Fitness maturation of the manipulated AAV capsid STAC-102

Abstract

This application relates to manipulating AAV capsids, such as STAC-102. In some embodiments, the manipulated AAV capsid mediates delivery to the central nervous system.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 466,579, filed on May 15, 2023, with the title "Engineered AAV Capsids for CNS Targeting", the entire content of which is incorporated herein by reference.

[0002] This application relates to engineering AAV capsids.

Background Art

[0003] The clinical translation of gene - based medicines for treating central nervous system (CNS) disorders has been limited by inefficient gene delivery. Administration of AAV into the cerebrospinal fluid enables access to the CNS with relatively low doses and limited exposure to existing anti - AAV antibodies. So far, the functional selection platform SIFTER (Selecting In vivo for Transduction and Expression of RNA) has been applied to identify capsids with improved CNS transduction after cerebrospinal fluid (CSF) administration (see U.S. Patent Application Publication No. 20200370137A1, which is incorporated herein by reference in its entirety). Engineered capsids STAC - 102 and STAC - 103 exhibited 10 - to - 100 - fold enrichment in both the in - vivo distribution of the vector genome and neuronal mRNA expression compared to AAV9 across important CNS regions. The fitness maturation of engineered capsids may further improve their important desirable properties. There is still a need to perform the fitness maturation of capsid STAC - 102 to identify second - generation STAC - 102 variants that mediate further improvement in CNS delivery in cynomolgus monkeys, an important genetic and anatomical model of human CNS delivery.

Summary of the Invention

[0004] STAC-102 is an engineered AAV capsid protein that exhibits enhanced in vivo distribution across the central nervous system (CNS) compared to wild-type AAV9. Modifications to the engineered AAV capsid protein STAC-102 are described herein. In some embodiments, these modifications result in further enhancement of in vivo distribution across the CNS compared to the unmodified STAC-102 capsid protein. In some embodiments, the modification involves substituting specific amino acids within the engineered AAV capsid STAC-102. In some embodiments, substituting specific amino acids within the STAC-102 capsid results in a subsequence within the capsid known as the STAC-102 variant sequence.

[0005] In some embodiments, an engineered AAV capsid protein is provided that contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NOs: 1-6.

[0006] In one embodiment, an engineered AAV capsid protein is provided that includes a diversification region such as in STAC-102 variant sequences A to F. In one embodiment, variant A includes SEQ ID NO: 1, variant B includes SEQ ID NO: 2, variant C includes SEQ ID NO: 3, variant D includes SEQ ID NO: 4, variant E includes SEQ ID NO: 5, or variant F includes SEQ ID NO: 6.

[0007] In one embodiment, an engineered AAV (e.g., STAC-102) sequence is provided which includes a variant diversification region of SEQ ID NO: 1, the diversification region having at position 5 one of amino acids K, M, N, Y, and P, and / or at position 8 one of amino acids M, H, N, S, T, A, I, L, F, Y, and P. [Table 1] 5th place: One of K, M, N, Y, or P 8th place: One of M, H, N, S, T, A, I, L, F, Y, or P

[0008] In one embodiment, an engineered AAV (e.g., STAC-102) sequence is provided which includes a variant diversification region of SEQ ID NO: 2, the diversification region having one of amino acids K, M, N, or L at position 7 and / or one of amino acids S, Q, or M at position 8. [Table 2] 7th place: One of K, M, N, or L 8th place: S, Q, or M

[0009] In one embodiment, an engineered AAV (e.g., STAC-102) sequence is provided which includes a variant diversification region of SEQ ID NO: 3, the diversification region having one of the amino acids K, R, F, Y, or G at position 4 and / or one of the amino acids D, K, N, or S at position 9. [Table 3] 4th place: One of K, R, F, Y, or G 9th place: One of D, K, N, or S

[0010] In one embodiment, an engineered AAV (e.g., STAC-102) sequence is provided which includes a variant diversification region of SEQ ID NO: 4, the diversification region having one of the amino acids K, S, L, Y, G, and P at position 11 and / or one of the amino acids T, D, E, and V at position 12. [Table 4] 11th place: One of K, S, L, Y, G, or P 12th place: One of T, D, E, or V

[0011] In one aspect, an engineered AAV (e.g., STAC-102) sequence containing the variant diversification region of SEQ ID NO: 5 is provided, and the diversification region contains, at position 4, any one of the amino acids T, M, D, E, Q, S, V, G, and / or, at position 7, any one of the amino acids P, H, R, A, V. [Table 5] Position 4: Any of T, M, D, E, Q, S, V, G Position 7: Any of P, H, R, A, V

[0012] In one aspect, an engineered AAV (e.g., STAC-102) sequence containing the variant diversification region of SEQ ID NO: 6 is provided, and the diversification region contains, at position 3, any one of the amino acids L, E, R, V, and / or, at position 4, any one of the amino acids T, H, E, A, P. [Table 6] Position 3: Any of L, E, R, V Position 4: Any of T, H, E, A, P [Brief Description of the Drawings]

[0013] [Figure 1] Figure 1. Strategies for fitness maturation. Each mutation position was altered for all possible amino acids, excluding cysteine and its original amino acid within STAC-102. A total of approximately 9,000 capsid sequences were designed and each was synthesized with three unique barcodes.

[0014] [Figure 2]Figure 2. Library design for functional capsid selection. Each capsid is linked to three unique barcodes using a bioinformatics lookup table. Hundreds to thousands of unique molecular identifiers (UMIs) per barcode are cloned, enabling further elucidation of distinct AAV transduction events. Capsid performance is evaluated based on barcode-encoded mRNA expression from the neuron-specific h synapsin I promoter.

[0015] [Figure 3] Figure 3. Library evaluation of production yield in HEK293. High-performance capsids have the following characteristics in the bubble plot: (1) large log2 fold enrichment (these data are normalized by the abundance of input (y-axis)), (2) small coefficient of variation (x-axis), (3) high percentage of sequenced samples in which the capsid is seen (large bubble size), (4) robust unique molecular identifier recovery (green). The parental capsid STAC-102 and prominent second-generation capsids with improved performance in cynomolgus monkeys are annotated.

[0016] [Figure 4] Figure 4. Transduction of mouse cortical neurons in vitro. Expression of the barcode in neurons was evaluated 5 days after transduction. The annotated second-generation variants exhibit up to an 8-fold increase in mRNA expression compared to STAC-102.

[0017] [Figure 5] Figure 5. Transduction of human iPSC-derived neurons in vitro. Expression of the barcode in neurons was evaluated 9 days after transduction. The annotated second-generation variants show up to a 25-fold increase in mRNA expression compared to STAC-102.

[0018] [Figure 6]Figure 6. Neuronal mRNA expression in whole brain sections. Two coronal sections from the forebrain and hindbrain were analyzed. Annotated second-generation variants show up to a 9-fold increase in mRNA expression compared to STAC-102.

[0019] [Figure 7] Figure 7. Neuronal mRNA expression in cortical brain punches. Brain punches from 23 cortical regions were analyzed and data pooled. Annotated second-generation variants show up to a 10-fold increase in mRNA expression compared to STAC-102.

[0020] [Figure 8] Figure 8. Neuronal mRNA expression in the spinal cord. Ceral, thoracic, and lumbar regions were analyzed. Annotated second-generation variants show up to an 8-fold increase in mRNA expression compared to STAC-102.

[0021] [Figure 9] Figure 9. Overview of the performance of second-generation STAC-102 capsids. Magnification changes are calculated compared to STAC-102. Bubble size is proportional to the magnification change, and UMI retrieval is indicated by color. Six second-generation variants were selected based on their consistently high performance in cynomolgus monkey CNS tissue and production yields similar to or better than the parent capsid STAC-102. Most of these variants similarly outperformed parent STAC-102 in both mouse cortical neurons and human iPSC-derived neurons in vitro. Deep brain structures, where transduction from the ICM pathway is more difficult, indicate a lower number of UMIs retrieved.

[0022] [Figure 10] Figure 10. Heatmap of capsids associated with variant A (sequence number 9). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue.

[0023] [Figure 11]Figure 11. Heatmap of capsids associated with variant B (sequence number 10). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue.

[0024] [Figure 12] Figure 12. Heatmap of capsids associated with variant C (sequence number 11). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue.

[0025] [Figure 13] Figure 13. Heatmap of capsids associated with variant D (sequence number 12). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue.

[0026] [Figure 14] Figure 14. Heatmap of capsids associated with variant E (sequence number 13). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue.

[0027] [Figure 15] Figure 15. Heatmap of capsids associated with variant F (sequence number 14). The log2 magnification changes relate to neuronal mRNA expression in cynomolgus monkey cortical tissue. [Modes for carrying out the invention]

[0028] In one embodiment, fitness maturation of the capsid STAC-102 led to the identification of the engineered STAC-102 variant sequence. In several embodiments, fitness maturation of the capsid STAC-102 led to the identification of the engineered STAC-102 variant sequence shown in Table 1. In several embodiments, the engineered STAC-102 variant sequence mediates enhanced delivery to the central nervous system (CNS).

[0029] In one embodiment, an engineered AAV capsid protein is provided. In this embodiment, the engineered AAV capsid protein comprises at least three consecutive amino acids from any of the diversification region sequences provided by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 as described herein. In some embodiments, the engineered AAV capsid protein comprises modifications of SEQ ID NOs: 1-6, such as one or two substitutions, to any amino acid within any of SEQ ID NOs: 1-6.

[0030] In another embodiment, an AAV capsid protein of the manipulated STAC-102 is provided. In this embodiment, the manipulated STAC-102 capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to, or containing, the sequence outside the diversification region of any one of SEQ ID NOs: 9 (Variant Sequence A), 10 (Variant Sequence B), 11 (Variant Sequence C), 12 (Variant Sequence D), 13 (Variant Sequence E), or 14 (Variant Sequence F) as described herein. In some embodiments, the manipulated STAC-102 capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to, or contains, one of the sequence numbers described herein: SEQ ID NO: 9 (Variant Sequence A), SEQ ID NO: 10 (Variant Sequence B), SEQ ID NO: 11 (Variant Sequence C), SEQ ID NO: 12 (Variant Sequence D), SEQ ID NO: 13 (Variant Sequence E), or SEQ ID NO: 14 (Variant Sequence F). In some embodiments, the manipulated STAC-102 capsid protein includes modifications of SEQ ID NOs: 9-14, such as one or two substitutions of any amino acid in any of SEQ ID NOs: 9-14. Manipulated AAV capsid protein

[0031] In one embodiment, compositions and formulations comprising an engineered AAV capsid protein, as well as methods for preparing and using them, are described herein. In some embodiments, the AAV capsid protein is enhanced in its directivity to cells or tissues, for example, for the delivery of genetic material to specific cells or tissues, such as CNS tissue or CNS cells.

