Engineered blood brain barrier penetrant aav capsids

Abstract

This application relates to engineering AAV capsids. In some embodiments, the engineered AAV capsids are capable of penetrating the blood brain barrier and transducing cells in the central nervous system.

Description

ENGINEERED BLOOD BRAIN BARRIER PENETRANT AAV CAPSIDSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 466,598 filed on May 15, 2023 and entitled “Engineered Blood Brain Barrier Penetrant AAV Capsids,” and U.S Provisional Patent Application No 63 / 606,012 filed on December 4, 2023 and entitled, “Engineered Blood Brain Barrier Penetrant AAV Capsids,” the entire contents of each of which are incorporated by reference herein.FIELD

[0002] This application relates to engineering AAV capsids.BACKGROUND

[0003] The clinical translation of genomic medicines to treat disorders of the central nervous system (CNS) has been limited by inefficient gene delivery. AAV capsids that cross the blood brain barrier (BBB) in rodents exhibit widespread CNS transduction and efficacy; however, capsids that cross the BBB in non-human primates have been challenging to engineer.

[0004] Attempts at providing AAV capsids with improved properties, e.g., improved tropism to a target cell or tissue upon systemic administration, have met with limited success. As such, there is a need for improved methods of producing AAV capsids and resulting AAV capsids for delivery of genetic material of interest to a target cell or tissue, e.g., a CNS cell or tissue, including delivery across the BBB.SUMMARY

[0005] In an aspect, CNS-targeting molecules, i.e., targeting peptides, are provided. In another aspect, an engineered AAV capsid protein is provided wherein a peptide sequence is inserted into a parent capsid at a peptide insertion site. In embodiments, the peptide sequence, the parent capsid, and the peptide insertion site are as indicated in a single row of Table 1.

[0006] In an aspect, an adeno-associated virus (AAV) capsid protein is provided comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any amino acid sequence set forth in any one of SEQ ID NO: 1-1232.

[0007] In embodiments, the amino acid sequence comprises, consists of, or consists essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 71.

[0008] In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of serotype AAV2. In embodiments, the serotype AAV2 comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1233. In embodiments, the amino acid sequence is inserted into the AAV2 capsid protein between amino acids 588 and 589. In embodiments, the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1233. In embodiments, the AAV capsid having SEQ ID NO 71 inserted therein protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1233.

[0009] In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of serotype AAV9. In embodiments, the serotype AAV9 comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1234. In embodiments, the amino acid sequence is inserted into the AAV9 capsid protein between amino acids 587 and 590. In embodiments, the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235. In embodiments, the AAV capsid having SEQ ID NO 71 inserted therein protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1235.

[0010] In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in any one of SEQ ID NO: 1-1232. In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 71.

[0011] In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11,AAVrhlO, AAVrh39, or AAVrh74. In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of AAV2 or AAV9.

[0012] In embodiments, the amino acid sequence is inserted into a surface exposed region of the AAV capsid.

[0013] In an aspect, an AAV capsid protein is provided comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of an amino acid sequence set forth in any of SEQ ID NO: 1-1232, wherein the amino acid sequence is inserted between amino acid positions 450 and 600 of the AAV capsid protein.

[0014] In embodiments, the amino acid sequence is inserted between amino acid positions 588 and 589 of the AAV capsid protein. In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of the sequence forth in SEQ ID NO: 1233. In embodiments, the amino acid sequence comprises the sequence set forth in SEQ ID NO: 71.

[0015] In embodiments, the amino acid sequence is inserted between amino acid positions 587 and 590 of the AAV capsid protein. In embodiments, the AAV capsid protein comprises consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1234. In embodiments, the amino acid sequence comprises, consists of, or consists essentially of SEQ ID NO: 71. In embodiments, the AAV capsid protein having SEQ ID NO 71 inserted therein comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235. In embodiments, the AAV capsid protein comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of SEQ ID NO: 71. In embodiments, the AAV capsid protein comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 1235.

[0016] In an aspect, a CNS-targeting molecule is provided comprising, consisting of, or consisting essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of an amino acid set forth in any of SEQ ID NO: 1-1232.

[0017] In embodiments, the amino acid sequence comprises, consists of, or consists essentially of at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of the amino acid sequence set forthin SEQ ID NO: 71. In embodiments, the amino acid sequence is fused or conjugated to a small molecule, antibody, scFV, ASO, siRNA, lipid, polymer, or recombinant protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 illustrates an example schematic of the barriers and selective pressure to identify AAV variants that exhibited enrichment in CNS tissue.

[0019] FIG. 2 illustrates examples of SIFTER (Selecting in vivo for transduction and expression of RNA) capsid libraries linked to barcodes. Each capsid is linked to multiple barcodes defined in a look-up table and created by pooled oligo synthesis. Different oligo pool designs allow for multiplexing libraries varying parental capsid identity or mutational strategy. Hundreds to thousands of unique molecular identifiers (UMIs) are cloned per barcode enabling detection of individual transduction events. Barcodes and UMIs are expressed under the control of either a neuron specific human synapsin I or ubiquitous CMV promoter, allowing the read out of capsid performance in multiple cell types.

[0020] FIG. 3 shows the diversities of the five round 2 sub-libraries (Library A-E) that were multiplexed and cloned under expression of a neuron specific human synapsin 1 (hSynl) promoter or a ubiquitous CMV promoter. The resulting 10 libraries were administered via intravenous injection, and an endpoint analysis to recover library transcripts was performed.

[0021] FIG. 4 provides data showing that the production of libraries is relatively uniform with most variants found + / - 32-fold change from the cloned library. As expected, variants that produce well are associated with more UMIs recovered. Bubbles circled in black outlines are variants that were selected for a third-round evaluation from NHP tissue samples. The position of the AAV9 in the library is highlighted.

[0022] FIGs. 5 and 6. The library was administered to two cynomolgus macaques and barcodes were recovered from CNS and peripheral tissues to assess biodistribution and expression of the library transcript. FIG. 5 shows data from in vitro induced pluripotent stem cell (iPS) neuron transduction. Overall, the performance of the library in vitro is not predictive of the performance in vivo as many recovered variants from the NHP samples are less enriched in cultured neurons. FIG. 6 shows data of in vivo library recovery from NHP samples. After filtering out variants that were poorly recovered, a total of 6,728 variants were identified from the human synapsin I library, and 4,005 variants from the CMV library. Based on consistencyin recovery across animals and fold enrichment, 810 variants were selected from the hSynl library and 426 variants from the CMV library, for a final evaluation in NHP.

[0023] Bubble Plot Legend for FIGs. 5 and 6. The graphs show the fold-change in variant enrichment normalized to the administered viral library on the y-axis and the coefficient of variation in the detection on the x-axis. The bubble size corresponds to the fraction of replicates the variant was recovered from. The color scale indicates the number of unique molecular identifiers (UMI) associated with each variant. The top graphs are the variants’ performance under the control of the neuron specific hSynl promoter and the bottom graphs are under the control of the ubiquitous CMV promoter. Bubbled circles in black outlines are variants that were selected for a third-round evaluation from NHP tissue samples.

[0024] FIG. 7. The round 3 library was administered to three cynomolgus macaques and barcodes were recovered from CNS tissues to assess on-target biodistribution and expression of the library transcript. The figure shows the performance of the 1236 variants in the round 3 library evaluation. The log2 fold change (log2FC) in expression of each variant from the hSynl and CMV promoters is shown on the y-axis and the coefficient of variation of the log2 fold change measurement is shown on the x- axis. The data represents assessment of whole brain transduction and mRNA expression mediated by each capsid in the library.

