Mutant AAV with central nervous system targeting motifs and compositions containing same

Abstract

Provided herein are compositions including brain, brain microvascular endothelial cell-targeting peptides and / or blood-brain barrier crossing (BBB) tissue-targeting peptides linked thereto or inserted in a targeting protein of a recombinant vector having at least one exogenous peptide comprising Xn – n-mer – Xm. Compositions providing such conjugates, targeting peptides, or recombinant vectors having an engineered capsid or envelope protein are provided as are uses thereof.

Description

[0001] MUTANT AAV WITH CENTRAL NERVOUS SYSTEM TARGETING

[0002] MOTIFS AND COMPOSITIONS CONTAINING SAME

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The electronic sequence listing filed herewith named “UPN-23-10399PCT.xml” with size of 290,951 bytes, created on date of June 4, 2024, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are incorporated herein by reference in entirety.

[0005] BACKGROUND OF THE INVENTION

[0006] The Adeno-Associated Virus (AAV) is a desirable gene therapy vector, since recombinant AAV vectors can deliver a transgene that is stably expressed long-term. However, AAV gene therapy is currently limited to a small number of diseases due to challenges in delivery and tropism. This is particularly true for disorders of the central nervous system (CNS). Direct delivery of AAV gene therapy vectors is possible, by injecting the vector directly into the cerebrospinal fluid (CSF), but this method typically transduces 1% or less of brain cells. Furthermore, most of that transduction is concentrated on the cells that are in direct contact with the CSF. Cells in the “deep brain” are rarely transduced. This has limited the number of CNS disorders treatable by gene therapy.

[0007] In contrast to the CSF network, the vascular system of the brain reaches nearly every cell in the CNS. This is because of a high demand these tissues have for glucose, oxygen, and other nutrients. However, cells in the brain and spinal cord are protected from the circulatory system by a specialized vascular unit, the Blood Brain Barrier (BBB). The BBB limits the diffusion of large molecules like viral vectors and proteins, and even many small molecule drugs through a complex network of tightly linked cells that surround the blood vessels of the brain and spinal cord. Thus, a grand challenge in gene therapy delivery to the CNS has been the engineering of an AAV variant capable of crossing the BBB at high efficiency and transducing cells in the deep brain. One AAV capsid developed at the California Institute of Technology (CalTech) has a seven amino acid peptide inserted into hypervariable loop 8 (HVR8) on the AAV9 capsid to generate a rAAV called AAV9-PHP.B which is reported to mediate interaction with Ly6a, a GPI-anchored receptor on the brain vasculature of some mouse strains. US Patent Published Application No. 2017 / 0166926A1. This interaction drives transport of AAV9- PHP.B across the BBB, resulting in ~50-fold higher transduction of brain cells than AAV9. However, this finding has not translated to larger animals or humans.

[0008] There remains a need for vectors which can specifically target selected tissue and cell types.

[0009] SUMMARY OF THE INVENTION

[0010] In one aspect provided herein is a recombinant adeno-associated virus particle (rAAV) comprising (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences. In another aspect provided herein is a recombinant adeno-associated virus particle (rAAV) comprising: (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences. In certain embodiments, AAV capsid comprises exogenous targeting peptide is inserted between any two contiguous amino acids in the hypervariable region VIII (HVRVIII) or IV (HVRIV) at a suitable location of a parental AAV capsid. In certain embodiments, the parental AAV capsid is selected from AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, and AAVrh91. In certain embodiments, the exogenous targeting peptide is inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 parental capsid as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 60, or an analogous position in the AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parental AAV capsid, optionally wherein the exogenous peptide is immediately preceded by “AQ.”

[0011] In another aspect, provided herein is a recombinant brain cell-targeting peptide, wherein the recombinant brain cell-targeting peptide comprises “Xn - n-mer - Xm”, wherein: (a) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (b) the n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (c) Xm is 0, 1, 2, or 3 amino acid / s independently selected from any amino acid; and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In another aspect, provided herein is a conjugate comprising: (a) a recombinant brain cell-targeting peptide, wherein the recombinant brain cell-targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (ii) the an n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid / s independently selected from any amino acid; and (b) a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, provided herein is a fusion polypeptide or protein comprising the recombinant brain cell-targeting peptide as described herein.

[0012] In another aspect, provided herein are compositions comprising rAAV, recombinant peptide, conjugate and / or fusion polypeptide or protein comprising exogenous targeting peptide as described herein, and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base. In another aspect, provided herein are recombinant nucleic acid molecule encoding recombinant brain cell-targeting peptide, and AAV capsid comprising exogenous targeting peptide. In another aspect, provided herein is a use of a stock of rAAV, recombinant brain cell targeting peptide, or fusion polypeptide or protein, as described herein, for use in delivering to a patient in a need thereof. In certain embodiments, a method is provided for targeting therapy to the brain, comprising administering to a patient in need thereof an rAAV as described herein. In certain embodiments, a method is provided for treating one or more of cognitive, neurodegenerative, and / or disease of the central nervous system by delivering to a subject in need thereof a stock of the rAAV as described herein. In some embodiments, a method is provided of treating of various infections, disease, and or disorders of the central nervous system.

[0013] In another aspect, provided herein are methods of use of rAAV as described herein, wherein the method is part of a co-therapeutic regimen which further comprises administering at least one or more of immunosuppressive agents comprising corticosteroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent. In certain embodiments, the method is part of a co-therapeutic regimen which further comprises administering one or more of: (a) a corticosteroid or combination of corticosteroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and / or (f) gamma interferon. In certain embodiments, the one or more of immunosuppressive agents is administered (i) prior to rAAV administration, (ii) post-rAAV administration, or (iii) prior to and post-rAAV administration. In certain embodiments, the method is part of a co-therapeutic regimen which further comprises co-administering a ligand which specifically prevents binding between human neonatal Fc receptor (FcRn) and the neutralizing antibodies without interfering with albumin binding to FcRn. In certain embodiments, the method is part of a co-therapeutic regimen which further comprises pretreating the patient or the subject by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid of the recombinant AAV vector.

[0014] These and other embodiments and advantages of the invention will be apparent from the specification, including, without limitation, the detailed description of the invention.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 A shows RNA levels in brain tissue post rAAV transduction, plotted as copy number (#) / 100ng RNA. FIG. IB shows DNA levels in brain tissue post rAAV transduction, plotted as GC / diploid cell. FIG. 1C shows RNAZDNA ratio (normalized to AAV9) for AAV9 and modified AAV9 various vectors as analyzed in brain tissue.

[0017] FIG. 2A shows RNA levels in liver tissue post rAAV transduction, plotted as copy number (#) / 100ng RNA. FIG. 2B shows DNA levels in liver tissue post rAAV transduction, plotted as GC / diploid cell. FIG. 2C shows RNAZDNA ratio (normalized to AAV9) for AAV9 and modified AAV9 various vectors as analyzed in liver tissue.

[0018] FIG. 3 shows an alignment of the specified region of the amino acid sequences of the various AAV capsid proteins of AA9 (amino acids 566 to 615 of AAV9 capsid; SEQ ID NO: 79), AAV8 (amino acids 565 to 614 of AAV8 capsid; SEQ ID NO: 80), AAV7 (amino acids 567 to 616 of AAV7; SEQ ID NO: 81), AAV6 (amino acids 550 to 599 of AAV6 capsid; SEQ ID NO: 82), AAV5 (amino acids 556 to 605 of AAV5; SEQ ID NO: 83), AAV4 (amino acids 558 to 607 of AAV4 capsid; SEQ ID NO: 84), AAV3B (amino acids 564 to 613 of AAV3B capsid; SEQ ID NO: 85), AAV2 (amino acids 566 to 615 of AAV2 capsid; SEQ ID NO: 86), and AAV1 (amino acids 566 to 615 of AAV1 capsid; SEQ ID NO: 87), which is focused on the region HVRVIII in which the targeting peptide may be inserted (based on structure analysis).

[0019] FIG. 4A shows RNA biodistribution, plotted as normalized RNA Transcript / lOOng, in NHP tissues. FIG. 4B shows DNA biodistribution, plotted as normalized GC per diploid cell.

[0020] FIG. 5 shows representative images of the biodistribution histology analysis (GFP- IHC) of brain tissues (cerebellum, hippocampus, parietal cortex, temporal cortex) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39).

[0021] FIG. 6 shows representative images of the biodistribution histology analysis (GFP- IHC) of liver tissue (left lobe) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39).

[0022] FIG. 7A shows representative images, at 4x and 20x magnification, of the biodistribution histology analysis (GFP-IHC) of kidney tissues (right kidney, left kidney) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39). FIG. 7B shows representative images, at 4x and lOx magnification, of the biodistribution histology analysis (GFP-IHC) of kidney tissues (right kidney, left kidney) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39).

[0023] FIG. 8A shows representative images of the biodistribution histology analysis (GFP-IHC) of heart tissue (right ventricle) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39). FIG. 8B shows representative images of the biodistribution histology analysis (GFP-IHC) of heart tissue (left ventricle) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39). FIG. 8C shows representative images, at 4x and lOx magnification, of the biodistribution histology analysis (GFP-IHC) of heart tissues (right ventricle, left ventricle) from NHP administered with rAAV9 comprising QGVKDPR (SEQ ID NO: 39).

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] In certain embodiments, a targeting peptide and nucleic acid sequences encoding same are provided herein. Also provided herein are fusion proteins, modified proteins, engineered viral capsids (e.g., recombinant adeno-associated virus (rAAV) capsid) and other moieties operably linked to an exogenous targeting peptide, wherein the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is an exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, X’” is K, P, R, Q, L, K, S, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. The exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88) may also be represented “W / Y / T / Q / R / N / E / D-G-L / I / M / K / V / R / T-K / P / R / Q / L / K / S-DPR” (SEQ ID NO: 88). In certain embodiments, the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7, or the full length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or the full length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Also provided herein are nucleic acid sequences encoding same. In certain embodiments, this exogenous motif modifies the native tissue specificity of the source (parental) protein, viral vector, viral capsid (e.g., AAV capsid), or other moiety. In certain embodiments, compositions having one or more of these exogenous targeting peptides have enhanced or altered Blood Brain Barrier (BBB), brain cell, and / or primary brain microvascular endothelial cell targeting. In certain embodiments, compositions having one or more of these exogenous targeting peptides have enhanced or altered cardiac (heart) cell targeting. In certain embodiments, compositions having one or more of these exogenous targeting peptides have enhanced or altered kidney (e.g., podocytes, proximal tubules, distal tubules, and collecting duct tubules) cell targeting. In certain embodiments, viral vectors having modified capsids containing this motif exhibit increased transduction of AAV production cells in vitro.