[0032] In some embodiments, engineered AAV capsid proteins have advantages over wild-type AAV capsid proteins. In some embodiments, these advantages include (i) enhanced cell or tissue targeting compared to the natural / wild-type AAV serotype, e.g., enhanced cell or tissue targeting to the central nervous system (CNS) compared to the natural / wild-type AAV serotype, (ii) increased blood-brain barrier penetration after administration to the target, (iii) broader distribution across multiple brain regions, e.g., the prefrontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus and / or hippocampus, and (iv) increased expression of the genetic material in multiple brain regions. In some embodiments, the engineered AAV capsid enhances the delivery of the genetic material to multiple brain regions, e.g., the prefrontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus and / or hippocampus, and (v) delivery of the genetic material of interest to a desired tissue, cell, or organelle.

[0033] In some embodiments, the engineered AAV capsid proteins and genetic material described herein may be delivered to one or more (e.g., 1 to 10) target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins have enhanced directivity to specific target cell types, tissues, or organs. As a non-limiting example, the engineered AAV capsid proteins have enhanced directivity to cells and tissues of the central nervous system or peripheral nervous system (CNS and PNS, respectively). In some embodiments, the engineered AAV capsid proteins are produced recombinantly and are adeno-associated virus (AAV) serotypes such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrh10, AAVrh39, or AAVrh74, or combinations thereof. In some embodiments, the manipulated AAV capsid proteins are produced by recombination and are based on any one or more (e.g., 1 to 15) AAV serotypes known in the art. In some embodiments, a library of AAV variants contains AAV variant capsid proteins derived from 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 at the time the individual serotype libraries are prepared. In some embodiments, the combined library is produced by modifying the nucleic acids encoding the 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.

[0034] Intracellularly manipulated AAV capsid protein

[0035] In some embodiments, the manipulated AAV capsid protein is contained within a cell. In some embodiments, the cell is derived from the central nervous system (CNS). In some embodiments, the cell is derived from the primary nervous system (PNS). In some embodiments, the cell is derived from the brain. In some embodiments, the cell is derived from the spinal cord. In some embodiments, the cell is derived from, among other things, the prefrontal cortex, sensory cortex, motor cortex, cerebellar cortex, cerebral cortex, brainstem, hippocampus, or thalamus.

[0036] Modified AAV capsid protein delivered to target cells

[0037] The engineered AAV capsid protein can be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid protein exhibits enhanced targeting to target cell types, tissues, or organs. As a non-limiting example, the engineered AAV capsid protein may exhibit enhanced targeting to cells and tissues of the central or peripheral nervous system, or to muscle cells and tissues. The engineered AAV capsid protein may, additionally or alternatively, reduce targeting to undesirable target cell types, tissues, or organs. As a non-limiting example, the engineered AAV capsid protein may exhibit enhanced targeting to B cells, hematopoietic cells, leukocytes, platelets, macrophages, megakaryocytes, monocytes, and / or T cells.

[0038] Fitness maturation of modified STAC-102 parent capsid

[0039] In some embodiments, the manipulated AAV capsid protein comprises a modified STAC-102 parental capsid. In embodiments, the manipulated STAC-102 capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to, or containing, the sequence of SEQ ID NO: 8. In some embodiments, the manipulated AAV capsid protein comprises a modified STAC-102 parental capsid comprising one or two amino acid modifications to SEQ ID NO: 8.

[0040] [Table 7]

[0041] In some embodiments, one or two modifications to SEQ ID NO: 8 include one or two substitutions to SEQ ID NO: 8. In some embodiments, one or two substitutions to SEQ ID NO: 8 occur in one or two of the following amino acids 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, and 597 (amino acids shown in bold in SEQ ID NO: 8). In some embodiments, amino acids 585-597 of the STAC-102 capsid include the diversification region of the STAC-102 parent capsid. In some embodiments, the diversification region of the STAC-102 parent capsid includes the following sequence: RGNMTLTRQERQA (SEQ ID NO: 7).

[0042] Amino acid modifications in SEQ ID NO: 8 (STAC-102 parent capsid)

[0043] In some embodiments, the STAC-102 parental capsid is modified, i.e., by substitution, insertion, deletion, or a combination thereof. In some embodiments, the STAC-102 parental capsid is modified at amino acid position 585. In some embodiments, the modification at position 585 includes substitution. In some embodiments, the STAC-102 parental capsid is modified at amino acid position 586. In some embodiments, the modification at position 586 includes substitution. In some embodiments, the STAC-102 parental capsid is modified at amino acid position 587. In some embodiments, the modification at position 587 includes substitution. In some embodiments, the STAC-102 parental capsid is modified at amino acid position 588. In some embodiments, the modification at position 588 includes substitution. In some embodiments, the diversification region of the STAC-102 parental capsid is modified at amino acid position 589. In some embodiments, the modification at position 589 includes substitution. In some embodiments, the diversification region of the STAC-102 parent capsid is modified at amino acid position 590. In some embodiments, the modification at position 590 includes substitution. In some embodiments, the STAC-102 parent capsid is modified at amino acid position 591. In some embodiments, the modification at position 591 includes substitution. In some embodiments, the STAC-102 parent capsid is modified at amino acid position 592. In some embodiments, the modification at position 592 includes substitution. In some embodiments, the STAC-102 parent capsid is modified at amino acid position 593. In some embodiments, the modification at position 593 includes substitution. In some embodiments, the STAC-102 parent capsid is modified at amino acid position 594. In some embodiments, the modification at position 594 includes substitution. In some embodiments, the STAC-102 parent capsid is modified at amino acid position 595. In some embodiments, the modification at position 595 includes substitution. In some embodiments, the diversification region of the STAC-102 parent capsid is modified at amino acid position 596. In some embodiments, the modification at position 596 includes substitution.In some embodiments, the diversification region of the STAC-102 parental capsid is modified at amino acid position 596. In some embodiments, the modification at position 596 includes substitution. In some embodiments, the STAC-102 parental capsid is modified at amino acid position 597. In some embodiments, the modification at position 597 includes substitution. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, and / or 597. In some embodiments, modifications at positions 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, and / or 597 include substitutions, insertions, deletions, or combinations thereof.

[0044] Combinations of positions that can be modified in the diversification region of the STAC-102 parental capsid are disclosed. Note that the amino acids referred to herein are in reference to the STAC-102 parental capsid, not to the manipulated STAC-102 variant sequence. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 589 and 592. In some embodiments, the modifications at positions 589 and 592 include one substitution at each of positions 589 and 592. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 591 and 592. In some embodiments, the modifications at positions 591 and 592 include one substitution at each of positions 591 and 592. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 593. In some embodiments, modifications at positions 588 and 593 include one substitution at each of positions 588 and 593. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 595 and 596. In some embodiments, modifications at positions 595 and 596 include one substitution at each of positions 595 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 591. In some embodiments, modifications at positions 588 and 591 include one substitution at each of positions 588 and 591. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 588. In some embodiments, modifications at positions 587 and 588 include one substitution at each of positions 587 and 588.

