Peptides targeting LDLR for blood-brain barrier crossing

Abstract

This application relates to targeting peptides and engineering AAV capsids. In some embodiments, the targeting peptides and engineered AAV capsids are capable of interacting with LDLR receptor to mediate delivery to a cell or tissue.

Description

PEPTIDES TARGETING LDLR FOR BLOOD-BRAIN BARRIER CROSSINGFIELD

[0001] This application relates to peptides and engineered AAV (adeno-associated virus) capsids that mediate blood-brain barrier crossing by interacting with LDLR (Low-Density Lipoprotein Receptor).CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 643,754, filed on May 7, 2024 and entitled “PEPTIDES TARGETING LDLR FOR BLOOD-BRAIN BARRIER CROSSING,” and U.S. Provisional Patent Application No. 63 / 643,687, filed on May 7, 2024 and entitled “PEPTIDES TARGETING TFRC FOR BLOOD-BRAIN BARRIER CROSSING,” the entire contents of which are incorporated by reference herein.BACKGROUND

[0003] The clinical translation of genomic medicines has been limited by inefficient gene delivery through viral vectors like AAV capsids.

[0004] Attempts at engineering AAV capsids with improved properties, e.g., improved tropism to a target cell or tissue upon systemic administration, have met with limited success due to the unpredictable nature of how AAV capsid modifications influence targeting of cellular receptors. As such, there is a need for improved methods of engineering AAV capsids for delivery of genetic material of interest to a target cell or tissue, e.g., a CNS cell or tissue, including delivery across the blood-brain barrier (BBB).

[0005] One approach to achieve this goal is to engineer AAV capsids that interact with receptors that are expressed in target cell and tissue types. LDLR (Low-Density Lipoprotein Receptor) is a receptor that is expressed on the blood-brain barrier and is involved in the transcytosis of low-density lipoprotein (LDL) across the BBB. However, LDLR is not known to be a target for AAV or to mediate transcytosis of AAV across the BBB.

[0006] There remains a need to engineer AAV capsids having enhanced tropism to a target cell or tissue through engineering novel receptor interactions.SUMMARY

[0007] In an aspect, targeting molecules, i.e., targeting peptides, are provided to enable targeting of a cell or a tissue, for example, the CNS. In some embodiments, a CNS-targeting molecule interacts with (e.g., binds to) LDLR, thereby enabling the CNS-targeting molecule to cross the BBB. In some embodiments, the CNS-targeting molecule comprises a peptide motif as indicated in a single row of Table 4. In some embodiments, the CNS-targeting molecule 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 Tables 1 to 3. In some embodiments, the amino acid sequence is fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, a method for identifying a CNS-targeting molecule that crosses the BBB is provided, comprising selecting for variant AAV capsids that interact with LDLR.

[0008] In some embodiments, an adeno-associated virus (AAV) capsid protein is provided that interacts with (e.g., binds to) LDLR, thereby enabling the AAV capsid protein to cross the BBB. In some embodiments, the AAV capsid protein comprises a peptide motif as indicated in a single row of Table 4, optionally wherein the peptide motif is located within a surface- exposed loop of a parent capsid. In some embodiments, the peptide motif is located within an insertion between amino acids 587 and 590 in AAV9 (SEQ ID NO: 640). In some embodiments, 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”) as shown in a single row in Tables 1 to 3. In some embodiments, the amino acid sequence is inserted into a parent capsid ("Parent Capsid”) at an insertion site (“Peptide Insertion Site”), optionally as shown in a single row in Tables 1 to 3. In some embodiments, the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 3, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row as shown in a single row in Tables 1 to 3. In some embodiments, the 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 (i.e., CNSRCV300 in US Application No.63 / 606, 012).

[0009] In some embodiments, an AAV capsid protein is provided, wherein the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in SEQ ID NOs: 641-649 designated as CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453.

[0010] In some embodiments, a nucleic acid molecule encoding an engineered AAV capsid protein described herein is provided. In some embodiment, a host cell comprising such a nucleic acid molecule is provided.

[0011] In some embodiments, a composition is provided, comprising: 1) an adeno- associated virus (AAV) capsid protein as described herein; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload. In some embodiments, the payload of interest is a therapeutic payload, and the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein. In some embodiments, the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.

[0012] In some embodiments, a method of delivering a payload of interest to a cell or a tissue is provided, wherein a coding sequence for the therapeutic payload is encapsidated in an AAV capsid protein as described herein, and wherein the AAV capsid protein interacts with LDLR. In some embodiments, delivering the payload of interest to the cell or the tissue comprises crossing a BBB.

[0013] In some embodiments, a method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell, comprising contacting the cell with a composition as described herein.

[0014] In some embodiments, a method of treating a disease in a subject is provided, comprising administering to the subject a composition as described herein.

[0015] In some embodiments, use of an AAV capsid protein, a nucleic acid construct, or a host cell as described herein is provided for the manufacture of a medicament in a method as described herein.

[0016] In some embodiments, a method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue is provided, comprising selecting for variant AAV capsids that interact with LDLR.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 illustrates a schematic of the approach used to pan AAV capsid libraries against an immobilized receptor. The receptor, in this case human LDLR, is biotinylated and then immobilized on a streptavidin coated bead. The AAV capsid libraries are then panned in parallel against the immobilized receptor or a streptavidin bead control without a receptor. AAV library genomes are extracted from each sample, amplified via PCR, and subjected to nextgeneration sequencing to quantify the enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the immobilized receptor condition relative to the bead only control.

[0018] FIG. 2 illustrates a schematic of the approach used to assess AAV library transduction in cells overexpressing a receptor, in this case human LDLR, relative to AAV library transduction in cells expressing a fluorescent protein transfection control. Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA is extracted from cells, reverse transcribed to cDNA, and then PCR is used to amplify the AAV cDNA. Next-generation sequencing analysis is used to quantify enrichment of capsid variants. Capsids that specifically interact with the receptor are identified by comparing enrichment in the receptor expression condition relative to the fluorescent protein transfection control.