[0026] Advantageously, in certain embodiments provided herein is a recombinant brain cell-targeting peptide (Blood Brain Barrier (BBB), brain cell, and / or primary brain microvascular endothelial cell targeting peptide, also referred to as “targeting peptide”, “exogenous targeting peptide”), wherein the recombinant exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments provided herein is a recombinant brain cell-targeting peptide (e.g., Blood Brain Barrier (BBB), brain cell, and / or primary brain microvascular endothelial cell targeting peptide, also referred to as “targeting peptide”, “exogenous targeting peptide”), wherein the recombinant exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments provided herein is a recombinant brain cell -targeting peptide (e g., Blood Brain Barrier (BBB), brain cell, and / or primary brain microvascular endothelial cell targeting peptide, also referred to as “targeting peptide”, “exogenous targeting peptide”), wherein the recombinant exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n- mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0027] Advantageously, in certain embodiments provided herein is an engineered rAAV capsid comprising the exogenous targeting peptide, wherein the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments provided herein is an engineered rAAV capsid comprising the exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments provided herein is an engineered rAAV capsid comprising the exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is selected from TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0028] In certain embodiments, the exogenous targeting peptide provided herein provide significant transduction advantages in the central nervous system (CNS) including Blood Brain Barrier (BBB), brain, spinal cord, and / or primary brain microvascular endothelial cell as compared to a parental capsid (e.g., AAV9, or another clade F capsid, or another clade capsid). In certain embodiments, the exogenous targeting peptide provided herein provide significant transduction advantages in the cardiac (heart) cell as compared to a parental capsid (e.g., AAV9, or another clade F capsid, or another clade capsid). In certain embodiments, the exogenous targeting peptide provided herein provide significant transduction advantages in the kidney cell as compared to a parental capsid (e.g., AAV9, or another clade F capsid, or another clade capsid). In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - QGVKDPR (SEQ ID NO: 39) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - NGLKDPR (SEQ ID NO: 33) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TGVKDPR (SEQ ID NO: 42) - Xm”, wherein the Xn is 0, I, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TGMRDPN (SEQ ID NO: 57) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TVMRDPR (SEQ ID NO: 54) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - RGVQDPR (SEQ ID NO: 44) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - DGMRDPR (SEQ ID NO: 43) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - QGVRDPR (SEQ ID NO: 45) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TGVLDPR (SEQ ID NO: 98) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - YGGPDPR (SEQ ID NO: 99) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - QGIQDPR (SEQ ID NO: 100) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TDMRDPR (SEQ ID NO: 101) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TGMRDPK (SEQ ID NO: 102) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - YGVKDPR (SEQ ID NO: 103) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TGMIDPR(SEQ ID NO: 104) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid. In certain embodiments, engineered rAAV capsids comprising the exogenous targeting peptide which comprises “Xn - TAMRDPR (SEQ ID NO: 105) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the brain cell, as compared to a parental capsid.

[0029] In certain embodiments, engineered rAAV capsid comprises the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, the n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers, Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the heart cell, as compared to a parental capsid.

[0030] In certain embodiments, engineered rAAV capsid comprises the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, the n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers, Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the kidney cell, as compared to a parental capsid.

[0031] In certain embodiments, the rAAV comprises a mutant AAV capsid having an exogenous targeting peptide as identified herein. In certain embodiments, the mutant AAV capsid comprises an exogenous targeting peptide which is immediately preceded by flanking amino acids which are mutated, as compared to parental AAV capsid. In certain embodiments, the mutated flanking amino acids, together with 1, 2, 3, 4, 5, or 6 inserted amino acids comprise the exogenous targeting peptide. In certain embodiments, the entirety of the exogenous targeting peptide is inserted into the parental AAV capsid. In still other embodiments, the sequence inserted into a capsid may comprise all or a fragment of the exogenous targeting peptide at the carboxy or amino terminus (i.e., via insertion of the 5' or 3' coding sequences therefor) and further comprises 0-3 flanking amino acid residues as provided in the above formulae.

[0032] The exogenous targeting peptide may be linked to a recombinant protein (e.g., for enzyme replacement therapy) or polypeptide (e.g., an immunoglobulin) to form a fusion protein or a conjugate to target a desired tissue (e.g., CNS, brain). Additionally, the targeting peptide may be linked to a liposome and / or a nanoparticle (a lipid nanoparticle, LNP) forming a peptide-coated liposome and / or LNP to target the desired tissue. Sequences encoding at least one copy of an exogenous targeting peptide and optional linking sequences may be fused in frame with the coding sequence for the recombinant protein and co-expressed with the protein or polypeptide to provide fusion proteins or conjugates. Alternatively, other synthetic methods may be used to form a conjugate with a protein, polypeptide or another moiety (e.g., DNA, RNA, or a small molecule). In certain embodiments, multiple copies of a targeting peptide are in the fusion protein / conjugate. Suitable methods for conjugating a targeting peptide to a recombinant protein include modifying the amino (N)-terminus and one or more residues on a recombinant human protein (e.g., an enzyme) using a first crosslinking agent to give rise to a first crosslinking agent modified recombinant human protein, modifying the amino (N)-terminus of a short extension linker region preceding a targeting peptide using a second crosslinking agent to give rise to a second crosslinking agent modified variant target peptide, and then conjugating the first crosslinking agent modified recombinant human protein to the second crosslinking agent modified variant targeting peptide containing a short extension linker. Other suitable methods for conjugating a targeting peptide to a recombinant protein include conjugating a first crosslinking agent modified recombinant human protein to one or more second crosslinking agent modified variant targeting peptides, wherein the first crosslinking agent modified recombinant protein comprises a recombinant protein characterized as having a chemically modified N-terminus and one or more modified lysine residues and the one or more second crosslinking agent modified variant targeting peptides comprise one or more variant targeting peptides comprising a modified N- terminal amino acid of a short extension linker preceding the targeting peptide. Still other suitable methods for conjugating a targeting peptide to a protein, polypeptide, nanoparticle, or another biologically useful chemical moiety may be selected. See, e.g., US Patent No. US 9,545,450 B2 (NHS-phosphine cross-linking agents; NHS-Azide cross-linking agents); US Published Patent Application No. US 2018 / 0185503 Al (aldehyde-hydrazide crosslinking).

[0033] In certain embodiments, the exogenous targeting peptide may be engineered (e.g., inserted) at a suitable site within a protein or polypeptide (e.g., a viral capsid protein, AAV capsid). In certain embodiments, a targeting peptide may be flanked at its amino (N-) (e.g., optional Xn) and / or carboxy (COO-) (e.g., optional Xm) terminus by a short extension linker. Such a linker may be about 1 to about 20 amino acid residues in length, or about 2 to about 20 amino acids residues, or about 1 to about 15 amino acid residues, or about 2 to about 12 amino acid residues, or about 2 to about 7 amino acid residues in length. The short extension linker can also be about 10 amino acids in length. The presence and length of a linker at the N-terminus is independently selected from a linker at the carboxyterminus, and the presence and length of a linker at the carboxy terminus is independently selected from a linker at the N-terminus. Suitable short extension linkers can be provided using an about 5-amino acid flexible GS extension linker (glycine-glycine-glycine-glycine- serine), an about 10-amino acid extension linker comprising 2 flexible GS linkers, an about 15-amino acid extension linker comprising 3 flexible GS linkers, an about 20-amino acid extension linker comprising 4 flexible GS linkers, or any combination thereof.

[0034] In certain embodiment, a composition is provided which is useful for targeting a CNS cell (e.g., brain cell). In certain embodiment, a composition is provided which is useful for targeting a heart cell. In certain embodiment, a composition is provided which is useful for targeting a kidney cell. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n- mer”) of X’-G-X”-X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S, wherein the n-mer is flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

[0035] In certain embodiments, the composition comprises an engineered capsid, fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6 consecutive amino acids of any one of the n-mers, flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid, fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6 consecutive amino acids of any one of the n-mers, flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of QGVKDPR (SEQ ID NO: 39) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of NGLKDPR (SEQ ID NO: 33) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TGVKDPR (SEQ ID NO: 42) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TGMRDPN (SEQ ID NO: 57) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TVMRDPR (SEQ ID NO: 54) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of RGVQDPR (SEQ ID NO: 44) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of DGMRDPR (SEQ ID NO: 43) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of QGVRDPR (SEQ ID NO: 45) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TGVLDPR (SEQ ID NO: 98) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of YGGPDPR (SEQ ID NO: 99) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of QGIQDPR (SEQ ID NO: 100) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TDMRDPR (SEQ ID NO: 101) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TGMRDPK (SEQ ID NO: 102) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of YGVKDPR (SEQ ID NO: 103) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TGMIDPR(SEQ ID NO: 104) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TAMRDPR (SEQ ID NO: 105) flanked at its amino terminus and / or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. Examples of suitable proteins, including enzymes, immunoglobulins, therapeutic proteins, immunogenic polypeptides, nanoparticles, DNA, RNA, and other moieties (e.g., small molecules, etc.) for targeting are described in more detail below. These and other biologic and chemical moieties are suitable for use with the exogenous targeting peptide(s) provided herein.

[0036] In certain embodiments, the targeting peptide core amino acid sequence is encoded by a nucleic acid sequence.

[0037] In certain embodiments, the targeting peptide core (e.g., “n-mer”) is selected from Table 1, below.

[0038] Table 1.

[0039] In certain embodiments, the targeting peptide core is X’-G-X”-X’”DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S In certain embodiments, the targeting peptide core is QGVKDPR (SEQ ID NO: 39). In certain embodiments, the targeting peptide core is NGLKDPR (SEQ ID NO: 33). In certain embodiments, the targeting peptide core is TGVKDPR (SEQ ID NO: 42). In certain embodiments, the targeting peptide core is TGMRDPN (SEQ ID NO: 57). In certain embodiments, the targeting peptide core is TVMRDPR (SEQ ID NO: 54). In certain embodiments, the targeting peptide core is RGVQDPR (SEQ ID NO: 44). In certain embodiments, the targeting peptide core is DGMRDPR (SEQ ID NO: 43). In certain embodiments, the targeting peptide core is QGVRDPR (SEQ ID NO: 45). In certain embodiments, the targeting peptide core is TGVLDPR (SEQ ID NO: 98). In certain embodiments, the targeting peptide core is YGGPDPR (SEQ ID NO: 99). In certain embodiments, the targeting peptide core is QGIQDPR (SEQ ID NO: 100). In certain embodiments, the targeting peptide core is TDMRDPR (SEQ ID NO: 101). In certain embodiments, the targeting peptide core is TGMRDPK (SEQ ID NO: 102). In certain embodiments, the targeting peptide core is YGVKDPR (SEQ ID NO: 103). In certain embodiments, the targeting peptide core is TGMIDPR (SEQ ID NO: 104). In certain embodiments, the targeting peptide core is TAMRDPR (SEQ ID NO: 105). In certain embodiments, more than one copy of a targeting peptide within this motif is provided in a conjugate or modified protein (e.g., a parvovirus capsid, rAAV capsid). In certain embodiments, two or more different targeting peptide cores are present.

[0040] In certain embodiments, a composition is provided which is useful for targeting a CNS cell. In certain embodiments, a composition is provided which is useful for targeting a brain cell. In certain embodiments, a composition is provided which is useful for targeting brain endothelial cell. In certain embodiments, a composition is provided which is useful for targeting a heart cell. In certain embodiments, a composition is provided which is useful for targeting a kidney cell.