[0045] In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 586. In some embodiments, the modifications at positions 585 and 586 include one substitution at each of positions 585 and 586. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 587. In some embodiments, the modifications at positions 585 and 587 include one substitution at each of positions 585 and 587. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 588. In some embodiments, the modifications at positions 585 and 588 include one substitution at each of positions 585 and 588. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 589. In some embodiments, modifications at positions 585 and 589 include one substitution at each of the 585 and 589 positions, and in some embodiments, the STAC-102 parent capsid is modified at amino acid positions 585 and 590. In some embodiments, modifications at positions 585 and 590 include one substitution at each of the 585 and 590 positions. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 585 and 591. In some embodiments, modifications at positions 585 and 591 include one substitution at each of the 585 and 591 positions. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 585 and 592. In some embodiments, modifications at positions 585 and 592 include one substitution at each of the 585 and 592 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 593. In some embodiments, the modifications at positions 585 and 593 include one substitution at each of the 585 and 593 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 594. In some embodiments, the modifications at positions 585 and 594 include one substitution at each of the 585 and 594 positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 595. In some embodiments, the modifications at positions 585 and 595 include one substitution at each of the 585 and 595 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 596. In some embodiments, the modifications at positions 585 and 596 include one substitution at each of the 585 and 596 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 585 and 597. In some embodiments, the modifications at positions 585 and 597 include one substitution at each of the 585 and 597 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 587. In some embodiments, the modifications at positions 586 and 587 include one substitution at each of positions 586 and 587. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 588. In some embodiments, the modifications at positions 586 and 588 include one substitution at each of positions 586 and 588. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 589. In some embodiments, the modifications at positions 586 and 589 include one substitution at each of positions 586 and 589. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 590. In some embodiments, the modifications at positions 586 and 590 include one substitution at each of positions 586 and 590. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 591. In some embodiments, the modifications at positions 586 and 591 include one substitution at each of these positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 592. In some embodiments, the modifications at positions 586 and 592 include one substitution at each of these positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 593. In some embodiments, the modifications at positions 586 and 593 include one substitution at each of positions 586 and 593. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 594. In some embodiments, the modifications at positions 586 and 594 include one substitution at each of positions 586 and 594. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 595. In some embodiments, the modifications at positions 586 and 595 include one substitution at each of positions 586 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 596. In some embodiments, the modifications at positions 586 and 596 include one substitution at each of the positions 586 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 586 and 597. In some embodiments, the modifications at positions 586 and 597 include one substitution at each of the positions 586 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 588. In some embodiments, the modifications at positions 587 and 588 include one substitution at each of the positions 587 and 588. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 589. In some embodiments, the modifications at positions 587 and 589 include one substitution at each of the positions 587 and 589. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 590. In some embodiments, the modifications at positions 587 and 590 include one substitution at each of the 587 and 590 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 591. In some embodiments, the modifications at positions 587 and 591 include one substitution at each of the 587 and 591 positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 592. In some embodiments, the modifications at positions 587 and 592 include one substitution at each of positions 587 and 592. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 593. In some embodiments, the modifications at positions 587 and 593 include one substitution at each of positions 587 and 593. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 594. In some embodiments, the modifications at positions 587 and 594 include one substitution at each of positions 587 and 594. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 587 and 595. In some embodiments, the modifications at positions 587 and 595 include one substitution at each of positions 587 and 595. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 587 and 586. In some embodiments, the modifications at positions 587 and 596 include one substitution at each of positions 587 and 596. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 587 and 597. In some embodiments, the modifications at positions 587 and 597 include one substitution at each of positions 587 and 597. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 588 and 589. In some embodiments, the modifications at positions 588 and 589 include one substitution at each of positions 588 and 589. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 590. In some embodiments, the modifications at positions 588 and 590 include one substitution at each of the 588 and 590 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 591. In some embodiments, the modifications at positions 588 and 591 include one substitution at each of the 588 and 591 positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 592. In some embodiments, the modifications at positions 588 and 592 include one substitution at each of the 588 and 592 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 593. In some embodiments, the modifications at positions 588 and 593 include one substitution at each of the 588 and 593 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 594. In some embodiments, the modifications at positions 588 and 594 include one substitution at each of the 588 and 594 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 588 and 595. In some embodiments, the modifications at positions 588 and 595 include one substitution at each of the positions 588 and 595. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 588 and 596. In some embodiments, the modifications at positions 588 and 596 include one substitution at each of the positions 588 and 596. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 588 and 597. In some embodiments, the modifications at positions 588 and 597 include one substitution at each of the positions 588 and 597. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 589 and 590. In some embodiments, the modifications at positions 589 and 590 include one substitution at each of the positions 589 and 590. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 589 and 591. In some embodiments, the modifications at positions 589 and 591 include one substitution at each of these positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 589 and 592. In some embodiments, the modifications at positions 589 and 592 include one substitution at each of these positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 589 and 593. In some embodiments, the modifications at positions 589 and 593 include one substitution at each of positions 589 and 593. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 589 and 594. In some embodiments, the modifications at positions 589 and 594 include one substitution at each of positions 589 and 594. Includes. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 589 and 595. In some embodiments, the modifications at positions 589 and 595 include one substitution at each of positions 589 and 595. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 589 and 596. In some embodiments, the modifications at positions 589 and 596 include one substitution at each of positions 589 and 596. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 589 and 597. In some embodiments, the modifications at positions 589 and 597 include one substitution at each of positions 589 and 597. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 590 and 591. In some embodiments, the modifications at positions 590 and 591 include one substitution at each of the positions 590 and 591. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 590 and 592. In some embodiments, the modifications at positions 590 and 592 include one substitution at each of the positions 590 and 592. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 590 and 593. In some embodiments, the modifications at positions 590 and 593 include one substitution at each of the positions 590 and 593. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 590 and 594. In some embodiments, the modifications at positions 590 and 594 include one substitution at each of the positions 590 and 594. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 590 and 595. In some embodiments, the modification at positions 590 and 595 includes one substitution at each of positions 590 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 590 and 596. In some embodiments, the modification at positions 590 and 596 includes one substitution at each of positions 590 and 596.In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 590 and 597. In some embodiments, the modifications at positions 590 and 597 include one substitution at each of positions 590 and 597. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 591 and 592. In some embodiments, the modifications at positions 591 and 592 include one substitution at each of positions 591 and 592. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 591 and 593. In some embodiments, the modifications at positions 591 and 593 include one substitution at each of positions 591 and 593. In some embodiments, the STAC-102 parent capsid is modified at amino acid positions 591 and 594. In some embodiments, the modifications at positions 591 and 594 include one substitution at each of positions 591 and 594. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 591 and 595. In some embodiments, the modifications at positions 591 and 595 include one substitution at each of positions 591 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 591 and 596. In some embodiments, the modifications at positions 591 and 596 include one substitution at each of positions 591 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 591 and 597. In some embodiments, the modifications at positions 591 and 597 include one substitution at each of positions 591 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 592 and 593. In some embodiments, the modifications at positions 592 and 593 include one substitution at each of the 592 and 593 positions. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 592 and 594. In some embodiments, the modifications at positions 592 and 594 include one substitution at each of the 592 and 594 positions.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 592 and 595. In some embodiments, the modifications at positions 592 and 595 include one substitution at each of positions 592 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 592 and 596. In some embodiments, the modifications at positions 592 and 596 include one substitution at each of positions 592 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 592 and 597. In some embodiments, the modifications at positions 592 and 597 include one substitution at each of positions 592 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 593 and 594. In some embodiments, the modifications at positions 593 and 594 include one substitution at each of positions 593 and 594. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 593 and 595. In some embodiments, the modifications at positions 593 and 595 include one substitution at each of positions 593 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 593 and 596. In some embodiments, the modifications at positions 593 and 596 include one substitution at each of positions 593 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 593 and 597. In some embodiments, the modifications at positions 593 and 597 include one substitution at each of positions 593 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 594 and 595. In some embodiments, the modifications at positions 594 and 595 include one substitution at each of positions 594 and 595. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 594 and 596. In some embodiments, the modifications at positions 594 and 596 include one substitution at each of positions 594 and 596.In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 594 and 597. In some embodiments, the modifications at positions 594 and 597 include one substitution at each of positions 594 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 595 and 596. In some embodiments, the modifications at positions 595 and 596 include one substitution at each of positions 595 and 596. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 595 and 597. In some embodiments, the modifications at positions 595 and 597 include one substitution at each of positions 595 and 597. In some embodiments, the STAC-102 parental capsid is modified at amino acid positions 596 and 597. In some embodiments, modifications at positions 596 and 597 include one substitution at each of positions 596 and 597. In some embodiments, one or more modifications to sequence number 8 result in one of the sequences shown in sequence numbers 9-14.

[0046] Generation of a manipulated AAV capsid protein library

[0047] In one embodiment, the preparation of a library encoding an engineered AAV capsid protein having desired characteristics compared to the natural / wild-type AAV serotype is disclosed herein. In one embodiment, the preparation of a library encoding an engineered AAV capsid protein having desired characteristics compared to the parental capsid STAC-102 is disclosed herein. Thus, an AAV capsid protein library having desired characteristics compared to the parental capsid STAC-102 is described herein. In some embodiments, the desired characteristic is enhanced cell or tissue tropism compared to the parental capsid STAC-102, for example, enhanced cell or tissue tropism to the central nervous system (CNS) compared to the parental capsid STAC-102. In some embodiments, the desired characteristic is increased permeability across the blood-brain barrier after administration to a subject. In some embodiments, the desired characteristic is broader distribution across multiple brain regions, such as the prefrontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus and / or hippocampus. In some embodiments, the desired feature is an increase in the expression of genetic material in multiple brain regions. In some embodiments, the desired feature is the delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0048] In some embodiments, each member of the library includes one or more of the following: a) a nucleic acid sequence encoding an AAV capsid protein containing an engineered STAC-102 variant sequence, b) a nucleic acid sequence encoding a barcode, c) a nucleic acid sequence encoding a promoter, 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 cells or tissues of interest. In some embodiments, each member of the library also includes a poly(A) sequence.

[0049] In some embodiments, each engineered AAV capsid protein was synthesized as an oligopool. In some embodiments, each member of the library contains one or more nucleic acid sequences encoding an AAV capsid protein, including a) one or more (e.g., 1 to 10) nucleic acid sequences encoding one or more (e.g., 1 to 10) barcodes; b) one or more (e.g., 1 to 10) nucleic acid sequences encoding one or more (e.g., 1 to 10) promoters; c) a nucleic acid sequence encoding a unique molecular identifier (UMI); or a combination thereof. In some embodiments, each member of the library also contains genetic material to be delivered to cells or tissues of interest. In some embodiments, each member of the library also contains a poly(A) sequence. In some embodiments, each of the one or more (e.g., 1 to 10) barcodes is associated with the identity of a single engineered AAV capsid protein. In some embodiments, each barcode is associated with the identity of a single engineered AAV capsid protein. In some embodiments, each barcode is linked to a UMI.

[0050] In some embodiments, nucleic acids containing barcodes are appended to the genome of each AAV capsid protein in the library. In some embodiments, the barcodes are bioinformatically ligated to STAC-102 variant sequences. In some embodiments, the DNA sequences encoding STAC-102 variant sequences are synthesized to further include random or specific barcodes. The barcodes may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides. In some embodiments, each targeting STAC-102 variant sequence is ligated to at least two distinct barcodes. In some embodiments, each barcode is ligated to one or more (e.g., 1 to 10) UMIs.

[0051] In some embodiments, each member of the library contains a nucleic acid comprising two or more barcode sequences (e.g., 1 to 10). In some embodiments, each member of the library contains two or more nucleic acids (e.g., 1 to 10) each comprising a barcode sequence. In some embodiments, each member of the library contains 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 and operably linked to a promoter. In some embodiments, each capsid is linked to one to three unique barcodes using a bioinformatics lookup table. In some embodiments, the capsid is linked to three unique barcodes, and 100 to 1,000, 100 to 2,000, 100 to 5,000, or 100 to 10,000 UMIs are cloned per barcode to enable further elucidation of distinct AAV transduction events. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from neuron-specific promoters. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from neuron-specific h synapsin I promoters.

[0052] In some embodiments, libraries were prepared containing engineered AAV capsid proteins that contained at least one mutation compared to the parent capsid STAC-102. In some embodiments, the engineered AAV capsid proteins contained two mutations compared to the parent capsid STAC-102. In some embodiments, the engineered AAV capsid proteins contained 1 to 10 mutations compared to the parent capsid STAC-102. In some embodiments, each mutation site was modified for all possible amino acids except cysteine ​​and its original amino acid in STAC-102.