[0019] FIG. 3 illustrates a schematic of the approach used to assess AAV library binding in cells overexpressing a receptor, in this case human LDLR, relative to AAV library binding in cells expressing a fluorescent protein transfection control. Cells are transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells are transduced with the AAV capsid libraries. Cells are incubated for 1 hour to allow for AAV binding to cells. DNAis extracted from cells and then PCR is used to amplify the AAV genome. Next-generation sequencing analysis is used to quantify enrichment of capsid variants. Capsidsthat specifically interact with the receptor are identified by comparing enrichment in the receptor expression condition relative to the fluorescent protein transfection control.

[0020] FIG. 4 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque LDLR. Three representative examples of capsid library screens that were conducted for LDLR are shown:1) Immobilized human receptor versus a bead only control. Data shown are for the round 2 library.2) Binding to cells overexpressing human LDLR versus a transfection control. Data shown are for the round 2 library.3) Binding to cells overexpressing macaque LDLR versus a transfection control. Data shown are for the round 2 library.

[0021] A subset of capsids that exhibited specific enrichment for human or macaque LDLR orthologs are colored in green. The marginal axis histograms represent capsids that were identified in only the receptor condition (y-axis) or the negative control (x-axis).

[0022] FIG. 5 The figure shows that AAV library screens can identify capsid variants that specifically target human or cynomolgus macaque LDLR. Two representative examples of capsid library screens that were conducted for LDLR are shown:1) Transduction of cells overexpressing human LDLR versus a transfection control. Data shown are for the round 2 library.2) Transduction of cells overexpressing macaque LDLR versus a transfection control. Data shown are for the round 2 library.

[0023] A subset of capsids that exhibited specific enrichment for human or macaque LDLR orthologs are colored in green. The marginal axis histograms represent capsids that were identified in only the receptor condition (y-axis) or the negative control (x-axis).

[0024] FIG. 6 shows the performance of five exemplary capsid variants that exhibit enrichment for binding to immobilized human LDLR, and transduction of cells overexpressing either human or cynomolgus macaque receptor.

[0025] FIG. 7(A)-(C) shows Tables 1 , 2, and 3.

[0026] FIG. 8 shows the relative mRNA expression mediated by engineered capsids in cells overexpressing a receptor, in this case human LDLR, relative to the transgene expression in wild-type cells. A stable CHO-K1 cell line expressing human LDLR was generated using alentiviral vector. Wild-type CH0-K1 cells or CH0-K1 cells engineered to express human LDLR were transduced with a barcoded pool of AAV capsids including the control capsid AAV9 as well as capsids engineered to target LDLR. Cells were incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA was extracted from cells, reverse transcribed to cDNA, and then PCR was used to amplify the barcoded AAV cDNA. Nextgeneration sequencing analysis was conducted to quantify enrichment of capsid variants in the pool relative to the input abundance of each capsid in the barcoded pool. Capsids engineered to target LDLR exhibit higher transduction in the engineered CH0-K1 cells overexpressing human LDLR compared to wild-type CH0-K1 cells. In contrast, the parental capsid AAV9 shows no significant increase in transgene expression in the engineered CH0-K1 cells overexpressing human LDLR compared to wild-type CH0-K1 cells. The size of each circle is proportional to the absolute value of the log2FC. The log2FC value is annotated above each circle. The coefficient of variation (CoV) percentile is represented by the circle opacity as shown in the legend, wherein a higher percentile score indicates a lower coefficient of variation.

[0027] FIG. 9 shows the transgene expression mediated by individual engineered capsids in cells overexpressing human or cynomolgus macaque LDLR, relative to the transgene expression in cells expressing a fluorescent protein transfection control. Representative engineered capsids, designed to target LDLR and described herein, are presented as illustrative examples. Neuro2A cells were transfected with a plasmid encoding a strong ubiquitous promoter that drives the expression of the receptor or a negative control fluorescent protein. Forty-eight hours after transfection, the cells were transduced with the AAV capsid. Cells were incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA was extracted from cells and RT-qPCR was conducted to quantify mRNA transgene expression. Data were normalized to expression of the housekeeping gene GAPDH. Capsids engineered to target LDLR exhibit higher transduction in cells overexpressing human, or macaque LDLR relative to cells overexpressing a fluorescent protein transfection control. In contrast, the parental capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or macaque LDLR relative to cells overexpressing a fluorescent protein transfection control.

[0028] FIG. 10 shows the fold change improvement in AAV mediated transgene expression for cells expressing LDLR relative to cells expressing the transfection control. Representative engineered capsids, designed to target LDLR and described herein, are presented as illustrative examples. Capsids engineered to target LDLR exhibit significantlyhigher transduction in cells overexpressing human or macaque LDLR relative to cells overexpressing a fluorescent protein transfection control. In contrast, the parent capsid AAV9 shows no significant increase in transgene expression in cells overexpressing human or macaque LDLR relative to cells overexpressing a fluorescent protein transfection control.

[0029] FIG. 11 shows the fold change improvement in transgene expression for capsids engineered to target LDLR relative to the parental capsid AAV9. Representative engineered capsids, designed to target LDLR and described herein, are presented as illustrative examples. The transduction mediated by the capsids engineered to target LDLR is significantly enhanced relative to AAV9 in cells expressing human or macaque LDLR.DETAILED DESCRIPTION

[0030] In an aspect, targeting molecules, i.e., targeting peptides, are provided for interacting with a specific cellular receptor, preferably where in the receptor is expressed on the blood brain barrier. In embodiments, the receptor is the low-density lipoprotein receptor, LDLR. In an aspect, delivery to and / or targeting of a cell or a tissue, preferably wherein the molecules are CNS (central nervous system)-targeting. In embodiments, CNS-targeting molecules comprising a targeting peptide sequence indicated in Tables 1-3 are provided. In another aspect, engineered AAV capsid proteins are provided. In embodiments, a targeting peptide is inserted into a parental AAV capsid, for example an AAV3B capsid protein (SEQ ID NO: 638), an AAV5 capsid protein (SEQ ID NO: 639), or an AAV9 capsid protein (SEQ ID NO: 640). In embodiments, the targeting peptide is any of the targeting peptides disclosed in Tables 1-3. In embodiments, the peptide sequence is inserted into the parental capsid at any of the peptide insertion sites disclosed in Tables 1-3. In embodiments, the peptide sequence that is inserted into the parental capsid contains a motif disclosed in Table 4. In some embodiments, the targeting peptide functions to target the CNS-targeting molecule to a specific target tissue (e.g., CNS tissue).Generation of an Engineered AAV Capsid Library

