Compositions and methods for enhanced CNS delivery through a novel protein interaction

Abstract

This application relates to compositions and methods for enhanced CNS delivery. In some embodiments, the compositions and methods comprise targeting moieties or molecules, for example, as used in or as a part of engineered AAV capsids, that bind or interact with CBLN1 to mediate delivery of a therapeutic or diagnostic agent throughout the CNS.

Description

[0001] Ref No.: P.0287. WO 1

[0002] COMPOSITIONS AND METHODS FOR ENHANCED CNS DELIVERY THROUGH A NOVEL PROTEIN INTERACTION

[0003] FIELD

[0004] This application relates to compositions and methods for enhanced CNS delivery via an interaction with CBLN1 (Cerebellin-1) protein, or with a complex of cerebellins optionally comprising CBLN1.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 720,687, filed on November 14, 2024 and entitled “COMPOSITIONS AND METHODS FOR ENHANCED CNS DELIVERY THROUGH A NOVEL PROTEIN INTERACTION” the entire contents of which are incorporated by reference herein.

[0007] BACKGROUND

[0008] The clinical translation of genomic medicines to treat disorders of the central nervous system (CNS) has been limited by inefficient gene delivery.

[0009] 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 generating compositions that exhibit enhanced delivery of genetic material of interest to a target cell or tissue, e.g., a CNS cell or tissue, including delivery across the blood-brain barrier (BBB).

[0010] One approach to achieve this goal is to engineer compositions, such as novel AAV capsids, that interact with proteins that are expressed in the CNS and involved in BBB transcytosis. STAC-BBB was previously identified as a novel AAV capsid variant (CNSRCV300 in PCT Application No. PCT / US2024 / 029507) that exhibits enhanced CNS delivery after intravenous administration. STAC -BBB is derived from the parental serotype AAV9.

[0011] There remains a need to characterize STAC-BBB binding to targets that enable enhanced CNS delivery.

[0012] 1

[0013] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0014] SUMMARY

[0015] The present disclosure provides a composition comprising a targeting molecule that binds or interacts with cerebellin-1 (CBLN1), or binds to or interacts with a complex of cerebellins optionally comprising CBLN1, wherein the targeting molecule enables enhanced delivery of the composition to the central nervous system (CNS), and further optionally wherein the targeting molecule is associated with a therapeutic or diagnostic agent. In some embodiments, the targeting molecule comprises a polymer, optionally wherein the targeting molecule is a polypeptide. In some embodiments, the targeting molecule is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, the composition comprises an AAV capsid. In some embodiments, the composition encapsidates a nucleotide encoding a therapeutic or diagnostic agent, optionally wherein the composition was derived from a parental AAV capsid protein. In some embodiments, the parental AAV capsid protein is selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74. In some embodiments, the composition encapsidates a nucleotide encoding a therapeutic or diagnostic agent in a STAC-BBB AAV capsid, optionally wherein the STAC-BBB AAV capsid is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO.l. In some embodiments, the targeting molecule binds or interacts with human, cynomolgus, or murine CBLN1. In some embodiments, CBLN1 acts as a receptor to mediate BBB transcytosis. In other embodiments, the present disclosure provides a method of delivering a therapeutic or diagnostic agent to the CNS, comprising fusing or conjugating the therapeutic or diagnostic agent to a targeting molecule described herein. In other embodiments, a method of delivering a therapeutic or diagnostic agent to the CNS is provided, comprising encapsidating a nucleic acid encoding the therapeutic or diagnostic agent in a capsid protein described herein.

[0016] In another aspect, an engineered adeno-associated virus (AAV) capsid protein is provided, wherein the capsid protein binds or interacts with CBLN1, or binds to or interacts with a complex of cerebellins optionally comprising CBLN1, and wherein the capsid protein transduces the CNS. In some embodiments, the capsid protein is a STAC-BBB AAV capsid, optionally wherein the STAC-BBB AAV capsid is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO.l. In

[0017] 2

[0018] 4896-3717-6698 Ref No.: P.0287. WO 1

[0019] other embodiments, a method of delivering a therapeutic or diagnostic agent to the CNS is provided, comprising fusing or conjugating the therapeutic or diagnostic agent to, or encapsidating a nucleic acid encoding the therapeutic or diagnostic agent in, a capsid protein described herein. In another aspect, a fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4, is provided, wherein the cerebellin enables enhanced delivery of the fusion protein to the central nervous system (CNS), and optionally wherein the cerebellin molecule is associated with a therapeutic or diagnostic agent.

[0020] In another aspect, a composition is provided, comprising: i) A) a cerebellin, optionally cerebellin 1, 2, 3, and / or 4; or B) a fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4; and, ii) a cerebellin targeting molecule, optionally wherein the cerebellin or the targeting molecule is associated with a therapeutic or diagnostic agent. In another aspect, a composition comprising a CBLN 1 protein and a CBLN 1 targeting molecule is provided, optionally wherein the CBLN1 targeting molecule is associated with a therapeutic or diagnostic agent. In some embodiments, an AAV capsid variant comprises the CBLN1 targeting molecule. In some embodiments, a STAC-BBB AAV capsid comprises the CBLN1 targeting molecule, optionally wherein the STAC-BBB AAV capsid is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO.l. In some embodiments, the CBLN1 targeting molecule is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, the CBLN1 targeting molecule binds or interacts with human, cynomolgus, or murine CBLN1. In some embodiments, the AAV capsid variant or the STAC-BBB AAV capsid encapsidates a nucleic acid encoding a therapeutic or diagnostic agent. In some embodiments, the AAV capsid variant is derived from a parental capsid selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74. In some embodiments, the composition provides for increased CNS delivery of the CBLN1 targeting molecule compared to administering the CBLN1 targeting molecule without the CBLN1 protein. In other embodiments, a method of delivering a therapeutic or diagnostic agent to the CNS is provided, comprising administering a composition described herein to a subject.

[0021] 3

[0022] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0023] In another aspect, a method for identifying an AAV capsid variant with increased transduction of the CNS is provided, comprising selecting for AAV capsid variants that bind or interact with CBLN1, or binds to or interacts with a complex of cerebellins optionally comprising CBLN1. In some embodiments, the method comprises modifying one or more amino acids within a hypervariable and / or surface exposed loop of a selected AAV capsid variant to create a modified AAV capsid variant, optionally wherein the modified AAV capsid variant is further screened for improved transduction of the CNS.

[0024] In another aspect, a method for identifying a targeting molecule that confers increased transduction of the CNS within a pool of compositions is provided, comprising selecting for compositions that bind or interact with CBLN1, or binds to or interacts with a complex of cerebellins optionally comprising CBLN1. In some embodiments, the targeting molecule is part of an AAV capsid variant.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 In vitro protein pulldown of STAC-BBB with human, cyno, or mouse CBLN1.

[0026] Human, cynomolgus macaque, or mouse CBLN1 were produced as Fc fusions in HEK293 cells and immobilized on Protein A magnetic beads. Purified STAC-BBB or AAV9 were incubated with CBLN1-FC coated magnetic beads. Beads were subjected to multiple washes to remove non-specifically bound proteins. SDS PAGE analysis was conducted and the integrated volume of capsid protein (VP1 / VP2 / VP3) pulled down by CBLN1 was quantified by densitometry analysis and normalized to the loading control. Human, cyno, and mouse orthologs of CBLN1 specifically bind the STAC-BBB capsid, but not the parental serotype AAV9.

[0027] FIG. 2 Overexpression of CBLN1 in HEK293 cells confers a gain-of-function for STAC-BBB transduction as assessed by RT-qPCR.

[0028] The figure shows AAV transgene expression normalized to transfection control for HEK293 cells over expressing cyno, human, or mouse CBLN1 tethered to the extracellular membrane. Purified STAC-BBB or AAV9 expressing mCherry under the control of the CMV promoter was added to cells at different multiplicity of infections (MOI) ranging from le3 to le5 AAV particles per cell. mCherry transgene expression was quantified by RT-qPCR and normalized to endogenous

[0029] 4

[0030] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0031] housekeeping gene, GAPDH, 72 hours post-transduction. Both STAC-BBB and AAV9 exhibit a dose-response in normalized transgene expression. However, only STAC-BBB displays an increase in transgene expression relative to the transfection control in cells expressing cyno, human, or mouse CBLN1. As expected, the mock transduction control shows no transgene expression.

[0032] FIG. 3 Fold change increase in transgene expression is observed for STAC-BBB in HEK293 cells overexpressing tethered CBLN1.

[0033] The figure shows the fold expression increase for STAC-BBB and AAV9 normalized to transfection control for HEK293 cells over expressing cyno, human, or mouse CBLN1 tethered to the extracellular membrane. Purified STAC-BBB or AAV9 expressing mCherry under the control of the CMV promoter was added to cells at different multiplicity of infections (MOI) ranging from le3 to le5 AAV particles per cell. mCherry transgene expression was quantified by RT-qPCR and a nonlinear regression model was applied to interpolate relative transgene expression values for each MOI. These values were then scaled to the transfection control value for each capsid and the midpoint was used to calculate the fold difference in transgene expression. STAC-BBB displays a 65.5 to 88.4 fold increase in transgene expression depending upon the ortholog tested. AAV9 displays no increase in transgene expression relative to the transfection control.

[0034] FIG. 4 Overexpression of CBLN1 in HEK293 cells confers a gain-of-function for STAC-BBB transduction as assessed by immunofluorescent microscopy.

[0035] The figure shows detection of native mCherry fluorescence after transduction of HEK293 cells overexpressing human, cyno, or mouse CBLN1 tethered to the extracellular membrane. Purified STAC-BBB or AAV9 expressing mCherry under the control of the CMV promoter was added to cells at a multiplicity of infection (MOI) of le5 AAV particles per cell. An increase in mCherry fluorescence was observed for STAC-BBB, but not AAV9, in cells overexpressing human, cyno, or mouse CBLN 1.

[0036] FIG. 5 Bio-Layer Interferometry (BLI) affinity measurements demonstrate STAC-BBB binds directly to CBLN1 with high affinity.

[0037] 5

[0038] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0039] The figure shows the Octet BLI sensorgrams of a concentration series of STAC-BBB and AAV9 binding to biotinylated CBLN1 immobilized on streptavidin biosensor tips. In this assay format, STAC-BBB bound immobilized CBLN1 with an ~11 pM dissociation constant (KD) while no binding to CBLN1 was observed for AAV9.

[0040] FIG. 6(A)-(G) shows Tables 1, 2, 3, 4, 5, 6, and 7.

[0041] DETAILED DESCRIPTION

[0042] In some embodiments, the present invention relates to compositions and methods for enhanced CNS delivery. In some embodiments, the compositions and methods comprise targeting moieties or molecules, for example as used in engineered AAV capsids, that bind or interact with CBLN1 to mediate delivery of a therapeutic or diagnostic agent throughout the CNS.

[0043] Cerebellin

[0044] Cerebellin is a family of proteins that function as synaptic organizers in the cerebellum and other parts of the brain. There are four family members, CBLN1, CBLN2, CBLN3 and CBLN4, that form complexes with other proteins to control both excitatory and inhibitory neurotransmission. Cerebellin-1 (CBLN1) is a secreted glycoprotein of the Clq / TNF superfamily that is released by neurons and operates in trans-synaptic complexes. Secreted CBLN1 is detectable in human plasma and serum. CBLN1 is proteolytically processed into a mature form containing an N-terminal cysteine-rich linker and a C-terminal Clq-like globular domain that assembles into trimers which dimerize via disulfides to form hexamers. Other CBLN family members include CBLN2, CBLN3, and CBLN4. Each of these family members are secreted Clq domain proteins that assemble as trimers and form homohexamers. In addition to forming homohexamers, heteromeric complexes can form amongst CBLN family members (Wei et al., 2012, Wei et al., 2013, and Rong et al., 2019). For example, CBLN1 can form heteromultimers with CBLN3 (Bao et al., 2005, Bao et al., 2006, lijima et al., 2007).