[0041] In certain embodiments, provided herein is a composition comprising a nucleic acid sequence molecule, wherein the nucleic acid molecule is a DNA molecule or RNA molecule, e.g., naked DNA, naked plasmid DNA, messenger RNA (mRNA), linked to a targeting peptide sequence motif linked to the nucleic acid molecule. In some embodiments, the nucleic acid molecule is further coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and / or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., WO2014 / 089486, US 2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085 Al, US9670152B2, and US 8,853,377B2, X. Su, et al., Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013 / 182683, WO 2010 / 053572 and WO 2012 / 170930, all of which are incorporated herein by reference. In certain embodiments, the targeting peptide motif is chemically linked to a nanoparticle surface, wherein the nanoparticle encapsulates a nucleic acid molecule. In some embodiments, the nanoparticle comprising the targeting peptide linked to the surface is designed for targeted tissuespecific delivery. In some embodiments two or more different targeting peptides are linked to the surface of the nanoparticle. Suitable chemical linking or cross-linking include those known to one skilled in the art. Capsids

[0042] In certain embodiments, a recombinant parvovirus is provided which has a modified parvovirus capsid, wherein the capsid includes amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, a recombinant parvovirus is provided which has a modified parvovirus capsid, wherein the capsid has amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or a n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, a recombinant parvovirus is provided which has a modified parvovirus capsid, wherein the capsid has amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n- mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Such a recombinant parvovirus may be a hybrid bocavirus / AAV or a recombinant AAV vector (rAAV). In other embodiments, other viral vectors may be generated having one or more exogenous targeting peptides in an exposed capsid protein to modulate and / or alter the targeting specificity of the viral vector as compared to the parental vector, wherein the one or more exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In other embodiments, other viral vectors may be generated having one or more exogenous targeting peptides in an exposed capsid protein to modulate and / or alter the targeting specificity of the viral vector as compared to the parental vector, wherein the one or more exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0043] The exogenous targeting peptide may be inserted (and / or engineered via mutation of sequences encoding flanking amino acid residue(s)) into a hypervariable loop (HVR) VIII (also referenced as HVR8) at any suitable location. For example, based on the numbering of the AAV9 capsid, the peptide is inserted with linkers of various lengths between amino acids 588 and 589 (Q-A) of the AAV9 capsid protein, based on the numbering of the AAV9 VP1 (also referenced as Vpl or vpl) amino acid sequence: SEQ ID NO: 60. See, also, WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, filed September 7, 2018. The amino acid residue locations (i.e., amino acid numbering reference) are identical in AAVhu68 (SEQ ID NO: 61). However, another site may be selected within HVRVIII. Alternatively, another exposed loop HVR (e.g., HVRIV) may be selected for the insertion. In certain embodiments, exogenous targeting peptide may be inserted (and / or engineered via mutation of sequences encoding flanking amino acid residue(s)) into a HVRIV. Comparable HVR regions may be selected in other capsids. In certain embodiments, the location for the HVRVIII and HVRIV is determined using an algorithm and / or alignment technique as described in US Patent No. US 9,737,618 B2 (column 15, lines 3-23), and US Patent No. US 10,308,958 B2 (column 15, line 46 - column 16, line 6), which are incorporated herein by reference in its entirety. In certain embodiments, the targeting peptide may be inserted (and / or engineered) into a hypervariable loop HVRVIII as described in International Patent Application No. PCT / US2021 / 061312, fded December 1, 2022, which is now published as WO2022 / 119871 A2 (published June 9, 2022) which is incorporated herein by reference in its entirety. In certain embodiments, AAV1 capsid protein is selected as a parental capsid, wherein the targeting peptide with linkers of various lengths is inserted in a suitable location of the HVRVIII region of amino acid 582 to 585, or HVRIV region of amino acid 456 to 459 based on vpl numbering (Gurda, BL., et al., Capsid Antibodies to Different Adeno- Associated Virus Serotypes Bind Common Regions, 2012, Journal of Virology, June 12, 2013, 87(16): 9111-91114). In certain embodiments, AAV8 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted in a suitable location of HVRVIII region of amino acid 586 to 591, or HVRIV region of amino acid 456 to 460, based on VP1 numbering (Gurda, BL., et al., Mapping a Neutralizing epitope onto the Capsid of Adeno-Associated Virus Serotype 8, 2012, Journal of Virology, May 16, 2012, 86(15):7739-7751).

[0044] In certain embodiments, parental AAV capsid is AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, or AAVrh91.

[0045] In certain embodiments, the exogenous targeting peptide is engineered and / or inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 parental capsid as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 60, or an analogous position in a AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parental AAV capsid.

[0046] In certain embodiments, exogenous targeting peptide has an amino acid sequence at its carboxy terminus and its amino terminus which is immediately preceded by “AQ” at position 588 of the parental capsid, which is a Clade F capsid of AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95 or AAVhu96.

[0047] In certain embodiments, an rAAV comprises AAV capsid, wherein the AAV capsid is modified (i.e., mutated) to comprise the exogenous targeting peptide motif X’-G-X”- X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S. In certain embodiments, an rAAV comprises AAV capsid, wherein the AAV capsid is modified to comprise the exogenous targeting peptide QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO:

[0048] 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105). In certain embodiments, an rAAV comprises AAV capsid, wherein the AAV capsid is modified to comprise the exogenous targeting peptide as described herein, optionally wherein the AAV capsid is modified at HVRVIII loop, at any suitable location. In certain embodiments, an rAAV comprises AAV capsid, wherein the AAV capsid is modified at HVRVIII loop to comprise the exogenous targeting peptide QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO:

[0049] 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105). In certain embodiments, an rAAV comprises AAV capsid, wherein the AAV capsid is modified at HVRVIII loop to comprise exogenous targeting peptide motif X’-G-X ”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S.

[0050] In certain embodiments, AAV9 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted (and / or engineered) in a suitable location of HVRVIII region of amino acid 588 and 589 (Q-A), based on VP1 numbering. In other embodiments, AAVhu68 or another clade F capsid is selected as the parental capsid. In certain embodiments, AAV8 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted in a suitable location of HVRVIII region of amino acid 590 and 591 (N-T), based on VP1 numbering. In certain embodiments, AAV7 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted in a suitable location of HVRVIII region of amino acid 589 and 590 (N-T), based on VP1 numbering. In certain embodiments, AAV6 is selected as parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 588 and 589 (S-T), based on VP1 numbering. In certain embodiments, AAV5 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 577 and 578 (T-T), based on VP1 numbering. In certain embodiments, AAV4 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 586 and 587 (S-N), based on VP1 numbering. In certain embodiments, AAV3 / 3B is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 588 and 589 (N-T), based on VP1 numbering. In certain embodiments, AAV2 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 587 and 589 (N-R), based on VP1 numbering. In certain embodiments, AAV1 is selected as parental capsid, wherein the targeting peptide with linkers of various length is inserted and / or engineered in a suitable location of HVRVIII region of amino acid 588 and 589 (S-T), based on VP1 numbering. See also, FIG. 3 which shows an alignment of the specified region of the amino acid sequences of the various AAV capsid proteins of AA9 (amino acids 566 to 615 of AAV9 capsid; SEQ ID NO: 79), AAV8 (amino acids 565 to 614 of AAV8 capsid; SEQ ID NO: 80), AAV7 (amino acids 567 to 616 of AAV7; SEQ ID NO: 81), AAV6 (amino acids 550 to 599 of AAV6 capsid; SEQ ID NO: 82), AAV5 (amino acids 556 to 605 of AAV5; SEQ ID NO: 83), AAV4 (amino acids 558 to 607 of AAV4 capsid; SEQ ID NO: 84), AAV3B (amino acids 564 to 613 of AAV3B capsid; SEQ ID NO: 85), AAV2 (amino acids 566 to 615 of AAV2 capsid; SEQ ID NO: 86), and AAV1 (amino acids 566 to 615 of AAV1 capsid; SEQ ID NO: 87), which is focused on the region HVRVIII in which the targeting peptide may be inserted (based on structure analysis).

[0051] In certain embodiments, the residues of the parental AAV capsid sequence is preserved (i.e., there are no substitutions and / or deletions in the 1, 2, and / or 3 amino acid residues of the parental AAV at the N-terminus and / or C-terminus of immediately preceding the target peptide inserted, insert, as compared to that of the parental AAV capsid amino acid sequence). In certain embodiments, there are no deletions in the 1, 2 and / or 3 amino acid residues as compared to that of the parental AAV capsid amino acid sequence at the N-terminus and / or C-terminus of immediately preceding the target peptide inserted, insert. In certain embodiments, one or more amino acid residues, as compared to that of the parental AAV capsid amino acid sequence, are modified at the N-terminus and / or C-terminus of immediately preceding the n-mer, and / or to provide the mutant AAV capsid with one or more residues of the n-mer sequence. In certain embodiments, one or more amino acid residues, as compared to that of the parental AAV capsid amino acid sequence, are modified at the positions at the N-terminus and / or C-terminus immediately flanking the n-mer, and / or to provide the mutant AAV with one or more residues of the n- mer sequence.

[0052] In certain embodiments, the parental capsid is modified to comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”- X’”DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise modulate targeting to CNS cell (e.g., brain cell, or brain endothelial cell). In certain embodiments, the parental capsid is modified to comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer selected is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n- mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise modulate targeting to CNS cell (e.g., brain cell, or brain endothelial cell). In certain embodiments, the parental capsid is modified to comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise modulate targeting to CNS cell (e.g., brain cell, or brain endothelial cell). See, e.g., WO 2020 / 223231, published November 5, 2020 (rh91, including table with deamidation pattern), US Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, US Provisional Patent Application No. 63 / 109,734, filed November 4, 2020, and International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, which is now published as WO 2022 / 036220, all of which are incorporated herein by reference in their entireties. In certain embodiments, AAV capsids having reduced capsid deamidation may be selected. See, e.g., PCT / US 19 / 19804 and PCT / US18 / 19861, both filed Feb 27, 2019, and incorporated by reference in their entireties. See also, International Patent Application No. PCT / US2021 / 055436, filed October 18, 2021, now publication No. WO 2022 / 082109, and International Patent Application No. PCT7US2022 / 077315, filed September 30, 2022, now publication No. WO 2023 / 056399 are incorporated herein by reference in their entireties.

[0053] In certain embodiments, the mutant capsids described herein are characterized by having a deamidation pattern similar to their parental AAV, e.g., such as described in US 2020 / 0056159, published Feb 20, 2020 (AAVhu68; highly deamidated in N57, N329, N452 and N512), with minor optional amounts of deamidation); US 2020 / 0407750, published Dec 31, 2020 (AAV9, highly deamidated in N57, N329, N452 and N512), each of which is incorporated herein by reference in its entirety.

[0054] In certain embodiments, provided herein is the parental capsid which is modified to comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, I, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise modulate targeting to heart cell. In certain embodiments, the parental capsid is modified to comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR(SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7, or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise modulate targeting to kidney cell (e.g., podocytes, proximal tubules, distal tubules, and collecting duct tubules).

[0055] In certain embodiments, a recombinant adeno-associated virus particle (rAAV) is provided comprising: (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product under control of sequences which direct expression thereof. In certain embodiments, a recombinant adeno- associated virus particle (rAAV) is provided comprising: (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45); and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product under control of sequences which direct expression thereof. In certain embodiments, a rAAV comprises capsid proteins having an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), and / or combination of any thereof. In certain embodiment, the rAAV comprising capsid proteins comprising exogenous targeting peptide as described herein (i.e., engineered rAAV capsid) has a greater CNS (e.g., brain cell, brain endothelial cell) specificity, targeting, and / or efficacy as compared to a parental rAAV capsid.

[0056] In certain embodiments, provided herein is a recombinant adeno-associated viral particle (rAAV) comprising an AAV capsid, wherein the AAV capsid is not an AAV2 capsid. In certain embodiments, the rAAV comprises an AAV capsid, wherein the AAV capsid is not a mutant AAV2 capsid comprising NDVRAVS (SEQ ID NO: 89) sequence. In certain embodiments, the rAAV comprises an AAV2 capsid wherein the AAV2 capsid protein comprises at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G- X”-X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV2 capsid wherein the AAV2 capsid protein comprises at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0057] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X ”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105); and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), and / or combination of any thereof. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises QGVKDPR (SEQ ID NO: 39). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises NGLKDPR (SEQ ID NO: 33). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TGVKDPR (SEQ ID NO: 42). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TGMRDPN (SEQ ID NO: 57). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TVMRDPR (SEQ ID NO: 54). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises RGVQDPR (SEQ ID NO: 44). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises DGMRDPR (SEQ ID NO: 43). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises QGVRDPR (SEQ ID NO: 45). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TGVLDPR (SEQ ID NO: 98). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises YGGPDPR (SEQ ID NO: 99). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises QGIQDPR (SEQ ID NO: 100). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TDMRDPR (SEQ ID NO: 101). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TGMRDPK (SEQ ID NO: 102). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises YGVKDPR (SEQ ID NO: 103). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TGMIDPR (SEQ ID NO: 104. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TAMRDPR (SEQ ID NO: 105).