[0025] FIG. 8. The figure shows the performance of the 1236 variants in the round 3 library evaluation. The vector genome biodistribution of each variant is shown. The data represents assessment of whole brain transduction mediated by each capsid in the library.

[0026] FIG. 9. The graph shows the log2 fold-change in neuronal (hSynl) mRNA expression of CNSRCV300 (also referred to as SEQ ID NO: 1235, STAC-BBB, STAC / BBB, or STACBBB) and AAV9 across six brain slices analyzed per animal. The box and whisker plots contains the rest of the round 3 library performance.

[0027] FIG. 10. The graph shows the log2 fold-change in ubiquitous (CMV) mRNA expression of CNSRCV300 and AAV9 across six brain slices analyzed per animal. The box and whisker plots contains the rest of the round 3 library performance.

[0028] FIG. 11. The graph shows the log2 fold-change in vector genome biodistribution of CNSRCV300 and AAV9 across four brain slices analyzed per animal. The box and whisker plots contains the rest of the round 3 library performance.

[0029] FIG. 12. The graph shows the log2 fold-change in mRNA expression and vector genome biodistribution of CNSRCV300 and AAV9 across all animals included in the study. The box and whisker plots contains the rest of the round 3 library performance.

[0030] FIG. 13. The graph shows the log2 fold change in neuronal (hSynl) mRNA expression of CNSRCV300 and AAV9 in all tissues punches analyzed across the cortex, hippocampal region, deep brain regions, cerebellum and brain stem. The neuronal (hSynl) mRNA expression in cervical, thoracic and lumbar spinal cord and dorsal root ganglia levels is also shown for CNSRCV300 and AAV9.

[0031] FIG. 14. The graph shows the log2 fold change in ubiquitous (CMV) mRNA expression of CNSRCV300 and AAV9 in all tissues punches analyzed across the cortex, hippocampal region, deep brain regions, cerebellum and brain stem. The ubiquitous (CMV) mRNA expression in cervical, thoracic and lumbar dorsal root ganglia levels is also shown for CNSRCV300 and AAV9. Lastly, peripheral tissues mRNA expression in peripheral tissues is also shown for CNSRCV300 and AAV9.

[0032] FIG. 15. The image shows that CNSRCV300 drives widespread and robust transgene expression throughout the brain at a dose of 2el3 vg / kg. Negative control tissue without AAV treatment shows no signal.

[0033] FIG. 16. The images show that CNSRCV300 exhibits widespread transduction across all cortical regions.

[0034] FIG. 17. The images show that CNSRCV300 mediates efficient transduction of neurons in a variety of cortical and subcortical regions.

[0035] FIG. 18. The images show that CNSRCV300 mediates efficient transduction of neurons in the dentate nucleus.

[0036] FIG. 19. The images show that CNSRCV300 mediates efficient transduction of neurons in the Thalamus.

[0037] FIG. 20. The images show that CNSRCV300 mediates efficient transduction of neurons in the Putamen.

[0038] FIG. 21. The images show that CNSRCV300 mediates efficient transduction of neurons in the substantia nigra.

[0039] FIG. 22. The images show that CNSRCV300 mediates efficient transduction of neurons in the lateral geniculate nucleus.

[0040] FIG. 23. The images show that CNSRCV300 mediates efficient transduction of neurons in the pons.

[0041] FIG. 24. The images show that CNSRCV300 mediates efficient transduction of neurons in the precentral gyrus.DETAILED DESCRIPTION

[0042] In an aspect, CNS (central nervous system)-targeting molecules, i.e., targeting peptides, are provided. In embodiments, CNS-targeting molecules comprising a targeting peptide seq indicated in Table 1 are provided. In another aspect, engineered AAV capsid proteins are provided. In embodiments, a targeting peptide is inserted into an AAV capsid, for example an AAV9 capsid protein or an AAV2 capsid protein. In embodiments, the targeting peptide is any of the targeting peptide disclosed in Table 1. In embodiments, the peptide sequence is inserted into the AAV9 or AAV2 capsid at any of the peptide insertion sites disclosed in Table 1. In some embodiments, the targeting peptide comprises a targeting peptide that functions to target the CNS-targeting molecule to a specific target tissue (e.g., CNS tissue).Libraries of AAV Capsid Proteins

[0043] In one aspect, disclosed herein is the development of libraries encoding AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype. Thus, described herein are libraries of AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, the desired characteristic isenhanced cell or tissue tropism as compared to the natural / wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural / wild-type AAV serotype In some embodiments, the desired characteristic is increased penetrance through the blood brain barrier following administration to a subject. In some embodiments, the desired characteristic is wider distribution throughout the multiple brain regions, e g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the desired characteristic is elevated genetic material expression in multiple brain regions. In some embodiments, the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0044] In some embodiments, each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising a targeting peptide inserted into a hypervariable and / or surface exposed loop of the capsid protein: b) a nucleic acid sequence encoding barcode: c) one or more nucleic acid sequences encoding one or more promoters: and d) a nucleic acid sequence encoding a unique molecular identifier (UMI). In some embodiments, each member of the library also includes genetic material to be delivered to a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence.

[0045] In some embodiments, the genetic material encodes one or more peptides or a polypeptides. In some embodiments, the genetic material encodes one or more antibodies or antibody fragments. In some embodiments the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNA.

[0046] In some embodiments, the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are noncoding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.

[0047] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.

[0048] In some embodiments, the genetic material encodes a gene therapy product. A gene therapy product can include a peptide, a polypeptide, a recombination donor template, or an RNA molecule that when expressed carries out a desired therapeutic effect. In some embodiments, the therapeutic effect is treating any one more diseases or disorders described herein.

[0049] In some embodiments, each member of the library is fused or coupled to an active agent. In some embodiments, each member of the library is fused or coupled to an active agent through conjugation. In some embodiments, the active agent comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises a DNA-binding and / or targeting composition, for example, a zinc finger protein (ZFP), TAL-effector domain, and / or a sgRNA of CRISPR / Cas system. In some embodiments, the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region). In some embodiments, the peptide is fused to a Fc region of an antibody. In some embodiments, the peptide is fused to the C-terminus of the Fc region. In some embodiments, the peptide is fused to the N-terminus of the Fc region. In some embodiments, the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the peptide is fused or coupled directly to at least one strand of the RNAi. In some embodiments, the peptide the peptide is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the peptide is fused or coupled to the sense strand of RNAi. In some embodiments, the peptide is fused or coupled to the antisense strand of RNAi. In some embodiments the active agent comprises a diagnostic agent. In some embodiments, the diagnostic agent comprises a detectable moiety such as a fluorophore. In some embodiments, the active agent is a small molecule.

[0050] In some embodiments, the promoter is operably linked to the genetic material to be delivered to the cell. In some embodiments, the one or more promoters comprises a tissue and / or cell specific promoter. In some embodiments, the one more promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others.

[0051] In some embodiments, the one or more promoters comprise a cell type and / or tissue specific type promoter. Exemplary cell type and / or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter(TTR), expressed in hepatocytes. Other non-limiting cell type and / or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific. Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), CAMK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes).

[0052] In embodiments, tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS. In embodiments, tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and / or Schwann cells.

[0053] In some embodiments, the one or more promoters are naturally occurring promoters. In some embodiments, the one or more promoters are synthetic. In some embodiments, the one or more promoters is derived from mammals, humans, viruses, or plants. In some embodiments, the one or more promoters is truncated. In some embodiments, the one or more promoters is mutated.