[0031] In one embodiment, disclosed herein is the development of libraries encoding engineered AAV capsid proteins, wherein members of the library encode engineered AAV capsid proteins having different sequences, and wherein some members of the library encode an AAV capsid protein having a desired characteristic compared to a natural / wild-type AAV serotype. In one embodiment, disclosed herein is the development of libraries encoding 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 is increased penetrance through the blood brain barrier following administration to a subject. In some embodiments, the desired characteristic is wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus. In some embodiments, the desired characteristic is elevated genetic material expression in multiple brain regions. In some embodiments, the desired characteristic is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0032] 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 promoter(s); d) a nucleic acid sequence encoding a unique molecular identifier (UMI); and combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to and expressed in a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence.

[0033] In some embodiments, each engineered AAV capsid protein was synthesized as an oligo pool. In some embodiments, each member of a library comprises one or more (such as 1- 10) of a nucleic acid sequence encoding an AAV capsid protein comprising a) nucleic acid sequences encoding one or more (such as 1-10) barcodes: b) nucleic acid sequences encoding one or more (such as 1-10) promoters; c) nucleic acid sequences encoding one or more (such as 1-10,000) unique molecular identifiers (UMIs); or combinations thereof. In some embodiments, each member of the library also includes genetic material to be delivered to a cell or tissue of interest. In some embodiments, each member of the library also includes a polyA sequence. In some embodiments, each of the one or more (such as 1-10) barcodes is linked to the identity of a single engineered AAV capsid protein. In some embodiments, each of the barcodes is linked to one or more (such as 1-10,000) UMIs.

[0034] 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.

[0035] 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.

[0036] 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 AAV3B, AAV5, or AAV9 (SEQ ID NOs: 638-640). In some embodiments, the engineered AAV capsid proteins contain a peptide sequence inserted within a parent capsid protein, for example, the parent capsid AAV3B, AAV5, or AAV9 (SEQ ID NOs: 638-640). 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 AAV3B, AAV5, or AAV9 (SEQ ID NOs: 638-640).

[0037] In some embodiments, the libraries are packaged in HEK293 cells where the helper functions (e.g. E2A, E4, VA, El A and E1B) are supplied in trans. In some embodiments, the AAV rep function comprises rep78, rep 68, rep 52, and rep40 genes. In some embodiments, therep genes are supplied in trans. In some embodiments, the start codon of the rep78 and / or the rep68 gene is altered from ACG to ATG to increase replication of the capsid library construct containing inverted terminal repeats (ITRs), thereby improving AAV library manufacturing yield. In some embodiments, the cap genes are supplied as genetic material that is packaged into the manufactured AAVs. In some embodiments, the capsid gene is controlled by the p40 promoter such that it is only expressed during manufacturing in HEK293 cells in the presence of helper virus functions.Methods of Screening Libraries of Engineered AAV Capsid Proteins

[0038] 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, 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.

[0039] In another embodiment, disclosed herein are methods for directed evolution of engineered AAV capsid proteins and identification of an engineered AAV capsid protein with a desired characteristic compared to a natural / wild-type AAV serotype. In some embodiments, the steps for directed evolution of engineered AAV capsid proteins to identify engineered AAV capsid proteins with a desired characteristic compared to a natural / wild-type AAV serotype comprise (i) modifying the natural / wild-type AAV serotype to create variant capsids; (ii) packaging of the variant AAVs in producer cells wherein adenovirus helper and AAV rep functions are supplied in trans; (iii) purification of viral capsid library pools; (iv) administration of the pools in vitro or in vivo; (v) recovery of engineered AAV capsid proteins from target tissues or cell lines; (vi) next-generation sequencing to determine the identity of the engineered variant capsid sequences; (vii) repeated rounds of in vitro or in vivo selection where variants are isolated from a target tissue or cell line; and (viii) full evaluation of enriched variants. In some embodiments, the desired characteristic includes enhanced tissue tropism as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includes enhanced tissue tropism for tissues of the peripheral nervous system as compared to the natural / wild-type AAV serotype. In some embodiments, the desired characteristic includesenhanced tissue tropism of the central nervous system as compared to the natural / wild-type AAV serotype.Engineered AAV Capsid Proteins

[0040] 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. LDLR. In some embodiments, the engineered AAV capsid proteins demonstrate binding to a receptor, e.g. LDLR. 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.

[0041] 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 AAV3B, AAV5, or AAV9 (SEQ ID NOs: 638-640). In some embodiments, the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 638). 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: 639). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 640).

[0042] 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 Tables 1 to 3. 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 anamino acid sequence (“Peptide Sequence”) as shown in a single row in Tables 1 to 3. In some embodiments, the amino acid sequence comprises a peptide sequence as indicated in a single row in Tables 1 to 3, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row. In some embodiments, an engineered AAV capsid protein sequence is provided comprising a peptide motif as indicated in a single row of Table 4, optionally wherein the peptide motif is located within an insertion between amino acids 587 and 590 in AAV9 (SEQ ID NO: 640).

[0043] 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 Tables 1 to 3.

[0044] In some embodiments, the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises any one of the sequences corresponding to SEQ ID NOs: 641-649 designated as CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453.

[0045] In some embodiments, the engineered AAV capsid proteins have advantages over wild-type AAV capsid proteins. In some embodiments, these advantages including (i) enhanced cell or tissue tropism as compared to the natural / wild-type AAV serotype, for example, enhanced cell or tissue tropism to the central nervous system (CNS) as compared to the natural / wild-type AAV serotype (ii) increased penetrance through the blood brain barrier following administration to a subject, (iii) wider distribution throughout the multiple brain regions, for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus, (iv) elevated expression of genetic material in multiple brain regions. In some embodiments, the engineered AAV capsids enhance the delivery of genetic material to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and / or hippocampus, (v) is delivery of genetic material of interest to a desired tissue, cell, or organelle.