[0058] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0059] - QGVKDPR (SEQ ID NO: 39) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising QGVKDPR (SEQ ID NO: 39).

[0060] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0061] - NGLKDPR (SEQ ID NO: 33) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising NGLKDPR (SEQ ID NO: 33).

[0062] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0063] - TGVKDPR (SEQ ID NO: 42) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TGVKDPR (SEQ ID NO: 42).

[0064] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0065] - TGMRDPN (SEQ ID NO: 57) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TGMRDPN (SEQ ID NO: 57).

[0066] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0067] - TVMRDPR (SEQ ID NO: 54) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TVMRDPR (SEQ ID NO: 54).

[0068] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0069] - RGVQDPR (SEQ ID NO: 44) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising RGVQDPR (SEQ ID NO: 44).

[0070] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0071] - DGMRDPR (SEQ ID NO: 43) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising DGMRDPR (SEQ ID NO: 43).

[0072] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0073] - QGVRDPR (SEQ ID NO: 45) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising QGVRDPR (SEQ ID NO: 45).

[0074] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0075] - TGVLDPR (SEQ ID NO: 98) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TGVLDPR (SEQ ID NO: 98).

[0076] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0077] - YGGPDPR (SEQ ID NO: 99) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising YGGPDPR (SEQ ID NO: 99).

[0078] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0079] - QGIQDPR (SEQ ID NO: 100) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising QGIQDPR (SEQ ID NO: 100).

[0080] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0081] - TDMRDPR (SEQ ID NO: 101) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TDMRDPR (SEQ ID NO: 101).

[0082] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0083] - TGMRDPK (SEQ ID NO: 102) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TGMRDPK (SEQ ID NO: 102).

[0084] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0085] - YGVKDPR (SEQ ID NO: 103) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising YGVKDPR (SEQ ID NO: 103).

[0086] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0087] - TGMIDPR (SEQ ID NO: 104) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TGMIDPR (SEQ ID NO: 104).

[0088] In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more of the exogenous peptides comprising “Xn

[0089] - TAMRDPR (SEQ ID NO: 105) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising at least one or more exogenous peptides comprising TAMRDPR (SEQ ID NO: 105).

[0090] In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif QGVKDPR (SEQ ID NO: 39) which is expressed from a nucleic acid sequence of SEQ ID NO: 67, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 68. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif NGLKDPR (SEQ ID NO: 33) which is expressed from a nucleic acid sequence of SEQ ID NO: 65, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 66. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif TGVKDPR (SEQ ID NO: 42) which is expressed from a nucleic acid sequence of SEQ ID NO: 73, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 74. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif TGMRDPN (SEQ ID NO: 57) which is expressed from a nucleic acid sequence of SEQ ID NO: 77, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 78. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif TVMRDPR (SEQ ID NO: 54) which is expressed from a nucleic acid sequence of SEQ ID NO: 75, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 76. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif RGVQDPR (SEQ ID NO: 44) which is expressed from a nucleic acid sequence of SEQ ID NO: 71, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 72. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif DGMRDPR (SEQ ID NO: 43) which is expressed from a nucleic acid sequence of SEQ ID NO: 63, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 64. In certain embodiments, provided herein is an engineered AAV9 capsid comprising exogenous targeting motif QGVRDPR (SEQ ID NO: 45) which is expressed from a nucleic acid sequence of SEQ ID NO: 69, or a sequence at least about 70% identical thereto which encodes the amino acid sequence of SEQ ID NO: 70.

[0091] In certain embodiments, the rAAV comprises an AAVhu68 capsid wherein the AAVhu68 capsid protein comprises at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAVhu68 capsid wherein the AAVhu68 capsid protein comprises at least one or more of the exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAVhu68 capsid wherein the AAVhu68 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), and / or combination of any thereof.

[0092] In certain embodiments, the rAAV comprises a mutant AAV9- NGLKDPR capsid or a mutant AAVhu68- NGLKDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the NGLKDPR (SEQ ID NO: 33) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- NGLKDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0093] In certain embodiments, the rAAV comprises a mutant AAV9- TGVKDPR capsid or a mutant AAVhu68- TGVKDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TGVKDPR (SEQ ID NO: 42) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TGVKDPR - VP proteins in which each the VP 1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0094] In certain embodiments, the rAAV comprises a mutant AAV9- DGMRDPR capsid or a mutant AAVhu68- DGMRDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the DGMRDPR (SEQ ID NO: 43) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- DGMRDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0095] In certain embodiments, the rAAV comprises a mutant AAV9- TGMRDPN capsid or a mutant AAVhu68- TGMRDPN capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TGMRDPN (SEQ ID NO: 57) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TGMRDPN - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0096] In certain embodiments, the rAAV comprises a mutant AAV9- QGVRDPR capsid or a mutant AAVhu68- QGVRDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the QGVRDPR (SEQ ID NO: 45) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- QGVRDPR - VP proteins in which each the VP 1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0097] In certain embodiments, the rAAV comprises a mutant AAV9- RGVQDPR capsid or a mutant AAVhu68- RGVQDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGVQDPR (SEQ ID NO: 44) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- RGVQDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0098] In certain embodiments, the rAAV comprises a mutant AAV9- TVMRDPR capsid or a mutant AAVhu68- TVMRDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TVMRDPR (SEQ ID NO: 54) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TVMRDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0099] In certain embodiments, the rAAV comprises a mutant AAV9- QGVKDPR capsid or a mutant AAVhu68- QGVKDPRcapsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the QGVKDPR (SEQ ID NO: 39) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- QGVKDPR - VP proteins in which each the VP 1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0100] In certain embodiments, the rAAV comprises a mutant AAV9- TGVLDPR capsid or a mutant AAVhu68- TGVLDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TGVLDPR (SEQ ID NO: 98) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TGVLDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0101] In certain embodiments, the rAAV comprises a mutant AAV9- YGGPDPR capsid or a mutant AAVhu68- YGGPDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the YGGPDPR (SEQ ID NO: 99) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- YGGPDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0102] In certain embodiments, the rAAV comprises a mutant AAV9- QGIQDPR capsid or a mutant AAVhu68- QGIQDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the QGIQDPR (SEQ ID NO: 100) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- QGIQDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0103] In certain embodiments, the rAAV comprises a mutant AAV9- TDMRDPR capsid or a mutant AAVhu68- TDMRDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TDMRDPR (SEQ ID NO: 101) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TDMRDPR - VP proteins in which each the VP 1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0104] In certain embodiments, the rAAV comprises a mutant AAV9- TGMRDPK capsid or a mutant AAVhu68- TGMRDPK capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TGMRDPK (SEQ ID NO: 102) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TGMRDPK - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0105] In certain embodiments, the rAAV comprises a mutant AAV9- YGVKDPR capsid or a mutant AAVhu68- YGVKDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the YGVKDPR (SEQ ID NO: 103) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- YGVKDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0106] In certain embodiments, the rAAV comprises a mutant AAV9- TGMIDPR capsid or a mutant AAVhu68- TGMIDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TGMIDPR (SEQ ID NO: 104) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TGMIDPR - VP proteins in which each the VP 1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0107] In certain embodiments, the rAAV comprises a mutant AAV9- TAMRDPR capsid or a mutant AAVhu68- TAMRDPR capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the TAMRDPR (SEQ ID NO: 105) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV- TAMRDPR - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the parental capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0108] In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous targeting peptide which is immediately preceded by “AQ”. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous targeting peptide wherein the exogenous peptide is flanked by “AQ” (e g., “AQ- QGVKDPR (SEQ ID NO: 39)”, “AQ- QGVKDPR (SEQ ID NO: 39)-AQ”, or “QGVKDPR (SEQ ID NO: 39)-AQ”). In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid proteins comprises exogenous peptide which is immediately preceded by the native residues of the parent AAV which may be unmodified at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide which is immediately preceded by the native residues of the parent AAV which may be mutated at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In certain embodiments, wherein the parental capsid is an AAV9 capsid or other Clade F capsid, the AAV9 parental capsid or other Clade F parental capsid is unmodified at the residues flanking the inserted exogenous targeting peptide. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid proteins comprise an exogenous peptide wherein the exogenous peptide is flanked by “SAQ” at the amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide wherein the exogenous peptide is flanked by “AQA” at the carboxy (COO-) terminus. In certain embodiments, wherein the parent capsid is an AAV9 capsid or other Clade F capsid, the AAV9 parent capsid or other Clade F parent capsid is modified (i.e., mutated) at the residues flanking the inserted exogenous targeting peptide. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises an exogenous peptide wherein the exogenous peptide is flanked by a mutated trimer “ENT” at amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid proteins comprise an exogenous peptide wherein the exogenous peptide is flanked by mutated trimer “SHQ”, SWQ”, SAI”, “GAQ”, “FAQ”, “QAQ”, “AAQ”, “SGQ”, “SGM” at amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid proteins comprise exogenous peptide wherein the exogenous peptide is flanked by mutated trimer “QQA”, “NQA”, “AMA”, “AQC”, “GQA”, “ARA”, “GRA” at carboxy (COO-) terminus. In other embodiments, the flanking residues may be modified, e.g., where a non-Clade F parental AAV is selected and / or to reduce the number of AAV residues inserted.

[0109] In certain embodiments, capsids from Clade F AAV such as AAVhu68 or AAV9 are selected for parental capsids. Methods of generating vectors having the AAV9 capsid or AAVhu68 capsid, and / or chimeric capsids derived from AAV9 have been described. See, e.g., US 7,906,111, which is incorporated by reference herein. See also, US Provisional Patent Application No. 63 / 093,275, filed October 18, 2020, which is incorporated herein by reference. Other AAV serotypes which transduce nasal cells or another suitable target (e.g., muscle or lung) may be selected as sources for capsids of AAV viral vectors including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rhlO, AAVrh64Rl, AAVrh64R2, rh8, AAVrh32.33 (See, e.g., US Published Patent Application No. 2007-0036760-Al; US Published Patent Application No. 2009-0197338-Al; and EP 1310571). See also, WO 2003 / 042397 (AAV7 and other simian AAV), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005 / 033321 (AAV9), and WO 2006 / 110689, or yet to be discovered, or a recombinant AAV based thereon, may be used as a source for the AAV capsid. See, e.g., WO 2020 / 223232 Al (AAV rh90), WO 2020 / 223231 Al International Application No. PCT / US21 / 45945, filed August 13, 2021 (AAV rh91), and WO 2020 / 223236 Al (AAV rh92, AAV rh93, AAV rh91.93), which are incorporated herein by reference in its entirety. These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. In some embodiments, an AAV capsid (cap) for use in the viral vector can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of the aforementioned AAV caps or its encoding nucleic acid. In some embodiments, the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vpl, Vp2, and Vp3 (also referred to as vpl, vp2, vp3, or VP1, VP2, VP3) monomers from two or three different AAVs or recombinant AAVs. In some embodiments, an rAAV composition comprises more than one of the aforementioned caps.

[0110] As used herein, the term “clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor- Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence. The Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of an AAV vpl capsid protein, one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun; 78(12): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005 / 033321.