[0054] In some embodiments, a nucleic acid comprising a barcode is added to the genome of each AAV capsid proteins in a library. In some embodiments, the barcode is bioinformatically linked to the targeting peptide introduced into the capsid protein. In some embodiments, the DNA sequences encoding the targeting peptide are synthesized to further comprise a random or specified barcode. The barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides. In some embodiments, each targeting peptide is linked to at least 2 distinct barcodes. In some embodiments, each barcode is linked to one or more UMI.

[0055] In some embodiments, each member of the library comprises a nucleic acid comprising more than one barcode sequences. In some embodiments, each member of the library comprises two or more nucleic acids each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter.

[0056] In some embodiments, the AAV capsid proteins are derived from AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9 serotypes. In some embodiments, the AAV variant capsid proteins are derived from less well characterized AAV serotypes, including but not limited to AAV4, AAV7, AAVrhlO, AAVrh39, and AAVrh74. In some embodiments, a library of AAV variants comprises AAV variant capsid proteins derived from a single AAV serotype. In some embodiments, a library of AAV variants comprises 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, the 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 once individual serotype libraries are developed. In some embodiments, combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.

[0057] In some embodiments, the AAV serotype includes the AAV2 serotype. In some embodiments, the AAV2 serotype includes a mutation of arginine to alanine at amino acid position 588. In some embodiments, the AAV2 serotype includes the sequence SEQ ID NO: 1233. In some embodiments, the AAV serotype includes the AAV9 serotype. In some embodiments, AAV9 serotype includes the sequence of SEQ ID NO: 1234.

[0058] In some embodiments, targeting peptides are introduced into aDNA sequence encoding an exposed loop in the capsid protein. In some embodiments, the targeting peptide is inserted into exposed loops (e.g. hypervariable regions) in the AAV capsid. In some embodiments, the targeting peptide comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the targeting peptide comprises between 9 and 16 amino acids. In some embodiments, the targeting peptide is 9 amino acids in length. In some embodiments, the targeting peptide is 16 amino acids in length. In some embodiments, the HI loop is targeted (mutated) while in others, the DE loop is targeted (mutated). In some embodiments, mutations (e.g., insertions, deletions and / or substitutions) are made in both loops. In further embodiments, targeting peptides are introduced into the VR region of a surface loop, including into VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and or VR-IX. In yet other embodiments, targeting peptides are made in VR-I, VR-IV, and or VR-VIII. In some embodiments, targeting peptides are introduced into the AAV capsid proteins VP1, VP2 or VP3, or in two of the capsid proteins in any combination, or in all three. In some embodiments, the targeting peptides are introduced into VP1. In some embodiments, the targeting peptidesare introduced into VP2. In some embodiments, the targeting peptides are introduced into VP3. In some embodiments, the targeting peptides are introduced into VP1 and VP2. In some embodiments, the targeting peptides are introduced into VP1 and VP3. In some embodiments, the mutations are introduced into VP2 and VP3. In some embodiments, the targeting peptides are introduced into VP1, VP2, and VP3. In some embodiments, the targeting peptide is introduced at a single site in a gene encoding a capsid protein.

[0059] In some exemplary embodiments, a targeting peptide is introduced into the variable regions VR-I, VR-IV, or VR-VIII of the capsid protein. In some embodiments, the targeting peptide is introduced at a location between positions 450 and 600 of the capsid protein. In some embodiments, the targeting peptide is inserted between positions 587 and 590 of the capsid protein. In some embodiments, the targeting peptide is inserted between positions 587 and 590 of the AAV9 capsid protein. In some embodiments, the targeting peptide is inserted between positions 588 and 589 of the capsid protein. In some embodiments, position 588 has been altered from an arginine to an alanine. In some embodiments, the targeting peptide is inserted between positions 588 and 589 of the AAV2 capsid protein.

[0060] In some embodiments, the targeting peptide comprises any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises at least 5, 6, 7, 8, or 9 contiguous amino acids of any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises a sequence that is at least 80% identical to any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises a sequence that is at least 80% identical, least 85% identical, at least 90% identical, at least 95%, or at least 99% identical to or comprises any one of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises SEQ ID NO: 71. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.Engineered AAV Capsid Proteins

[0061] Development of engineered AAV capsid proteins

[0062] Described are compositions comprising an engineered AAV capsid proteins and methods of making and using the same.

[0063] Disclosed are methods and compositions to develop engineered AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype. For example,capsid proteins are useful in delivering peptides (e g., targeting peptides) across the blood brain barrier following administration to a subject. In some embodiments, the engineered AAV capsid proteins are useful in achieving is wider distribution of the genetic material throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the engineered AAV capsid proteins are useful in elevating genetic material expression in multiple brain regions. In some embodiments, the engineered AAV capsid proteins are used to deliver genetic material of interest to a desired tissue, cell, or organelle.

[0064] Gene editing system

[0065] In some embodiments, the genetic material of interest comprises a gene editing system or portions of a gene editing system. In some embodiments, the gene editing system is capable of inducing single or double-stranded breaks into nucleic acid sequences. In some embodiments, the gene editing system is capable of inserting, substituting, or deleting a base or a sequence of bases into nucleic acid sequences. In some embodiments, the gene editing system includes a CRISPR-Cas system. In some embodiments, the gene editing system includes a TALEN. In some embodiments, the gene editing system includes a zinc finger nuclease.

[0066] Engineered AAV capsid proteins within a cell

[0067] In some embodiments, engineered AAV capsid proteins are contained within a cell. In some embodiments, the cell is derived from the CNS. In some embodiments, the cell is derived from the 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 any of the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the cerebral cortex, the brain stem, the hippocampus, or the thalamus, amongst others.

[0068] Engineered AAV capsid proteins delivered to a target cell

[0069] The engineered AAV capsid proteins may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (PNS), or cells and tissues of a muscle. The engineeredAAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins may have enhanced tropism for B cells, hematopoietic cells, leukocytes, platelets, macrophages, megakaryocytes, monocytes and / or T cells.

[0070] Methods of detecting engineered AAV capsid proteins

[0071] In some embodiments, a method of identifying an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype is provided comprising: (i) contacting a cell, cell line, or tissue in vitro or in vivo with any one of the libraries of engineered AAV capsid proteins, (ii) allowing the engineered AAV capsid proteins in said library to transduce the cell, cell line, or tissue; (iii) recovering from the cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.

[0072] In another aspect, disclosed herein are methods for directed evolution of engineered AAV capsid proteins and identification of an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype comprise (i) insertion of targeting peptides into hypervariable and / or surface-exposed loops in capsid proteins from one or more AAV serotypes creating libraries of modified variant capsids for each AAV serotype; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where variants are isolated from a target tissue or cell line; and (viii) full evaluation of enriched variants. In some embodiments, the desired characteristic includes enhanced tissue tropism as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism of the central nervous system as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced ability to cross the blood brain barrier as compared to thenatural / wild-type AAV serotype. In some embodiments, the targeting peptides that are inserted into the hypervariable and / or surface-exposed loops include any of the sequences set forth in SEQ ID NO: 1-1232. In some embodiments, SEQ ID NO: 71 is inserted into the hypervariable and / or surface exposed loop.

[0073] Capsid proteins

[0074] In some embodiments, capsid proteins, for example AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9 are chosen for starting points. In some embodiments, capsid proteins from less well characterized AAV serotypes are chosen, including but not limited to AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, and AAVrh74. In some embodiments, a library of AAV variants comprises AAV variant capsid proteins derived from a single AAV serotype. In some embodiments, a library of AAV variants comprises 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, the 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 once individual serotype libraries are developed. In some embodiments, combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.