[0046] 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, ororganisms. 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 are produced recombinantly and are based on any one or more (such as 1-15) AAV serotypes known in the art.Adeno-associated virus (AAV)

[0047] 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. LDLR. 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, LDLR.Modified AAV Serotypes

[0048] 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 aregenerated 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.

[0049] In some embodiments, different AAV serotypes are different in their ability to direct or modulate an AAV particle to a particular cell or tissue. In some embodiments, the AAV serotype can be modified to interact with a receptor, e.g. LDLR. 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).

[0050] 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 regions, for example, 2 brain regions, 3 brain regions, 4 brain regions, 5 brain regions, 6 brain regions, 7 brain regions, 8 brain regions, 9 brain regions, or 10 brain regions. In 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.

[0051] 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.

[0052] In some embodiments, the modified AAV serotype comprises a peptide motif described herein (e.g., as shown in Table 4). 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 allcontiguous amino acids of a Peptide Sequence shown in Tables 1-3. In some embodiments, the inserted sequence is inserted into a parent capsid serotype as shown in Tables 1-3. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-3. In some embodiments, the modified AAV serotype comprises an inserted peptide sequence described herein (e.g., a Peptide Sequence shown in Tables 1-3) and / or a peptide motif described herein (e.g., Table 4). In some embodiments, the modified AAV serotype comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences corresponding to CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453. In some embodiments, the modified AAV sequence comprises any one of the sequences corresponding to CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453.Structure of AAV

[0053] 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.

[0054] 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.AAV particles

[0055] In some embodiments, the engineered AAV capsid proteins assemble to form AAV particles. In some embodiments, the engineered AAV capsid proteins interact with a receptorexpressed at the blood brain barrier, for example, LDLR. 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.Delivery of AAV particles

[0056] 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.

[0057] 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.AAV viral genomes

[0058] 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.

[0059] 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, theITR regions are derived from a serotype known to the art. ITR regions may be between 100 and 150 nucleotides in length.Targeting Peptide Sequences

[0060] In embodiments, targeting peptide sequences (e.g., targeting molecules or sequences) are disclosed herein. In some embodiments, the sequences enhance or enable interaction with LDLR. In some embodiments, the sequences can function to target a cell or a tissue, for example, as a general CNS-targeting molecule or sequence.

[0061] 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, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, any one of the targeting sequences or sequence motifs described herein may be fused or conjugated to a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein. In some embodiments, CNS-targeting sequences may be utilized to enable a small molecule, an antibody, zinc finger protein, scFV, ASO (antisense oligonucleotide), siRNA, lipid, polymer or recombinant protein to cross the blood brain barrier.

[0062] 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.

[0063] In some embodiments, the targeting sequences enable the binding of an AAV capsid protein to a specific receptor, e.g. LDLR. 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. LDLR. In some embodiments, the targeting sequences modulate the binding affinity of an AAV capsid protein to a specific receptor, e.g. LDLR.

[0064] In some embodiments, the sequences may increase tropism of an AAV capsid protein to a cell or tissue of the CNS. In some embodiments, the cell of the CNS is a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial cells (e.g., microglia, astrocytes, oligodendrocytes) and / or asupporting 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.

[0065] 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).

[0066] 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).

[0067] 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 Tables 1 to 3. In some embodiments, the sequence comprises a motif sequence as shown in Table 4. In some embodiments, the sequence comprises the amino acid sequence (“Peptide Sequence”) as shown in a single row in Tables 1 to 3.

[0068] 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 1-3. In some embodiments, the inserted sequence is inserted into a parent capsid sequence at or near the Peptide Insertion Site as shown in Tables 1-3. 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, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, or STAC-BBB.

[0069] In some embodiments the peptide sequence is inserted within or near amino acids 588 through 589 corresponding to the sequence of AAV3B (SEQ ID NO: 638). 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: 639). In some embodiments the peptide sequence is inserted within or near amino acids 587 through 590 corresponding to the sequence of AAV9 (SEQ ID NO: 640).

[0070] Administering Engineered AAV Capsid Proteins to Subjects

[0071] In some embodiments, when administered to subjects, AAV capsid proteins containing the targeting peptides described herein mediate enhanced delivery to cells and tissues relative to AAV capsid proteins that lack the targeting peptides. In some embodiments, the AAV capsid protein administered to subjects comprises an engineered AAV sequence described herein.Genetic Material

[0072] 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 embodiment, 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.

[0073] In some embodiments, the genetic material can include sequences that are coding sequences. In some embodiments, the genetic material can include sequences that are noncoding sequences. In some embodiments, the genetic material can include sequences that are both coding sequences and non-coding sequences. In some embodiments, the expression of the genetic material is capable of being regulated. In some embodiments, the genetic material comprises elements that are regulatable.

[0074] In some embodiments, mRNA is encoded in the genetic material. In some embodiments, the mRNA is codon optimized.

[0075] 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.

[0076] 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 specificpromoter. In some embodiments, the one more promoters comprise a ubiquitous promoter. Examples of ubiquitous promoters include cytomegalovirus (CMV), chicken P-actin (CBA), ubiquitin C (UBC), and elongation factor la-subunit (EFl -a), amongst others. In some embodiments, the promoter comprises a cell type and / or tissue specific type promoter. Exemplary cell type and / or tissue specific promoters include the human synapsin promoter (hSynl), only expressed in neurons, or the transthyretin promoter (TTR), expressed in hepatocytes. Other non-limiting cell type and / or tissue specific promoters for use in the methods and compositions of the invention include cytokeratin 18 and 19 (epithelial cell specific, Other cell-specific promoters include GFAP promoter (astrocytes), TBG promoter (liver), MHCK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes). In embodiments, tissue specific or cell specific promoters can restrict expression to tissues or cells of the CNS or PNS. In embodiments, tissue specific or cell specific promoters can be used to restrict expression to neurons of the sympathetic system, the parasympathetic system, astrocytes, microglia, oligodendrocytes, and / or Schwann cells.