[0111] As used herein, an “AAV9 capsid” is a self-assembled AAV capsid composed of multiple AAV9 vp proteins. The AAV9 vp proteins are typically expressed as alternative splice variants encoded by a nucleic acid sequence which encodes the vpl amino acid sequence of GenBank accession: AAS99264. These splice variants result in proteins of different length. In certain embodiments, “AAV9 capsid” includes an AAV having an amino acid sequence which is 99% identical to AAS99264 or 99% identical thereto. See, also, WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9. See, also US7906111 and WO 2005 / 033321. When specified, “AAV9 variants” may include those described in, e.g., W02016 / 049230, US 8,927,514, US 2015 / 0344911, and US 8,734,809.

[0112] A rAAVhu68 is composed of an AAVhu68 capsid and a vector genome. An AAVhu68 capsid is an assembly of a heterogenous population of vpl, a heterogenous population of vp2, and a heterogenous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term “heterogenous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. See, also, PCT / US2018 / 019992, WO 2018 / 160582, entitled “Adeno-Associated Virus (AAV) Clade F Vector and Uses Therefor”, and which are incorporated herein by reference in its entirety.

[0113] In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 90, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 33 (NGLKDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 91, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 42 (TGVKDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 92, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 43 (DGMRDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 93, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 57 (TGMRDPN). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 94, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 45 (QGVRDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 95, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 44 (RGVQDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 96, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 54 (TVMRDPR). In certain embodiments, the mutant AAV capsid is produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 97, or a sequence 95% to 100%, or at least 99%, identical thereto encoding SEQ ID NO: 39 (QGVKDPR). In certain embodiments, the exogenous peptide is inserted between amino acids

[0114] 588 and 589 in the AAVhu68 capsid (as based on amino acid sequence of SEQ ID NO: 61). In other embodiments, exogenous peptide is inserted between amino acids 588 and

[0115] 589 in the AAV9 capsid (as based on amino acid sequence of SEQ ID NO: 60). Still other suitable locations for these inserts may be determined. In still other embodiments, these peptides may be used in other vectors or compositions for targeting.

[0116] In certain embodiments, the coding sequence of a mutant AAV9 capsid having the exogenous targeting peptide inserted in the hypervariable region between amino acids 588 and 589 in the AAV9 parental capsid is: SEQ ID NO: 63 (DGMRDPR), SEQ ID NO: 65 (NGLKDPR); SEQ ID NO: 67 (QGVKDPR); SEQ ID NO: 69 (QGVRDPR); SEQ ID NO: 71 (RGVQDPR); SEQ ID NO: 73 (TGVKDPR); SEQ ID NO: 75 (TVMRDPR); or SEQ ID NO: 77 (TGMRDPN)

[0117] In other embodiments, a mutant AAV9 is encoded by any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 64 (DGMRDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66 (NGLKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68 (QGVKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 70 (QGVRDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 72 (RGVQDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 74 (TGVKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76 (TVMRDPR mutant VP1), or any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 78 (TGMRDPN mutant VP1).

[0118] In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 63, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 64 (DGMRDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 65, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 66 (NGLKDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 67, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 68 (QGVKDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 69, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 70 (QGVRDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 71, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 72 (RGVQDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 73, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 74 (TGVKDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 75, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 76 (TVMRDPR mutant VP1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 77, or a sequence 95% to 100% identical, or at least 99% identical, thereto encoding SEQ ID NO: 78 (TGMRDPN mutant VP1).

[0119] For other recombinant viral vectors, suitable exposed portions of the viral capsid or envelope protein which is responsible for targeting specificity are selected for insertion of the targeting peptide. For example, in an adenovirus, it may be desirable to modify the hexon protein. In a lentivirus, an envelope fusion protein may modified comprise one or more copies of the targeting motif. For vaccinia virus, the major glycoprotein may be modified to comprise one or more copies of the targeting motif. Suitably, these recombinant viral vectors are replication-defective for safety purposes.

[0120] Expression Cassette and Vectors

[0121] Vector genomic sequences which are packaged into an AAV capsid and delivered to a host cell are typically composed of, at a minimum, a transgene and its regulatory sequences (e.g., expression cassette), and AAV inverted terminal repeats (ITRs). Both single-stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components (also referred to as regulatory sequences, regulatory control sequences, control sequences, regulatory elements, expression control sequences, expression control elements) in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue.

[0122] As used herein, the term “regulatory sequence”, or “regulatory control sequences “or “expression control sequence” refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, promoter sequences, intron sequences, and polyA signal sequences which direct, enable, induce, repress, or otherwise control the transcription, translation and / or expression of protein encoding nucleic acid sequences to which they are operably linked.

[0123] The AAV sequences of the vector typically comprise the cis-acting AAV 5’ and AAV 3’ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 base pairs (bp) in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5’ and 3’ AAV ITR sequences (also referred to as “5' ITR and 3' ITR”, “AAV 5’ ITR”, or “5’ ITR”). In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5’ ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type (WT) length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full-length AAV 5’ and 3’ ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

[0124] In certain embodiments, the provided herein is rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5' and / or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR. In certain embodiments, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences which enables expression of transgene in a target cell, (e.g., by directing transcription, translation and / or expression).

[0125] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV- 5' ITR - an optional enhancer - a promoter - an optional intron - coding sequence (e.g., test transgene) - polyadenylation (polyA) signal sequence - AAV3' - ITR. In other embodiments, the orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e.g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and / or the 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

[0126] In addition to the major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements necessary which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. Examples of other suitable transgenes are provided herein. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0127] In certain embodiments, the regulatory sequences comprise one or more of a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signals such as splicing and polyadenylation signals (poly A), a sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), and sequences that enhance translation efficiency (i.e., Kozak consensus sequence). In certain embodiments the selected promoter is a constitutive promoter. In certain embodiments, the promoter is a ubiquitous promoter.

[0128] In certain embodiments, the regulatory sequences are selected such that the total rAAV vector genome is about 2.0 to about 5.5 kilobases in size. In one embodiment, it is desirable that the rAAV vector genome approximate the size of the native AAV genome. Thus, in one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 4.7 kb in size. In another embodiment, the total rAAV vector genome is less about 5.2kb in size. The size of the vector genome may be manipulated based on the size of the regulatory sequences including the promoter, enhancer, intron, poly A, etc. See, Wu et al, Mol Ther, Jan 2010 18(l):80-6, which is incorporated herein by reference.

[0129] The regulatory control elements typically contain a promoter sequence as part of the expression control sequences, e.g., located between the selected 5’ ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [see, e.g., WO 2011 / 126808 and WO 2013 / 04943], tissue specific promoters, or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein.

[0130] Examples of constitutive promoters suitable for controlling expression of the therapeutic products include, but are not limited to chicken beta (P)-actin (CB) promoter, CB7 promoter (a CB7 hybrid promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta-actin promoter, and a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor), human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the P-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006- 10010 (1989)), the long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, the thymidine kinase promoter of Herpes Simplex Virus and other constitutive promoters known to those of skill in the art. Examples of tissue- or cellspecific promoters suitable for use in the present invention include, but are not limited to, endothelin-I (ET -I) and Flt-I, which are specific for endothelial cells, FoxJl (that targets ciliated cells). In certain embodiments, the promoter is a tissue-specific (e.g., neuronspecific) promoter. In certain embodiments, a suitable promoter may include without limitation, an elongation factor 1 alpha (EFl alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91 (2):217-23), a synapsin 1 promoter (see, e.g., Kugler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), a shorted synapsin promoter, a neuron-specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or a CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol. Biotechnol. 2016 Jan;58(l):30-6. doi: 10.1007 / sl2033- 015-9899-5). In certain embodiments, the promoter is a tissue-specific promoter which is kidney-specific (e.g., nephron-specific) promoter (e.g., kidney-specific cadherin (KSPC) gene promoter, parathyroid hormone receptor ‘kidney-specific’ promoter, podocytespecific promoter, human nephrin promoter, putative kidney-specific promoters from myoinositol oxygenase (MIOX), acyl-CoA synthetase medium-chain family member 2 (Acsm2), uromodulin (UMOD), kidney androgen-regulated protein (Kap) and solute carrier family 34 (sodium phosphate), member 1 (Slc34al))). See also, Asico, L.D., et al., Nephron segment-specific gene expression using AAV vectors, Biochemical and Biophysical Research Communications, 26 February 2018, 497(1): 19-24; Schreiber, C.A., et al., Hitting the Kidney from All Angles: Using Direct Intrarenal Pelvis Injection Combined with Serotype and Promoter Targeting to Achieve Renal AAV Gene Delivery, Molecular Therapy, AAV VECTORS II, 24 (SUPPLEMENT 1) S124, MAY 2016; R. M. Deviatiirov, et al, “Human library of cardiac promoters and enhancers”, bioRxiv, pp. 1-27, June 15, 2020, which are incorporated herein by reference in their entirety. In certain embodiments, the promoter is cardiac specific promoter, e.g., cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2) promoters. See also, Pacak, C.A., et al., Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice, Genetic Vaccines and Therapy 2008, 6:13. In certain embodiments, the expression cassette comprises a promoter which is a chicken cardiac Troponin T promoter (also referred to as chicken TnT or chTnT). In certain embodiments, the promoter is a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer and a chicken cardiac troponin T (chicken cTnT or chTnT) promoter. See also, International Patent Application No. PCT / US2022 / 082384, filed December 24, 2022, which is incorporated herein by reference in its entirety. In certain embodiments, and enhancer is a a-myosin heavy-chain enhancer. Preferably, such promoters are of human origin.

[0131] Examples of inducible promoters suitable for controlling expression of the therapeutic product include promoters responsive to exogenous agents (e.g., pharmacological agents) or to physiological cues. These response elements include, but are not limited to a hypoxia response element (HRE) that binds HIF-Ia and 0, a metal-ion response element such as described by Mayo et al. (1982, Cell 29:99-108); Brinster et al. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5: 1480-1489); or a heat shock response element such as described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., ppI67-220, 1991).

[0132] In certain embodiments, expression of the gene product is controlled by a regulatable promoter that provides tight control over the transcription of the sequence encoding the gene product, e.g., a pharmacological agent, or transcription factors activated by a pharmacological agent or in alternative embodiments, physiological cues. Promoter systems that are non-leaky and that can be tightly controlled are preferred. Examples of regulatable promoters which are ligand-dependent transcription factor complexes that may be used in the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in US Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006 / 0014711, 2007 / 0161086, and International Published Application No. WO 01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, and International Published Application Nos. WO 01 / 70816, WO 02 / 066612, WO 02 / 066613, WO 02 / 066614, WO 02 / 066615, WO 02 / 29075, and WO 2005 / 108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).