[0045] In some embodiments, cerebellin-1 (CBLN1), or a complex of cerebellins optionally comprising CBLN1, enables enhanced delivery of a composition to the central nervous system (CNS), further

[0046] 6

[0047] 4896-3717-6698 Ref. No.: P0287.WO1

[0048] optionally wherein the composition is associated with a therapeutic or diagnostic agent. In some embodiments, the composition comprises an AAV capsid. In some embodiments, ‘associated with a therapeutic or diagnostic agent’ comprises covalent links or associations, including both cleavable and non-cleavable links or associations. In some embodiments, ‘associated with a therapeutic or diagnostic agent’ comprises covalent links or associations that are peptide or nonpeptide based. In some embodiments, ‘associated with a therapeutic or diagnostic agent’ comprises non-covalent associations. In some embodiments, the complex of cerebellins further comprise(s) cerebellin 2, 3 and / or 4. In some embodiments, cerebellin-1 (CBLN1), or a complex of cerebellins optionally comprising CBLN1, is directly fused or conjugated to a small molecule, antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer, or recombinant protein. In some embodiments, cerebellin-1 (CBLN1), or a complex of cerebellins optionally comprising CBLN1, is co-delivered with a small molecule, antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer, or recombinant protein.

[0049] In some embodiments, a method of enhanced CNS or BBB delivery comprises directly fusing, conjugating, associating, or co-delivering cerebellin-1 (CBLN1), or a complex of cerebellins optionally comprising CBLN1, to or with another composition, for example, an AAV capsid, a small molecule, antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer, or recombinant protein.

[0050] Identifying Proteins that Bind Cerebellin

[0051] In some embodiments, disclosed herein are proteins that bind or interact with cerebellin 1, 2, 3, and / or 4, and methods for generating or identifying such proteins. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are AAVs. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are peptides. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are small protein fragments. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are antibodies. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are nanobodies or single-domain antibodies. In some embodiments, the proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 are single-chain variable fragments, scFv, or Fab. In some

[0052] 7

[0053] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0054] embodiments, the antibodies that bind or interact with cerebellin 1, 2, 3, and / or 4 have altered CDR loops.

[0055] In some embodiments, a library is generated comprising proteins that may bind or interact with cerebellin 1, 2, 3, and / or 4. In some embodiments, the libraries are phage display libraries. In some embodiments, the libraries are yeast display libraries. In some embodiments, such a library is screened to identify proteins that bind or interact with cerebellin 1, 2, 3, and / or 4 by pulldown assays in vitro.. In some embodiments, proteins that may bind or interact with cerebellin 1, 2, 3, and / or 4 are designed in silico using de novo sequences. In some embodiments, the molecules designed in silico may be screened in vitro. In some embodiments, proteins that may bind or interact with cerebellin 1, 2, 3, and / or 4 are screened by biolayer interferometry (BLI) to measure binding affinity.

[0056] Generation of an Engineered AAV Capsid Library

[0057] In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins, wherein members of the library encode engineered AAV capsid proteins having different sequences, and wherein some members of the library encode an AAV capsid protein having a desired characteristic compared to a natural / wild-type AAV serotype or patent serotype. In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins with a desired characteristic compared to a parent capsid. Thus, described herein are libraries of AAV capsid proteins with a desired characteristic compared to a parent capsid. In some embodiments, the desired characteristic is enhanced cell or tissue tropism as compared to the parent capsid, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the parent capsid. In some embodiments, the desired characteristic comprises binding to and / or interacting with cerebellin 1, 2, 3, and / or 4. 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

[0058] 8

[0059] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0060] embodiments, the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0061] In some embodiments, each member of a library comprises one or more of a) a nucleic acid sequence encoding an AAV capsid protein comprising an engineered variant AAV sequence; b) a nucleic acid sequence encoding barcode; c) nucleic acid sequence(s) encoding a promoters); d) a nucleic acid sequence encoding a unique molecular identifier (UMI); and combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to and expressed in a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence.

[0062] In some embodiments, each engineered AAV capsid protein is synthesized as an oligo pool. In some embodiments, each member of a library comprises one or more (such as 1-10) of a nucleic acid sequence encoding an AAV capsid protein comprising a) nucleic acid sequences encoding one or more (such as 1-10) barcodes: b) nucleic acid sequences encoding one or more (such as 1-10) promoters; c) nucleic acid sequences encoding one or more (such as 1-10,000) unique molecular identifiers (UMIs); or combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence. In some embodiments, each of the one or more (such as 1-10) barcodes is linked to the identity of a single engineered AAV capsid protein. In some embodiments, each of the barcodes is linked to one or more (such as 1-10,000) UMIs.

[0063] In some embodiments, a nucleic acid comprising a barcode is added to the genome of each AAV capsid in a library. In some embodiments, a unique barcode is bioinformatically linked to each different variant sequence that is represented within the library, for example, each different variant AAV sequence. In some embodiments, the DNA sequences encoding an AAV variant sequence are synthesized to further comprise a random or specified barcode. The barcode may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides. In some embodiments, each AAV variant sequence is linked to at least 2 distinct barcodes. In some embodiments, each barcode is linked to one or more (such as 1-10,000) UMIs.

[0064] 9

[0065] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0066] In some embodiments, each member of the library comprises a nucleic acid comprising more than one barcode sequences (such as 1-10). In some embodiments, each member of the library comprises two or more nucleic acids (such as 1-10) each comprising a barcode sequence. In some embodiments, each member of the library comprises a first nucleic acid comprising a first barcode and a second nucleic acid comprising a second barcode. In some embodiments, the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode are different. In some embodiments, each of the first nucleic acid comprising the first barcode and the second nucleic acid comprising the second barcode is independently operatively linked to a promoter. In some embodiments, each capsid is linked to at least one unique barcode. In some embodiments, each capsid is linked to at least two unique barcodes using a bioinformatic look-up table. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the neuron specific human Synapsin 1 promoter. In some embodiments, capsid performance is evaluated based on barcoded mRNA expression from the ubiquitous CMV promoter.

[0067] In some embodiments, libraries are created encoding engineered AAV capsid proteins that comprise at least one mutation relative to a parent capsid, for example, the parent capsid being AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:1). In some embodiments, the engineered AAV capsid proteins contain a peptide sequence inserted within a parent capsid protein, for example, the parent capsid AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:1). In some embodiments, the engineered AAV capsid proteins contain a peptide sequence inserted within a surface exposed loop of a parent capsid protein, for example, the parent capsid AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), STAC-BBB (SEQ ID NO:1).

[0068] 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 AAV rep function comprises rep78, rep 68, rep 52, and rep40 genes. In some embodiments, the rep genes are supplied in trans. In some embodiments, the start codon of the rep78 and / or the rep68 gene is altered from

[0069] 10

[0070] 4896-3717-6698 Ref No.: P.0287. WO 1

[0071] ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield. In some embodiments, the cap genes are supplied as genetic material that is packaged into the manufactured AAVs. In some embodiments, the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.

[0072] Methods of Screening Libraries of Engineered AAV Capsid Proteins

[0073] 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 an immobilized receptor protein (e.g., cerebellin 1, 2, 3, and / or 4 protein), a cell, a 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 immobilized receptor protein, cell, cell line, or tissue the AAV variant; and (iv) identifying the engineered AAV capsid protein with the desired characteristic.

[0074] In another embodiment, disclosed herein are methods for directed evolution of engineered AAV capsid proteins and identification of an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, a desired characteristic is binding to or interacting with cerebellin 1, 2, 3, and / or 4. In some embodiments, the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype comprise (i) modifying the natural / wild-type AAV serotype to create variant capsids; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where variants are isolated from a target tissue or cell line; and (viii) full evaluation of enriched variants. In some embodiments, the desired characteristic includes enhanced tissue tropism as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the

[0075] 11

[0076] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0077] 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.

[0078] Engineered AAV Capsid Proteins

[0079] In one embodiment, described herein are compositions comprising engineered AAV capsid proteins and methods of making and using the same. In some embodiments, the engineered AAV capsid proteins interact with a receptor, e.g., cerebellin 1, 2, 3, and / or 4. In some embodiments, the engineered AAV capsid proteins demonstrate binding to a receptor, e.g. cerebellin 1, 2, 3, and / or 4. The engineered AAV capsid proteins may be delivered to one or more of target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid protein has enhanced tropism for a cell or tissue, e.g., for the delivery of genetic material to a specific cell or tissue, for example a CNS tissue or a CNS cell, or cells and tissues of a muscle. The engineered AAV capsid proteins may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ. As a non-limiting example, the engineered AAV capsid proteins that are desired to have tropism for CNS cells may have enhanced tropism for neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, Schwann cells, and reduced tropism for liver and dorsal root ganglion.

[0080] In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of a peptide sequence inserted within a parent capsid protein. In some embodiments, the peptide sequence is inserted within or near a surface-exposed loop of the parent capsid protein. In a preferred embodiment, the parent capsid is AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:1). In some embodiments, the peptide sequence is inserted within or near amino acids 453 through 454 corresponding to the sequence of AAV1 (SEQ ID NO: 3). In some embodiments, the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV1 (SEQ ID NO: 3). In some embodiments the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 5). In some embodiments the peptide sequence is inserted within or near amino acids 453 through 454 corresponding to the sequence of

[0081] 12

[0082] 4896-3717-6698 Ref. No.: P.0287. WO 1

[0083] AAV6 (SEQ ID NO: 4). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 4). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 6). In some embodiments the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV9 (SEQ ID NO: 2). In some embodiments, an engineered AAV capsid protein is provided wherein the capsid is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a parent sequence outside the inserted peptide sequence, for example wherein the parent capsid is AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:1). In some embodiments, percent identity for amino acid sequences may be defined as the ratio of the number of identical amino acid residues between two aligned sequences to the total length of the alignment, expressed as a percentage

[0084] In some embodiments, an amino acid sequence is inserted into a parent capsid ("Parent Capsid”) at an insertion site (“Peptide Insertion Site”). In some embodiments, the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7. In some embodiments, an engineered AAV capsid protein is provided comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7. In some embodiments, the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 7, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row.

[0085] 13

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[0087] In some embodiments, an amino acid sequence is inserted into a parental capsid, optionally wherein the parental capsid is selected from any one of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB. In some embodiments, the inserted amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7.

[0088] In some embodiments, the engineered AAV capsid proteins have advantages over wild-type AAV capsid proteins. In some embodiments, these advantages include (i) enhanced cell or tissue tropism as compared to the natural / wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural / wild-type AAV serotype (ii) increased penetrance through the blood brain barrier following administration to a subject, (iii) wider distribution throughout the multiple brain regions, for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus, (iv) elevated expression of genetic material in multiple brain regions. In some embodiments, the engineered AAV capsids enhance the delivery of genetic material to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus, (v) is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0089] In embodiments, the engineered AAV capsid proteins and genetic material described herein may be delivered to one or more (such as 1-10) target cells, tissues, organs, or organisms. In some embodiments, the engineered AAV capsid proteins have enhanced tropism for a specific target cell type, tissue or organ. As a non-limiting example, the engineered AAV capsid protein has enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, engineered AAV capsid proteins are produced recombinantly and are an adeno-associated virus (AAV) serotype such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, AAVrh74, or STAC-BBB, or a combination thereof. In some embodiments, engineered AAV capsid proteins

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[0092] are produced recombinantly and are based on any one or more (such as 1-15) AAV serotypes known in the art.