[0077] 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.Gene Editing System

[0078] 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 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.Epigenetic Regulation System

[0079] 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 ofmodifying 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.Active Agents

[0080] In some embodiments, engineered AAV sequences described herein are fused or coupled to an active agent. In some embodiments, a sequence is fused or coupled to an active agent through conjugation. In some embodiments, the active agent comprises a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibody or a portion of an antibody (e.g., Fc region). In some embodiments, the sequence is fused to a Fc region of an antibody. In some embodiments, the sequence is fused to the C-terminus of the Fc region. In some embodiments, the sequence is fused to the N-terminus of the Fc region. In some embodiments, the therapeutic agent comprises an RNAi agent (e.g., siRNA, shRNA, IncRNA, piRNA, snoRNA, or miRNA). In some embodiments, the sequence is fused or coupled directly to at least on strand of the RNAi. In some embodiments, the sequence is fused or coupled to at least one strand of RNAi using a linker. In some embodiments, the sequence is fused or coupled to the sense strand of RNAi. In some embodiments, the sequence is fused or coupled to the antisense strand of RNAi.

[0081] In some embodiments the active agent comprises a diagnostic agent. In some embodiments, the diagnostic agent comprises a detectable moiety such as a fluorophore. In some embodiments, the active agent is a small molecule.Pharmaceutical Compositions and Dosage Forms

[0082] Compositions herein e.g., 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 diluentsto (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.

[0083] 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.

[0084] In some embodiments, the compositions described herein (e.g., 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.

[0085] In some embodiments, the pharmaceutical compositions described herein function to increase the stability, increase transduction or transfection efficiency, impact biodistribution, increase expression of the protein, and / or alter the release profile.Methods of Delivery and Treatment

[0086] 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.

[0087] 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.

[0088] In some embodiments, the compositions described herein are administered through systemic administration. In some embodiments, systemic administration includes intravenous administration.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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 areadministered 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.

[0093] 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.

[0094] 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.

[0095] 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.Equivalents and Scope

[0096] 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, andtechniques 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 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.

[0097] 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.

[0098] 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.

[0099] 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.Exemplary Embodiments

[0100] Non-limiting exemplary embodiments of the present disclosure are described below.Embodiment 1. An adeno-associated virus (AAV) capsid protein that interacts with Low-Density Lipoprotein Receptor (LDLR), thereby enabling delivery of the AAVcapsid protein to a cell or a tissue, optionally wherein interaction with LDLR enables the AAV capsid protein to cross the blood-brain barrier (BBB).Embodiment 2. The AAV capsid protein of embodiment 1, comprising a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3), optionally wherein the peptide motif is located within a surface-exposed loop of the AAV capsid protein.Embodiment 3. The AAV capsid protein of embodiment 2, wherein the peptide motif is located within an insertion between amino acids 587 and 590 in AAV9 (SEQ ID NO: 640).Embodiment 4. The AAV capsid protein of embodiment 1, 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”) set forth in any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.Embodiment 5. The AAV capsid protein of embodiment 4, 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 in Tables 1 to 3.Embodiment 6. The AAV capsid protein of embodiment 5, wherein the amino acid sequence comprises a peptide sequence according to any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row as shown in Tables 1 to 3.Embodiment 7. The AAV capsid protein of any one of embodiments 1-6, wherein the 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.Embodiment 8. An AAV capsid protein, wherein the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in any one of SEQ ID NOs: 641-649 designated as CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453.Embodiment 9. A nucleic acid molecule encoding an AAV capsid protein of any one of embodiments 1-8.Embodiment 10. A host cell comprising the nucleic acid molecule of embodiment 9.Embodiment 11. A composition comprising: 1) an AAV capsid protein of any one of embodiments 1-8; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload.Embodiment 12. The composition of embodiment 11, wherein the payload of interest is a therapeutic payload, and wherein the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.Embodiment 13. The composition of embodiment 11 or embodiment 12, wherein the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR- associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.Embodiment 14. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an AAV capsid protein according to any one of embodiments 1-8, and wherein the AAV capsid protein interacts with LDLR.Embodiment 15. The method of embodiments 14, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.Embodiment 16. A method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell, comprising contacting the cell with a composition of any one of embodiments 11-13.Embodiment 17. A method of treating a disease in a subject, comprising administering to the subject a composition of any one of embodiments 11-13.Embodiment 18. Use of an AAV capsid protein of any one of embodiments 1-8, a nucleic acid construct of embodiment 9, or a host cell of embodiment 10 for the manufacture of a medicament in a method of any one of embodiments 14-17.Embodiment 19. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that interact with LDLR.Embodiment 20. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that: i) comprise a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3); or ii) comprise 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 any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.Embodiment 21. A targeting molecule that interacts with LDLR, thereby enabling delivery of the targeting molecule to a cell or a tissue, optionally wherein the interaction with LDLR enables the targeting molecule to cross the blood-brain barrier (BBB).Embodiment 22. The targeting molecule of embodiment 21, comprising a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3).Embodiment 23. The targeting molecule of embodiment 21, 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”) according to any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.Embodiment 24. The targeting molecule of any one of embodiments 21 to 23, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.Embodiment 25. A composition comprising: 1) a targeting molecule of any one of embodiments 21-23; and 2) a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload.Embodiment 26. The composition of embodiment 25, wherein the payload of interest encodes or comprises a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.Embodiment 27. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is associated with a targeting molecule according to any one of embodiments 21-24, and wherein the targeting molecule interacts with LDLR.Embodiment 28. The method of embodiments 27, wherein delivering the payload of interest to the cell or the tissue comprises crossing the BBB.Embodiment 29. A method of delivering a therapeutically relevant gene or protein of interest to a cell, comprising contacting the cell with the composition of any one of embodiments 25-26.Embodiment 30. A method of modulating the expression or activity of a therapeutically relevant gene of interest or protein in a cell, comprising contacting the cell with the composition of any one of embodiments 25-26.Embodiment 31. A method of treating a disease in a subject, comprising administering to the subject the composition of any one of embodiments 25-26.Embodiment 32. A method for identifying a targeting molecule that targets a cell or a tissue, optionally wherein the targeting molecule crosses the BBB, comprising selecting for targeting molecules that interact with LDLR.Embodiment 33. A method for identifying a targeting molecule that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for targeting molecules that: i) comprise a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3); or ii) comprise 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 any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.EXAMPLESExample 1. Methods1.1 AAV capsid library generation

[0101] Capsid libraries used in round 1 screening were constructed by insertion of peptides into the exposed loops of the capsid proteins. Gibson assembly was used to generate the capsid libraries where the introduced peptides were encoded by the primers used to amplify the assembly fragments. Two PCR products from the capsid gene sequence were amplified (left and right fragments) that had 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).