[0053] In some embodiments, the library is packaged within HEK293 cells, where helper functions (e.g., E2A, E4, VA, ElA, and ElB) are supplied trans. In some embodiments, the rep functions of the AAV include the rep78, rep68, rep52, and rep40 genes. In some embodiments, the rep genes are supplied trans. In some embodiments, the start codons of the rep78 and / or rep68 genes are changed from ACG to ATG to increase replication of the capsid library construct containing the inverted terminal sequence (ITR), thereby improving the AAV library production yield. In some embodiments, the cap genes are supplied as genetic material to the produced AAV. In some embodiments, the capsid genes are controlled by the p40 promoter so that they are expressed only during production in HEK293 cells in the presence of helper virus functions.

[0054] Performance of manipulated AAV capsid proteins

[0055] In some embodiments, high-performance capsids exhibit the following characteristics in a bubble plot: (1) a large log2 enrichment ratio (these data are normalized by the abundance of the input (y-axis)), (2) a small coefficient of variation (x-axis), (3) a large proportion of sequenced samples in which the capsid is observed (large bubble size), (4) robust unique molecular identifier recovery (green), (5) and combinations thereof.

[0056] In some embodiments, barcode expression in neurons was evaluated 1–5, 1–10, 5–10, or 5 days after transduction. In some embodiments, second-generation variants (i.e., capsids with one mutation or 1–2 or 1–10 mutations compared to the parental capsid STAC-102) exhibited up to a 5–25-fold increase in mRNA expression compared to the parental capsid STAC-102. In some embodiments, coronal sections from the forebrain and / or hindbrain were analyzed for mRNA expression of the modified STAC-102 parental capsid compared to the parental capsid STAC-102. In some embodiments, second-generation variants (i.e., capsids with one mutation or 1–2 or 1–10 mutations compared to the parental capsid STAC-102) exhibited a 2–15-fold increase in mRNA expression compared to the parental capsid STAC-102. In some embodiments, brain punches from cortical regions were pooled, and mRNA expression of second-generation variants relative to parental STAC-102 was evaluated. Annotated second-generation variants exhibited up to a 5- to 20-fold increase in mRNA expression compared to parental STAC-102. In some embodiments, mRNA expression of second-generation variants relative to parental STAC-102 was analyzed in the cervical, thoracic, and lumbar regions. Second-generation variants exhibited up to a 5- to 15-fold increase in mRNA expression compared to STAC-102.

[0057] Adeno-associated virus (AAV) AAV can infect a wide range of cells, including quiescent and dividing cells. In some embodiments, AAV may be modified to contain components necessary for the assembly of functional recombinant viruses or viral particles. In some embodiments, AAV is engineered to target specific tissues and / or cells. In some embodiments, AAV is engineered to deliver specific genetic material to tissues and / or cells.

[0058] Modified AAV cellotype

[0059] In some embodiments, the AAV is based on any natural or recombinant AAV serotype. Different AAV serotypes have different characteristics, such as different packaging, directivity, and transduction profiles. In some embodiments, the engineered AAV capsid protein is based on a wild-type AAV serotype. In some embodiments, the AAV serotype includes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the AAV serotype includes less well-characterized AAV serotypes such as AAV3, AAV4, AAV7, AAV11, AAVrh10, AAVrh39, or AAVrh74. In some embodiments, the engineered AAV capsid protein is derived from multiple wild-type AAV serotypes, e.g., 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 at the time the individual serotype libraries are constructed. In some embodiments, the combined library is produced by modifying the 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.

[0060] In some embodiments, different AAV serotypes differ in their ability to guide or modulate AAV particles to specific cells or tissues. In some embodiments, AAV serotypes may be modified to increase the directivity of AAV particles to cells or tissues of the central nervous system (CNS). In some embodiments, AAV serotypes may be modified to increase the directivity of AAV particles to cells or tissues of the peripheral nervous system (PNS).

[0061] In some embodiments, modified AAV serotypes have desirable characteristics compared to natural / wild-type AAV serotypes. In some embodiments, modified AAV serotypes enable increased blood-brain barrier penetration after administration to a subject. In some embodiments, modified AAV serotypes cause increased in vivo distribution to brain regions. In some embodiments, brain regions include the prefrontal cortex, sensory cortex, motor cortex, cerebellar cortex, hippocampus, thalamus, or putamen. In some embodiments, the brain includes any brain region known in the art. In some embodiments, modified AAV serotypes cause increased in vivo distribution to multiple brain regions, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 brain regions. In some embodiments, modified AAV serotypes cause increased in vivo distribution to 1 to 10 brain regions. In some embodiments, modified AAV serotypes are useful for increasing gene expression in multiple brain regions. In some embodiments, the modified AAV serotype is used to deliver the genetic material of interest to a desired tissue, cell, or organelle.

[0062] In some embodiments, the modified AAV serotype causes an increase in intracellular distribution to spinal cord regions. In some embodiments, the spinal cord regions include any of the thoracic, lumbar, and / or cervical spinal cord regions. In some embodiments, the spinal cord regions include any region of the spinal cord known in the art. In some embodiments, the modified AAV serotype increased intracellular distribution to multiple spinal cord regions, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 spinal cord regions. In some embodiments, the modified AAV serotype increased intracellular distribution to 1 through 10 spinal cord regions.

[0063] In some embodiments, the modified AAV serotype is modified with a peptide containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of any peptide (i.e., variant sequence) described in any of SEQ ID NOs: 1-6. In some embodiments, the modified AAV serotype is modified with a peptide (i.e., variant sequence) described in any of SEQ ID NOs: 1-6. In some embodiments, the modified AAV serotype is modified with any of the peptides described herein.

[0064] AAV structure

[0065] In some embodiments, the AAV genome comprises single-stranded DNA (ssDNA) molecules approximately 4.5 kb to 5.0 kb in length, for example, approximately 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, or 5.0 kb. In some embodiments, the AAV genome contains inverted end sequences (ITRs) adjacent to the 5' and 3' ends of the AAV molecule. In some embodiments, the ITRs contain the replication origin of the viral genome. In some embodiments, the length of the ITRs is approximately 145 bp, for example, approximately 130 bp to 160 bp.

[0066] In some embodiments, the AAV genome includes at least three genes. In some embodiments, the at least three genes include rep, cap, and X. In some embodiments, the AAV genome nucleotides include nucleotide sequences encoding four non-structural Rep proteins (Rep78, Rep68, Rep52, and Rep40 encoded by the Rep gene). In some embodiments, the AAV viral genome includes nucleotide sequences encoding three capsids, or structural proteins (i.e., VPl, VP2, and VP3 encoded by the capsid gene or cap gene). In some embodiments, the rep protein is used for replication and packaging. In some embodiments, the capsid proteins are assembled to create the AAV protein shell.

[0067] AAV particles

[0068] In some embodiments, the engineered AAV capsid protein is packaged within the AAV particle. In some embodiments, AAV particles with enhanced directivity to target tissues (e.g., CNS and PNS) are provided. In some embodiments, the AAV particle contains an engineered STAC-102 variant sequence that alters directivity to specific cell types, tissues, organs, or organisms in vivo, ex vivo, or in vitro. In some embodiments, the AAV particle can penetrate the blood-brain barrier.

[0069] AAV particle delivery

[0070] AAV particles can be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, AAV particles exhibit enhanced directivity to target cell types, tissues, or organs. As a non-limiting example, AAV particles may exhibit enhanced directivity to cells and tissues of the central nervous system or peripheral nervous system (CNS and PNS, respectively), or to muscle cells and tissues. Additionally or alternatively, AAV particles may exhibit reduced directivity to undesirable target cell types, tissues, or organs.

[0071] In some embodiments, AAV particles are used to deliver the viral genome to tissues or cells, such as cells or tissues of the central nervous system (CNS) or primary nervous system (PNS).

[0072] The delivered viral genome may include genetic material of interest, such as, for example, genetic material encoding an antibody or enzyme, or regulatory RNA. In some embodiments, the viral genome includes at least one ITR sequence. In some embodiments, the viral genome includes two ITR sequences. In some embodiments, the ITR sequences are adjacent to the genetic material of interest. In some embodiments, the ITR sequences are complementary to each other. In some embodiments, the ITR regions are derived from the same serotype as the capsid protein. The ITR region may be 100 to 150 nucleotides long.

[0073] In some embodiments, AVV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replication. In some embodiments, the viral genome contains components necessary for the assembly of functional recombinant viruses or viral particles, which are loaded or manipulated to target specific tissues and express or deliver genetic material of interest to those tissues.

[0074] AAV Diversification Region Array In some embodiments, sequences of AAV diversification regions are disclosed herein. In some embodiments, the sequences may function as general CNS targeting molecules.

[0075] In some embodiments, CNS targeting molecules are fused to or conjugated to small molecules, antibodies, scFVs, ASOs (antisense oligonucleotides), siRNAs, lipids, polymers, or recombinant proteins. In some embodiments, any of SEQ ID NOs: 1-6 are fused to or conjugated to small molecules, antibodies, scFVs, ASOs (antisense oligonucleotides), siRNAs, lipids, polymers, or recombinant proteins. In some embodiments, CNS targeting molecules can be used to enable small molecules, antibodies, scFVs, ASOs (antisense oligonucleotides), siRNAs, lipids, polymers, or recombinant proteins to cross the blood-brain barrier.

[0076] In some embodiments, the sequence is part of the engineered AAV capsid protein. In some embodiments, the engineered AAV capsid protein is any engineered AAV capsid protein disclosed herein.

[0077] In some embodiments, the sequence may increase the directivity of the AAV capsid protein to CNS cells or tissues. In some embodiments, CNS cells are supporting cells of the brain such as neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes), and / or immune cells (e.g., T cells). In some embodiments, CNS tissue is the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, or entorhinal cortex.

[0078] In some embodiments, the sequence increases the targeting of the AAV capsid protein to cells, regions, or tissues of the PNS. In some embodiments, the cells or tissues of the PNS are dorsal root ganglia (DRGs).

[0079] In some embodiments, the sequence includes at least four consecutive amino acids of the sequence shown in SEQ ID NOs: 1 to 6.

[0080] In some embodiments, the sequence includes a variant of the amino acid sequence described in SEQ ID NOs: 1-6. In some embodiments, the variant refers to one or more substitutions, deletions, or additions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In some embodiments, the variant includes one, two, three, or four substitutions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In some embodiments, the variant includes one, two, three, or four deletions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In some embodiments, the variant includes one, two, three, or four additions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In some embodiments, the variant includes any combination of the above substitutions, deletions, or additions. In some embodiments, the variant amino acid sequence includes one or more conservative substitutions of amino acids in the amino acid sequences described in SEQ ID NOs: 1-6.