[0075] In some embodiments, the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, E1A and E1B) are supplied in trans. In some embodiments, the AA V rep function comprises rep78, rep68, rep52, and rep40 genes. In some embodiments, the rep genes are supplied in trans. In some embodiments, the start codon of the rep78 and / or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield. In some embodiments, the cap genes are supplied as genetic material to the manufactured AAVs. In some embodiments, the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.CNS (Central Nervous System)- Targeting Molecules (Targeting Peptides)

[0076] Described herein are CNS-targeting molecules, i.e, targeting peptides. In some embodiments, the CNS-targeting molecules have enhanced tropism for a cell or tissue, such asthe delivery of genetic material of interest to said cell or tissue, for example a CNS tissue or PNS tissue or a CNS cell or PNS cell.

[0077] In some embodiments, the CNS-targeting molecule comprises a sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises at least 5, 6, 7, 8, or 9 contiguous amino acids of a sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises the sequence set forth in SEQ ID NO: 71. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.

[0078] In some embodiments, the CNS-targeting molecule comprises variants of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In some embodiments, the variant comprises 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In some embodiments, the variant comprises 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In some embodiments, the variant comprises 1, 2, 3, or 4 insertions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In some embodiments, the variant comprises any combination of the substitutions, deletions, or insertions described above.

[0079] In embodiments, a variant refers to a variant in the nucleotide sequence that encodes any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 substitutions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 deletions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, or 4 insertions to any of the amino acids of any of the amino acid sequences set forth in SEQ ID NO: 1-1232. In embodiments, the variant in the nucleotide sequence encodes any combination of the substitutions, deletions, or insertions described above.

[0080] In some embodiments, the CNS-targeting molecule is fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein. In some embodiments, any of SEQ ID NO: 1-1232 are fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein. In some embodiments, SEQ ID NO: 71 is fused or conjugated to a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein. In some embodiments, CNS-targeting molecules may be utilized to enable a small molecule, an antibody, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.

[0081] In some embodiments, the CNS-targeting molecules are part of an engineered AAV capsid protein. In some embodiments the engineered capsid protein comprises any of the serotypes of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9. In embodiments, the capsid protein comprises the serotype AAV2 or AAV9. In some embodiments, the AAV2 serotype comprises SEQ ID NO: 1233. In some embodiments, the AAV2 serotype comprises a variant of SEQ ID NO: 1233. In some embodiments, the AAV9 serotype comprises SEQ ID NO: 1234. In some embodiments, the AAV9 serotype comprises a variant of SEQ ID NO: 1234. In embodiments, a variant refers to any one or more of a substitution, deletion, or addition to any of the amino acids in either of the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234. 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 either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234. 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 deletions of any of the amino acids in either of the amino acid sequences set forth of SEQ ID NO: 1233 or SEQ ID NO: 1234. 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 insertions of any of the amino acids in either of the amino acid sequences set forth SEQ ID NO: 1233 or SEQ ID NO: 1234.

[0082] In some embodiments, the variant comprises an amino acid sequence that comprises at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234 or SEQ ID NO:1233, and at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of SEQ ID NOS: 1- 1232.

[0083] In some embodiments, the AAV2 serotype comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO:1233. In some embodiments, the AAV9 serotype comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO:1234.

[0084] In embodiments, a variant refers to a variant in the nucleotide sequence that encodes the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234. In some embodiments, the variant in the nucleotide sequences results in encoding any one or more of a substitution, deletion, or addition to any of the amino acids of either of the amino acid sequences set forth in SEQ ID NO: 1233 or SEQ ID NO: 1234 In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions of any of the amino acids in either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234. In 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 deletions of any of the amino acids in either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234. 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 insertions of any of the amino acids in either of the amino acid sequences of SEQ ID NO: 1233 or SEQ ID NO: 1234.

[0085] In some embodiments, the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234 or SEQ ID NO: 1233, and at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of SEQ ID NOS: 1-1232

[0086] In some embodiments, a nucleotide sequence encodes an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1233. In some embodiments, a nucleotide sequence encodes an amino acidsequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1234.

[0087] In some embodiments, any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into an AAV. In some embodiments, any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into AAV2. In some embodiments, AAV2 comprises the sequence set forth in SEQ ID NO: 1233. In some embodiments, SEQ ID NO: 71 is inserted into SEQ ID NO: 1233. SEQ ID NO: 1233 is AAV2 with an amino acid substitution. In some embodiments, any of the CNS-targeting molecules set forth in any of SEQ ID NO: 1-1232 is inserted into AAV9. In some embodiments, AAV9 comprises the sequence set forth in SEQ ID NO: 1234. In some embodiments, SEQ ID NO: 71 is inserted into SEQ ID NO: 1234. SEQ ID NO: 1234 is AAV9. SEQ ID NO: 71 inserted into SEQ ID NO: 1234 can result in SEQ ID NO: 1235.

[0088] In some embodiments, insertion of SEQ ID NO: 71 into AAV9 results in the sequence set forth in SEQ ID NO: 1235 (CNSRCV300). In some embodiments, insertion of SEQ ID NO: 71 into AAV9 results in a variant sequence of SEQ ID NO: 1235. 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, insertions, and / or deletions of any of the amino acids in the amino acid sequence of SEQ ID NO: 1235. In some embodiments, the variant comprises at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with SEQ ID NO: 1235 or comprises SEQ ID NO: 1235. In embodiments, the variant comprises at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.

[0089] In some embodiments, the variant refers to a variant in the nucleotide sequence that encodes the amino acid sequences set forth in SEQ ID NO: 1235. In embodiments, the variant in the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions, insertions, and / or deletions of any of the amino acids in the amino acid sequence of SEQ ID NO: 1235. In some embodiments, the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identitywith SEQ ID NO: 1235. In embodiments, the variant in the nucleotide sequence encodes an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1235, and at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 71.

[0090] AAV particles

[0091] In some embodiments, the CNS-targeting molecules are part of an engineered AAV capsid protein, and the engineered AAV capsid proteins are packaged into AAV particles. In some embodiments, the AAV particles that have enhanced tropism for a target tissue (e.g., CNS and PNS) are provided. CNS-targeting molecules may be inserted into an AAV capsid protein sequence to alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. In some embodiments, the AAV particles are capable of penetrating the blood brain barrier.

[0092] Delivery of AAV Particles

[0093] The AAV particles may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively), or cells and tissues of a muscle. The AAV particles may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.

[0094] In some embodiments, the AAV particles are used to deliver a viral genome to a tissue or cells such as CNS or PNS cell or tissue.

[0095] The delivered viral genome may include genetic material of interest, such as, for example, an antibody, an enzyme, or regulatory RNA, amongst others. In some embodiments, the viral genome includes at least one ITR sequence. In some embodiments, the viral genome includes 2 ITR sequences. In some embodiments, the ITR sequences flank the genetic material of interest. In some embodiments, the ITR sequences are complementary to each other. In some embodiments, the ITR regions are derived from the same serotype as the capsid protein. ITR regions may be between 100 and 150 nucleotides in length.

[0096] In some embodiments, the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating In some embodiments, the genome of the virus contains the components required for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver genetic material of interest to the particular tissue.

[0097] AAV capsid proteins comprising CNS-targeting molecules (targeting peptides)

[0098] In some embodiments, the CNS-targeting molecules, i.e., targeting peptides, are part of a recombinant AAV capsid protein. In some embodiments, AAV capsid proteins described herein may be produced recombinantly and may be based on adeno-associated virus (AAV) wild type sequence.