[0102] AAV9 capsid libraries with insertions between 587 and 590 with replacement of 588 or 589 used in round 1 screening were created by inserting trimer-19 synthesized codon block oligos using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs catalog number E2621).

[0103] For round 2 selection, capsid variants for library screening were synthesized as an oligo pool. Each capsid peptide was synthesized with unique nucleotide sequences encoding the peptide, and each peptide was linked to at least three distinct barcodes. The oligo pool was cloned into a linearized intermediate plasmid, followed by cloning of a constant donor sequence to separate the barcode and peptide region and generate the full AAV vector construct. Expression of barcodes was driven by a ubiquitous CMV promoter. The peptide sequences listed in Table 1 were inserted into AAV3B (SEQ ID NO: 638) between amino acids 588 and 589. The peptide sequences listed in Table 2 were inserted into AAV5 (SEQ ID NO: 639) between amino acids 577 and 578. The peptide sequences listed in Table 3 were inserted intoAAV9 (SEQ ID NO: 640) between amino acids 454 and 455 or between 588 and 589 or between 587 and 590 with replacement of 588 or 589, as indicated.

[0104] AAV capsid libraries were manufactured in HEK293 cells. Briefly, AAV libraries were 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 were purified by cesium density centrifugation and buffer exchanged into PBS plus 0.001% PF-68 by dialysis. DNase-resistant viral genomic titers were measured by quantitative real time PCR.1.2 Biotinylation of recombinant human LDLR

[0105] Recombinant human LDLR was diluted to 2 mg / mL in PBS (without calcium and magnesium). EZ-Link NHS-LC-Biotin (Thermo Fisher Catalog #21336) was added to a final concentration of 10 mM and the mixture was incubated on ice for 2 hours. After incubation, the biotinylation reaction was quenched by adding 500 mM glycine to a final concentration of 50 mM and incubating on ice for 30 minutes. The same procedure was applied to biotinylate bovine serum albumin for use as a blocking agent.1.3 In vitro panning with recombinant human LDLR

[0106] To block non-specific interactions 1 mg of DynaBeads M-280 Streptavidin beads were rotated for an hour at room temperature with 4el3 molecules of biotinylated bovine serum albumin. The beads were then rotated for an hour at room temperature with 4el3 vg of AAV capsid library. After the one-hour rotation, the pre-cleared AAV capsid library that did not bind to the streptavidin beads or biotinylated bovine serum albumin was separated using a tube magnet. One mg of Dynabeads M-280 Streptavidin beads were then rotated at room temperature with 4el3 molecules of biotinylated human LDLR. The pre-cleared AAV capsid library was added to the receptor-coated beads and rotated for an hour at room temperature. The unbound capsids were separated from the bead bound capsids using a tube magnet. The beads were washed three times with SuperBlock (PBS) Blocking Buffer (ThermoFisher Catalog #37515). The receptor bound AAV capsids were then eluted from the beads with Pierce IgG Elution Buffer pH 2.0 (ThermoFisher Catalog #21028). Vector genomes were extracted using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega Catalog # AS 1330) and samples were prepared for NGS using Kapa HiFi Hotstart ReadyMix (Roche Catalog #KK2602). Amplification of library specific amplicons was 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 for30 sec followed by 72°C for 1 minute. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 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 were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2. Following NGS of library amplicons the reads were demultiplexed and features were extracted using a custom bioinformatic pipeline.

[0107] In parallel to sequencing, variants interacting with recombinant human LDLR were recovered from the bead bound fraction and re-cloned in a subsequent capsid library, produced and re-screened for binding to recombinant human LDLR. Binding variants went through a total of two selections before being included in the pooled round 2 library for further confirmation both against recombinant human LDLR and in cells overexpressing human or macaque LDLR.1.4 Capsid library screening in cells overexpressing LDLR

[0108] Neuro2A cells were seeded in 10 cm dishes coated with poly-D-lysine (PDL) at a density of 3E6 cells per dish. 24 hours later cells were transfected with 1 microgram of plasmid encoding human or macaque LDLR under the control of the ubiquitous cytomegalovirus (CMV) promoter. In parallel a transfection control plate was transfected with 1 microgram of plasmid encoding a fluorescent protein (GFP or mRuby) under the control of the ubiquitous cytomegalovirus (CMV) promoter. Lipofectamine 3000 was used for transfection of the plasmid DNA. Cell culture media was changed 24 hours post-transfection to remove transfection reagents. 48 hours after transfection of plasmid DNA, the cells were 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 was assessed with transduction and binding assays.

[0109] For cell culture transduction assays the media was changed 24 hours posttransduction to fully supplemented media (10% FBS). 72 hours post-transduction, the plates were washed one time with phosphate buffered saline (with calcium and magnesium), and RNA was extracted using the Qiagen RNeasy kit following the manufacturers protocol andquantified by NanoDrop 8000 spectrophotometer. RNA was reverse transcribed to cDNA using the NEB Induro Reverse Transcriptase kit.

[0110] For cell culture binding assays the cells were 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 was extracted using the Qiagen DNeasy kit following the manufacturers protocol and quantified by NanoDrop 8000 spectrophotometer.