[0093] Adeno-associated virus (AAV)

[0094] AAV are capable of infecting a wide range of cells including quiescent cells and dividing cells. In some embodiments, AAV can be modified so that it contains the components necessary for the assembly of a functional recombinant virus or viral particle. In some embodiments, the AAV is engineered to interact with a specific receptor, e.g., cerebellin 1, 2, 3, and / or 4. In some embodiments, the AAV is engineered to target a specific tissue and / or cell, for example, CNS tissue and / or cell. In some embodiments, the AAV is engineered to deliver specific genetic material to a tissue and / or cell. In some embodiments, the AAV is engineered to target a blood brain barrier receptor, for example, cerebellin 1, 2, 3, and / or 4.

[0095] Modified AAV Serotypes

[0096] In some embodiments, an engineered AAV may be based on any natural or recombinant AAV serotype. Different AAV serotypes have different characteristics such as different packaging, tropism, and transduction profiles. In some embodiments, the engineered AAV capsid proteins are based on a wild-type AAV serotype. In some embodiments, the AAV serotype comprises AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the AAV serotype comprises less well-characterized AAV serotypes such as AAV3, AAV4, AAV7, AAV11, AAVrhlO, AAVrh39, or AAVrh74. In some embodiments, the AAV serotype is an engineered AAV serotype such as STAC-BBB. In some embodiments, the engineered AAV capsid protein is derived from multiple AAV serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes. In some embodiments, AAV variant capsid proteins derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more AAV serotypes are combined to create chimeric capsids. In some embodiments, combinatorial libraries are generated by modifying nucleic acids encoding AAV capsid proteins from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes in the same pool.

[0097] In some embodiments, different AAV serotypes are different in their ability to direct or modulate an AAV particle to a particular cell or tissue. In some embodiments, the AAV serotype can be

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[0100] modified to interact with a receptor, e.g. cerebellin 1, 2, 3, and / or 4. In some embodiments, the AAV serotype modified to interact with a receptor has an altered tropism. In some embodiments, the AAV serotype can be modified to increase the tropism of the AAV particle to cells or tissues of the central nervous system (CNS). In some embodiments, the AAV serotype can be modified to increase tropism of the AAV particle to cells or tissues of the peripheral nervous system (PNS).

[0101] In some embodiments, the modified AAV serotype has a desired characteristic compared to a parental AAV serotype. In some embodiments, the modified AAV serotype allows for increased penetration of the blood brain barrier following administration to a subject. In some embodiments, the modified AAV serotype causes increased biodistribution to a brain region. In some embodiments, the brain region comprises the frontal cortex, the sensory cortex, the motor cortex, the cerebellar cortex, the hippocampus, the thalamus, or the putamen. In some embodiments, the brain comprises any brain region known in the art. In some embodiments, the modified AAV serotype causes increased biodistribution to more than one brain region, for example, 2 brain regions, 3 brain regions, 4 brain regions, 5 brain regions, 6 brain regions, 7 brain regions, 8 brain regions, 9 brain regions, or 10 brain regions. In some embodiments, the modified AAV serotype causes increased biodistribution to 1- 10 brain regions. In some embodiments, the modified AAV serotype are useful in elevating genetic material expression in multiple brain regions. In some embodiments, the modified AAV serotype are used to deliver genetic material of interest to a desired tissue, cell, or organelle.

[0102] In some embodiments, the modified AAV serotype causes increased biodistribution to regions of the spinal cord. In some embodiments, the region of the spinal cord comprises any of the thoracic spinal cord region, the lumbar spinal cord region, and / or the cervical spinal cord region. In some embodiments, the region of the spinal cord includes any region of the spinal cord known in the art.

[0103] In some embodiments, the modified AAV serotype comprises an inserted sequence having at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of a Peptide Sequence shown in Tables 1 -7. In some embodiments, the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7. In some embodiments,

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[0106] the modified AAV serotype comprises an inserted peptide sequence described herein (e g., a Peptide Sequence shown in Tables 1-7).

[0107] Structure of AAV

[0108] In some embodiments, the genome of the AAV comprises a single-strand DNA (ssDNA) molecule that is approximately between about 2.5 kb and about 5.0 kb in length. In some embodiments, the genome of the AAV comprises a self-complementary DNA (scDNA) molecule that is approximately between about 0.5 kb and about 2.5 kb in length. In some embodiments, the AAV genome contains inverted terminal repeats (ITRs) that flank the 5’ and 3’ ends of the AAV molecule. In some embodiments, the ITRs contain origins of replication for the viral genome. In some embodiments, the length of the ITRs is about 145 bp in length, for example, between about 130 bp in length and 160 bp in length.

[0109] In some embodiments, the AAV genome comprises the rep and cap genes. In some embodiments, the AAV genome nucleotide includes nucleotide sequences that encode four non-structural Rep proteins (Rep 78, Rep68, Rep52, Rep40, encoded by Rep genes). In some embodiments, the AAV viral genome includes nucleotide sequences that encode the three capsid, or structural, proteins (i.e., VP1, VP2, VP3, encoded by the cap gene). In some embodiments, the rep proteins are used for replication and packaging. In some embodiments, the capsid proteins are assembled to create the protein shell of the AAV.

[0110] AAV Particles

[0111] In some embodiments, the engineered AAV capsid proteins assemble to form AAV particles. In some embodiments, the engineered AAV capsid proteins interact with a receptor expressed at the blood brain barrier, for example, cerebellinl, 2, 3 and / or 4. In some embodiments, the AAV particles that have enhanced tropism for a target tissue (e.g., CNS and PNS) are provided. In some embodiments, the AAV particles include engineered AAV variant sequences that alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro. In some embodiments, the AAV particles are capable of penetrating the blood brain barrier.

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[0114] Delivery of AAV Particles

[0115] 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.

[0116] In some embodiments, the AAV particles can be used to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating. In some embodiments, the AAV particles are used to deliver any cargoes of interest, or example, therapeutic cargoes.

[0117] AAV Viral Genomes

[0118] In some embodiments, the AAV particles are used to deliver a viral genome (i.e., a genetic payload) to a tissue or cells such as CNS or PNS cell or tissue.

[0119] The delivered viral genome may include genetic material of interest, such as, for example, genetic material that encodes an engineered DNA recombinase protein, a fusion protein comprising a DNA-binding domain (e.g., a zinc finger or a TALE protein) fused to a functional domain (e.g., to modulate DNA function or to cleave DNA), an antibody, an enzyme, regulatory RNA, a CRISPR protein, or a cDNA, amongst others. In some embodiments, the viral genome includes 2 ITR sequences. In some embodiments, the ITR sequences flank the genetic material of interest. In some embodiments, the ITR sequences are complementary to each other. In some embodiments, the ITR sequences are not complementary to each other. In some embodiments, one ITR sequence is a self-complementary ITR. In some embodiments, the ITR regions are derived from the same serotype as the capsid protein. In some embodiments, the ITR regions are derived from AAV2 serotype. In some embodiments, the ITR regions are derived from a serotype known to the art. ITR regions may be between 100 and 150 nucleotides in length.

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[0122] Targeting Peptide Sequences

[0123] In embodiments, targeting peptide sequences (e.g., targeting molecules, targeting moi eties, or targeting sequences) are disclosed herein. In some embodiments, the sequences enhance or enable interaction with cerebellin 1, 2, 3, and / or 4. In some embodiments, the sequences can function to target a cell or a tissue, for example, as a general CNS-targeting molecule or sequence.

[0124] In embodiments, the targeting sequences function as a general CNS-targeting molecule sequence. In some embodiments, the CNS-targeting sequence is fused or conjugated to a small molecule, a polypeptide, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, any one of the targeting sequences described herein may be fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, CNS-targeting sequences may be utilized to enable a small molecule, an antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.

[0125] In some embodiments, the targeting sequences are part of an engineered AAV capsid protein. In some embodiments, the engineered AAV capsid protein is any engineered AAV capsid protein disclosed herein.

[0126] In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor, e.g., cerebellin 1, 2, 3, and / or 4. In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor expressed at the blood brain barrier, e.g., cerebellin 1, 2, 3, and / or 4. In some embodiments, the targeting sequences modulate the binding affinity of an AAV capsid protein to a specific receptor, e.g., cerebellin 1, 2, 3, and / or 4.

[0127] In some embodiments, the sequences may increase tropism of an AAV capsid protein to a cell or tissue of the CNS. In some embodiments, the cell of the CNS is a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial

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[0130] cells (e.g., microglia, astrocytes, oligodendrocytes) and / or a supporting cells of the brain such as immune cells (e.g., T cells). In some embodiments, the CNS tissue is the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, caudate nucleus, hippocampus, putamen, basal ganglia, entorhinal cortex, cerebellum, or spinal cord.

[0131] In some embodiments, the sequences increase tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).

[0132] In some embodiments, the sequences decrease tropism of an AAV capsid protein to a cell, region, or tissue of the PNS. In some embodiments, the cell or tissue of the PNS is dorsal root ganglion (DRG).

[0133] In some embodiments, the targeting sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7. In some embodiments, the sequence comprises the amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7.

[0134] In some embodiments, the targeting peptide sequence is inserted within a parent AAV capsid sequence. In some embodiments, the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-7. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-7. In some embodiments, the targeting peptide sequence is inserted within or near a surface-exposed loop of a parent AAV capsid sequence. In some embodiments, the parent capsid sequence is any of the serotypes AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:l).In some embodiments, the peptide sequence is inserted within or near amino acids 453 through 454 corresponding to the sequence of AAV1 (SEQ ID NO: 3). In some embodiments, the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV1 (SEQ ID NO: 3). In some embodiments the peptide sequence is inserted within or near amino acids 577 through 578 corresponding to the sequence of AAV5 (SEQ ID NO: 5). In some embodiments the peptide sequence is inserted within

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[0137] or near amino acids 453 through 454 corresponding to the sequence of AAV6 (SEQ ID NO: 4). In some embodiments the peptide sequence is inserted within or near amino acids 589 through 590 corresponding to the sequence of AAV6 (SEQ ID NO: 4). In some embodiments the peptide sequence is inserted within or near amino acids 590 through 591 corresponding to the sequence of AAV8 (SEQ ID NO: 6). In some embodiments the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV9 (SEQ ID NO: 2). In some embodiments, an engineered AAV capsid protein is provided wherein the capsid is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a parent sequence outside the inserted peptide sequence, for example wherein the parent capsid is AAV1 (SEQ ID NO:3), AAV5 (SEQ ID NO:5), AAV6 (SEQ ID NO:4), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:2), or STAC-BBB (SEQ ID NO:1).

[0138] Disclosed herein is a composition comprising a targeting moiety or molecule that binds to cerebellin-1 (CBLN1), or binds to a complex of cerebellins wherein the targeting moiety or molecule is characterized by the unifying technical features of (a) enables enhanced delivery of the composition to the central nervous system; (b) exhibiting an equilibrium dissociation constant KD between 0.001-100 nM under physiological buffer; and (c) has a targeting sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) as shown in a single row in the second column of Tables 1 to 7.