[0081] In embodiments, a variant refers to a variant in a nucleotide sequence that encodes any of the amino acid sequences described in SEQ ID NOs: 1-6. In embodiments, a variant in a nucleotide sequence consequently encodes one or more substitutions, deletions, or additions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In embodiments, a variant in a nucleotide sequence encodes one, two, three, or four substitutions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In embodiments, a variant in a nucleotide sequence encodes one, two, three, or four deletions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In embodiments, a variant in a nucleotide sequence encodes one, two, three, or four additions to any of the amino acids in any of the amino acid sequences described in SEQ ID NOs: 1-6. In embodiments, a variant in a nucleotide sequence encodes any combination of the above substitutions, deletions, or additions.

[0082] In some embodiments, the capsid protein comprises one of the serotypes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. In embodiments, the capsid protein comprises serotype AAV2 or AAV9.

[0083] In some embodiments, the capsid protein comprises a variant of the STAC-102 parent capsid. In some embodiments, the capsid protein comprises a variant of the sequence described in Sequence ID No. 8. In embodiments, the variant refers to one or more substitutions, deletions, or additions to any of the amino acids in the amino acid sequence described in Sequence ID No. 8. In 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 in the amino acid sequence of Sequence ID No. 8. In embodiments, the variant includes 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 in the amino acid sequence described in Sequence ID No. 8. In embodiments, the variant includes 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 in the amino acid sequence described in Sequence ID No. 8.

[0084] In some embodiments, a variant refers to a variant in a nucleotide sequence that encodes the amino acid sequence described in Sequence ID No. 8. In some embodiments, the variant in a nucleotide sequence consequently encodes one or more substitutions, deletions, or additions to any of the amino acids in the amino acid sequence described in Sequence ID No. 8. In some embodiments, the variant in a 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 in the amino acid sequence of Sequence ID No. 8. In the embodiment, 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 deletions of any amino acid in the amino acid sequence of SEQ ID NO: 8. 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 additions to any of the amino acids in the amino acid sequence of SEQ ID NO: 8.

[0085] Administration to subjects with AAV containing diversified region sequences

[0086] In some embodiments, capsid proteins containing the manipulated diversification region sequence described herein are expressible at a higher level in neurons compared to capsid proteins lacking the manipulated diversification region sequence when administered to a subject. In some embodiments, the capsid protein administered to the subject contains the manipulated STAC-102 sequence described herein. In some embodiments, capsid proteins containing the manipulated variant sequence (e.g., variant STAC-102) are expressible at least twice as highly in neurons compared to capsid proteins lacking the manipulated variant sequence (e.g., variant STAC-102). In some embodiments, capsid proteins containing the manipulated variant sequence (e.g., variant STAC-102) are expressible at a 5 to 10 times higher level in neurons compared to capsid proteins lacking the manipulated variant sequence (e.g., variant STAC-102).

[0087] Length and structure of the diversification region (modified sequence within the manipulated AAV)

[0088] The divergence region within the manipulated AAV (e.g., STAC-102) variant sequence may vary in length. In some embodiments, the divergence region is about 3 to about 20 amino acids long. In some embodiments, the divergence region is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids long. In some embodiments, the divergence region is about 8 to 10, 10 to 12, 12 to 14, 14 to 16, or 16 to 18 amino acids long. In some embodiments, the divergence region is about 13 amino acids long.

[0089] In some embodiments, the diversification region may comprise one amino acid sequence from SEQ ID NOs: 1 to 6, as shown in Table 1. In some embodiments, the diversification region may comprise four consecutive amino acid sequences from SEQ ID NOs: 1 to 6, as shown in Table 1. In some embodiments, the peptide is isolated and, for example, recombinant.

[0090] In some embodiments, the diversification region includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids of any sequence described in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 3 consecutive amino acids of any sequence shown in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 4 consecutive amino acids of any sequence shown in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 5 consecutive amino acids of any sequence described in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 6 consecutive amino acids of any sequence described in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 7 consecutive amino acids of any sequence described in SEQ ID NOs: 1-6. In some embodiments, the diversification region includes at least 8 consecutive amino acids of any sequence described in SEQ ID NOs: 1-6.

[0091] In some embodiments, the diversification region contains 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%, 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 of SEQ ID NOs: 1 to 6.

[0092] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the diversification region includes an amino acid sequence that has at least one, two, or three modifications, such as substitutions (e.g., conservative substitutions), insertions, or deletions, compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence includes at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 1.

[0093] In some embodiments, SEQ ID NO: 1 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position N5 of SEQ ID NO: 1 is modified. In some embodiments, the modification at position N5 includes substitution. In some embodiments, position N8 of SEQ ID NO: 1 is modified. In some embodiments, the modification at position N8 includes substitution. In some embodiments, both positions N5 and N8 are modified in SEQ ID NO: 1. In some embodiments, the modifications at positions N5 and N8 include substitution at both positions N5 and N8.

[0094] In some embodiments, position N5 of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 contains amino acid K. In some embodiments, position N5 contains amino acid M. In some embodiments, position N5 contains amino acid N. In some embodiments, position N5 contains amino acid Y. In some embodiments, position N5 contains amino acid P. In some embodiments, position N8 contains amino acid M. In some embodiments, position N8 contains amino acid H. In some embodiments, position N8 contains amino acid N. In some embodiments, position N8 contains amino acid S. In some embodiments, position N8 contains amino acid T. In some embodiments, position N8 contains amino acid A. In some embodiments, position N8 contains amino acid I. In some embodiments, position N8 contains amino acid L. In some embodiments, position N8 contains amino acid F. In some embodiments, position N8 contains amino acid Y. In some embodiments, position N8 contains amino acid P. In some embodiments, positions N5 and N8 contain any combination as described above.

[0095] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the diversification region includes an amino acid sequence that, compared to the amino acid sequence of SEQ ID NO: 2, contains at least one, two, or three amino acids, such as substitutions (e.g., conservative substitutions), insertions, or deletions. In some embodiments, the amino acid sequence contains at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 2.

[0096] In some embodiments, SEQ ID NO: 2 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, position N7 of SEQ ID NO: 2 is modified. In some embodiments, the modification at position N7 includes substitution. In some embodiments, position N8 of SEQ ID NO: 2 is modified. In some embodiments, the modification at position N8 includes substitution. In some embodiments, both positions N7 and N8 are modified in SEQ ID NO: 2. In some embodiments, the modifications at positions N7 and N8 include substitution at both positions N7 and N8.

[0097] In some embodiments, the N7 position of SEQ ID NO: 2 or a variant of SEQ ID NO: 2 contains amino acid K. In some embodiments, the N7 position contains amino acid M. In some embodiments, the N7 position contains amino acid N. In some embodiments, the N7 position contains amino acid L. In some embodiments, the N8 position contains amino acid S. In some embodiments, the N8 position contains amino acid Q. In some embodiments, the N8 position contains amino acid M. In some embodiments, the N7 and N8 positions contain any of the aforementioned combinations.

[0098] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the diversification region includes an amino acid sequence that has at least one, two, or three modifications, such as substitutions (e.g., conservative substitutions), insertions, or deletions, compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence includes at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 3.

[0099] In some embodiments, SEQ ID NO: 3 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, the N4 position of SEQ ID NO: 3 is modified. In some embodiments, the modification at the N4 position includes substitution. In some embodiments, the N9 position of SEQ ID NO: 3 is modified. In some embodiments, the modification at the N9 position includes substitution. In some embodiments, both the N4 and N9 positions are modified in SEQ ID NO: 3. In some embodiments, the modifications at the N4 and N9 positions include substitution at both the N4 and N9 positions.

[0100] In some embodiments, the N4 position of SEQ ID NO: 3 or a variant of SEQ ID NO: 3 contains amino acid K. In some embodiments, the N4 position contains amino acid R. In some embodiments, the N4 position contains amino acid F. In some embodiments, the N4 position contains amino acid Y. In some embodiments, the N4 position contains amino acid G. In some embodiments, the N9 position contains amino acid D. In some embodiments, the N9 position contains amino acid K. In some embodiments, the N9 position contains amino acid N. In some embodiments, the N9 position contains amino acid S. In some embodiments, the N4 and N9 positions contain any combination described above.

[0101] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the diversification region includes an amino acid sequence that has at least one, two, or three modifications, such as substitutions (e.g., conservative substitutions), insertions, or deletions, compared to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amino acid sequence includes at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 4.

[0102] In some embodiments, SEQ ID NO: 4 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, the N11 position of SEQ ID NO: 4 is modified. In some embodiments, the modification at the N11 position includes substitution. In some embodiments, the N12 position of SEQ ID NO: 4 is modified. In some embodiments, the modification at the N12 position includes substitution. In some embodiments, both the N11 and N12 positions are modified in SEQ ID NO: 4. In some embodiments, the modifications at the N11 and N12 positions include substitution at both the N11 and N12 positions.

[0103] In some embodiments, the N11 position of SEQ ID NO: 4 or a variant of SEQ ID NO: 4 contains amino acid K. In some embodiments, the N11 position contains amino acid S. In some embodiments, the N11 position contains amino acid L. In some embodiments, the N11 position contains amino acid Y. In some embodiments, the N11 position contains amino acid G. In some embodiments, the N11 position contains amino acid P. In some embodiments, the N12 position contains amino acid T. In some embodiments, the N12 position contains amino acid D. In some embodiments, the N12 position contains amino acid E. In some embodiments, the N12 position contains amino acid V. In some embodiments, the N11 and N12 positions contain any combination as described above.

[0104] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence includes at least one, two, or three modifications, such as substitutions (e.g., conservative substitutions), insertions, or deletions, compared to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence includes at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 5.

[0105] In some embodiments, SEQ ID NO: 5 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, the N4 position of SEQ ID NO: 5 is modified. In some embodiments, the modification at the N4 position includes substitution. In some embodiments, the N7 position of SEQ ID NO: 5 is modified. In some embodiments, the modification at the N7 position includes substitution. In some embodiments, both the N4 and N7 positions are modified in SEQ ID NO: 5. In some embodiments, the modifications at the N4 and N7 positions include substitution at both the N4 and N7 positions.