[0099] CNS-targeting molecules may be inserted into an AAV capsid protein sequence to alter tropism relative to the natural AAV capsid protein, to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. Stated another way, CNS-targeting molecules, which refer to the targeting peptides, that are inserted into the capsid protein, allow the capsid protein to penetrate the blood brain barrier.

[0100] In some embodiments, the targeting peptide is used for enhanced or improved transduction of a target cell or tissue (e.g., cells or tissues of the central nervous system (CNS) or peripheral nervous system (PNS)). In some embodiments, the targeting peptide is used to facilitate the AAV capsid protein across the blood brain barrier following administration to a subject. In some embodiments, the targeting peptide is used for enhanced or improved distribution of the genetic material throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the targeting peptide is used for enhanced or improved genetic material expression in multiple brain regions. In some embodiments, the targeting peptide is used for enhanced or improved delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0101] In some embodiments, the targeting peptide increases tropism of the AAV capsid to a cell, region, or tissue of the CNS. Examples of CNS cells include but are not limited to neurons (e.g., excitatory neurons, inhibitory neurons, and motor neurons) and glial cells (e.g.,ependymal cells, astrocytes, oligodendrocytes. Examples of CNS tissue include but are not limited to the cortex (e.g., frontal cortex, parietal cortex, occipital cortex, temporal cortex), thalamus, hypothalamus, striatum, hippocampus, entorhinal cortex, and basal ganglia.

[0102] In some embodiments, the AAV capsid protein comprising a targeting peptide is capable of increased tropism by at least 1.1 -, 1.2-, 1.3-, 1.4-, 1.5-fold, relative to an AAV capsid protein that lacks a targeting peptide. In some embodiments, the AAV capsid protein comprising a targeting peptide is capable of increased tropism by over 1.5-fold, relative to an AAV capsid protein that lacks a targeting peptide.

[0103] In some embodiments, the AAV capsid protein comprising the targeting peptide facilitates increased expression of delivered genetic material (e.g., a therapeutic cargo) by at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-fold in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a targeting peptide. In some embodiments, the AAV capsid protein comprising the targeting peptide facilitates increased expression of delivered genetic material (e.g., a therapeutic cargo) by more than 1.5-fold in a specific cell, region, or tissue, relative to an AAV capsid protein that lacks a targeting peptide.

[0104] In some examples, the targeting peptide is between 6 amino acids and 20 amino acids in length, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some examples, the targeting peptide is between 9 and 16 amino acids in length. In some examples, the targeting peptide is 9 amino acids in length. In some examples, the targeting peptide is 16 amino acids in length.

[0105] In some embodiments, the targeting peptide comprises an amino acid sequence of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71. In some embodiments, the targeting peptide is part of an AAV vector. In some embodiments, the targeting peptide is part of a capsid protein of the AAV vector. In some embodiments, nucleic acid sequences encode targeting peptides.

[0106] In some embodiments, the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises an amino acid sequence of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of any sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the AAV capsid protein comprise a nucleic acid sequence encoding a peptide that comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.

[0107] Insertion of Targeting Peptides into Capsid Proteins

[0108] In some embodiments, a targeting peptide is a part of a capsid protein, and the targeting peptide is inserted at a location between amino acid residues 450 and 600 of the capsid protein. In some embodiments, the amino acid sequence is inserted at a location between amino acid residues 587 and 590 of the AAV9 capsid protein. In some embodiments, the amino acid sequence is inserted at location between amino acid residues 588 and 589 of the AAV2 capsid protein.

[0109] In some embodiments, a peptide sequence that comprises any of the sequences set forth in SEQ ID NO: 1-1232 is inserted into the capsid protein. In some embodiments, the peptide sequence comprises 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of any of the sequences set forth in SEQ ID NO: 1-1232. In some embodiments, disclosed is a peptide sequence that comprises the sequence set forth in SEQ ID NO: 71. In some embodiments, the peptide sequence comprises 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 71.

[0110] In some embodiments, a targeting peptide is inserted into any of the AAV capsid protein comprises any of the AAV serotypes AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8 and AAV9. In some embodiments, the AAV capsid protein comprises the AAV2 serotype. In some embodiments, the AAV2 serotype includes a mutation at position 588 from arginine to alanine. In some embodiments, the AAV2 serotype comprises the sequence of SEQ ID NO: 1233. In some embodiments, an amino acid sequence is inserted between positions 450 and 600 of SEQ ID NO: 1233. In some embodiments, an amino acid sequence is inserted between positions 588 and 589 of SEQ ID NO: 1233. In some embodiments, the AAV capsid proteincomprises the AAV9 serotype. In some embodiments, the AAV9 serotype includes the sequence of SEQ ID NO: 1234. In some embodiments, an amino acid sequence is inserted between positions 450 and 600 of SEQ ID NO: 1234. In some embodiments, the amino acid sequence is inserted between positions 587 and 590 of SEQ ID NO: 1234. In some embodiments, the amino acid sequence inserted into the AAV2 or AAV9 serotypes comprises an amino acid sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the amino acid sequence inserted into the AAV2 or AAV9 serotypes comprises an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, insertion of the amino acid sequence set forth in SEQ ID NO: 71 into an AAV9 serotype results in the sequence set forth in SEQ ID NO: 1235 (CNSRCV300).[OHl] In some embodiments, a targeting peptide is inserted into an AAV capsid protein. Any targeting peptide described herein may be inserted into a parent AAV capsid protein in any location that results in fully functional AAV particles. The targeting peptide may be inserted into capsid proteins VP1, VP2 and / or VP3. In some embodiments, a targeting peptide, is inserted in a hypervariable region of the AAV capsid protein. Non-limiting examples of such hypervariable and / or surface exposed loop of the AAV capsid protein. In some embodiments, the targeting peptide is inserted into the Hl loop. In some embodiments, the targeting peptide is inserted into the DE loop. In some embodiments, the targeting sequencing is inserted into the variable region of the surface exposed loop, for example any of VR-I, VR-II, VR-III, VR- IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX. In some embodiments, the targeting peptide comprises any of SEQ ID NO: 1-1232. In some embodiments, the targeting peptide comprises SEQ ID NO: 71.

[0112] In some embodiments, the AAV capsid proteins described herein have enhanced tropism for a specific cell or tissue, for example, a CNS or PNS cell or tissue. In some embodiments, the enhanced tropism for a specific cell or tissue is due to the insertion of any of the sequences set forth in SEQ ID NO: 1-1232 into the AAV capsid protein. In some embodiments, the enhanced tropism for a specific cell or tissue is due to the insertion of SEQ ID NO: 71 in to the AAV capsid protein. In some embodiments, the AAV capsid proteins are capable of penetrating the blood brain barrier. In some embodiments, the AAV capsid proteins described herein are capable of penetrating the blood brain barrier due to the insertion of any of the sequences set forth in SEQ ID NO: 1-1232 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of penetrating the bloodbrain barrier due to the insertion of SEQ ID NO: 71 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions including, but not limited to the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, and hippocampus. In some embodiments, the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions due to the insertion of any of the SEQ ID NO: 1-1232 into the AAV capsid protein. In some embodiments, the AAV capsid proteins described herein are capable of distributing throughout multiple brain regions due to the insertion of SEQ ID NO: 71 into the AAV capsid protein.Pharmaceutical Compositions and Dosage Forms

[0113] Compositions described herein including CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV particles, and AAV vectors can be included in pharmaceutical compositions. In some embodiments, the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material, (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and / or (7) allow for regulatable expression of the genetic material.

[0114] The pharmaceutical compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period. For example, pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, every three years, every five years, every 10 years, every 20 years, or a one-time administration.