[0139] Administering Engineered AAV Capsid Proteins to Subjects

[0140] In some embodiments, when administered to subjects, AAV capsid proteins containing the targeting peptides described herein mediate enhanced delivery to cells and tissues. In some embodiments, the enhancement is compared to (i) AAV capsid proteins that lack the targeting peptides; (ii) AAV capsid proteins without the targeting peptides administered to subjects; (iii) a selected AAV capsid variant without a modification in one or more amino acids within a hypervariable and / or surface exposed loop; (iv) a parental capsid that does not associate with cerebellin 1, 2, 3, and / or 4; (v) a composition without the targeting moiety or molecule and combinations thereof. In some embodiments, the enhancement is by at least 1.5, 2, 3, 4, 5, 10, 50, 100 or greater fold compared to AAV capsid proteins that lack the targeting peptides. In some

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[0143] embodiments, the enhancement is by at least 1.5, 2, 3, 4, 5, 10, 50, 100 or greater fold compared to AAV capsid proteins without the targeting peptides administered to subjects. In some embodiments, the enhancement is by at least 1.5, 2, 3, 4, 5, 10, 50, 100 or greater fold compared to a selected AAV capsid variant without a modification in one or more amino acids within a hypervariable and / or surface exposed loop. In some embodiments, the enhancement is by at least 1.5, 2, 3, 4, 5, 10, 50, 100 or greater fold compared to a parental capsid that does not associate with cerebellin 1, 2, 3, and / or 4. In some embodiments, the enhancement is by at least 1.5, 2, 3, 4, 5, 10, 50, 100 or greater fold compared to a composition without the targeting moiety or molecule. In some embodiments, the AAV capsid protein administered to subjects comprises an engineered AAV sequence described herein.

[0144] Genetic Material

[0145] In some embodiments, the engineered AAV capsid proteins described herein encapsidate genetic material of interest to be delivered to a cell of interest. In some embodiments, the genetic material of interest may be a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload. As such, in embodiments the engineered AAV capsid proteins described herein enable delivery of genetic material to a cell of interest. In embodiments, the genetic material may encode a research, diagnostic, and / or therapeutic payload. In embodiments, the genetic material encodes a zinc finger protein, a TALE protein, a recombinase protein, and / or a CRISPR protein, or fragments thereof. In embodiments, the genetic material encodes one or more antibodies or an antibody fragments. In some embodiments the genetic material encodes one or more regulatory RNA, such as RNAi agents or microRNAs.

[0146] In some embodiments, the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are non-coding 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.

[0147] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.

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[0150] In some embodiments, the genetic material encodes a gene therapy product. A gene therapy product can include a peptide, a polypeptide, or an RNA molecule that when expressed carries out a desired therapeutic effect. In some embodiments, the therapeutic effect is treating any one or more diseases or disorders described herein.

[0151] In some embodiments, a promoter is operably linked to the genetic material to be delivered to the cell. In some embodiments, the promoter comprises a tissue and / or cell specific promoter. In some embodiments, the promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken 0-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others. In some embodiments, the promoter comprises a cell type and / or tissue specific type promoter. Exemplary cell type and / or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes. Other non-limiting cell type and / or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific, Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), MHCK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes). In embodiments, tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS. In embodiments, tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and / or Schwann cells.

[0152] In some embodiments, the promoters are naturally occurring promoters. In some embodiments, the promoter is synthetic. In some embodiments, the promoter is derived from mammals, humans, viruses, or plants. In some embodiments, the promoters are truncated. In some embodiments, the promoter is mutated.

[0153] Gene Editing System

[0154] 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 at one or more site of interest. 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 at one or more site of interest. In some 23

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[0156] 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. In some embodiments, the gene editing system includes a modified recombinase protein.

[0157] Epigenetic Regulation System

[0158] In some embodiments, the genetic material of interest comprises an epigenetic regulation system or components of an epigenetic regulation system for general or targeted gene regulation. In some embodiments, the epigenetic regulation system is capable of modifying chromatin structure or altering epigenetic marks on nucleic acid sequences. In some embodiments, the epigenetic regulation system is capable of promoting or repressing gene expression without altering the underlying DNA sequence. In some embodiments, the epigenetic regulation system includes a CRISPR-dCas system fused to epigenetic effector domains. In some embodiments, the epigenetic regulation system includes a transcription activator or repressor domain tethered to a programmable DNA-binding protein. In some embodiments, the epigenetic regulation system includes hi stone-modifying enzymes or DNA methyltransferases targeted to a specific genomic locus. In some embodiments, the epigenetic regulation system includes a TALEN. In some embodiments, the epigenetic regulation system includes a zinc finger protein fused to an epigenetic effector domain, for example, a zinc finger repressor or a zinc finger activator.

[0159] Active Agents

[0160] In some embodiments, the targeting molecules or engineered AAV sequences described herein are fused or coupled to, or associated with, an active agent. In some embodiments, the active agent is a therapeutic or diagnostic agent. In some embodiments, ‘associated with’ an active agent or a therapeutic or diagnostic agent comprises covalent links or associations, including both cleavable and non-cleavable links or associations. In some embodiments, ‘associated with’ an active agent or a therapeutic or diagnostic agent comprises covalent links or associations that are peptide or non-peptide based. In some embodiments, ‘associated with’ an active agent or a therapeutic or diagnostic agent comprises non-covalent associations. In some embodiments, a sequence is fused or coupled to an active agent through conjugation. In some embodiments, the active agent comprises a therapeutic agent. In some embodiments, the active agent comprises a polypeptide. In

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[0163] some embodiments, the active agent comprises an oligonucleotide. Tn some embodiments, the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region). In some embodiments, the sequence is fused to a Fc region of an antibody. In some embodiments, the sequence is fused to the C-terminus of the Fc region. In some embodiments, the sequence is fused to the N-terminus of the Fc region. In some embodiments, the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the sequence is fused or coupled directly to at least on strand of the RNAi. In some embodiments, the sequence is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the sequence is fused or coupled to the sense strand of RNAi. In some embodiments, the sequence is fused or coupled to the antisense strand of RNAi.

[0164] 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.

[0165] Pharmaceutical Compositions and Dosage Forms

[0166] Compositions herein (e.g., targeting molecules, engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) can be included in pharmaceutical compositions. In some embodiments, the pharmaceutical compositions can include one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the genetic material; (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and / or (7) allow for regulatable expression of the genetic material.

[0167] The pharmaceutical compositions described herein can be administered periodically, such as once or twice a day, or any other suitable time period. For example, pharmaceutical compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, or every three years.

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[0170] In some embodiments, the compositions described herein (e.g., targeting molecules, engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical. Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients. The compositions described herein can also be formulated for injection, insufflation, infusion, or intradermal exposure. For instance, an injectable formulation may comprise the disclosed compositions in an aqueous or non-aqueous solution at a suitable pH and tonicity. The compositions can be included liquid dosage form for oral administration, such as suspensions, emulsions, or syrups.

[0171] 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.

[0172] Methods of Delivery and Treatment

[0173] In some embodiments, methods for introducing the compositions described herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins) into cells and / or tissues are provided. In some embodiments, the methods comprise introducing into cells and / or tissues any of the compositions described herein in an amount sufficient to modulate, e.g., increase, the production of a target mRNA and / or protein in the cells and / or tissues.

[0174] 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.

[0175] In some embodiments, the compositions described herein are administered through systemic administration. In some embodiments, systemic administration includes intravenous administration.

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[0178] In some embodiments, the compositions described herein are administered to the central nervous system of via intraventricular administration and / or intrathecal administration. In some embodiments, the compositions described herein are administered to the central nervous system via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the compositions described herein are administered to the central nervous system via administration into the cerebrospinal fluid.

[0179] In some embodiments the compositions can be delivered to target cells or target tissue including, but not limited to, the CNS, heart, lung, trachea, esophagus, muscle, bone, cartilage, stomach, pancreas, intestine, liver, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testes, prostate, eye, blood, lymph, or oral mucosa. In some embodiments, the target 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.

[0180] 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.

[0181] In some embodiments, the compositions described herein are delivered to the central nervous system through the cerebral spinal fluid pathway. In some embodiments, compositions described herein are administered to the central nervous system via intraparenchymal delivery. In some embodiments, the compositions described herein are administered to the central nervous system via intracranial delivery. In some embodiments, the compositions described herein are delivered to the central nervous system via intraocular delivery. In some embodiments, the compositions described herein are administered to the brain. In some embodiments, the compositions described herein are administered to the brain via injection into the brain. In some embodiments, the compositions described herein are administered to the brain via intrahippocampal injection.

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[0184] In some embodiments, the compositions described herein are administered as part of a composition that allows for extended release. In some embodiments, the compositions comprises a formulation that includes a depot.

[0185] Disclosed herein are methods of treatment using any of the compositions described herein (engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins). In embodiments, the disclosed compositions can be used to treat any one or more of muscular or neuromuscular disorders, neurooncological disorders, neurological diseases / disorders, and neurodegenerative disorders, amongst others. In embodiments, the disclosed compositions can be used to treat any one or more of Alzheimer's disease, Huntington's disease; autism; Parkinson's disease; Spinal muscular atrophy, Friedreich's ataxia. In embodiments, the disclosed compositions are used in treatments through any of the methods of delivery described herein.

[0186] In some embodiments, disclosed are methods for treating, or ameliorating a disease or condition associated with abnormal gene and / or protein in a subject in need of treatment, the methods comprising administering to the subject any effective amount of at least one of the compositions described herein (e.g., engineered AAV capsid sequences, AAV particles, and engineered AAV capsid proteins), delivering the compositions described herein into targeted cells, inhibiting or activating the gene expression and protein production, and ameliorating symptoms of the disease or condition in the subject.

[0187] Equivalents and Scope

[0188] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of neurology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and 28

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[0190] plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0191] 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.

[0192] It is assumed that words of this disclosure are for the purpose of description and not limitation. Changes to words in the claims can be made, while still retaining the scope of the disclosure in its broad embodiment. Specific embodiments of the disclosure have been described herein. However, these embodiments are not intended to be limiting of the broad scope of this disclosure.

[0193] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

[0194] Exemplary Embodiments

[0195] Numbered non-limiting exemplary embodiments of the present disclosure are described below.

[0196] 1. A composition comprising a targeting moiety or molecule that binds to cerebellin-1 (CBLN1), or binds to a complex of cerebellins optionally comprising CBLN1, wherein the targeting moiety or molecule enables enhanced delivery of the composition to the central nervous system (CNS) compared to a composition without the targeting moiety or molecule, and

[0197] 29

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[0199] optionally wherein the targeting moiety or molecule is associated with a therapeutic or diagnostic agent.

[0200] 2. A composition comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4, wherein the cerebellin enables enhanced delivery of the composition to the central nervous system (CNS), and optionally wherein the cerebellin molecule is associated with a therapeutic or diagnostic agent.

[0201] 3. A fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4, wherein the cerebellin enables enhanced delivery of the fusion protein to the central nervous system (CNS), and optionally wherein the cerebellin molecule is associated with a therapeutic or diagnostic agent.

[0202] 4. A composition comprising:

[0203] i) A) a cerebellin, optionally cerebellin 1, 2, 3, and / or 4; or B) a fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4;

[0204] and

[0205] ii) a targeting molecule that binds to cerebellin- 1 (CBLN1), or binds to a complex of cerebellins optionally comprising CBLN1;

[0206] optionally wherein the cerebellin or the targeting molecule is associated with a therapeutic or diagnostic agent.

[0207] 5. The composition of embodiment 1 or embodiment 4, wherein the targeting molecule binds to CBLN1 with a KD of 0.001-100 nM, optionally wherein the KD is measured by a BLI assay or wherein the targeting molecule binds to CBLN1 with a KD of 0.001 nM or higher, optionally wherein the KD is measured by a BLI assay.

[0208] 6. The composition of embodiment 1 or embodiment 4, wherein the targeting molecule comprises a polymer, optionally wherein the targeting molecule is a polypeptide.