[0106] In some embodiments, the N4 position of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 contains amino acid T. In some embodiments, the N4 position contains amino acid M. In some embodiments, the N4 position contains amino acid D. In some embodiments, the N4 position contains amino acid E. In some embodiments, the N4 position contains amino acid E. In some embodiments, the N4 position contains amino acid Q. In some embodiments, the N4 position contains amino acid S. In some embodiments, the N4 position contains amino acid V. In some embodiments, the N4 position contains amino acid G. In some embodiments, the N7 position contains amino acid P. In some embodiments, the N7 position contains amino acid H. In some embodiments, the N7 position contains amino acid R. In some embodiments, the N7 position contains amino acid A. In some embodiments, the N7 position contains amino acid V. In some embodiments, the N14 and N17 positions contain any combination as described above.

[0107] In some embodiments, the diversification region includes the amino acid sequence of SEQ ID NO: 6 and includes an amino acid sequence that, compared to the amino acid sequence of SEQ ID NO: 6, includes at least one, two, or three modifications, such as substitutions (e.g., conservative substitutions), insertions, or deletions. In some embodiments, the amino acid sequence includes at least one, two, or three different amino acids compared to the amino acid sequence of SEQ ID NO: 6.

[0108] In some embodiments, SEQ ID NO: 6 contains 13 amino acids, each amino acid containing consecutive amino acid positions N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, and N13. In some embodiments, the N3 position of SEQ ID NO: 6 is modified. In some embodiments, the modification at the N3 position includes substitution. In some embodiments, the N4 position of SEQ ID NO: 6 is modified. In some embodiments, the modification at the N4 position includes substitution. In some embodiments, both the N3 and N4 positions are modified in SEQ ID NO: 6. In some embodiments, the modifications at the N3 and N4 positions include substitution at both the N3 and N4 positions.

[0109] In some embodiments, the N3 position of SEQ ID NO: 6 or a variant of SEQ ID NO: 6 contains amino acid L. In some embodiments, the N3 position contains amino acid E. In some embodiments, the N3 position contains amino acid R. In some embodiments, the N3 position contains amino acid V. In some embodiments, the N4 position contains amino acid T. In some embodiments, the N4 position contains amino acid H. In some embodiments, the N4 position contains amino acid E. In some embodiments, the N4 position contains amino acid A. In some embodiments, the N4 position contains amino acid P. In some embodiments, the N3 and N4 positions contain any combination as described above.

[0110] Functional properties of manipulated variant AAV sequences In some embodiments, an engineered variant AAV sequence (e.g., variant STAC-102) containing a diversification region sequence described herein is used to enhance or improve the transduction of target cells or tissues (e.g., cells or tissues of the central nervous system (CNS) or peripheral nervous system (PNS)). In some embodiments, the diversification region is used to facilitate the crossing of the blood-brain barrier of the AAV capsid protein after administration to the subject. In some embodiments, the diversification region is used to enhance or improve the distribution of genetic material across multiple brain regions, such as the prefrontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus. In some embodiments, the diversification region is used to enhance or improve genetic material expression in multiple brain regions. In some embodiments, the diversification region is used to enhance or improve the delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0111] In some embodiments, the diversification region is contained within the manipulated AAV capsid protein, increasing the directivity of the AAV capsid protein to cells, regions, or tissues 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 tissues include, but are not limited to, the cortex (e.g., prefrontal cortex, parietal cortex, occipital cortex, temporal cortex), thalamus, hypothalamus, striatum, hippocampus, entorhinal cortex, and basal ganglia.

[0112] In some embodiments, AAV capsid proteins containing the diversification region described herein can increase AAV capsid protein expression by at least 0.08 times in specific cells, regions, or tissues compared to AAV capsid proteins lacking the targeting diversification region. In some embodiments, AAV capsid proteins containing the diversification region can increase expression by 0.08 to 10 times, for example, 0.08 to 2 times, 2 to 3 times, 4 to 5 times, 5 to 6 times, 6 to 7 times, 7 to 8 times, 8 to 9 times, or 9 or 10 times compared to AAV capsid proteins lacking the diversification region. In some embodiments, AAV capsid proteins containing the diversification region can increase AAV capsid protein expression by more than 10 times in specific cells, regions, or tissues compared to AAV capsid proteins lacking the diversification region.

[0113] genetic material In some embodiments, the manipulated AAV capsid protein described herein encapsulates genetic material to be delivered to cells of interest. Thus, in embodiments, the manipulated AAV capsid protein described herein enables the delivery of genetic material to cells of interest. In embodiments, the genetic material encodes zinc finger protein, TALE protein, and / or CRISPR protein, or fragments thereof. In embodiments, the genetic material encodes one or more antibodies or antibody fragments. In some embodiments, the genetic material encodes one or more regulatory RNAs, such as RNAi reagents or microRNAs.

[0114] In some embodiments, the genetic material may include sequences that are coding sequences. In some embodiments, the genetic material may include sequences that are non-coding sequences. In some embodiments, the genetic material may include sequences that are both coding and non-coding sequences. In some embodiments, the expression of the genetic material can be regulated. In some embodiments, the genetic material includes regulating elements.

[0115] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, mRNA is codon-optimized.

[0116] In some embodiments, the genetic material encodes a gene therapy product. The gene therapy product may include a peptide, polypeptide, or RNA molecule that, when expressed, exerts a desired therapeutic effect. In some embodiments, the therapeutic effect is to treat any one or more diseases or disorders described herein.

[0117] In some embodiments, the promoter is operablely linked to the genetic material delivered to the cell. In some embodiments, the promoter includes a tissue and / or cell-specific promoter. In some embodiments, one or more promoters include a ubiquitous promoter. Examples of ubiquitous promoters include, among others, cytomegalovirus (CMV), chicken β-actin (CBA), ubiquitin C (UBC), and elongation factor 1α-subunit (EF1-α). In some embodiments, the promoter includes a cell-type and / or tissue-specific promoter. Exemplary cell-type and / or tissue-specific promoters include the human synapsin promoter (hSynl), which is expressed only in neurons, or the transthyretin promoter (TTR), which is expressed in hepatocytes. Other non-limiting cell type and / or tissue-specific promoters for use in the methods and compositions of the present invention include cytokeratin 18 and 19 (epithelial cell-specific), and other cell-specific promoters include the GFAP promoter (astrocytocyte), TBG promoter (liver), CAMK promoter (skeletal muscle), and MYH6 promoter (cardiomyocyte). 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 nervous system, parasympathetic nervous system, astrocytocytes, microglia, oligodendrocytes, and / or Schwann cells.

[0118] In some embodiments, the promoter is a natural promoter. In some embodiments, the promoter is synthetic. In some embodiments, the promoter is derived from a mammal, human, virus, or plant. In some embodiments, the promoter is cleaved. In some embodiments, the promoter is mutated.

[0119] Active drug In some embodiments, the manipulated variant AAV sequence described herein (e.g., variant STAC-102) is fused to or bound to an active agent. In some embodiments, the sequence is fused to or bound to the active agent by 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., the Fc region). In some embodiments, the sequence is fused to the Fc region of the 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 reagent (e.g., siRNA, shRNA, lncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the sequence is fused to or bound directly on at least one strand of the RNAi. In some embodiments, the sequence is fused to or bound to at least one strand of the RNAi using a linker. In some embodiments, the sequence is fused to or bound to the sense strand of the RNAi. In some embodiments, the sequence is fused to or bound to the antisense strand of the RNAi.

[0120] In some embodiments, the active agent includes a diagnostic agent. In some embodiments, the diagnostic agent includes a detectable portion such as a fluorophore. In some embodiments, the active agent is a small molecule.

[0121] Pharmaceutical compositions and dosage forms The compositions of this specification (e.g., engineered STAC-102 variant sequences, AAV particles, and engineered AAV capsid proteins) may be incorporated into pharmaceutical compositions. In some embodiments, the pharmaceutical composition may contain one or more excipients or diluents to (1) increase stability, (2) increase cell transfection or transduction, (3) allow sustained or delayed release of genetic material, (4) alter in vivo distribution (e.g., to target the composition to a particular tissue or cell type), (5) increase translation of the encoded protein, (6) alter the release profile of the encoded protein, and / or (7) enable regulated expression of genetic material.

[0122] The pharmaceutical compositions described herein may be administered regularly, such as once or twice a day, or at any other suitable interval. For example, the pharmaceutical compositions may be administered to the target subject as needed, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, once every nine months, once a year, once every eighteen months, once every two years, once every thirty months, or once every three years.

[0123] In some embodiments, the compositions described herein (e.g., manipulated STAC-102 variant sequences, manipulated AAV capsid proteins, and modified STAC-102 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 use. Each dosage form may contain various solubilizers, disintegrants, surfactants, fillers, thickeners, binders, wetting agents, or other pharmaceutically acceptable excipients. The compositions described herein may also be formulated for injection, infusion, infusion, or intradermal exposure. For example, an injectable formulation may include a composition disclosed in an aqueous or non-aqueous solution at an appropriate pH and tonicity. The composition may include a liquid dosage form for oral administration, such as a suspension, emulsion, or syrup.

[0124] In some embodiments, the pharmaceutical compositions described herein function to increase stability, increase transduction or transfection efficiency, affect in vivo distribution, increase protein expression, and / or alter the release profile.

[0125] Gene editing systems In some embodiments, the genetic material of interest includes a gene editing system or a portion of a gene editing system. In some embodiments, the gene editing system can induce single-strand or double-strand breaks in a nucleic acid sequence. In some embodiments, the gene editing system can insert, substitute, or delete bases or sequences of bases in a nucleic acid sequence. 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.

[0126] Intracellularly manipulated AAV capsid protein In some embodiments, the manipulated AAV capsid protein is contained within a cell. In some embodiments, the cell is derived from the central nervous system (CNS). In some embodiments, the cell is derived from the primary nervous system (PNS). In some embodiments, the cell is derived from the brain. In some embodiments, the cell is derived from the spinal cord. In some embodiments, the cell is derived from, among other things, the prefrontal cortex, sensory cortex, motor cortex, cerebellar cortex, cerebral cortex, brainstem, hippocampus, or thalamus.