[0115] In some embodiments, the compositions described herein (CNS-targeting (targeting peptides), AAV capsid proteins, AAV particles, and AAV vectors) can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical. Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients. The compositions describedherein can also be formulated for injection, insufflation, infusion, or intradermal exposure. For instance, an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity. The compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.

[0116] In some embodiments, the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and / or alter the release profile.Methods of Delivery of and Treatment using the CNS-Targeting Molecules

[0117] In some embodiments, methods for introducing the compositions described herein (CNS -targeting molecules (targeting peptides), AAV capsid proteins, AAV particles, and AAV vectors) into cells and / or tissues are provided. In some embodiments, the methods comprise introducing into cells and / or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and / or protein in the cells and / or tissues.

[0118] In some embodiments, the compositions described herein are delivered via a localized delivery route. In some embodiments, the localized delivery route includes any one or more of intramuscular administration, intraparenchymal administration, and intracerebral administration, amongst others. In some embodiments, the compositions described herein are administered via a localized delivery route through a bolus infusion.

[0119] In some embodiments, the compositions described herein are administered through systemic administration. In some embodiments, systemic administration includes intravenous administration. In some embodiments, intravenous administration includes subcutaneous administration. In some embodiments, the systemic administration includes intraventricular administration.

[0120] In some embodiments, the compositions described herein are administered to the central nervous system of via intraventricular administration and / or intravenous administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the CNS-targeting molecules described herein are administered to the central nervous system via intraventricular administration.

[0121] In some embodiments the compositions can be delivered to target cell or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell or tissue includes, but is not limited to CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target cell or target tissue is a CNS cell or tissue. In some embodiments, the target cell or tissue is liver cell or tissue.

[0122] In some embodiments, the target cell includes, but is not limited to, neurons, glial cells, astrocytes, oligodendroglia, microglia, Schwann cells, ependymal cells, hepatocytes, stellate fat storing cells, Kupffer cells, liver endothelial cells, epithelial cells, cardiomyocytes, smooth muscle cells, T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.

[0123] In some embodiments, the compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via inj ection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.

[0124] In some embodiments, disclosed is a method of delivering a nucleic acid to a target cell or tissue of a subject, comprising: administering a composition comprising an AAV vector, wherein the AAV vector further comprises a capsid protein comprising a targeting peptide comprising at least 5, 6, 7, 8, or 9 contiguous amino acids of an amino acid sequence set forth in SEQ ID NO: 1-1232. In some embodiments, the capsid protein comprises the targeting peptide set forth in any one of SEQ ID NO: 1-1232.

[0125] In some embodiments, the compositions described herein are administered as part of a composition that allows for extended release. In some embodiments, the compositions comprise a formulation that includes a depot.

[0126] Disclosed herein are methods of treatment using any of the compositions described herein (CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV vectors, and AAV particles). In embodiments, the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases / disorders, and neurodegenerative disorders, amongst others. In embodiments, the disclosed compositions can be used to treat any one or more of Alzheimer's disease, Huntington's disease; autism; Parkinson's disease; Spinal muscular atrophy, Friedreich's ataxia. In embodiments, the disclosed compositions are used in treatments through any of the methods of delivery described herein.

[0127] In some embodiments, disclosed are methods for treating, or ameliorating a disease or condition associated with abnormal gene and / or protein in a subject in need of treatment, the methods comprising administering to the subject any effective amount of at least one of the compositions described herein (CNS-targeting molecules (targeting peptides), AAV capsid proteins, AAV vectors, and AAV particles), delivering the compositions described herein into targeted cells, inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject.Equivalents and Scope

[0128] The disclosure includes many equivalents to the specific embodiments described herein. A person of skill in the art will be able to ascertain equivalents to the specific embodiments, through routine experimentation.

[0129] It is assumed that words of this disclosure are for the purpose of description and not limitation. Changes to words in the claims can be made, while still retaining the scope of the disclosure in its broad aspect. Specific embodiments of the disclosure have been described herein. However, these embodiments are not intended to be limiting of the broad scope of this disclosure. While some embodiments comprise / include the disclosed features and may therefore include additional features not specifically described, other embodiments may beessentially free of or completely free of non-disclosed elements - that is, non-disclosed elements may optionally be essentially omitted or completely omitted.

[0130] The disclosure is further illustrated by the following examples, which are intended to be purely illustrative and not limiting of the disclosure herein.EXAMPLESExample l.Methods

[0131] 1.1. AAV capsid library generation

[0132] Capsid variants for library screening were synthesized as an oligo pool. Each capsid peptide was synthesized with unique nucleotide sequences encoding the peptide, and each peptide was linked to at least two distinct barcodes. The oligo pool was cloned into a linearized intermediate plasmid, followed by cloning of a constant donor sequence to separate the barcode and peptide region and generate the full AAV vector construct. Two separate constant donor sequences were used, generating the barcoded library transcript under the control of a neuron specific promoter or a ubiquitous promoter. This enables an assessment of which capsids drive the most functional mRNA expression in neurons as well as all cell types transduced. Moreover, each barcode is linked to a unique molecular identifier (UMI). Based on the overall size of the cloned library each barcode is appended to hundreds to thousands of UMIs. The utility of the UMI is to additionally assess how many distinct AAV transduction events give rise to the NGS read counts that are measured.

[0133] The peptide sequences listed in Table 1 were inserted into variable region 8 of AAV serotypes 2 and 9. Where indicated in Table 1, peptides are inserted into AAV9 (SEQ ID 1234) between 587 / 590, the insertions are after amino acid 587 and before 590 replacing amino acids 588 and 589. Other peptides inserted into AAV9 indicated in Table 1 are inserted between amino acids 588 / 589. Peptides inserted into AAV2, were inserted between positions 588 / 589 and the wildtype arginine at position 588 is altered to alanine (R588A) (Seq ID 1233).

[0134] These AAV plasmid libraries were manufactured in HEK293 cells. Briefly, libraries were produced by transient transfection including supplementation of Rep in trans, capsids were purified by cesium density centrifugation, and buffer exchanged into PBS by Amicon filtration. DNase-resistant viral genomic titers were measured by quantitative real time PCR.

[0135] 1.2. Administration of AAV libraries to cynomolgus macaques

[0136] The AAV library was administered into cynomolgus macaques as an intravenous bolus injection with a volume < 5 mL per kg of animal body weight.

[0137] 1.3. Tissue Collection and Processing

[0138] At the time of sacrifice, 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 sliced at a 4 mm coronal slice thickness. All slices were hemisected along the mid-sagittal plane. The brain slices were processed and stored according to the following table.

[0139] 1.4. Brain Tissue Collection for Molecular Analysis (mRNA and DNA)

[0140] Brain slices collected for mRNA and DNA molecular analysis (NGS) were placed in chilled RNA Later and refrigerated (1 to 8°C) for approximately 24 to 72 hours to preserve mRNA integrity. After storage, 2 mm punches were collected according to the Sponsor- provided brain template. The brain punch template is maintained in the raw data. Following removal from RNA Later and prior to obtaining the punches, the brain slices were placed on a flat surface and cross reference labels added to the brain slice and photographed (photographs are maintained with the raw data). All brain punch samples were placed into clean vials, frozen on dry ice and stored at -60°C or below until shipment. The residual brain slices, following sample collection, were frozen and stored at -60°C or below until shipment.

[0141] The residual brain slices, following sample collection, were frozen and stored at -60°C or below until shipment.