[0209] 7. The composition of any one of embodiments 1-2 or embodiments 4-6 or the fusion protein of embodiment 3, wherein the targeting molecule or cerebellin is associated with, fused, or conjugated to a small molecule, antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer, or recombinant protein.

[0210] 30

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[0212] 8. The composition of any one of embodiments 1-2 or embodiments 4-7 or the fusion protein of embodiment 3 or embodiment 7, wherein the composition or fusion protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.

[0213] 9. The composition or fusion protein of embodiment 8, wherein the composition or fusion protein comprises all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.

[0214] 10. The composition of any one of embodiments 1-2 or embodiments 4-9 wherein the composition comprises an AAV capsid protein.

[0215] 11. The composition of embodiment 10, wherein the composition encapsidates a nucleotide encoding a therapeutic or diagnostic agent, optionally wherein the composition was derived from a parental AAV capsid protein.

[0216] 12. The composition of embodiment 10, wherein the AAV capsid protein is STAC-BBB, optionally wherein the STAC-BBB AAV capsid protein is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO.l.

[0217] 13. The composition of embodiment 10, wherein the parental AAV capsid protein is selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74.

[0218] 14. The composition of any one of embodiments 10-13, wherein the AAV capsid protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.

[0219] 15. The composition of embodiment 14, wherein the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”) as shown in a single row (third and fourth columns, respectively) in Tables 1 to 7.

[0220] 16. The composition of any one of embodiments 10-15, wherein the amino acid sequence comprises a peptide sequence of any one of SEQ ID NOs: 7-4109 as shown in a single row and

[0221] 31

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[0223] second column in Tables 1 to 7, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row (third and / or fourth columns, respectively) as shown in Tables 1 to 7.

[0224] 17. An AAV capsid protein comprising, an insertion sequence of Formula I between residues 453 / 454 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):

[0225] X1X2X3X4X5X6X7 (I);

[0226] wherein, Xi is an amino acid selected from K or R; X2is an amino acid selected from A, G, H, L, N, P, Q, S, T, or V; X3 is an amino acid selected from A, D, H, N, Q, S, T, or V; X4 is an amino acid selected from A, D, E, G, N, P, Q, S, or T; X5 is an amino acid selected from A, D, E, N, Q, S, T, or V; Xe is an amino acid selected from A, D, E, G, N, P, or Q; and X7 is an amino acid selected from A, D, H, N, P, Q, or S;

[0227] and wherein the AAV capsid protein comprises a targeting molecule.

[0228] 18. An AAV capsid protein comprising, an insertion sequence of Formula II located between residues 453 / 454 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):

[0229] XiX2P (II);

[0230] wherein, Xi is an amino acid selected from D or E; and X2is an amino acid selected from A, H, K, P, Q, R, S, or T;

[0231] and wherein the AAV capsid protein comprises a targeting molecule.

[0232] 19. An AAV capsid protein comprising, an insertion sequence of Formula III located between residues 589 / 590 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):

[0233] XIX2X3GQ (III);

[0234] wherein, Xi is an amino acid selected from A, E, I, L, N, P, Q, or S; X2is an amino acid selected from A, G, L, M, N, Q, R, S, or T; and X3 is an amino acid selected from E, H, L, N, R, S, or T;

[0235] and wherein the AAV capsid protein comprises a targeting molecule.

[0236] 20. An AAV capsid protein comprising, an insertion sequence of Formula IV located between residues 577 / 578 of AAV5 (SEQ ID NO. 5):

[0237] RXIX2X3X4DX5P (IV);

[0238] 32

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[0240] wherein, Xi is an amino acid selected from H, I, K, L, Q, R, T, or V;X2 is an amino acid selected from K, N, Q, R, or S; X3 is an amino acid selected from A, E, F, H, I, P, S, or V; X4 is an amino acid selected from A, D, M, Q, S, or T; and X5 is an amino acid selected from D, F, L, M, N, Q, S, T, W;

[0241] and wherein the AAV capsid protein comprises a targeting molecule.

[0242] 21. An AAV capsid protein comprising, an insertion sequence of Formula V contained between residues 577 / 578 of AAV5 (SEQ ID NO. 5):

[0243] XiTDLP (V);

[0244] wherein, Xi is an amino acid selected from A, H, P, or V;

[0245] and wherein the AAV capsid protein comprises a targeting molecule.

[0246] 22. An AAV capsid protein comprising, an insertion sequence of Formula VI contained between residues 588 / 589 of AAV9 (SEQ ID NO. 2):

[0247] RX1X2X3X4P (VI);

[0248] wherein, Xi is an amino acid selected from D, G, L, P, S, T, or V;X2 is an amino acid selected from D, F, L, M, Q, S, T, or V; X3 is an amino acid selected from A, D, G, L, N, P, S, or V; andX4 is an amino acid selected from D, E, G, H, L, P, S, or T;

[0249] and wherein the AAV capsid protein comprises a targeting molecule.

[0250] 23. An AAV capsid protein comprising, an insertion sequence of Formula VII contained between residues 588 / 589 of AAV9 (SEQ ID NO. 2):

[0251] X1X2X3X4P (VII);

[0252] wherein, Xi is an amino acid selected from A, G, K, L, M, P, R, or T;X2 is an amino acid selected from A, F, K, L, Q, R, S, T, or V; X3 is an amino acid selected from A, D, H, I, L, N, P, Q, R, S, or V; and X4 is an amino acid selected from D, or E;

[0253] and wherein the AAV capsid protein comprises a targeting molecule.

[0254] 24. The composition of any one of embodiment 1 or embodiments 4-23, wherein the targeting molecule binds human, cynomolgus, or murine CBLN1.

[0255] 33

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[0257] 25. The composition of any one of embodiments 1-24, wherein the cerebellin protein is derived from human, cynomolgus, or murine CBLN 1.

[0258] 26. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising fusing or conjugating the therapeutic or diagnostic agent to , or associating the therapeutic or diagnostic agent with, the targeting molecule or cerebellin according to any one of embodiments 1-25. 27. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising or encapsi dating a nucleic acid encoding the therapeutic or diagnostic agent in, fusing or conjugating the therapeutic or diagnostic agent to, or associating the therapeutic or diagnostic agent with, an AAV capsid protein according to any one of embodiments 10-23.

[0259] 28. An engineered adeno-associated virus (AAV) capsid protein, wherein the capsid protein binds to one or more of cerebellin 1-4, and wherein the capsid protein has enhanced delivery to the CNS compared to a parental capsid that does not associate with cerebellin 1, 2, 3, and / or 4.

[0260] 29. The capsid protein of embodiment 28, wherein the capsid protein comprises an AAV capsid protein according to any one of embodiments 10-23, optionally wherein the capsid protein encapsi dates a nucleic acid encoding a therapeutic or diagnostic agent.

[0261] 30. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising fusing or conjugating the therapeutic or diagnostic agent to, associating the therapeutic or diagnostic agent with, or encapsidating a nucleic acid encoding the therapeutic or diagnostic agent in, the capsid protein of embodiment 28 or embodiment 29.

[0262] 31. A method for identifying an AAV capsid variant with increased transduction of the CN S, comprising selecting for AAV capsid variants that bind to cerebellin 1, 2, 3, and / or 4.

[0263] 32. The method of embodiment 31, comprising modifying one or more amino acids within a hypervariable and / or surface exposed loop of a selected AAV capsid variant to create a modified AAV capsid variant, optionally wherein the modified AAV capsid variant is further screened for improved transduction of the CNS compared to an AAV capsid without a modification in one or more amino acids within a hypervariable and / or surface exposed loop.

[0264] 33. A method for identifying a targeting molecule that confers increased transduction of the CNS within a pool of compositions, comprising selecting for compositions that bind cerebellin 1,

[0265] 34

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[0267] 2, 3, and / or 4, optionally wherein the targeting molecule comprises or is selected from peptides, small protein fragments, antibodies, or AAV capsids.

[0268] 34. The method of embodiment 33, wherein the targeting molecule is part of an AAV capsid variant.

[0269] 35. A composition comprising a cerebellin 1, 2, 3, and / or 4 protein and a cerebellin 1, 2, 3, and / or 4 targeting molecule, optionally wherein the cerebellin 1, 2, 3, and / or 4 targeting molecule is associated with a therapeutic or diagnostic agent.

[0270] 36. A composition comprising a cerebellin 1, 2, 3, and / or 4 protein, optionally wherein the cerebellin 1, 2, 3, and / or 4 protein is associated with a therapeutic or diagnostic agent.

[0271] 37. The composition of embodiment 35, wherein an AAV capsid variant comprises the cerebellin 1, 2, 3, and / or 4 targeting molecule.

[0272] 38. The composition of embodiment 35 or embodiment 36, wherein a capsid protein according to any one of embodiments 10-23 comprises the cerebellin 1, 2, 3, and / or 4 targeting molecule.

[0273] 39. The composition of any one of embodiments 35-38, wherein the cerebellin 1, 2, 3, and / or 4 protein or the cerebellin 1, 2, 3, and / or 4 targeting molecule is fused or conjugated to, or is associated with, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.

[0274] 40. The composition of any one of embodiments 35-39, wherein the cerebellin 1, 2, 3, and / or 4 protein or the cerebellin 1, 2, 3, and / or 4 targeting molecule binds human, cynomolgus, or murine cerebellin 1, 2, 3, and / or 4.

[0275] 41. The composition of embodiment 37 or embodiment 38, wherein the AAV capsid variant or the capsid protein encapsidates a nucleic acid encoding a therapeutic or diagnostic agent. 42. The composition of embodiment 37, wherein the AAV capsid variant is derived from a parental capsid selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74.

[0276] 35

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[0278] 43. The composition of embodiment 35, wherein the composition provides for increased CNS delivery of the cerebellin 1, 2, 3, and / or 4 targeting molecule compared to administering the cerebellin 1, 2, 3, and / or 4 targeting molecule without the cerebellin 1, 2, 3, and / or 4 protein. 44. The composition of embodiment 36, wherein the composition provides for increased CNS delivery of the therapeutic or diagnostic agent compared to administering the therapeutic or diagnostic agent without being associated with the cerebellin 1, 2, 3, and / or 4 protein.

[0279] 45. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising administering the composition of any one of embodiments 35-44 to a subject.

[0280] EXAMPLES

[0281] Example 1. Methods

[0282] 1.1. In vitro protein pull-down of STAC-BBB with immobilized CBLN1

[0283] DNA encoding human, cyno or mouse CBLN1 (NCBI accession numbers human: 869, cyno: 102118708, and mouse: 12404) fused to a human IgGl Fc domain are transfected into HEK293T cells (20 pg per 100 mm dish with lipofectamine 3000 reagent) in complete DMEM media with 10% FBS. 16 hr post-transfection, the media is removed and replaced with 10 mb complete DMEM media with 5% FBS. Media containing the secreted Fc tagged CBLN1 fusion proteins is collected 48 hrs post media change and incubated rotating end-over-end at 4°C with 35 pL of Protein A beads (ThermoFisher, 1000 ID) for 16 hrs. Beads are washed three times with 200 pL of DPBS containing 0.05% Tween-20 and resuspended in 35 pL DPBS containing 0.05% Tween-20.

[0284] 12.5 pL of washed beads are transferred to low protein binding microcentrifuge tubes (ThermoFisher, 90411) and mixed with 1.5ell AAV vector genomes overnight rotating end-over-end at 4°C. Beads are washed three times with 100 pL of DPBS containing 0.05% Tween-20, resuspended in 10 pL of DPBS containing 0.05% Tween-20 and 5 pL was analyzed by SDS-PAGE. The band intensity of capsid proteins (VP1 / VP2 / VP3) and CBLNl-Fc fusion proteins are quantified using ImageJ software.