[0127] Modified AAV capsid protein delivered to target cells The engineered AAV capsid protein can be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid protein exhibits enhanced targeting to target cell types, tissues, or organs. As a non-limiting example, the engineered AAV capsid protein may exhibit enhanced targeting to cells and tissues of the central or peripheral nervous system, or to muscle cells and tissues. The engineered AAV capsid protein may, additionally or alternatively, reduce targeting to undesirable target cell types, tissues, or organs. As a non-limiting example, the engineered AAV capsid protein may exhibit enhanced targeting to B cells, hematopoietic cells, leukocytes, platelets, macrophages, megakaryocytes, monocytes, and / or T cells.

[0128] Method for detecting manipulated AAV capsid proteins In some embodiments, a method is provided for identifying engineered AAV capsid proteins having desired characteristics compared to natural / wild-type AAV serotypes, the method comprising: (i) contacting a cell, cell line, or tissue in vivo or in vitro with one of a library of engineered AAV capsid proteins; (ii) enabling the engineered AAV capsid proteins in the library to transduce the cell, cell line, or tissue; (iii) recovering the AAV variant from the cell, cell line, or tissue; and (iv) identifying an engineered AAV capsid protein having desired characteristics.

[0129] In another embodiment, the desired evolution of the engineered AAV capsid protein and a method for identifying engineered AAV capsid proteins with desired characteristics compared to the natural / wild-type AAV serotype are disclosed herein. In some embodiments, steps for the desired evolution of an engineered AAV capsid protein, such as identifying an engineered AAV capsid protein having desired features compared to a natural / wild-type AAV serotype, include: (i) modifying a natural / wild-type AAV serotype to produce a variant capsid; (ii) packaging the variant AAV in a producing cell that supplies the adenovirus helper and the rep function of AAV in trans; (iii) purifying a pool of viral capsid libraries; (iv) administering the pool in vitro or in vivo; (v) recovering the engineered AAV capsid protein from a target tissue or cell line; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequence; (vii) repeated rounds of selection in vitro or in vivo, where the variant is isolated from a target tissue or cell line; and (viii) a complete evaluation of the enriched variant. In some embodiments, the desired feature includes enhanced tissue-directivity compared to the natural / wild-type AAV serotype. In some embodiments, the desired feature includes enhanced tissue-directivity to peripheral nervous system tissues compared to the natural / wild-type AAV serotype. In some embodiments, the desired feature includes enhanced tissue-directivity to central nervous system tissues compared to the natural / wild-type AAV serotype. In some embodiments, modification of the AAV capsid protein results in an AAV capsid protein containing one of the STAC-102 sequences described herein.

[0130] Delivery and treatment methods In some embodiments, methods are provided for introducing the compositions described herein (e.g., engineered STAC-102 variant sequences, AAV particles, and engineered AAV capsid proteins) into cells and / or tissues. In some embodiments, the method includes introducing one of the compositions described herein into cells and / or tissues in an amount sufficient to modulate, for example, increase, the production of target mRNA and / or proteins in the cells and / or tissues.

[0131] In some embodiments, the compositions described herein are delivered via a local delivery route. In some embodiments, the local delivery route includes, among other things, one or more of intramuscular, intraparenchymal, and intracerebral delivery. In some embodiments, the compositions described herein are administered via a local delivery route by bolus injection.

[0132] In some embodiments, the compositions described herein are administered systemically. In some embodiments, systemic administration includes intravenous administration. In some embodiments, intravenous administration includes subcutaneous administration. In some embodiments, systemic administration includes intraventricular administration.

[0133] In some embodiments, the compositions described herein are administered to the central nervous system via intracerebroventricular and / or intravenous administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, systemic administration is intravenous (IV) injection. In some embodiments, the compositions described herein are administered to the central nervous system via intracerebroventricular administration.

[0134] In some embodiments, the composition can be delivered to target cells or tissues, including but not limited to the CNS, heart, lungs, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testis, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cells or tissues include but are not limited to the CNS, heart, lungs, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testis, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cells or tissues are CNS cells or tissues. In some embodiments, the target cells or tissues are liver cells or tissues.

[0135] In some embodiments, target cells include, but are not limited to, neurons, glial cells, astrocytes, oligodendrocytes, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat-storing cells, Kupffer cells, hepatic endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T cells, B cells, hematopoietic stem cells, and embryonic stem cells.

[0136] In some embodiments, the compositions described herein are delivered to the central nervous system via the cerebrospinal fluid pathway. In some embodiments, the 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.

[0137] In some embodiments, the compositions described herein are administered as part of a composition that enables sustained release. In some embodiments, the compositions include formulations comprising a depot.

[0138] This specification discloses therapeutic methods using any of the compositions described herein (engineered STAC-102 variant sequences, AAV particles, and engineered AAV capsid proteins). In embodiments, the disclosed compositions may be used to treat, among other things, one or more of the following: muscle disorders or neuromuscular disorders, neurooncological disorders, neurological diseases / disorders, and neurodegenerative disorders. In embodiments, the disclosed compositions may be used to treat one or more of the following: Alzheimer's disease, Huntington's disease, autism, Parkinson's disease, spinal muscular atrophy, and Friedreich's ataxia. In embodiments, the disclosed compositions may be used for therapeutic purposes via any of the delivery methods described herein.

[0139] In some embodiments, methods are disclosed for treating or improving diseases or conditions related to abnormal genes and / or proteins in subjects requiring treatment, the methods comprising administering any effective amount of at least one of the compositions described herein (e.g., engineered STAC-102 variant sequences, AAV particles, and engineered AAV capsid proteins) to a subject, delivering the compositions described herein to target cells, inhibiting or activating gene expression and protein production, and improving the symptoms of the disease or condition in the subject.

[0140] Equivalents and range This disclosure includes many equivalents to the specific embodiments described herein. Those skilled in the art will be able to identify equivalents to the specific embodiments through conventional experimentation.

[0141] The language of this disclosure is intended for illustrative purposes only and not to limit it. While the language of the claims may be modified, the scope of this disclosure remains in its broadest form. Specific embodiments of this disclosure are described herein. However, these embodiments are not intended to limit the broad scope of this disclosure.

[0142] This disclosure is further illustrated by the following embodiments, which are intended purely for illustrative purposes and not to limit the disclosures herein. [Examples]

[0143] Example 1. Method Fitness maturation of AAV capsid STAC-102. Fitness maturation was performed by creating a library of AAV capsids with one or two amino acid mutations introduced into the parent capsid STAC-102. In Figure 1, amino acids 586, 587, and 588 are the three 5' amino acids of the 7-mer peptide MTLTRQE inserted into STAC-102, while amino acids 589, 590, and 591 are immediately 3' of the peptide. Each mutation site was modified for all possible amino acids in STAC-102 except for cysteine ​​and its original amino acid.

[0144] AAV Capsid Library Generation. Capsid variants for library screening were synthesized as oligopools. Each capsid peptide was synthesized with three unique nucleotide sequences encoding the peptide, and each peptide was ligated to a separate barcode. The oligopools were cloned into linearized intermediate plasmids, and then stationary donor sequences were cloned to separate the barcode and peptide regions, generating complete AAV vector constructs. The resulting constructs contained neuron-specific promoters that drove barcode expression, with each barcode associated with the identity of a single capsid. This allows for evaluation of which capsid drives the most functional mRNA expression in neurons. Furthermore, each barcode is ligated to a unique molecular identifier (UMI). Based on the overall size of the cloned library, each barcode is ligated to hundreds to thousands of UMIs. The usefulness of UMIs lies in additionally evaluating the number of separate AAV transduction events that produce the measured number of NGS reads.

[0145] These AAV plasmid libraries were prepared in HEK293 cells. Briefly, the libraries were produced by transient transfection including the addition of Rep in trans, the capsids were purified by cesium density centrifugation, and the buffer was exchanged to PBS by Amicon filtration. Deoxyribonuclease-resistant viral genome titers were measured by quantitative real-time PCR.

[0146] Administration of AAV library to cynomolgus monkeys. The AAV capsid library was administered to cynomolgus monkeys as a slow bolus injection into the cisterna magna. 1 mL of the library test material was administered at a rate of 1 mL / min.

[0147] Tissue collection and preservation. Two weeks after administration, the animals were euthanized and 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 ​​into 4 mm coronal sections using the brain matrix. Each brain section was immersed in RNAlater (ratio 1:10) and refrigerated at 4°C for 24 hours. After removal from RNAlater, the sections were briefly washed in cooled ribonuclease-free phosphate-buffered saline, and 2 mm punches were taken from different brain regions. The punches and remaining tissue sections were frozen at ≤-65°C. The same workflow was used to process spinal cord and dorsal root ganglion tissue.

[0148] Tissue lysis for RNA isolation. Punch samples of brain and spinal cord to be processed were placed on dry ice. Approximately half of each tissue sample was excised and transferred to an Eppendorf tube pre-filled with 600 μL of TRI reagent and two 3.2 mm steel beads. The sample tubes were placed in a Retsch MM300 Tissue-Lyser and homogenized for 1.5 minutes over 6 rounds at a frequency of 25.1 Hz, with a 1-minute pause between rounds to prevent overheating.

[0149] RNA isolation from brain punches, spinal cord, and dorsal root ganglia. Total RNA isolation and purification from homogenized tissue punches preserved in RNAlater were performed using the MagMAX96 Total RNA Isolation Kit with the KingFisher Flex Purification System, according to the manufacturer's protocol. Briefly, bromochloropropane (BCP) was added, and the homogeneity was separated into aqueous and organic phases by centrifugation. The aqueous phase containing partially purified RNA was then transferred to the wells of a 96-well KingFisher-treated plate. Isopropanol (100%) was added to each well, followed by the addition of magnetic RNA-binding beads. Subsequent processing was performed using the KingFisher Flex Purification System. Briefly, the RNA-binding beads were captured magnetically, and deoxyribonuclease digestion and several washes were performed on the beads. The purified RNA was eluted in 100 μL of low-salt elution buffer. The KingFisher-treated plate was then transferred to a tabletop magnetic stand. The eluate (approximately 90 μL) was separated from any remaining beads for downstream processing and transferred to a 96-well PCR plate. RNA yield and purity were determined using a NanoDrop 8000 spectrophotometer.