[0133] Still other promoter systems may include response elements including but not limited to a tetracycline (tet) response element (such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or a hormone response element such as described by Lee et al. (1981, Nature 294:228-232); Hynes et al. (1981, Proc. Natl. Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736); and Israel & Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and other inducible promoters known in the art. Using such promoters, expression of the soluble hACE2 construct can be controlled, for example, by the Tet-on / off system (Gossen et al., 1995, Science 268: 1766-9; Gossen et al., 1992, Proc. Natl. Acad. Sci. USA., 89(12): 5547-51); the TetR-KRAB system (Urrutia R., 2003, Genome Biol., 4(10):231 ; Deuschle U et al., 1995, Mol Cell Biol. (4): 1907-14); the mifepristone (RU486) regulatable system (Geneswitch; Wang Y et al., 1994, Proc. Natl. Acad. Sci. USA., 91 (17): 8180-4; Schillinger et al., 2005, Proc. Natl. Acad. Sci. U S A.102(39): 13789-94); and the humanized tamoxifen-dep regulatable system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

[0134] In another aspect, the gene switch is based on heterodimerization of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogs. Examples of such systems, include, without limitation, the ARGENT™ Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595, U.S. Publication No. 2002 / 0173474, U.S. Publication No. 200910100535, U.S. Patent No. 5,834,266, U.S. Patent No. 7,109,317, U.S. Patent No. 7,485,441, U.S. Patent No. 5,830,462, U.S. Patent No. 5,869,337, U.S. Patent No. 5,871,753, U.S. Patent No. 6,011,018, U.S. Patent No. 6,043,082, U.S. Patent No. 6,046,047, U.S. Patent No. 6,063,625, U.S. Patent No. 6,140,120, U.S. Patent No. 6,165,787, U.S. Patent No. 6,972,193, U.S. Patent No. 6,326,166, U.S. Patent No. 7,008,780, U.S. Patent No. 6,133,456, U.S. Patent No. 6,150,527, U.S. Patent No. 6,506,379, U.S. Patent No. 6,258,823, U.S. Patent No. 6,693,189, U.S. Patent No. 6,127,521, U.S. Patent No. 6,150,137, U.S. Patent No. 6,464,974, U.S. Patent No. 6,509,152, U.S. Patent No. 6,015,709, U.S. Patent No. 6,117,680, U.S. Patent No. 6,479,653, U.S. Patent No. 6,187,757, U.S. Patent No. 6,649,595, U.S. Patent No. 6,984,635, U.S. Patent No. 7,067,526, U.S. Patent No. 7,196,192, U.S. Patent No. 6,476,200, U.S. Patent No. 6,492,106, WO 94 / 18347, WO 96 / 20951, WO 96 / 06097, WO 97 / 31898, WO 96 / 41865, WO 98 / 02441, WO 95 / 33052, WO 99110508, WO 99110510, WO 99 / 36553, WO 99 / 41258, WO 01114387, ARGENT™ Regulated Transcription Retrovirus Kit, Version 2.0 (9109102), and ARGENT™ Regulated Transcription Plasmid Kit, Version 2.0 (9109 / 02), each of which is incorporated herein by reference in its entirety. The Ariad system is designed to be induced by rapamycin and analogs thereof referred to as "rapalogs". Examples of suitable rapamycins are provided in the documents listed above in connection with the description of the ARGENT™ system. In one embodiment, the molecule is rapamycin [e.g., marketed as Rapamune™ by Pfizer], In another embodiment, a rapalog known as AP21967 [ARIAD] is used. Examples of these dimerizer molecules that can be used in the present invention include, but are not limited to rapamycin, FK506, FK1012 (a homodimer of FK506), rapamycin analogs ("rapalogs") which are readily prepared by chemical modifications of the natural product to add a "bump" that reduces or eliminates affinity for endogenous FKBP and / or FRAP. Examples of rapalogs include, but are not limited to such as AP26113 (Ariad), API 510 (Amara, J.F., et al., 1997, Proc Natl Acad Sci USA, 94(20): 10618-23) AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692 and AP1889, with designed 'bumps' that minimize interactions with endogenous FKBP. Still other rapalogs may be selected, e.g., AP23573 [Merck], In certain embodiments, rapamycin or a suitable analog may be delivered locally to the A AV-transfected cells of the nasopharynx. This local delivery may be by intranasal injection, topically to the cells via bolus, cream, or gel. See, US Patent Application US 2019 / 0216841 Al, which is incorporated herein by reference.

[0135] In certain embodiments, the expression cassette further comprises an enhancer sequence as part of the expression control sequences. Suitable enhancers include those that are appropriate for a desired target tissue indication. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. For example, an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences. In still another embodiment, the expression cassette further comprises an intron, e.g., the chicken beta-actin intron. Examples of other suitable introns include those described in WO 2011 / 126808, the human beta globin IVS2 (Kelly et al, Nucleic Acids Research, 43(9):4721-32 (2015)); the Promega chimeric intron (Almond, B. and Schenborn, E. T. A Comparison of pCI-neo Vector and pcDNA4 / HisMax Vector); and the hFIX intron. Various introns suitable herein are known in the art and include, without limitation, those found at bpg.utoledo.edu / ~afedorov / lab / eid.html, which is incorporated herein by reference. See also, Shepelev V., Fedorov A. Advances in the Exon-Intron Database. Briefings in Bioinformatics 2006, 7: 178-185, which is incorporated herein by reference. In still another embodiment, the expression cassette further comprises a polyadenylation signal sequence. Examples of suitable polyadenylation (poly A) signal sequences include, e.g., rabbit beta globin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic poly As. Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605- 619).

[0136] In certain embodiments, the expression cassette may include one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE), human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE); or a synthetic post-transcriptional regulatory element. These expressionenhancing elements are particularly advantageous when placed in a 3’ UTR and can significantly increase mRNA stability and / or protein yield. In certain embodiments, the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B vims X (WUX) protein, including, for example, mutations in the start codon of the WHX gene. In certain embodiments, modified WPRE element is engineered to eliminate expression of the WHX protein, wherein the modified WPRE is a mutated version that contains five-point mutations in the putative promoter region of the WHX gene, along with an additional mutation in the start codon of the WHX gene (ATG mutated to TTG). This mutant WPRE is considered sufficient to eliminate expression of truncated WHX protein based on sensitive flow cytometry analyses of various human cell lines transduced with lentivirus containing a WPRE-GFP fusion construct (Zanta-Boussif et al., 2009). See also, Kingsman S.M., Mitrophanous K., & Olsen J.C. (2005), “Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (WPRE).” Gene Ther. 12(l):3-4; and Zanta-Boussif M. A., Charrier S., Brice-Ouzet A., Martin S., Opolon P., Thrasher A. J., Hope T.J., & Galy A. (2009), Validation of a Mutated Pre-Sequence Allowing High and Sustained Transgene Expression While Abrogating Whv-X Protein Synthesis: Application to the Gene Therapy of Was, Gene Ther. 16(5):605-19, both of which are incorporated herein by reference in its entirety. In other embodiments, enhancers are selected from a non-viral source. In certain embodiments, no WPRE sequence is present.

[0137] An AAV vector genome may include a sequence encoding multiple gene products (e.g., encoding one or more a protein, peptide, miR, miR seed or target). In certain embodiments, the transgene may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. Alternatively, the transgene may provide a product to a cell which is not natively expressed in the cell type or in the host. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide which is expressed in a host cell. The invention further includes using multiple transgenes. In certain situations, a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet- derived growth factor, or a dystrophin protein. In certain situations, a different transgene may be used to encode each subunit of a protein (e.g., an immunoglobulin domain, an immunoglobulin heavy chain, an immunoglobulin light chain). In one embodiment, a cell produces the multi-subunit protein following infected / transfection with the virus containing each of the different subunits. In another embodiment, different subunits of a protein may be encoded by the same transgene. An IRES is desirable when the size of the DNA encoding each of the subunits is small, e.g., the total size of the DNA encoding the subunits and the IRES is less than five kilobases. As an alternative to an IRES, the DNA may be separated by sequences encoding a 2A peptide, which self-cleaves in a post- translational event. See, e.g., ML Donnelly, et al, (Jan 1997) J. Gen. Virol., 78(Pt 1): 13-21; S. Furler, S et al, (June 2001) Gene Ther., 8(11 ): 864-873 ; H. Klump, et al., (May 2001) Gene Then, 8(10) : 811-817. This 2A peptide is significantly smaller than IRES, making it well suited for use when space is a limiting factor. More often, when the transgene is large, consists of multi-subunits, or two transgenes are co-delivered, rAAV carrying the desired transgene(s) or subunits are co-administered to allow them to concatamerize in vivo to form a single vector genome. In such an embodiment, a first AAV may carry an expression cassette which expresses a single transgene and a second AAV may carry an expression cassette which expresses a different transgene for co-expression in the host cell. However, the selected transgene may encode any biologically active product or other product, e.g., a product desirable for study.

[0138] In certain embodiments, an expression cassette further comprises at least one miRNA target sequences operably linked to a selected transgene, optionally in its 3’ UTR and / or its 5’ UTR. In certain embodiments, the mutant rAAV comprises a vector genome (comprising an expression cassette) which further comprises at least one miRNA seed, binding site or full sequence. MicroRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. In some embodiments, a microRNA sequence comprises a seed region, e.g., a sequence in the region of positions 2-8 of the mature microRNA, which has Watson-Crick sequence fully or partially complementarity to the miRNA target sequence of the nucleic acid. Such at least one miRNA may be used in combinations, including in an expression cassette or a vector genome also comprising a coding sequence for therapeutic protein, enzyme, or other moiety, and which is operably linked to the coding sequence. In certain embodiments, the vector genome may contain one miRNA to eight miRNA sequences, which are the same or different. Optionally, the vector genome does not contain therapeutic transgenes other than miRNA sequences.

[0139] In certain embodiments, the miRNA is a dorsal root ganglion (drg)-specific miRNA target sequence. In some embodiments, the miRNA binding site is complementary to a miRNA expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, and / or a miR182, binding site. In some embodiments, the miR binding site complementary to a miR expressed in expressed in a DRG neuron comprises a nucleotide sequence disclosed, e.g., in WO2020 / 132455, and in WO 2023 / 087019, the contents of which are incorporated by reference herein in its entirety.

[0140] In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three or preferably at least four tandem repeats of dorsal root ganglion (drg)-specific miRNA target sequences. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight tandem repeats of dorsal root ganglion (drg)-specific miRNA target sequences. See, e.g., PCT / US19 / 67872, filed December 20, 2019, and now published as WO 2020 / 132455, which are incorporated herein by reference. See, also, International Patent Application No. PCT / US21 / 32003, filed May 12, 2021, and now published WO2021 / 231579A1, which are incorporated herein by reference. See also, US International Patent Application No. PCT / US2022 / 079881, filed November 15, 2022, which is incorporated herein by reference in its entirety.

[0141] As another non-limiting example, a vector genome (expression cassette) may comprise miR- 122 miRNA to modulate, e.g., reduce, the expression of a gene product in the liver. In some embodiments, the vector genome (expression cassette) may comprise a miRNA, e.g., a miR-142-3p, to modulate, e.g., reduce, the expression, of the gene product in a cell or tissue of the hematopoietic lineage, including for example immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B- lymphocytes). Other suitable miRNA may include, e.g., miR-206 (skeletal muscle), miR- 018b, miR-431.See. e.g., Kim., H.K., Muscle-specific microRNA miR-206 promotes muscle differentiation, The Journal of Cell Biology, 2006, 174(5):677-687; WO 2019 / 035690A1; WO 2019 / 035690A1; WO 2022 / 147181A1, and Brazilian Patent Publication No. BR102018067702A2, which are all incorporate herein by reference.

[0142] Several different vector genomes were generated in the studies described herein. However, it will be understood by the skilled artisan that other genomic configurations, including other regulatory sequences may be substituted for the promoter, enhancer and other coding sequences may be selected. rAAV Vector Production

[0143] For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a production (packaging) host cell. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art. In certain embodiments, the production host cell is a human cell or insect cell. In certain embodiments, the production host cell in is HEK293 cell, HuH-7 cell, BHK cell, or Vero cell. In certain embodiments, production host cell is in a suspension cell culture.

[0144] In certain embodiments, a host cell is stably or transiently transfected with a genetic element (e.g., a plasmid or other nucleic acid molecule) which expresses a mutant AAV capsid as provided herein. In certain embodiments, such a genetic element comprises a nucleic acid sequence encoding a mutant AAV VP1 coding sequence comprising the mutant peptide(s) inserted therein, operably linked to expression control sequences which enable expression of the AAV capsid proteins in the packaging host cell. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 33 (NGLKDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 90 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 42 (TGVKDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 91 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 43 (DGMRDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 92 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 57 (TGMRDPN), which is optionally a nucleic acid sequence of SEQ ID NO: 93 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 45 (QGVRDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 94 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 44 (RGVQDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 95 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 54 (TVMRDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 96 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 39 (QGVKDPR), which is optionally a nucleic acid sequence of SEQ ID NO: 97 or a sequence 95% to 100% identical, or at least 99% identical, thereto.