[0285] 1.2. Transduction assay in HEK293 cells overexpressing CBLN1

[0286] HEK293T cells are seeded in 96-well PDL coated plates (Corning, 356461) at a density of 4E4 cells per well. 24 hours later cells are transfected with 100 ng of plasmid encoding human, macaque

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[0289] or mouse CBLN1 under the control of the ubiquitous cytomegalovirus (CMV) promoter with lipofectamine 3000 reagent. In parallel, control wells are transfected with 100 ng of plasmid encoding GFP under the control of the CMV promoter. Cell culture media was changed 24 hours post-transfection to remove transfection reagents. 48 hours post-transfection of plasmid DNA, the cells are transduced with AAV9- or STAC-BBB-CMV-mCherry at le3 to le5 AAV particles per cell. Plates are imaged 72 hrs post transduction with a 4X objective on an EVOS FL Auto 2 (ThermoFisher, AMAFD2000). Cells are harvested and reverse transcription was performed using the C2CT kit following the manufacturer’s instructions. Taqman qPCR is used to measure the levels of transgene expression and a reference gene (GAPDH). Transgene expression is normalized to the mean expression level of housekeeping gene GAPDH.

[0290] Example 2. STAC-BBB binds directly to CBLN1

[0291] CBLN1 orthologs from human, cyno, and mouse are cloned as fusions to human IgGl Fc domain separated by a HRV3C protease cleavage site and under the control of the ubiquitous Cytomegalovirus (CMV) promoter. HEK293T cells are transfected to overexpress secreted CBLNl-Fc fusions. Media from transfected cells containing CBLNl-Fc is used to coat Protein A magnetic beads. Coated beads are incubated with purified STAC-BBB or AAV9 capsid lots and capsid proteins and CBLNl-Fc are visualized by SDS-PAGE. Data shown in FIG 1 are the band intensities for STAC-BBB or AAV9 relative to the loading control. STAC-BBB demonstrated specific interaction with all three orthologs of CBLN1 tested. The parental capsid of STAC-BBB, AAV9, does not detectably bind to CBLN 1.

[0292] Example 3. STAC-BBB exhibits a gain of function for transduction in HEK293 cells overexpressing tethered CBLN1

[0293] CBLN1 orthologs from human, cyno, and mouse are cloned as fusions to a membrane tethering domain separated by a glycine and serine linker. Expression of the fusion product is under the control of CMV promoter and results in CBLN1 display at the cytoplasmic membrane. HEK293 cells are transfected with a transfection control expressing eGFP, or the CBLN 1 tethered construct.

[0294] 48 hours after transfection, cells are transduced with STAC-BBB or AAV9 expressing mCherry under the control of the CMV promoter at five different multiplicities of infection (MOI) ranging from le3 to le5 vector genome containing AAVs per cell. 72 hours post transduction, total RNA

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[0297] is harvested and mCherry transgene expression and GAPDH housekeeping gene expression is quantified by RT-qPCR. mCherry transgene expression is normalized to housekeeping control for all conditions tested. FIG 2 shows the gain of function in STAC-BBB transduction mediated by overexpression of tethered CBLN1 from the three orthologs tested. No gain of function in transduction is observed for AAV9, the parental serotype of STAC-BBB. Dose response curves were fit to a non-linear regression model and the midpoint was used to determine the fold increase in transgene expression shown in FIG 3. The gain of function is also shown in FIG 4, which shows mCherry fluorescence in HEK293 cells overexpressing human, cyno, or mouse CBLN1 tethered to the extracellular membrane. STAC-BBB or AAV9 expressing mCherry under the control of the CMV promoter was added to cells at a multiplicity of infection (MOI) of le5 AAV particles per cell. An increase in mCherry fluorescence was observed for STAC-BBB, but not AAV9, in cells overexpressing human, cyno, or mouse CBLN1.

[0298] Example 4. Measuring binding kinetics of AAV interaction with CBLN1 by Octet BLI Binding of AAV capsids to human CBLN1 protein is measured by bio-layer interferometry on an Octet Red96e instrument. All steps are performed at 30°C shaking at 1,000 rpm. Purified human CBLN1 protein is biotinylated at room temperature followed by a desalting step. Streptavidin biosensors are equilibrated in PBS with 0.05% Tween20 and 0.1% BSAbefore loading biotinylated CBLN1 protein. AAV capsids are diluted in PBS with 0.05% Tween20 and 0.1% BSA across a 2-fold capsid dilution series ranging in concentration from 16-1038 pM. Capsid association to each sensor is performed for 300 seconds followed by a 600 second dissociation step. Sensorgrams in FIG 5 shows STAC-BBB binding specifically to CBLN1 in a dose dependent manner, while no binding to AAV9 was observed. For kinetic analysis, a global 2: 1 heterogeneous ligand model was used for sensorgram curve fitting. The dissociation constant measured for STAC-BBB binding CBLN1 was approximately 11 pM.

[0299] Example 5. Identifying AAV capsid variants that bind CBLN1

[0300] 5.1 AAV capsid library generation

[0301] AAV capsid libraries are collections of AAV capsid variants that are screened to identify new properties. Capsid libraries are used to identify capsid variants that bind CBLN1. Capsid libraries are constructed through amino acid substitutions, insertions, or deletions in the AAV capsid

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[0304] proteins. In particular, capsid modifications are introduced in individual or multiple variable regions. These include VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII, or VR IX. For example, capsid libraries are constructed by mutagenesis of the VR1 region between amino acid position 266 and 270. The VR4 region is mutated between positions 452 and 460. Peptides are inserted between 454 and 455 in the VR4 region. The VR8 loop is mutated between positions 584 and 599. Gibson assembly is used to generate capsid libraries where the introduced amino acid modifications are encoded by the primers used to amplify the assembly fragments. Two PCR products from the capsid gene sequence are amplified (left and right fragments) that have an overlap region to facilitate assembly using the Gibson assembly procedure into a plasmid backbone (see e.g. Gibson et al (2009) Nat Meth 6(5):343-345). Alternatively, synthesized oligo pools are used to clone the libraries using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs catalog number E2621).

[0305] The resulting capsid libraries are manufactured in HEK293 cells. Briefly, AAV capsid libraries are produced by triple transient transfection of the capsid library plasmid, pXX6 helper (encodes essential adenovirus genes E4, E2A, and VA), and with supplementation of Rep in trans. Capsids are purified by cesium density centrifugation, and buffer exchanged into PBS plus 0.001% PF-68 by dialysis. DNase-resistant viral genomic titers are measured by quantitative real-time PCR.

[0306] 5.2 In vitro capsid library screening with CBLN1 protein

[0307] Capsid libraries are screened against recombinant CBLN1 protein to identify capsid variants that bind CBLN1. To produce recombinant CBLN1 protein a plasmid encoding CBLN1 fused to a human IgGl Fc domain is transfected into HEK293T cells (20 pg per 100 mm dish with lipofectamine 3000 reagent) in complete DMEM media with 10% FBS. In parallel, plasmid DNA encoding a control human IgGl Fc domain without a CBLN1 fusion is also transfected with the same method. 16 hours after transfection the media is removed and replaced with 10 mL complete DMEM media with 5% FBS. Media containing the secreted Fc tagged CBLN1 fusion proteins or Fc domain without CBLN1 is collected 48 hours post-media change and incubated rotating endover-end at 4°C with 35 pL of Protein A beads (ThermoFisher, 10001D) for 16 hours. The beads are washed three times with 200 pL of DPBS containing 0.05% Tween-20 and resuspended in 35 pL DPBS containing 0.05% Tween-20. 12.5 pL of the washed beads is transferred to low protein binding microcentrifuge tubes (ThermoFisher, 90411) and mixed with 1.5el0 vector genomes of

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[0310] AAV capsid library and rotated end-over-end overnight at 4°C. The beads are washed three times with 100 pL of DPBS containing 0.05% Tween-20. The unbound capsids are separated from the capsids interacting with the immobilized CBLNl-Fc fusion or Fc using a tube magnet. Vector genomes are extracted using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega Catalog AS1330) and samples are prepared for NGS using Kapa HiFi Hotstart ReadyMix (Roche Catalog KK2602). Amplification of library specific amplicons is performed with the following cycling conditions: 95°C for 3:00 min; 25 cycles at 98°C for 20 sec; 58°C for 15 sec; 72°C for 30 sec followed by 72°C for 1 minute. Amplification is qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification is used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices are added to the amplicons with 10 cycles of amplification: 95°C for 3:00 min; 10 cycles at 98°C for 20 sec; 60°C for 15 sec; 72°C for 30 sec followed by 72°C for 1 minute. Finally, samples are pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples are sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2. Following next-generation sequencing of library amplicons, the reads are demultiplexed and features (the barcode or peptide sequence) are extracted using a custom bioinformatic pipeline. For barcoded libraries, the extracted barcode is used to query a pre-determined lookup table and return the identity of the corresponding capsid variant. Finally, the log2 fold change enrichment of each capsid variant is normalized to its relative abundance in the administered AAV library. To identify AAV capsids that interact with CBLN1, bioinformatic analysis is used to compare the relative log2 fold change enrichment of capsids bound to a CBLNl-human IgGl Fc fusion protein versus human IgGl Fc alone.

[0311] 5.3. Capsid library screening in cells overexpressing CBLN1

[0312] HEK293 cells are seeded in 10 cm dishes coated with poly-D-lysine (PDL) at a density of 3E6 cells per dish. 24 hours later, cells are transfected with 1 microgram of plasmid encoding human, cyno, or mouse CBLN 1 under the control of the ubiquitous cytomegalovirus (CMV) promoter. In parallel, a transfection control plate is transfected with 1 microgram of plasmid encoding a fluorescent protein (GFP or mRuby) under the control of the ubiquitous cytomegalovirus (CMV) promoter. Lipofectamine 3000 is used for transfection of the plasmid DNA. Cell culture media is changed 24 hours post-transfection to remove transfection reagents. 48 hours after transfection of

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[0315] plasmid DNA, cells are transduced with the AAV capsid library at a multiplicity of infection of 3E4 vector genomes (vg) per cell in reduced serum media (0.5% FBS). Capsid delivery is assessed with transduction and binding assays.

[0316] For cell culture transduction assays, the media is changed 24 hours post-transduction to fully supplemented media (10% FBS). 72 hours post-transduction, the plates are washed one time with phosphate buffered saline (with calcium and magnesium), and RNA is extracted using the Qiagen RNeasy kit following the manufacturer's protocol and quantified by NanoDrop 8000 spectrophotometer. RNA is reverse transcribed to cDNA using the NEB Induro Reverse Transcriptase kit.

[0317] For cell culture binding assays, cells are incubated with the AAV capsid library for 1 hour at 37°C and washed three times with phosphate buffered saline (with calcium and magnesium). DNA is extracted using the Qiagen DNeasy kit following the manufacturer's protocol and quantified by NanoDrop 8000 spectrophotometer.