[0150] RNA isolation from whole coronary brain sections. The excised brain sections were weighed and placed in 50 mL Bigprep Lysing Matrix D tubes. These tubes were filled with 10 mL of TRIZOL reagent per gram of tissue. The tissue samples were homogenized at 4.0 m / s for 30 seconds, with a 2-minute pause. Tissue homogenization was repeated four times. The lysates were centrifuged at 4,300 × g for 5 minutes at 4°C. The clarified lysates were transferred to a new tube, centrifuged a second time, and the clarified lysates were transferred to a new tube. 5 mL aliquots were further treated by adding 0.2 mL of molecular-grade chloroform per 1 mL of Trizol. The samples were mixed by inversion and vortexing for 30 seconds, and then centrifuged at 4,300 g for 30 minutes at 4°C. Approximately 50% of the lysate volume, or approximately 2.5 mL of aqueous phase, was distributed into 2 mL microcentrifuge tubes. 0.5 mL of molecular-grade isopropanol was added to 1 mL of Trizol in a tube and mixed by inversion. The sample was incubated on ice for 10 minutes, followed by centrifugation at 12,000 g for 10 minutes at 4°C. The RNA pellet was resuspended in 1 mL of 75% ethanol per 1 mL of Trizol, vortexed, and centrifuged at 12,000 g for 5 minutes at 4°C. The RNA pellet was air-dried for 5 minutes and dissolved in 0.2 mL of ribonuclease-free DEPC-treated water per 1 mL of Trizol. RNA yield and purity were determined using a spectrophotometer. The samples were spot-checked for RNA integrity using the Agilent RNA 6000 nanokit and Bioanalyzer 2100.

[0151] Reverse transcription and next-generation sequencing. Isolated RNA was reverse transcribed using the Qiagen QuantiTect Reverse Transcription Kit with gene-specific primers. The synthesized complementary DNA (cDNA) was then advanced for next-generation sequencing (NGS).

[0152] The primers were designed to amplify regions of interest on cDNA, and these primers also contained sequences that bound to the NGS adapter. Using Phusion® HotStart Flex DNA polymerase, a PCR was set up to amplify the region of interest on the cDNA or vector genomic DNA, and a second PCR was set up to bind the adapter required for barcoding the NGS sample. These amplicons were advanced for NGS and analyzed using a custom bioinformatics pipeline.

[0153] Example 2. Results Fitness maturation of STAC-102 identified second-generation variants exhibiting 5- to 10-fold higher neuronal mRNA expression in cynomolgus monkeys after CSF administration. Library evaluation suggests these capsids have similar or better production yields compared to STAC-102. Bioinformatics analysis of enrichment ratios, coefficient of variation, and UMI recovery was used to select the primary capsids. Second-generation STAC-102 capsids will be individually evaluated in cynomolgus monkeys and represent promising candidates for therapeutic applications of the CNS.

[0154] Multiple evaluations of the STAC-102 fitness maturity library were performed in both in vitro and in vivo environments.

[0155] In vitro evaluation of the performance and production productivity of novel AAV variants. The following library data are presented as bubble plots, where the log2 factor change in enrichment of each capsid variant is normalized against its relative abundance in the administered test material. The coefficient of variation represents the consistency of capsid performance across multiple sequencing reactions. Bubble size is proportional to the percentage of sequenced samples in which the capsid is observed. The number of unique molecular identifiers is represented by color according to the color legend.

[0156] Capsids exhibiting desirable performance have the following characteristics in the bubble plot (see Figures 3-5): 1) The log2 factor of the enrichment is large, and these data are normalized by the abundance of the input (y-axis). 2) Small coefficient of variation (x axis) 3) A large proportion of sequenced samples show a capsid (large bubble size). 4) Robust UMI recovery (green)

[0157] The parent capsid STAC-102 and notable second-generation capsids with improved CNS delivery in cynomolgus monkeys are annotated according to the table below. [Table 8]

[0158] Conclusions of in vitro evaluation: The second-generation STAC-102 variants, annotated as A-F, were selected primarily based on their effective CNS delivery in cynomolgus monkeys. In particular, all of these capsids can be constructed as well as, or better than, the parent STAC-102 sequence. With the exception of variant D, the second-generation variants also show improved potency in vitro compared to the parent sequence.

[0159] Evaluation of novel AAV performance in the brains of cynomolgus monkeys. The performance of library AAV variants was evaluated by quantifying the enrichment of AAV library mRNA transcripts expressed in neurons in macaque CNS tissue.

[0160] Capsids exhibiting desirable performance have the following characteristics in the bubble plot (see Figures 6-8): 1) The log2 factor of the enrichment is large, and these data are normalized by the abundance of the input (y-axis). 2) Small coefficient of variation (x axis) 3) A large proportion of sequenced samples show a capsid (large bubble size). 4) Robust UMI recovery (green)

[0161] The parent capsid STAC-102 and notable second-generation capsids that function well with respect to CNS delivery in cynomolgus monkeys have been annotated.

[0162] Figure 9. Overview of the performance of the second-generation STAC-102 capsid. Magnification change is calculated compared to STAC-102. The bubble size is proportional to the magnification change, and the UMI count is shown in color. The cortex includes the anterior orbital gyrus, middle frontal gyrus, superior frontal gyrus, anterior cingulate gyrus, posterior cingulate gyrus, inferior frontal gyrus, lateral orbital gyrus, precentral gyrus, superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, supramarginal gyrus, postcentral gyrus, insula, cuneiform gyrus, precuneus, lingual gyrus, superior parietal lobule, angular gyrus, occipital gyrus, entorhinal cortex, and fusiform gyrus. The hippocampal region includes the hippocampus, parahippocampal gyrus, hippocampal base, and amygdala. The deep brain region includes the caudate nucleus, putamen, substantia nigra, globus pallidus, hypothalamus, thalamus, and lateral geniculate nucleus. The brainstem includes the midbrain, medulla, and pons. The spinal cord includes the cervical, thoracic, and lumbar levels. The dorsal root ganglia include the cervical, thoracic, and lumbar levels.

[0163] Conclusions from in vivo evaluation in cynomolgus monkeys: Second-generation STAC-102 variants annotated as A-F exhibit substantially higher neuronal mRNA expression in multiple CNS regions compared to the parent STAC-102 sequence.

[0164] Analysis of mutation hotspots in variants A–F. Based on library design and fitness maturation approaches, we were able to additionally evaluate the performance of many capsids closely related to variants A–F. In the heatmaps shown in Figures 10–15, squares with black ovals indicate amino acid identity within variant A at each location, which is also shown in the amino acid sequence on the right side of the heatmap. The diagonal lines are proportional to the coefficient of variation. The top of the heatmap shows the different amino acids and their biochemical properties evaluated at each location. The color of each square is proportional to the log2 factor of enrichment according to the provided color legend. Location refers to the position of each amino acid within the diversification motif. In summary, the heatmap provides an overview of which mutations perform better at each mutation location.

[0165] Conclusions from the variant analysis: Variants A-F represent the highest-performing sequences within each cluster, but several alternative sequences for each capsid exist that function similarly or at a slightly lower level.

[0166] STAC-102 (Sequence 8) And the complete capsid amino acid sequences of second-generation variants A-F (SEQ ID NOs: 9-14). In the following sequences, the diversification regions of STAC-102 and its variants are shown in bold. Mutations against STAC-102 are underlined.

[0167] [Table 9]

[0168] [Table 10]

[0169] [Table 11]

[0170] Parent capsid STAC-102 (sequence number 11, variant C) with sequence number 3

[0171] Table 12

[0172] Table 13

[0173] Table 14

[0174] Parent capsid STAC-102 (sequence number 14, variant F) with sequence number 6

[0175] Table 15

Claims

1. A modified AAV capsid protein containing at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs: 1-6.

2. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 1, wherein the variant sequence comprises (i) any amino acid K, M, N, Y, or P at amino acid position 5 of SEQ ID NO: 1, and / or (ii) any amino acid M, H, N, S, T, A, I, L, F, Y, or P at amino acid position 8 of SEQ ID NO: 1, and optionally the AAV capsid protein comprises SEQ ID NO:

1.

3. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 2, wherein the variant sequence comprises (i) any amino acid K, M, N, and L at amino acid position 7 of SEQ ID NO: 2, and / or (ii) any amino acid S, Q, and M at amino acid position 8 of SEQ ID NO: 2, and optionally the AAV capsid protein comprises SEQ ID NO:

2.

4. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 3, wherein the variant sequence comprises (i) any amino acid K, R, F, Y, and G at amino acid position 4 of SEQ ID NO: 3, and / or (ii) any amino acid D, K, N, and S at amino acid position 9 of SEQ ID NO: 3, and optionally the AAV capsid protein comprises SEQ ID NO:

3.

5. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 4, wherein the variant sequence comprises (i) any of amino acids K, S, L, Y, G, and P at amino acid position 11 of SEQ ID NO: 4, and / or (ii) any of amino acids T, D, E, and V at amino acid position 12 of SEQ ID NO: 4, and optionally the AAV capsid protein comprises SEQ ID NO:

4.

6. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 5, wherein the variant sequence comprises (i) any of amino acids T, M, D, E, Q, S, V, and G at amino acid position 4 of SEQ ID NO: 5, and / or (ii) any of amino acids P, H, R, A, and V at amino acid position 7 of SEQ ID NO: 5, and optionally the AAV capsid protein comprises SEQ ID NO:

5.

7. The manipulated AAV capsid protein according to claim 1, comprising a variant sequence of SEQ ID NO: 6, wherein the variant sequence comprises (i) any amino acid L, E, R, and V at amino acid position 3 of SEQ ID NO: 6, and / or (ii) any amino acid T, H, E, A, and P at amino acid position 4 of SEQ ID NO: 6, and optionally the AAV capsid protein comprises SEQ ID NO:

6.

8. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

1.

9. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

2.

10. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

3.

11. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

4.

12. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in Sequence ID No.

5.

13. The manipulated capsid protein according to claim 1, comprising the amino acid sequence described in Sequence ID No.

6.

14. A modified AAV capsid protein, wherein the modified AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO: 8, and optionally has one or two amino acid substitutions at any one or two amino acid positions 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, and 597 of SEQ ID NO:

8.

15. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO:

9.

16. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO:

10.

17. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO:

11.

18. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO:

12.

19. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO:

13.

20. The manipulated AAV capsid protein according to claim 14, wherein the manipulated AAV capsid protein comprises a variant sequence that is at least 80% identical to SEQ ID NO: 14.

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