[0111] Samples from transduction and binding assays were prepared for next-generation sequencing (NGS) using Kapa HiFi Hotstart ReadyMix (Roche Catalog #KK2602). PCR amplification of library specific amplicons was 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 10 sec followed by 72°C for 1 minute. Amplification was qualitatively confirmed by agarose gel electrophoresis and relative apparent amplification was used to determine the dilution of amplicons needed for indexing. Illumina plate level i5 and well level i7 indices were added to the amplicons with 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 15 sec followed by 72°C for 1 minute. Finally, samples were pooled and purified using Qiagen GeneRead Size Selection Kit following the manufacturer’s protocol. Samples were sequenced on the Illumina MiSeq platform using a MiSeq Reagent Kit v2. Following next-generation sequencing of library amplicons the reads were demultiplexed and features (the barcode or peptide sequence) were extracted using a custom bioinformatic pipeline. For barcoded libraries the extracted barcode was used to query a pre-determined lookup table and return the identity of the corresponding capsid variant. Finally, the log2 fold change (log2FC) enrichment of each capsid variant was normalized to its relative abundance in the administered AAV library.Example 2. Results from round 2 capsid library screening

[0112] Capsid variants that were enriched for binding to recombinant human LDLR were synthesized as a pooled round 2 library. For the round 2 evaluation capsids were evaluated in cells overexpressing the human or macaque ortholog of LDLR. Both a transduction and a binding assay were completed to assay cell entry and binding, respectively. The fold change enrichment of each capsid was determined by next-generation sequencing and normalized to capsid abundance in the administered library. The average log2FC for each assay is shown in Tables 1, 2, and 3 for peptides inserted into AAV3B, AAV5, and AAV9, respectively. Emptycells indicate that the capsid was not detected in that assay, potentially due to limited sample recovery or sequencing depth.Table 1. Round 2 library performance for peptides inserted into the parent capsid AAV3B Table 1 is shown in Figure 7.Table 2. Round 2 library performance for peptides inserted into the parent capsid AAV5 Table 2 is shown in Figure 7.Table 3. Round 2 library performance for peptides inserted into the parent capsid AAV9 Table 3 is shown in Figure 7.Example 3. Enrichment of peptide motifs in capsids interacting with LDLR

[0113] Bioinformatic analysis of the round 1 and 2 library results highlighted the enrichment of specific peptide motifs in capsids that interacted with LDLR in protein binding and / or cell culture transduction assays. A subset of these enriched peptide motifs (or motif sequences) are listed in Table 4 (e.g., as motif number 1-3).Table 4. Motifs enriched in peptides inserted into AAV9Example 4. Evaluation of a barcoded pool of capsids engineered to target LDLR

[0114] AAV capsids were each manufactured individually with a unique barcoded transgene expression cassette and then pooled to create a barcoded pool of AAV capsids including the control capsid AAV9 as well as capsids engineered to target LDLR. A stable CHO-K1 cell line expressing human LDLR was generated using a lentiviral vector. Wild-type CHO-K1 cells or CHO-K1 cells engineered to express human LDLR were seeded in a 6-well plate and then transduced 24 hours later with the barcoded pool of AAV capsids at a multiplicity of infection of 1E5. Cells were incubated for 72 hours to allow for AAV-mediated expression of transgene mRNA. RNA was extracted from cells, reverse transcribed to cDNA, and thenPCR was used to amplify the barcoded AAV cDNA. Next-generation sequencing analysis was conducted to quantify enrichment of capsid variants in the pool relative to the input abundance of each capsid in the barcoded pool. Capsids engineered to target LDLR exhibit enhanced transduction of cells expressing human LDLR relative to wild-type cells.Example 5. Evaluation of capsids engineered to target LDLR5.1. Methods for individual evaluation of receptor-targeted capsids in cells overexpressing human or macaque LDLR

[0115] Neuro2A cells were seeded in 96-well tissue culture plates at a density of 3E4 cells / well and transfected with 100 ng / well of a LDLR overexpression construct or a fluorescent protein transfection control using Lipofectamine 3000. The media was changed 24 hours post transfection. 48 hours post transfection, the cells were transduced with AAV in reduced serum EMEM media (2% FBS) at a multiplicity of infection of 3E3, 1E4, 3E4, or 1E5 viral genomes per cell. AAV capsids contained an expression cassette encoding a GFP fluorescent protein under the control of the neuron specific (hSynapsinl) promoter. 72 hours post transduction, the plates were washed one time with phosphate buffered saline (with calcium and magnesium) and cDNA was generated using a Cells-to-CT kit (Thermo Fisher Catalog #21336). Transgene expression was quantified by RT-qPCR using a Qiagen QuantiNova Probe PCR kit. A mRNA-specific primer probe was used to assess transgene expression and normalized to expression of the GAPDH housekeeping gene. Data normalization and analysis was performed using the Bio-Rad CFX Maestro software.5.2. Results of individual capsid evaluation

[0116] Capsids engineered to target LDLR were evaluated individually to assess capsid transduction in cells expressing LDLR. Representative engineered capsids, designed to target LDLR and described herein, are presented as illustrative examples. Figures 9 through 11 show that these engineered capsids exhibit enhanced transduction in cells transfected with the human or cynomolgus macaque ortholog of LDLR relative to a transfection control expressing a fluorescent protein. Enhanced transduction was evaluated by measuring the increase in transgene mRNA levels using RT-qPCR quantification.Sequences

[0117] The amino acid sequences of the parent capsids are defined as follows:

[0118] AAV3B capsid amino acid sequence SEQ ID NO: 638:

[0119] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLP GYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGV GKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNN EGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNH YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQND GTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGS QAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYL YYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNN SNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAEL DNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFI TQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIG TRYLTRNL

[0120] AAV5 capsid amino acid sequence SEQ ID NO: 639:

[0121] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGY NYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLAD DTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKP STS SDAEAGPSGSQQLQIPAQPAS SLGADTMS AGGGGPLGDNNQGADGVGNASGDW HCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYF DFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTV QVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLE YFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGV QFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASY QVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNR VAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAH FHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0122] AAV9 capsid amino acid sequence SEQ ID NO: 640:

[0123] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEF AWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDV YLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGT RYLTRNL

[0124] The amino acid sequences of individual capsids are defined as follows (inserted Peptide Sequence shown in bold):

[0125] CNSRCV440 capsid amino acid sequence SEQ ID NO: 641 :

[0126] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAQDAMPRGYFQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0127] CNSRCV441 capsid amino acid sequence SEQ ID NO: 642:

[0128] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEF AWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAETSHVRYPAQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0129] CNSRCV442 capsid amino acid sequence SEQ ID NO: 643:

[0130] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSANQMPRTTLDQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0131] CNSRCV443 capsid amino acid sequence SEQ ID NO: 644:

[0132] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEF AWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSASAMPRDHEEQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0133] CNSRCV444 capsid amino acid sequence SEQ ID NO: 645:

[0134] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEF AWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAKMIMPRGAEQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0135] CNSRCV445 capsid amino acid sequence SEQ ID NO: 646:

[0136] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQ AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSTSTLMPRGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVT QNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0137] CNSRCV446 capsid amino acid sequence SEQ ID NO: 647:

[0138] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSTSMPRAVGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0139] CNSRCV447 capsid amino acid sequence SEQ ID NO: 648:

[0140] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSANLLSPRQSAQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

[0141] CNSRCV453 capsid amino acid sequence SEQ ID NO: 649:

[0142] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG KSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEG ADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNA YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS Q AVGRS SF YCLEYFP SQMLRTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQ YL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAALEIRVHASQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFN KDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An adeno-associated virus (AAV) capsid protein that interacts with Low-Density Lipoprotein Receptor (LDLR), thereby enabling delivery of the AAV capsid protein to a cell or a tissue, optionally wherein interaction with LDLR enables the AAV capsid protein to cross the blood-brain barrier (BBB).

2. The AAV capsid protein of claim 1, comprising a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3), optionally wherein the peptide motif is located within a surface-exposed loop of the AAV capsid protein.

3. The AAV capsid protein of claim 2, wherein the peptide motif is located within an insertion between amino acids 587 and 590 in AAV9 (SEQ ID NO: 640).

4. The AAV capsid protein of claim 1, 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”) set forth in any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.

5. The AAV capsid protein of claim 4, 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 in Tables 1 to 3.

6. The AAV capsid protein of claim 5, wherein the amino acid sequence comprises a peptide sequence according to any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3, and optionally wherein the parent capsid and / or the insertion site is / are as indicated in the same single row as shown in Tables 1 to 3.

7. The AAV capsid protein of any one of claims 1-6, wherein the 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.

8. An AAV capsid protein, wherein the engineered AAV capsid protein is at least 80%, 85%, 90%, 95%, or 99% identical to or comprises a sequence set forth in any one of SEQ ID NOs: 641-649 designated as CNSRCV440, CNSRCV441, CNSRCV442, CNSRCV443, CNSRCV444, CNSRCV445, CNSRCV446, CNSRCV447, or CNSRCV453.

9. A nucleic acid molecule encoding an AAV capsid protein of any one of claims 1-8.

10. A host cell comprising the nucleic acid molecule of claim 9.

11. A composition comprising: 1) an AAV capsid protein of any one of claims 1-8; and 2) an expression construct comprising a coding sequence for a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload.

12. The composition of claim 11, wherein the payload of interest is a therapeutic payload, and wherein the therapeutic payload comprises a DNA binding domain, optionally wherein the therapeutic payload comprises a fusion protein.

13. The composition of claim 11 or claim 12, wherein the payload of interest comprises a therapeutic protein, a zinc finger protein, a CRISPR-associated DNA binding protein, a TALE protein, an antibody, an enzyme, a regulatory RNA, a Bxbl serine recombinase, or a DNA recombinase protein.

14. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is encapsidated in an AAV capsid protein according to any one of claims 1-8, and wherein the AAV capsid protein interacts with LDLR.

15. The method of claims 14, wherein delivering the payload of interest to the cell or the tissue comprises crossing a BBB.

16. A method of activating, expressing, repressing, or modulating the expression of a therapeutically relevant gene of interest in a cell, comprising contacting the cell with a composition of any one of claims 11-13.

17. A method of treating a disease in a subject, comprising administering to the subject a composition of any one of claims 11-13.

18. Use of an AAV capsid protein of any one of claims 1-8, a nucleic acid construct of claim 9, or a host cell of claim 10 for the manufacture of a medicament in a method of any one of claims 14-17.

19. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that interact with LDLR.

20. A method for identifying an AAV capsid variant that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for AAV capsids that: i) comprise a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3); or ii) comprise 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 any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.

21. A targeting molecule that interacts with LDLR, thereby enabling delivery of the targeting molecule to a cell or a tissue, optionally wherein the interaction with LDLR enables the targeting molecule to cross the blood-brain barrier (BBB).

22. The targeting molecule of claim 21, comprising a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3).

23. The targeting molecule of claim 21, 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”) according to any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.

24. The targeting molecule of any one of claims 21 to 23, wherein the targeting molecule is fused or conjugated to an AAV, a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.

25. A composition comprising: 1) a targeting molecule of any one of claims 21-23; and 2) a payload of interest, optionally wherein the payload of interest is a research, diagnostic, and / or therapeutic payload.

26. The composition of claim 25, wherein the payload of interest encodes or comprises a small molecule, an antibody, zinc finger protein, Cas protein, exosome, scFV, ASO (antisense oligonucleotide), siRNA, lipid, lipid nanoparticle, polymer, virus-like particle (VLP), bocavirus, dendrimer, aptamer, or recombinant protein.

27. A method of delivering a payload of interest to a cell or a tissue, wherein a coding sequence for the payload of interest is associated with a targeting molecule according to any one of claims 21-24, and wherein the targeting molecule interacts with LDLR.

28. The method of claims 27, wherein delivering the payload of interest to the cell or the tissue comprises crossing the BBB.

29. A method of delivering a therapeutically relevant gene or protein of interest to a cell, comprising contacting the cell with the composition of any one of claims 25-26.

30. A method of modulating the expression or activity of a therapeutically relevant gene of interest or protein in a cell, comprising contacting the cell with the composition of any one of claims 25-26.

31. A method of treating a disease in a subject, comprising administering to the subject the composition of any one of claims 25-26.

32. A method for identifying a targeting molecule that targets a cell or a tissue, optionally wherein the targeting molecule crosses the BBB, comprising selecting for targeting molecules that interact with LDLR.

33. A method for identifying a targeting molecule that crosses the BBB or exhibits enhanced delivery to a cell or a tissue, comprising selecting for targeting molecules that: i) comprise a peptide motif as indicated in a single row of Table 4 (e.g., peptide motif number 1, 2, or 3); orii) comprise 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 any one of SEQ ID NOs: 1-637 as shown in a single row in Tables 1 to 3.

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