[0318] Samples from transduction and binding assays are prepared for next-generation sequencing (NGS) using Kapa HiFi Hotstart ReadyMix (Roche Catalog KK2602). Amplification of library specific amplicons is performed with the following cycling conditions: 95°C for 3:00 min; 25 cycles at 98°C for 20 sec; 58°C for 15 sec; 72°C for 30 sec followed by 72°C for 1 minute. Amplification is qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification is used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices are added to the amplicons with 10 cycles of amplification: 95°C for 3:00 min; 10 cycles at 98°C for 20 sec; 60°C for 15 sec; 72°C for 30 sec followed by 72°C for 1 minute. Finally, samples are pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples are sequenced on the Illumina MiSeq platform using a MiSeq Reagent Kit v2. Following next-generation sequencing of library amplicons, the reads are demultiplexed and features (the barcode or peptide sequence) are extracted using a custom bioinformatic pipeline. For barcoded libraries, the extracted barcode is used to query a pre-determined lookup table and return the identity of the corresponding capsid variant. Finally, the log2 fold change enrichment of each capsid variant is normalized to its relative abundance in the administered AAV library. To identify AAV capsids that interact with CBLN1, bioinformatic analysis is used to compare the

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[0321] relative log2 fold change enrichment of capsid transduction in cells overexpressing CBLN1 versus the fluorescent protein transfection control.

[0322] Example 6. Identifying peptides that bind CBLN1

[0323] 6.1. Phage library construction

[0324] A combinatorial phage display library is generated using an M13 phagemid vector system. DNA fragments encoding targeting peptides are cloned into the gene III (pill) region of the Ml 3 genome to allow presentation on the phage surface. The ligated plasmid constructs are electroporated into Escherichia coli TGI cells to achieve a library complexity greater than 109independent transformants. Following infection with the helper phage M13KO7, recombinant phages propagate overnight at 30°C in 2*YT medium containing ampicillin (100 pg / mL) and kanamycin (50 pg / mL). Phages are recovered from the culture supernatant by precipitation with 20% polyethylene glycol (PEG 8000) and 2.5 M NaCl.

[0325] 6.2. CBLN1 preparation and immobilization

[0326] A plasmid encoding recombinant CBLN1 protein with a hexahistidine tag is transfected into HEK293T cells (20 pg per 100 mm dish with lipofectamine 3000 reagent) in complete DMEM media with 10% FBS. 16 hours after transfection the media is removed and replaced with 10 mb complete DMEM media with 5% FBS. Media containing the secreted hexahistidine tagged CBLN1 is collected 48 hours post-media change. Recombinant CBLN1 is purified with a HisTrap HP (GE Healthcare) affinity chromatography column using an AKTA chromatography system. For library screening, For library screening, recombinant CBLN1 is immobilized onto 96-well MaxiSorp microtiter plates at a concentration of 1-10 pg / mL in carbonate-bicarbonate buffer (0.05 M, pH 9.6) overnight at 4°C. Wells are blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 hour at room temperature to prevent non-specific binding.

[0327] 6.3. Phage panning and selection

[0328] Approximately 1011plaque-forming units (pfu) of the phage library are incubated with the immobilized CBLN1 for 1 hour at ambient temperature with gentle agitation. Unbound phages are removed through repeated washes with PBS containing 0.1% Tween-20 (PBST). Bound phages are eluted using 0.1 M glycine-HCl (pH 2.2) for 10 minutes and neutralized immediately with 1 M Tris-HCl (pH 9.0). The eluted phages are amplified by infecting mid-log phase E. coli TGI cultures and are rescued with helper phage for subsequent selection rounds. Three to four 42

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[0330] sequential panning rounds are conducted to enrich for phage clones displaying high-affinity binders.

[0331] Example 7. Identifying antibodies that bind CBLN1

[0332] 7.1. Phage antibody library construction

[0333] An antibody library is constructed by cloning variable region gene segments encoding antibody fragments (e.g., single-chain variable fragments, scFv, or Fab) into a phagemid vector for display on the surface of M13 bacteriophage particles. The gene segments are amplified from immune or naive B-cell sources using PCR and ligated into a phagemid vector that fuses the antibody fragment to the coat protein (pill). The recombinant vectors are transformed into Escherichia coli TGI cells to generate a library with a diversity exceeding 109independent clones. Following infection with M13KO7 helper phage, the library is rescued to produce phage particles displaying antibody fragments on their surfaces.

[0334] 7.2. CBLN1 preparation and immobilization

[0335] A plasmid encoding recombinant CBLN1 protein with a hexahistidine tag is transfected into HEK293T cells (20 pg per 100 mm dish with lipofectamine 3000 reagent) in complete DMEM media with 10% FBS. 16 hours after transfection the media is removed and replaced with 10 mb complete DMEM media with 5% FBS. Media containing the secreted hexahistidine tagged CBLN1 is collected 48 hours post-media change. Recombinant CBLN1 is purified with a HisTrap HP (GE Healthcare) affinity chromatography column using an AKTA chromatography system. For library screening, recombinant CBLN1 is immobilized onto 96-well MaxiSorp microtiter plates at a concentration of 1-10 pg / mL in carbonate-bicarbonate buffer (0.05 M, pH 9.6) overnight at 4°C. Wells are blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 hour at room temperature to prevent non-specific binding.

[0336] 7.3. Phage panning and selection

[0337] Approximately 1011plaque-forming units (pfu) of the phage antibody library are incubated with immobilized CBLN1 for 1 hour at ambient temperature with gentle agitation. Unbound phage particles are removed by multiple washes with PBS containing 0.1% Tween-20 (PBST), and bound phages are eluted using either trypsin digestion or a low-pH elution buffer (0.1 M glycine-HCl, pH 2.2), followed by neutralization with 1 M Tris-HCl (pH 9.0). Eluted phages are amplified by infecting mid-log phase E. coli TGI cells, which are subsequently rescued with helper phage to 43

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[0339] produce enriched phage pools. This panning and amplification cycle is repeated for three to four rounds, increasing the washing stringency in each successive round to favor the selection of higher-affinity antibody fragments.

[0340] 7.4. Yeast display antibody library construction

[0341] Variable regions identified by phage display screening are cloned into a yeast surface display vector (e.g., pCT or pDNL-6), which enables fusion of the antibody fragment to the Aga2p protein on the surface of Saccharomyces cerevisiae strain EBY100. Yeast cells are transformed via electroporation, generating a library comprising 107to 109unique clones. Transformed yeast cells are grown in selective media (SD-CAA) and induced in SG-CAA media by replacing glucose with galactose to promote antibody fragment expression on the cell surface.

[0342] 7.5. CBLN1 fluorescent labeling

[0343] 100 pg of purified CBLN 1 at 1 mg / mL is buffer exchanged into phosphate-buffered saline (PBS), pH 7.4. Alexa Fluor 488 NHS ester dye is dissolved in anhydrous dimethyl sulfoxide (DMSO) to a 10 mM stock concentration before labeling. Add 1 / 10 volume of 6* labeling buffer to the protein solution and mix gently by pipetting. Add Alexa Fluor NHS ester dye at a molar ratio of approximately 3:1 (dye:protein). Incubate the reaction mixture at room temperature, protected from light, for 1 hour. After incubation, free dye is quenched by adding 50 mM Tris pH 7.4 and removed by size exclusion using 7 kDa Zeba (Thermo Scientific) dye removal spin columns.

[0344] 7.6. Yeast display library screening by flow cytometry

[0345] Induced yeast displaying antibody fragments are incubated with fluorescently labeled CBLN1 at concentrations ranging from low nanomolar to micromolar, depending on anticipated affinity. Incubation occurs at room temperature for 30 60 minutes in PBS containing 0.5% BSAto reduce nonspecific interactions. Subsequently, cells are washed to remove unbound CBLN1.

[0346] To monitor surface display levels and normalize antigen binding signals, yeast cells are co-stained for the display tag (c-Myc) using a primary antibody and a fluorescent secondary antibody. Cells are analyzed and sorted by fluorescence-activated cell sorting (FACS), gating to select yeast populations exhibiting high affinity antigen binding coupled with robust display levels.

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[0349] Multiple rounds of sorting (3-5) are performed, with progressively decreasing antigen concentrations or inclusion of competitive non-labeled antigen to enrich clones with higher affinity and specificity.

[0350] Example 8. In silico protein design for de novo binders to CBLN1

[0351] 8.1. In Silico design of de novo binders to CBLN1

[0352] The crystal structure of CBLN1 (PDBID: 5KC5) is trimmed to reduce GPU memory usage. Binder lengths between 60 and 100 residues are hallucinated on the structure using AlphaFold 2 multimer. Sequence optimization of non-interface regions is performed using MPNNsoi. The optimized designs are validated and filtered using AlphaFold 2 monomer into final designs that pass scoring metrics such as predicted local distance difference test (pLDDT), interface predicted template modelling score (ipTM), and other physics based metrics. Passing structures are selected for experimental validation.

[0353] 8.2. E. Coli production of de novo binders to CBLN1

[0354] De novo binder amino acid sequences produced by in silico computation are back translated and fused to N- or C- terminal hexahistidine tags for gene synthesis. Protein coding sequences are cloned into a pSOL expression vector for purification in Nico21 cells. Cells in log phase growth are induced with 0.2% (w / v) L-rhamnose at 16°C for 24 hours. Cell pellets are lysed and clarified lysates are purified with a HisTrap HP (GE Healthcare) affinity chromatography column using an AKTA chromatography system.

[0355] 8.3 Measuring binding kinetics of de novo binder interaction with CBLN1 by Octet BLI Binding of in silico designed de novo binders to human CBLN1 protein is measured by bio-layer interferometry on an Octet Red96e instrument. All steps are performed at 30°C shaking at 1,000 rpm. Purified human CBLN1 protein is biotinylated at room temperature followed by a desalting step. Streptavidin biosensors are equilibrated in PBS with 0.05% Tween20 and 0.1% BSA before loading biotinylated CBLN1 protein. Purified binders are diluted in PBS with 0.05% Tween20 and 0.1% BSA across a 2-fold dilution series and association to each sensor is performed for 300 seconds followed by a 600 second dissociation step.

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[0358] SEQUENCES STAC-BBB capsid amino acid sequence SEQ ID NO. 1:

[0359] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGN LGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNF GQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDS QWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHC HFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMTPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNN FQF S YEFENVPFHS S YAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKF S VAGPSN MAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHK EGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAY VNIMDDMDQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEI QYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL AAV9 capsid amino acid sequence SEQ ID NO. 2:

[0360] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGN LGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNF GQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDS QWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHC HFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNN FQF S YEFENVPFHS S YAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKF SVAGPSN MAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHK EGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ AQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPP QILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYY KSNNVEFAVNTEGVYSEPRPIGTRYLTRNL AAV1 capsid amino acid sequence SEQ ID NO. 3:

[0361] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFN GLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNL GRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNF GQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHC DSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFH CHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSE YQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG NNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGS PAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMA SHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQ

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[0364] S S STDPATGD VHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNP PPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSN YAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL AAV6 capsid amino acid sequence SEQ ID NO. 4:

[0365] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFN GLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNL GRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFG QTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDS TWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCH F SPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTTNDGVTTIANNLT STVQ VF SD SEYQ LPYVLGSAHQGCLPPFPADVFM1PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNF TFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAG MSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHK DDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSS TDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQ ILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAK SANVDFTVDNNGLYTEPRPIGTRYLTRPL AAV5 capsid amino acid sequence SEQ ID NO. 5:

[0366] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGL DRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKA VFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQ QLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTK STRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRL INNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTE GCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEV PFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPM GRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENT MIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYN LQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPG NITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDS TGEYRTTRPIGTRYLTRPL AAV8 capsid amino acid sequence SEQ ID NO. 6:

[0367] MAADGYLPDWLEDNESEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFN GLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNL GRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNF GQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHC DSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRF HCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG NNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGG

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[0370] PNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGTAM ATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQ QQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHP PPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN YYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL

[0371] 48

[0372] 4896-3717-6698

Claims

Ref. No.: P.0287. WO 1WHAT IS CLAIMED IS:

1. A composition comprising a targeting moiety or molecule that binds to cerebellin-1 (CBLN1), or binds to a complex of cerebellins optionally comprising CBLN1, wherein the targeting moiety or molecule enables enhanced delivery of the composition to the central nervous system (CNS) compared to a composition without the targeting moiety or molecule, and optionally wherein the targeting moiety or molecule is associated with a therapeutic or diagnostic agent.