[0145] In certain embodiments, a host cell is stably or transiently transfected with a genetic element (e.g., a plasmid or other nucleic acid molecule) which expresses a mutant AAV capsid as provided herein. In certain embodiments, such a genetic element comprises a nucleic acid sequence encoding a mutant AAV VP1 coding sequence comprising the mutant peptide(s) (i.e., the exogenous targeting peptide(s)) inserted therein, operably linked to expression control sequences which enable expression of the AAV capsid proteins in the packaging host cell. In certain embodiments, the mutant peptides are engineered between amino acids 588 and 589 in the AAVhu68 capsid. In other embodiments, these peptides are inserted between amino acids 588 and 589 in the AAV9 capsid. Still other suitable locations for these inserts may be determined. In still other embodiments, these peptides may be used in other vectors or compositions for targeting. In certain embodiments, the coding sequence of a mutant AAV9 capsid having the exogenous targeting peptide inserted in the hypervariable region between amino acids 588 and 589 in the AAV9 parental capsid is: SEQ ID NO: 63 (DGMRDPR), SEQ ID NO: 65 (NGLKDPR), SEQ ID NO: 67 (QGVKDPR), SEQ ID NO: 69 (QGVRDPR), SEQ ID NO: 71 (RGVQDPR), SEQ ID NO: 73 (TGVKDPR), SEQ ID NO: 75 (TVMRDPR), or SEQ ID NO: 77 (TGMRDPN).

[0146] In other embodiments, a mutant AAV9 is encoded by any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 64 (DGMRDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66 (NGLKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68 (QGVKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 70 (QGVRDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 72 (RGVQDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 74 (TGVKDPR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76 (TVMRDPR mutant VP1), or any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 78 (TGMRDPN mutant VP1).

[0147] In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 1 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 1 (i.e., comprising AAV2 rep and AAV9- DGMRDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 4 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 4 (i.e., comprising AAV2 rep and AAV9- NGLKDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 7 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 7 (i.e., comprising AAV2 rep and AAV9- QGVKDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 8 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 8 (i.e., comprising AAV2 rep and AAV9- QGVRDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 9 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 9 (i.e., comprising AAV2 rep and AAV9- RGVQDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 17 or a sequence at least 90%, 95%, 98%, 99%.

[0148] 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 17 (i.e., comprising AAV2 rep and AAV9- TGVKDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 19 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 19 (i.e., comprising AAV2 rep and AAV9- TVMRDPR-modified cap). In certain embodiments, nucleic acid sequence encoding an AAV capsid used for production of a recombinant AAV comprising rep sequences and cap sequences comprises SEQ ID NO: 22 or a sequence at least 90%, 95%, 98%, 99%. 99.9%, 100% (or any values therebetween) identical to SEQ ID NO: 22 (i.e., comprising AAV2 rep and AAV9- TGMRDPN-modified cap).

[0149] In certain embodiments, provided herein is a production host cell comprising a recombinant nucleic acid molecule as described herein, a nucleic acid sequence encoding an AAV capsid protein, and sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NOs: 1-23. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 1. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 4. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 7. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 8. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 9. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 17. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19. In certain embodiment nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 22.

[0150] Methods of preparing AAV-based vectors (e.g., having an AAV9 or another AAV capsid) are known. See, e.g., US Published Patent Application No. 2007 / 0036760 (February 15, 2007), which is incorporated by reference herein. The invention is not limited to the use of AAV9 or other clade F AAV amino acid sequences, but encompasses peptides and / or proteins containing the terminal 0-galactose binding generated by other methods known in the art, including, e.g., by chemical synthesis, by other synthetic techniques, or by other methods. The sequences of any of the AAV capsids provided herein can be readily generated using a variety of techniques. Suitable production techniques are well known to those of skill in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY). Alternatively, peptides can also be synthesized by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc., 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These methods may involve, e.g., culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the minigene into the AAV capsid protein. These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

[0151] The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contains the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0152] These rAAVs are particularly well suited to gene delivery for therapeutic purposes and for preventing infection. Further, the compositions of the invention may also be used for production of a desired gene product in vitro. For in vitro production, a desired product (e.g., a protein) may be obtained from a desired culture following transfection of host cells with a rAAV containing the molecule encoding the desired product and culturing the cell culture under conditions which permit expression. The expressed product may then be purified and isolated, as desired. Suitable techniques for transfection, cell culturing, purification, and isolation are known to those of skill in the art. Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. For packaging a transgene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassettes. The cap and rep genes can be supplied in trans.

[0153] In one embodiment, the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production a viral vector. In one embodiment, the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made. Alternatively, the expression cassettes may be used to generate a viral vector other than AAV, or for production of mixtures of antibodies in vitro. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0154] The term “AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.

[0155] The recombinant AAV described herein may be generated using techniques which are known. See, e.g., WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0156] In certain embodiment, the rAAV are generated (manufactured) using triple transfection techniques. In certain embodiments the rAAV are generated using a stable mammalian cell line. In certain embodiments, the stable cell line comprises one or more of: (a) a first plurality of polynucleotide molecules which comprise a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for production of a replication-defective rAAV vector (Rep52 and Rep78), wherein said rep proteins coding sequences are operably linked to a doxycycline-inducible promoter which directs expression of the rep proteins in the cell line; (b) at least a second plurality of polynucleotide molecules each encoding adenovirus (Ad) helper proteins necessary for production of a replication-defective rAAV vector comprising at least an Ad E2A DNA Binding Protein (DBP) coding sequence, and Ad E4ORF6 coding sequence, wherein the Ad E2A DBP coding sequences and the Ad E4ORF6 coding sequences are operably linked to a doxycycline-inducible promoter which direct expression of the Ad helper proteins in the cell line; (c) a nucleic acid molecule comprising an Ad El coding sequence operably linked to a constitutive promoter which directs expression of the Ad El in the cell line; (d) at least a third plurality of nucleic acid molecules each of which comprises an AAV VP1 coding sequence which encodes AAV VP1 proteins, AAV VP2 proteins and AAV VP3 proteins which self-assemble to form an AAV capsid following expression in the cell, said AAV VP1 coding sequence being operably linked to a promoter which directs expression of the VP1 coding sequences in the cell line.

[0157] In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293 cells). Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors. In some embodiments, the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV genome and the gene of interest for packaging into the capsid, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media. The harvested vector-containing cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929, which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which is incorporated herein by reference in its entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0158] The crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafdtration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0159] A two-step affinity chromatography purification at high salt concentration followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT / US2016 / 065970, filed December 9, 2016, and US 11,098,286 B2, entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No.

[0160] PCT / US2016 / 065976, filed December 9, 2016, and US 11,015,174 B2, entitled “Scalable Purification Method for AAV8”, which are incorporated herein by reference. Purification methods for rhlO, International Patent Application No. PCT / US16 / 066013, filed December 9, 2016, and US 11,028,372 B2, entitled “Scalable Purification Method for AAVrhlO”, which are incorporated herein by reference. Purification methods for AAV1, International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and US 11,015,173 B2, entitled “Scalable Purification Method for AAV1”, which are incorporated herein by reference. See also, International Patent Application No. PCT7US2018 / 019992, filed February 27, 2018, now published WO 2018 / 160582, and International Patent Application No. PCT / US2021 / 055436, filed October 18, 2021, now published WO 2022 / 082109, which are incorporated herein by reference in their entireties. Other suitable methods may be selected.

[0161] To calculate empty and full particle content, vp3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where number of GC = number of particles) are plotted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate the number of particles in the band volumes of the test article peaks. The number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) / mb. Pt / mL divided by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty pt / mL. Empty pt / mL divided by pt / mL and x 100 gives the percentage of empty particles.

[0162] Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; and Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS- polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0163] Additionally, another example of measuring empty to full particle ratio is also known in the art. Sedimentation velocity, as measured in an analytical ultracentrifuge (AUC) can detect aggregates, other minor components as well as providing good quantitation of relative amounts of different particle species based upon their different sedimentation coefficients. This is an absolute method based on fundamental units of length and time, requiring no standard molecules as references. Vector samples are loaded into cells with 2-channel charcoal-epon centerpieces with 12mm optical path length. The supplied dilution buffer is loaded into the reference channel of each cell. The loaded cells are then placed into an AN-60Ti analytical rotor and loaded into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors. After full temperature equilibration at 20 °C the rotor is brought to the final run speed of 12,000 rpm. A280 scans are recorded approximately every 3 minutes for ~5.5 hours (110 total scans for each sample). The raw data is analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resultant size distributions are graphed and the peaks are integrated. The percentage values associated with each peak represent the peak area fraction of the total area under all peaks and are based upon the raw data generated at 280nm; many labs use these values to calculate empty: full particle ratios. However, because empty and full particles have different extinction coefficients at this wavelength, the raw data can be adjusted accordingly. The ratio of the empty particle and full monomer peak values both before and after extinction coefficient-adjustment is used to determine the empty-full particle ratio.

[0164] In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2- fold or higher. Typically, proteinase K treatment is about 0.2 mg / mL, but may be varied from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay. Quantification also can be done using ViroCyt or flow cytometry.

[0165] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0166] In certain embodiments, the manufacturing process for rAAV as described herein (e.g., comprising engineered rAAV) involves method as described in US Provisional Patent Application No. 63 / 371,597, filed August 16, 2022, and US Provisional Patent Application No. 63 / 371,592, filed August 16, 2022, which are incorporated herein by reference in its entirety.

[0167] Therapeutic Proteins and Delivery Systems

[0168] In certain embodiments, provided here are fusion partners, conjugate partners and recombinant vectors containing the exogenous targeting peptide are provided herein, comprising Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, which are useful with a variety of different therapeutic proteins, polypeptides, nanoparticles, and delivery systems. Fusion partners, conjugate partners and recombinant vectors containing the exogenous targeting peptide are also provided herein, comprising Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n- mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or the full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, which are useful with a variety of different therapeutic proteins, polypeptides, nanoparticles, and delivery systems. Examples of proteins and compounds useful in compositions provided herein and targeted delivery are described below. It will be understood that the viral vectors (e.g., rAAV), nanoparticles and other delivery systems contain sequences encoding the selected proteins (or conjugates) for expression in vivo.

[0169] In some embodiments, provided herein is an rAAV having a modified capsid with one or more exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X -G-X ”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV comprises vector genome comprising the desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. In some embodiments, the rAAV having a modified capsid with one or more exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - n- mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), QGVRDPR (SEQ ID NO: 45), TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV comprises vector genome comprising the desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. Such formulation involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid.

[0170] In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors (rAAV) and nanoparticles, comprising the exogenous targeting peptide provided herein, are useful in treatment of one or more of central nervous system (CNS)-based disorders. In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors (rAAV) and nanoparticles, comprising the exogenous targeting peptide provided herein, are useful in treatment of one or more of heart-based disorders. In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors (rAAV) and nanoparticles, comprising the exogenous targeting peptide provided herein, are useful in treatment of one or more of kidney-based disorders.

[0171] Examples of proteins (e.g., therapeutic proteins) and compounds useful in compositions (i.e., therapeutic indications) provided herein and targeted delivery include the following listed below. It will be understood that the rAAV comprise sequences encoding the selected proteins for expression in vivo.

