 of SEQ ID NO: 5 and / or (ii) any of amino acids L, V, F, G, S, H, M, W, Y, P, A, E, N, K, T, D, I, or R at amino acid position 12 of SEQ ID NO: 5; optionally wherein the AAV capsid protein comprises SEQ ID NO: 5.

21. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 6, wherein the variant sequence comprises (i) any of amino acids T, H, M, V, N, G, W, D, F, E, R, I, L, or K at amino acid position 1 of SEQ ID NO: 6 and / or (ii) any of amino acids F, Q, I, S, W, Y, V, A, M, H, or T at amino acid position 5 of SEQ ID NO: 6; optionally wherein the AAV capsid protein comprises SEQ ID NO: 6.964899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO122. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 7, wherein the variant sequence comprises (i) any of amino acids M, Y, I, L, or F at amino acid position 4 of SEQ ID NO: 7.

23. The engineered AAV capsid protein of claim 14 or claim 15, comprising a variant sequence of SEQ ID NO: 8, wherein the variant sequence comprises (i) any of amino acids N, W, K, I, H, G, F, S, Q, V, P, M, R, T, E, L, A, or Y at amino acid position 11 of SEQ ID NO: 8 and / or (ii) any of amino acids H, V, P, R, W, E, S, F, L, Y, G, N, Q, I, K, D, T, or M at amino acid position 13 of SEQ ID NO: 8; optionally wherein the AAV capsid protein comprises SEQ ID NO: 8.

24. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises a sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 18-25 or 81-122, optionally comprising a substitution of at least one or two amino acids at any one or two amino acid positions 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, and / or 598 of any one of SEQ ID NOs: 18-25 or 81-122.

25. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, or 7 contiguous amino acids of a variant sequence of SEQ ID NO: 5443.

26. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids N, V, A, P, L, H, F, Y, or T at amino acid position 1 of SEQ ID NO: 5443.

27. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids A, N, G, Q, K, or V at amino acid position 2 of SEQ ID NO: 5443.

28. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids S or T at amino acid position 3 of SEQ ID NO: 5443.

29. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids T, A, V, S, L, I, H, or G at amino acid position 4 of SEQ ID NO: 5443.

30. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids R, K, L, or D at amino acid position 5 of SEQ ID NO: 5443.

31. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises amino acid A at amino acid position 6 of SEQ ID NO: 5443.

32. The engineered AAV capsid protein of claim 25, wherein the variant sequence comprises any of amino acids F, K, M, or H at amino acid position 7 of SEQ ID NO: 5443.

33. An engineered AAV capsid protein comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids of an amino acid sequence of any one of SEQ ID NOs: 9-17 or 37.974899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO134. The engineered capsid protein of claim 33, comprising a variant amino acid sequence of a sequence set forth in SEQ ID NO: 9-17 or 37.

35. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 9, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 9 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 9; optionally wherein the AAV capsid protein comprises SEQ ID NO: 9.

36. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 10, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 10 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 10; optionally wherein the AAV capsid protein comprises SEQ ID NO: 10.

37. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 11, wherein the variant sequence comprises (i) any of amino acids V, A, M, Q, T, P, I, G, N, W, R, H, L, K, or Y at amino acid position 2 of SEQ ID NO: 11 and / or (ii) any of amino acids N, L, T, I, S, M, A, V, H, R, G, K, W, D, or E at amino acid position 4 of SEQ ID NO: 11 ; optionally wherein the AAV capsid protein comprises SEQ ID NO: 11.

38. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 12, wherein the variant sequence comprises (i) any of amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F at amino acid position 1 of SEQ ID NO: 12 and / or (ii) any of amino S, N, T, A, H, V, L, Y, P, W, G, D, R, or I at amino acid position 4 of SEQ ID NO: 12; optionally wherein the AAV capsid protein comprises SEQ ID NO: 12.

39. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 13, wherein the variant sequence comprises (i) any of amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K at amino acid position 4 of SEQ ID NO: 13 and / or (ii) any of amino K, A, H, S, T, R, P, M, V, W, N, Y, G, or lat amino acid position 6 of SEQ ID NO: 13; optionally wherein the AAV capsid protein comprises SEQ ID NO: 13.

40. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 14, wherein the variant sequence comprises (i) any of amino acids W, K, T, H, A, R, V, S, L, G, N, M, I, or P at amino acid position 4 of SEQ ID NO: 14 and / or (ii) any of amino E, N, F, I, H, G, R, K, P, A, M, L, or V at amino acid position 7 of SEQ ID NO: 14; optionally wherein the AAV capsid protein comprises SEQ ID NO: 14.984899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO141. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 15, wherein the variant sequence comprises (i) any of amino acids S, N, A, Q, V, H, T, D, Y, P, R, K, M, I, L, E, or F at amino acid position 1 of SEQ ID NO: 15 and / or (ii) any of amino S, N, T, A, H, V, L, Y, P, W, G, D, R, or I at amino acid position 4 of SEQ ID NO: 15; optionally wherein the AAV capsid protein comprises SEQ ID NO: 15.

42. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 16, wherein the variant sequence comprises (i) any of amino acids L, S, T, I, A, G, N, F, K, V, M, R, Y, E, D, W, H, or P at amino acid position 4 of SEQ ID NO: 16 and / or (ii) any of amino K, H, A, S, T, R, V, M, W, Q, L, F, G, D, or E at amino acid position 5 of SEQ ID NO: 16; optionally wherein the AAV capsid protein comprises SEQ ID NO: 16.

43. The engineered AAV capsid protein of claim 33 or claim 34, comprising a variant sequence of SEQ ID NO: 17, wherein the variant sequence comprises (i) any of amino acids L, T, S, R, H, W, A, I, F, D, V, N, P, Y, G, E, or K at amino acid position 4 of SEQ ID NO: 17 and / or (ii) any of amino K, A, H, S, T, R, P, M, V, W, N, Y, G, or lat amino acid position 6 of SEQ ID NO: 17; optionally wherein the AAV capsid protein comprises SEQ ID NO: 17.

44. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises a sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 26-34 or 80, optionally comprising a substitution of at least one or two amino acids at any one or two amino acid positions 453, 454, 455, 456, 457, 458, and / or 459 of any one of SEQ ID NOs: 26-34 or 80.

45. An engineered AAV capsid protein, wherein the engineered AAV capsid protein comprises: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of claims 1-23); and optionally ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of claims 25-43) .

46. The engineered AAV capsid protein of claim 45, wherein the engineered AAV capsid protein comprises: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of claims 1-23); and ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of claims 25-43).

47. An engineered AAV capsid protein, comprising diversified region sequences as shown in a single row in Table 8.

48. An engineered AAV capsid protein, comprising a diversified region sequence as shown in Table 4 or Table 7.994899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO149. The engineered AAV capsid protein of claim 48, wherein the diversified region sequence is modified as shown in Table 5 or Table 6.

50. A central nervous system targeting molecule comprising: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of claims 1-23); and optionally ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of claims 25-43).

51. The central nervous system targeting molecule of claim 50, comprising: i) SEQ ID NO: 35 or a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of claims 1- 23); and ii) a variant sequence of SEQ ID NO: 5443 as described herein (e.g., according to any one of claims 25-43).

52. The central nervous system targeting molecule of any one of claims 50-51, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.

53. An engineered AAV capsid protein comprising a variant of SEQ ID NO: 35 as described herein (e.g., according to any one of claims 1-23), wherein the engineered AAV capsid protein further comprises an amino acid insert immediately adjacent at the C-terminal end of the variant sequence; optionally wherein the amino acid is alanine (A).

54. The engineered AAV capsid protein of claim 53, wherein the engineered AAV capsid protein comprises an amino acid insert between amino acid positions 598 and 599 of a sequence set forth in any one of SEQ ID NOs: 18-25 or 81-122; optionally wherein the amino acid is alanine (A).

55. A nucleic acid molecule encoding an engineered AAV capsid protein of any one of claims 1-50 and 53-54.

56. A host cell comprising the nucleic acid molecule of claim 55.

57. A pharmaceutical composition comprising an engineered AAV capsid protein of any one of claims 1-50 and 53-54, or a central nervous system targeting molecule according to any one of claims 50-52.

58. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an engineered AAV capsid protein according to any one of claims 1-50 and 53-54.1004899-7730-5762Attorney Docket No.: | 91355.12316 P.0288.WO159. A method of delivering a payload of interest to a cell or a tissue, wherein the payload of interest is fused or conjugated to a central nervous system targeting molecule according to any one of claims 50-52.

60. A method of treating a disease in a subject, comprising administering to the subject a composition comprising an engineered AAV capsid protein according to any one of claims 1-50 and 53-54, or a central nervous system targeting molecule according to any one of claims 50-52.

61. Use of an engineered AAV capsid protein of any one of claims 1-50 and 53-54, a central nervous system targeting molecule of any one of claims 50-52, a nucleic acid molecule of claim 55, or a host cell of claim 56 for the manufacture of a medicament in the method of any one of claims 58-60.1014899-7730-5762

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