2. A composition comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4, wherein the cerebellin enables enhanced delivery of the composition to the central nervous system (CNS), and optionally wherein the cerebellin molecule is associated with a therapeutic or diagnostic agent.

3. A fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4, wherein the cerebellin enables enhanced delivery of the fusion protein to the central nervous system (CNS), and optionally wherein the cerebellin molecule is associated with a therapeutic or diagnostic agent.

4. A composition comprising:i) A) a cerebellin, optionally cerebellin 1, 2, 3, and / or 4; or B) a fusion protein comprising a cerebellin, optionally cerebellin 1, 2, 3, and / or 4;andii) a targeting molecule that binds to cerebellin-1 (CBLN1), or binds to a complex of cerebellins optionally comprising CBLN1 ;optionally wherein the cerebellin or the targeting molecule is associated with a therapeutic or diagnostic agent.

5. The composition of claim 1 or claim 4, wherein the targeting molecule binds to CBLN1 with a KD of 0.001-100 nM, optionally wherein the KD is measured by a BLI assay.

6. The composition of claim 1 or claim 4, wherein the targeting molecule comprises a polymer, optionally wherein the targeting molecule is a polypeptide.494896-3717-6698Ref. No.: P.0287. WO 17. The composition of any one of claims 1-2 or claims 4-6 or the fusion protein of claim 3, wherein the targeting molecule or cerebellin is associated with, fused, or conjugated to a small molecule, antibody, zinc finger protein, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, polymer, or recombinant protein.

8. The composition of any one of claims 1-2 or claims 4-7 or the fusion protein of claim 3 or claim 7, wherein the composition or fusion protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.

9. The composition or fusion protein of claim 8, wherein the composition or fusion protein comprises all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.

10. The composition of any one of claims 1-2 or claims 4-9 wherein the composition comprises an AAV capsid protein.

11. The composition of claim 10, wherein the composition encapsidates a nucleotide encoding a therapeutic or diagnostic agent, optionally wherein the composition was derived from a parental AAV capsid protein.

12. The composition of claim 10, wherein the AAV capsid protein is STAC-BBB, optionally wherein the STAC-BBB AAV capsid protein is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO.1.

13. The composition of claim 10, wherein the parental AAV capsid protein is selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74.

14. The composition of any one of claims 10-13, wherein the AAV capsid protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all contiguous amino acids of an amino acid sequence (“Peptide Sequence”) set forth in SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7.504896-3717-6698Ref. No.: P.0287. WO 115. The composition of claim 14, wherein the amino acid sequence is inserted into a parent capsid (“Parent Capsid”) at an insertion site (“Peptide Insertion Site”) as shown in a single row (third and fourth columns, respectively) in Tables 1 to 7.

16. The composition of any one of claims 10-15, wherein the amino acid sequence comprises a peptide sequence of any one of SEQ ID NOs: 7-4109 as shown in a single row and second column in Tables 1 to 7, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row (third and / or fourth columns, respectively) as shown in Tables 1 to 7.

17. An AAV capsid protein comprising, an insertion sequence of Formula I between residues 453 / 454 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):X1X2X3X4X5X6X7 (I);wherein, Xi is an amino acid selected from K or R; X2 is an amino acid selected from A, G, H, L, N, P, Q, S, T, or V; X3 is an amino acid selected from A, D, H, N, Q, S, T, or V; X4 is an amino acid selected from A, D, E, G, N, P, Q, S, or T; X5 is an amino acid selected from A, D, E, N, Q, S, T, or V; Xe is an amino acid selected from A, D, E, G, N, P, or Q; and X7 is an amino acid selected from A, D, H, N, P, Q, or S;and wherein the AAV capsid protein comprises a targeting molecule.

18. An AAV capsid protein comprising, an insertion sequence of Formula II located between residues 453 / 454 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):X1X2P (II);wherein, Xi is an amino acid selected from D or E; and X2 is an amino acid selected from A, H, K, P, Q, R, S, or T;and wherein the AAV capsid protein comprises a targeting molecule.

19. An AAV capsid protein comprising, an insertion sequence of Formula III located between residues 589 / 590 of AAV1 (SEQ ID NO. 3) or AAV6 (SEQ ID NO. 4):X1X2X3GQ (III);514896-3717-6698Ref. No.: P.0287. WO 1wherein, Xi is an amino acid selected from A, E, I, L, N, P, Q, or S; X2 is an amino acid selected from A, G, L, M, N, Q, R, S, or T; and X3 is an amino acid selected from E, H, L, N, R, S, or T;and wherein the AAV capsid protein comprises a targeting molecule.

20. An AAV capsid protein comprising, an insertion sequence of Formula IV located between residues 577 / 578 of AAV5 (SEQ ID NO. 5):RX1X2X3X4DX5P (IV);wherein, Xi is an amino acid selected from H, I, K, L, Q, R, T, or V;X2 is an amino acid selected from K, N, Q, R, or S; X3 is an amino acid selected from A, E, F, H, I, P, S, or V; X4 is an amino acid selected from A, D, M, Q, S, or T; and X5 is an amino acid selected from D, F, L, M, N, Q, S, T, W;and wherein the AAV capsid protein comprises a targeting molecule.

21. An AAV capsid protein comprising, an insertion sequence of Formula V contained between residues 577 / 578 of AAV5 (SEQ ID NO. 5):XiTDLP (V);wherein, Xi is an amino acid selected from A, H, P, or V;and wherein the AAV capsid protein comprises a targeting molecule.

22. An AAV capsid protein comprising, an insertion sequence of Formula VI contained between residues 588 / 589 of AAV9 (SEQ ID NO. 2):RX1X2X3X4P (VI);wherein, Xi is an amino acid selected from D, G, L, P, S, T, or V;X2 is an amino acid selected from D, F, L, M, Q, S, T, or V; X3 is an amino acid selected from A, D, G, L, N, P, S, or V; andX4 is an amino acid selected from D, E, G, H, L, P, S, or T;and wherein the AAV capsid protein comprises a targeting molecule.

23. An AAV capsid protein comprising, an insertion sequence of Formula VII contained between residues 588 / 589 of AAV9 (SEQ ID NO. 2):X1X2X3X4P (VII);524896-3717-6698Ref No.: P.0287. WO 1wherein, Xi is an amino acid selected from A, G, K, L, M, P, R, or T;X2 is an amino acid selected from A, F, K, L, Q, R, S, T, or V; X3 is an amino acid selected from A, D, H, I, L, N, P, Q, R, S, or V; and X4 is an amino acid selected from D, or E;and wherein the AAV capsid protein comprises a targeting molecule.

24. The composition of any one of claim 1 or claims 4-23, wherein the targeting molecule binds human, cynomolgus, or murine CBLN1.

25. The composition of any one of claims 1-24, wherein the cerebellin protein is derived from human, cynomolgus, or murine CBLN1.

26. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising fusing or conjugating the therapeutic or diagnostic agent to , or associating the therapeutic or diagnostic agent with, the targeting molecule or cerebellin according to any one of claims 1-25.

27. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising or encapsidating a nucleic acid encoding the therapeutic or diagnostic agent in, fusing or conjugating the therapeutic or diagnostic agent to, or associating the therapeutic or diagnostic agent with, an AAV capsid protein according to any one of claims 10-23.

28. An engineered adeno-associated virus (AAV) capsid protein, wherein the capsid protein binds to one or more of cerebellin 1-4, and wherein the capsid protein has enhanced delivery to the CNS compared to a parental capsid that does not associate with cerebellin 1, 2, 3, and / or 4.

29. The capsid protein of claim 28, wherein the capsid protein comprises an AAV capsid protein according to any one of claims 10-23, optionally wherein the capsid protein encapsidates a nucleic acid encoding a therapeutic or diagnostic agent.

30. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising fusing or conjugating the therapeutic or diagnostic agent to, associating the therapeutic or diagnostic agent with, or encapsidating a nucleic acid encoding the therapeutic or diagnostic agent in, the capsid protein of claim 28 or claim 29.

31. A method for identifying an AAV capsid variant with increased transduction of the CNS, comprising selecting for AAV capsid variants that bind to cerebellin 1, 2, 3, and / or 4.534896-3717-6698Ref. No.: P.0287. WO 132. The method of claim 31 , comprising modifying one or more amino acids within a hypervariable and / or surface exposed loop of a selected AAV capsid variant to create a modified AAV capsid variant, optionally wherein the modified AAV capsid variant is further screened for improved transduction of the CNS compared to an AAV capsid without a modification in one or more amino acids within a hypervariable and / or surface exposed loop.

33. A method for identifying a targeting molecule that confers increased transduction of the CNS within a pool of compositions, comprising selecting for compositions that bind cerebellin 1, 2, 3, and / or 4, optionally wherein the targeting molecule comprises or is selected from peptides, small protein fragments, antibodies, or AAV capsids.

34. The method of claim 33, wherein the targeting molecule is part of an AAV capsid variant.

35. A composition comprising a cerebellin 1, 2, 3, and / or 4 protein and a cerebellin 1, 2, 3, and / or 4 targeting molecule, optionally wherein the cerebellin 1, 2, 3, and / or 4 targeting molecule is associated with a therapeutic or diagnostic agent.

36. A composition comprising a cerebellin 1, 2, 3, and / or 4 protein, optionally wherein the cerebellin 1, 2, 3, and / or 4 protein is associated with a therapeutic or diagnostic agent.

37. The composition of claim 35, wherein an AAV capsid variant comprises the cerebellin 1, 2, 3, and / or 4 targeting molecule.

38. The composition of claim 35 or claim 36, wherein a capsid protein according to any one of claims 10-23 comprises the cerebellin 1, 2, 3, and / or 4 targeting molecule.

39. The composition of any one of claims 35-38, wherein the cerebellin 1, 2, 3, and / or 4 protein or the cerebellin 1, 2, 3, and / or 4 targeting molecule is fused or conjugated to, or is associated with, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFv, oligonucleotide, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.

40. The composition of any one of claims 35-39, wherein the cerebellin 1, 2, 3, and / or 4 protein or the cerebellin 1, 2, 3, and / or 4 targeting molecule binds human, cynomolgus, or murine cerebellin 1, 2, 3, and / or 4.544896-3717-6698Ref No.: P.0287. WO 141. The composition of claim 37 or claim 38, wherein the AAV capsid variant or the capsid protein encapsidates a nucleic acid encoding a therapeutic or diagnostic agent.

42. The composition of claim 37, wherein the AAV capsid variant is derived from a parental capsid selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAVrhlO, AAVrh39, or AAVrh74.

43. The composition of claim 35, wherein the composition provides for increased CNS delivery of the cerebellin 1, 2, 3, and / or 4 targeting molecule compared to administering the cerebellin 1, 2, 3, and / or 4 targeting molecule without the cerebellin 1, 2, 3, and / or 4 protein.

44. The composition of claim 36, wherein the composition provides for increased CNS delivery of the therapeutic or diagnostic agent compared to administering the therapeutic or diagnostic agent without being associated with the cerebellin 1, 2, 3, and / or 4 protein.

45. A method of delivering a therapeutic or diagnostic agent to the CNS, comprising administering the composition of any one of claims 35-44 to a subject.554896-3717-6698

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