[0142] 1.5. Spinal Cord and Dorsal Root Ganglia

[0143] The spinal cord with dorsal root ganglia (DRG) attached were divided into four segments (cervical, thoracic, lumbar and sacral). From the central portion of each segment, a single 2-2.5 cm cross-sections were collected for potential evaluation. The cross-sections were placed in RNA Later and refrigerated (1 to 8°C) for approximately 24 to 72 hours after which the samples were frozen at -60°C or below until shipment. Right and left DRG pairs associated with the portion of each segment was removed and frozen (left and right saved together) on dry ice and stored at -60°C or below until shipment.

[0144] 1.6. Biodistribution-Other Tissues

[0145] An approximate 500-600 mg sample of tissue from the liver, testes, pancreas, lung, skeletal muscle (quadriceps), heart, kidney, lymph node, and spleen were collected for analysis. Samples were frozen on dry ice and stored at -60°C or below until shipment.

[0146] 1.7. Tissue Lysis and Total RNA Isolation for Analysis of Library Expression from Punches

[0147] Brain punches to be processed were placed on dry ice. For spinal cord, DRG and liver, approximately 35 mg of each tissue sample was excised on dry ice. Each tissue sample was then transferred to an Eppendorf tube pre-filled with 600 pL of TRIZOL and two 3.2 mm steel beads Sample tubes were placed into a Retsch MM300 Tissue-Lyser and 8 rounds of homogenization were performed at a frequency of 25.1 Hz for 1 1 / 2 minutes with a 2-minute pause between rounds to prevent overheating.

[0148] Isolation and purification of total RNA from homogenized tissue was performed using the MagMAX™-96 Total RNA Isolation Kit in conjunction with the KingFisher™ Flex Purification System according to the manufacturer’s protocol. Briefly, bromochloropropane (BCP) was added and then centrifugation was performed to separate the homogenate into aqueous and organic phases. The aqueous phase containing partially purified RNA was then transferred to a 96 well KingFisher processing plate. Isopropanol (100%) was added to each well followed by addition of magnetic RNA binding beads. Subsequent processing was performed on the KingFisher™ Flex Purification System. Briefly, the RNA binding beads were magnetically captured and an on-bead DNase digestion and several washes were performed. Purified RNA was eluted in 100 pL of low salt elution buffer. The KingFisher processing plate was then transferred to a magnetic stand on the benchtop. The eluants (~90 pL) were transferred away from any residual beads into a 96-well PCR plate for downstream processing. The yield and purity of the RNA was determined using a NanoDrop 8000 spectrophotometer.

[0149] 1.8. Tissue Lysis and Total RNA Isolation for Analysis of Library Expression from Brain Slices

[0150] Hemisected brain slices in cassettes to be processed were placed on dry ice. Each brain slice was removed from the cassette, weighted (up to 3 g), and placed into 50 mL Bigprep Lysing Matrix D tubes. The tubes were filled with TRIZOL Reagent at 10 mL per g of tissue. The samples were then placed in a CoolBigPrep adapter for 2x 50 mL tubes and into a FastPrep-2 Classic bead beating grinder and lysis system. The tissue samples were homogenized at 4.0 meters / second for 30 seconds with 2 minutes pause. The tissue homogenization was repeated 4 times. The lysate was centrifuged at 4,300 xg for 5 minutes at 4°C. The clarified lysate was removed to a new tube and a second round of centrifugation and removal of clarified lysate to a fresh tube was conducted. Two 5 mL aliquots were transferred to separate tubes. The remaining sample was frozen at -80°C if present. The 5 mL aliquots were processed by adding 0.2 mL per mL Trizol of molecular grade chloroform. The sample was mixed by inversion and vortexing for 30 seconds then centrifuged at 4,300 xg for 30 minutes at 4°C. The aqueous phase, approximately 50 % of the lysate volume or ~2.5 mL was removed equally to 2 mL microcentrifuge tubes. 0.5 mL per mL Trizol of molecular grade isopropanol was added to the tubes and mixed by inversion. The samples were incubated for 10 minutes on ice followed by centrifugation at 12,000 xg for 10 minutes at 4°C. The RNA pellet was resuspended in 75% ethanol at 1 mL per mL Trizol, vortexed, and centrifuged at 12,000 xg for 5 minutes at 4°C. The RNA pellet was air dried for 5 minutes and dissolved in 0.2 mL per mL Trizol with RNAse free DEPC treated water. The yield and purity of the RNA was determined using a NanoDrop 8000 spectrophotometer. Samples were spot checked for RNA integrity using the Agilent RNA 6000 Nano kit and Bioanalyzer 2100.

[0151] 1.9. Purification of mRNA from total RNA

[0152] mRNA was purified from select brain slice derived total RNA using Dynabeads mRNA Purification Kit following the manufacturer’s instructions. Samples were spot checked for RNA integrity using the Agilent RNA 6000 Nano kit and Bioanalyzer 2100.

[0153] 1.10. Reverse Transcription of total RNA and mRNA to cDNA

[0154] The maximum allowable RNA input per kit was used following the manufacturer’s protocol. QuantiTect Reverse Transcription Kit was used with a library transcript specific RT oligo using total RNA or purified mRNA at template. The manufacturer’s protocol was used.

[0155] 1.11. PCR amplification of library barcodes, Illumina NGS, and bioinformatic analysis

[0156] All cDNA from various sources were treated similarly. Vector genomes from the test articles were extracted using QIAamp MinElute Virus Spin Kit. Library barcodes were amplified using transcript specific oligos with KAPA HiFi HotStart ReadyMix. Amplificationof library specific amplicons was performed with the following cycling conditions: 98°C for 2:00 min; 25 cycles at 98°C for 15 sec; 58°C for 25 sec; 72°C for 30 sec followed by 72°C for 3 minutes. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 12 cycles of amplification: 98°C for 30 sec; 12 cycles at 98°C for 15 sec; 60°C for 25 sec, 72°C for 30 sec followed by 72°C for 10 minutes. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit kit following the manufacturer’s protocol. Samples were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2.

[0157] Following NGS of library amplicons the reads were demultiplexed and features were extracted using a custom bioinformatic pipeline. Extracted barcodes were used to query a predetermined lookup table and return the identity of the corresponding variant. Finally, the log2 fold change enrichment of each variant was normalized to its relative abundance in the administered test article.Example 2. Results from round 2 library evaluation

[0158] Previously, a first-round SIFTER (Selecting in vivo for transduction and expression of RNA) library was conducted that identified -65,000 AAV variants included in this round 2 library. The round 2 library of -65,000 variants each under the control of neuron specific human synapsin 1 (hSynl) promoter or a ubiquitous CMV promoter was administered to two NHPs. Library barcodes were recovered from cDNA reverse transcribed from total RNA extracted from CNS tissue. Approximately 14,000 variants from this round 2 SIFTER library were recovered. After filtering out variants that were poorly recovered, a total of 6,728 variants were identified from the hSynl library, and 4,005 variants from the CMV library. A total of 1,236 variants were selected for evaluation in a final selection to nominate lead capsids. Several of the variants exhibited substantial improvement over AAV9 in CNS transduction.

[0159] The fold change enrichment in each tissue sample collected was determined by NGS and normalized to the abundance in the administered test article. The average log2FC across all the tissue samples analyzed from the animals per group is shown in Table 1.Table 1.

[0160] Seq ID 1233: AAV2 R588AMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPAR KRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGN SSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNL TSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFY CLEYFP SQMLRTGNNFTF S YTFED VPFHS S YAHSQSLDRLMNPLIDQ YLYYL SRTNTP SGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATK YHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEI RTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKI PHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSV EIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0161] Seq ID 1234: AAV9MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPA KKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSS SGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPW GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANN LTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFY CLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTING SGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSW ALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEI KTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAK IPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVS VEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNLExample 3. Results from round 3 library evaluation

[0162] The 1,236 capsid variants selected for further evaluation from the round 2 library were administered to three cynomolgus macaques in a final selection to nominate lead capsids. The fold change enrichment in brain wide CNS transduction was determined by next-generation sequencing and normalized to the abundance in the administered test article. The average log2 fold enrichment across all CNS tissue samples and all cynomolgus macaques is shown in Table 2. Enrichment in neuronal mRNA expression was assessed using the neuron specific human synapsin 1 (hSynl) promoter. Enrichment in mRNA expression in all CNS cells was assessed using the ubiquitous cytomegalovirus (CMV) promoter. #N / A indicates that the capsid was not detected. Many of the capsid variants exhibited substantial improvement over AAV9 in CNS transduction. These data are plotted in FIGs. 7 and 8.Table 2.Example 4. Immunohistochemistry data showing the ability of CNSRCV300 to cross the Blood Brain BarrierBackground:

[0163] Round 2 and 3 library evaluation results highlighted the exceptional ability of the capsid CNSRCV300 to cross the blood-brain barrier and mediate transgene expression throughout the central nervous system. Based on these results, next an individual evaluation of capsid CNSRCV300 in cynomolgus macaques was conducted. CNSRCV300 was manufactured in HEK293 cells with a vector encoding the ubiquitous CAG promoter and a nuclear localized GFP reporter. CNSRCV300 was administered intravenously in three cynomolgus macaques at a dose of 2E13 vector genomes per kilogram.Methods:

[0164] Brain tissue was collected at necropsy on Day 19 after intravenous administration of CNSRCV300. Brain slices of 4 mm thickness were designated for immunohistochemistry evaluation and placed in labelled cassettes, immersion fixed in 10% paraformaldehyde, refrigerated at 4° C for 16-24 hrs, transferred to phosphate buffered saline (lx PBS) with 0.01% sodium azide, and refrigerated at 4°C until processing.I l l

[0165] To prepare brain slices for processing, brains were treated overnight with 20% glycerol and 2% dimethyl sulfoxide to avoid freeze artifacts. Slices were embedded in a gelatin matrix. Blocks were rapidly frozen, after curing by immersion in 2-methylbuteane chilled with crushed dry ice and mounted on a freezing stage of a AO 860 sliding microtome.

[0166] Blocks were sectioned coronally at 40 pM thickness on the microtome. All sections were cut through the entire length of the specimen segment and collected sequentially into a series of 24 containers, pre-filled with Antigen Preserve solution (50% PBS, pH 7.0, 50% ethylene glycol, 1% polyvinyl pyrrolidone).

[0167] For IHC revealing GFP, free floating sections were stained. All incubation solution from the blocking serum onward used TRIS-buffered saline (TBS) with Triton-X-100 as the vehicle; all rinses were with TBS. After a hydrogen peroxide treatment and blocking serum, the sections were immune-stained with the primary antibody (Rabbit monoclonal anti-GFP antibody; 1 :50; Cat # G10362) overnight at room temperature. Vehicle solutions contained Triton-X-100 for permeabilization. Following rinses, a biotinylated secondary antibody (antirabbit IgG) was applied. After further rinses, ABC solution (avidin-biotin-HRP complex; VECTASTAIN® Elite ABC, Vector Labs, Burlingame, CA) was applied. The sections were again rinsed, then treated with diaminobenzidine tetrahydochloride (DAB) and Nickel sulphate to create a visible (black) reaction product. Following further rinses, the sections were mounted on gelatin-coated glass-slides and air dried.

[0168] FIGs. 15-24 show that capsid CNSRCV300 is able to cross the blood-brain barrier and mediate transgene expression throughout the central nervous system.

[0169] Seq ID 1235: CNSRCV300MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP GNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPA KKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSS SGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPW GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANN LTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFY CLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTING SGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSW ALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEI KTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQDRDVY LQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

Claims

WHAT IS CLAIMED IS:

1. An adeno-associated virus (AAV) capsid protein comprising at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 1-1232.

2. The AAV capsid protein of claim 1, wherein the amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 71 (YVNIMDDMD)3. The AAV capsid protein of claim 1 or claim 2, wherein the AAV capsid protein comprises serotype AAV2.

4. The AAV capsid protein of claim 3, wherein the serotype AAV2 comprises SEQ ID NO: 1233.

5. The AAV capsid protein of claim 4, wherein the amino acid sequence is inserted into the AAV2 capsid protein between amino acids 588 and 589.

6. The AAV capsid protein of claim 1 or claim 2, wherein the AAV capsid protein comprises serotype AAV9.

7. The AAV capsid protein of claim 6, wherein the serotype AAV9 comprises a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234.

8. The AAV capsid protein of claim 7, wherein the amino acid sequence is inserted into the AAV9 capsid protein between amino acids 587 and 590.

9. The AAV capsid protein of claim 6, wherein the AAV capsid protein comprises a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1235.

10. The AAV capsid protein of claim 9, wherein the AAV capsid protein comprises the sequence set forth in SEQ ID NO: 1235.

11. The AAV capsid protein of claim 1, wherein the AAV capsid protein comprises an amino acid sequence set forth in any one of SEQ ID NO: 1-1232.

12. The AAV capsid protein of claim 2, wherein the AAV capsid protein comprises an amino acid sequence set forth in SEQ ID NO: 71 (YVNIMDDMD).

13. The AAV capsid protein of claim 1 or claim 2, wherein the amino acid sequence is inserted into any of the parental capsids AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74.

14. The AAV capsid protein of claim 13, wherein the amino acid sequence is inserted into any of the parental capsids AAV2 or AAV9.

15. The AAV capsid protein of claim 1, wherein the amino acid sequence is inserted into a surface exposed region of the AAV capsid.

16. An AAV capsid protein, comprising at least 3, 4, 5, 6, 7, 8, 9 or all contiguous amino acids of an amino acid sequence set forth in any of SEQ ID NO: 1-1232, wherein the amino acid sequence is inserted between amino acid positions 450 and 600 of the AAV capsid protein.

17. The AAV capsid protein of claim 16, wherein the amino acid sequence is inserted between amino acid positions 588 and 589 of the AAV capsid protein.

18. The AAV capsid protein of claim 17, wherein the AAV capsid protein comprises a sequence having at least 80% sequence identity to the sequence forth in SEQ ID NO: 1233.

19. The AAV capsid protein of any of claims 16-18, wherein the amino acid sequence comprises the sequence set forth in SEQ ID NO: 71 (YVNIMDDMD).

20. The AAV capsid protein of claim 16, wherein the amino acid sequence is inserted between amino acid positions 587 and 590 of the AAV capsid protein.

21. The AAV capsid protein of claim 20, wherein the AAV capsid protein comprises a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1234.

22. The AAV capsid protein of claim 20 or claim 21, wherein the amino acid sequence comprises SEQ ID NO: 71 (YVNIMDDMD).

23. The AAV capsid protein of claim 19, wherein the AAV capsid protein comprises a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1235.

24. The AAV capsid protein of claim 23, wherein the AAV capsid protein comprises the sequence set forth in SEQ ID NO: 1235.

25. A CNS-targeting molecule, comprising at least 3, 4, 5, 6, 7, 8, 9, or all contiguous amino acids of an amino acid sequence set forth in any of SEQ ID NO: 1-1232.

26. The CNS-targeting molecule of claim 25, wherein the amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 71 (YVNIMDDMD).

27. A CNS-targeting molecule, of claim 25 or claim 26, wherein the amino acid sequence is fused or conjugated to a small molecule, antibody, scFV, ASO, siRNA, lipid, polymer, or recombinant protein.

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