[0172] In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors and nanoparticles, comprising the targeting motif provided herein, are useful in treatment of one or more of cognitive disorders and / or neurodegenerative disorders. Such disorders may include, without limitation, transmissible spongiform encephalopathies (e.g., Creutzfeld- Jacob disease), Parkinson’s disease, amyotropic lateral sclerosis (ALS), multiple sclerosis, Alzheimer’s Disease, Huntington disease, Canavan’s disease, traumatic brain injury, spinal cord injury (ATI335, anti-nogol by Novartis), migraine (ALD403 by Alder Biopharmaceuticals; LY2951742 by Eli; RN307 by Labrys Biologies), bovine spongiform encephalopathy, Gerstmann-Straussler- Scheinker syndrome, fatal familial insomnia, kuruysosomal storage diseases, stroke, and infectious disease affecting the central nervous system.

[0173] In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors and nanoparticles, comprising the targeting motif provided herein, are useful in delivery of antibodies against various infections of the central nervous. Such infectious diseases may include fungal diseases such as cryptoccocal meningitis, brain abscess, spinal epidural infection caused by, e.g., Cryptococcus neoformans, Coccidioides immitis, order Mucorales, Aspergillus spp, and Candida spp; protozoal, such as toxoplasmosis, malaria, and primary amoebic meningoencepthalitis, caused by agents such as, e.g., Toxoplasma gondii, Taenia solium, Plasmodium falciparus, Spirometra mansonoides (sparaganoisis), Echinococcus spp (causing neuro hydatosis), and cerebral amoebiasis; bacterial, such as, e.g., tuberculosis, leprosy, neurosyphilis, bacterial meningitis, lyme disease (Borrelia burgdorferi), Rocky Mountain spotted fever (Rickettsia rickettsia), CNS nocardiosis (Nocardia spp), CNS tuberculosis (Mycobacterium tuberculosis), CNS listeriosis (Listeria monocytogenes), brain abscess, and neuroborreliosis; viral infections, such as, e.g., viral meningitis, Eastern equine encephalitis (EEE), St Louis encepthalitis, West Nile virus and / or encephalitis, rabies, California encephalitis virus, La Crosse encepthalitis, measles encephalitis, poliomyelitis, which may be caused by, e.g., herpes family viruses (HSV), HSV-1, HSV-2 (neonatal herpes simplex encephalitis), varicella zoster virus (VZV), Bickerstaff encephalitis, Epstein-Barr virus (EBV), cytomegalovirus (CMV, such as TCN-202 is in development by Theraclone Sciences), human herpesvirus 6 (HHV-6), B virus (herpesvirus simiae), Flavivirus encephalitis, Japanese encephalitis, Murray valley fever, JC virus (progressive multifocal leukoencephalopathy), Nipah Virus (NiV), measles (subacute sclerosing panencephalitis); and other infections, such as, e.g., subactuate sclerosing panencephalitis, progressive multifocal leukoencephalopathy; human immunodeficiency virus (acquired immunodeficiency syndrome (AIDS)); streptococcus pyogenes and other P- hemolytic Streptococcus (e.g., Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infection, PANDAS) and / or Syndenham’s chorea, and Guillain-Barre syndrome, and prions.

[0174] In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors and nanoparticles, comprising the targeting motif provided herein, are useful in treatment of one or more of kidney disorders and / or disorder implicating kidney function (e.g., implicated “gene”). Such disorders may include, without limitation, Liddle Syndrome, Potter Syndrome, Primary Distal Renal Tubular Acidosis, COL4A1 / A2 -Related Disorders, Focal Segmental Glomerulosclerosis, Gitelman Syndrome, Dent Disease, WNT4 Deficiency, Nephrogenic Systemic Fibrosis, Autosomal Dominant Polycystic Kidney Disease, Barakat Syndrome, C3 Glomerulopathy: Dense Deposit Disease and C3 Glomerulonephritis, Atypical Hemolytic Uremic Syndrome, Urofacial Syndrome, Mayer-Rokitansky-Kuster-Hauser Syndrome, Galloway-Mowat Syndrome, WAGR Syndrome / 1 Ip Deletion Syndrome, Senior Loken Syndrome, Denys- Drash Syndrome, Branchiootorenal Spectrum Disorders, Congenital Hepatic Fibrosis, Retroperitoneal Fibrosis, Renal Cell Carcinoma, STEC Hemolytic Uremic Syndrome, Fraser Syndrome, Renal Glycosuria, Hepatorenal Syndrome, Alport Syndrome, Bartter Syndrome, IgA Nephropathy, Nail Patella Syndrome, Primary Hyperoxaluria, Acquired Lipodystrophy, Henoch-Schbnlein Purpura, Oculo-Auriculo-Vertebral Spectrum, Wilms’ Tumor, N-Acetylglutamate Synthetase Deficiency, Argininosuccinic Aciduria, Citrullinemia Type 1, Carbamoyl Phosphate Synthetase 1 Deficiency, Goodpasture Syndrome, Granulomatosis with Polyangiitis, Autosomal Recessive Polycystic Kidney Disease, Medullary Sponge Kidney, Blue Diaper Syndrome, Cystinuria, Cystinosis, Sindrome urofacial, Chronic Kidney Disease, Fanconi syndrome. In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors and nanoparticles, comprising the targeting motif provided herein, are useful in treatment of one or more of kidney disorders and / or disorder implicating kidney function (e.g., implicated “gene”), including Chronic Kidney Disease (LRP2, CUBN), Cystinosis (CTNS), Autosomal Dominant Polycystic Kidney Disease (PKD1, PKD2, GANAB), Autosomal Recessive Polycystic Kidney Disease (PKHD1), Alport syndrome (COL4A3, COL4A4, COL4A5), Autosomal Dominant Tubulointerstitial Kidney Disease / Medullary cystic kidney disease (MUC1 (type I), UMOD, REN, HNF1B, SEC61A1), Nephronophthisis (NPHP1), Bartter Syndrome (SLC12A1, KCNJ1 (type II), CLCNKB (type III and IV), BSND (type IV), CLCNKA (type IV), MAGED2 (type V)), Von Hippel- Lindau syndrome (VHL), Gitelman syndrome (SLC12A3), CLCNKB), Congenital nephrotic syndrome (NPHS1, NPHS2), Primary hyperoxaluria (AGXT (type I), GRHPR (type II), H0GA1 (type III)), Dent disease (CLCN5 (type I), OCRL (type II)), Thin Basement Membrane Nephropathy (COL4A3, COL4A4, COL4A5), Cystinuria (SLC3A1, SLC7A9), Liddle syndrome (SCNN1A, SCNN1B, SCNN1G), Papillorenal syndrome (PAX2), Cystinosis (CTNS). See also, Rocca, C. J., et al., Optimization of Kidney- Targeted Gene Delivery for Cystinosis Using AAV, Molecular Therapy, 20 (SUPPLEMENT 1) S145, MAY 2012; Rubin J.D., et al., Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections, Hum Gene Ther. December 2019; 30(12): 1559-1571; Rubin, J. D., and Barry, M. A., Improving Molecular Therapy in the Kidney, Mol Diagn Ther. 2020 Aug; 24(4): 375-396; Davis, L., and Park, F., Gene therapy research for kidney diseases, Physiol Genomics 51: 449-461, 2019; Devuyst, O., et al., Rare inherited kidney diseases: challenges, opportunities, and perspectives, Lancet. 2014 May 24; 383(9931): 1844-1859; kidney.org / category / rare-diseases; rarediseases.org / non- member-patient / national -kidney-foundation / ; which are incorporated herein by reference in their entireties.

[0175] In certain embodiments, proteins, polypeptides, nanoparticles, and / or delivery systems including viral vectors (rAAV) and nanoparticles, comprising the exogenous targeting peptide provided herein, are useful in treatment of one or more of cardiac musclebased (heart) disorders. Such disease and / or disorders may include, without limitation, auto immune disease, cancer, a neuro-muscular disease, a sugar or glycogen storage disease, cardiomyopathy, infectious disease affecting the cardiac muscle cell. More specifically, such disease and / or disorders may include, without limitation, Huntington’s disease, a Myotonic Dystrophy (Type 1 or Type 2), Facioscapulohumeral muscular dystrophy (FSHD), Barth syndrome, MPS type III disease, Pompe disease, Charcot-Marie-Tooth disease, Friedreich’s Ataxia, dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, Myotubular myopathy, Primary merosin deficiency, Dannon disease, idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the LMNA gene. Examples of genes and proteins those associated with disease and / or disorders, e.g., Friedrichs Ataxia (e.g., frataxin), cardiomyopathy (LMNA), Charcot-Marie-Tooth disease (MFN2), Fabry (hGLA). See also, US Provisional Patent Application No. 63 / 293,680, filed December 24, 2021, International Patent Application No. PCT / US2021 / 041406, filed July 13, 2021, now Publication No. WO 2022 / 015715A1, International Patent Application No. PCT / US2022 / 076939, filed September 23, 2022, International Patent Application No. PCT / US2020 / 066167, filed December 18, 2020, now Publication No. WO 2021 / 127533A1, International Patent Application No.

[0176] PCT / US2022 / 025879, filed April 22, 2022, now Publication No. WO 2022 / 226263 Al, and International Patent Application No. PCT / US2021 / 054145, filed October 8, 2021, now Publication No. W02022 / 076803 which are all incorporated herein by reference in their entireties.

[0177] In certain embodiments, the protein is MCT8 protein (SLC16A2 gene) and other compounds for treating of Allan-Hemdon-Dudley disease and the symptoms thereof. In certain embodiments, the protein is selected from a disease associated with a transport defect such as, e.g., cystic fibrosis (a cystic fibrosis transmembrane regulator), alpha- 1 -antitrypsin (hereditary emphysemsa), FE (hereditary hemaochromatosis), tyrosinase (oculocutaneous albinism), Protein C (protein C deficiency), Complement C inhibitor (type I hereditary angioedema), alpha-D-galactosidase (Fabry disease), beta hexosaminidase (Tay-Sachs), sucrase-isomaltase (congenital sucrase-isomaltase deficiency), UDP-glucoronosyl-transferase (Crigler-Najjar type II), insulin receptor (diabetes mellitus), growth hormone receptor (laron syndrome), among others. Examples of other genes and proteins those associated with, e.g. spinal muscular atrophy (SMA, SMN1), Huntingdon’s Disease, Rett Syndrome (e.g., methyl-CpG-binding protein 2 (MeCP2); UniProtKB - P51608), Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedrichs Ataxia (e.g., frataxin), ATXN2 associated with spinocerebellar ataxia type 2 (SCA2) / ALS; TDP-43 associated with ALS, progranulin (PRGN) (associated with non- Alzheimer’s cerebral degenerations, including, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia), CDKL5 deficiency, Angelman syndrome, N-glycanase 1 deficiency, Alzheimer’s disease, Fragile X syndrome, Neimann Pick disease (including types A and B (ASMD or Acid Sphingomyelinase Deficiency), and type c (NPC), mucopolysaccharidoses (MPS), Wolman disease, among others. See, e.g., orpha.net / consor / cgi- bin / Disease_Search_List.php; rarediseases.info.nih.gov / diseases. Further illustrative genes which may be delivered via the rAAV include, without limitation, glucose-6-phosphatase, associated with glycogen storage disease or deficiency type 1A (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK), associated with PEPCK deficiency; cyclin- dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9) associated with seizures and severe neurodevelopmental impairment; galactose-1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase (PAH), associated with phenylketonuria (PKU); gene products associated with Primary Hyperoxaluria Type 1 including Hydroxy acid Oxidase 1 (G0 / HA01) and AGXT, branched chain alpha-ketoacid dehydrogenase, including BCKDH, BCKDH-E2, BAKDH- Ela, and BAKDH-Elb, associated with Maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium chain acyl CoA dehydrogenase, associated with medium chain acetyl CoA deficiency; ornithine transcarbamylase (OTC), associated with ornithine transcarbamylase deficiency; argininosuccinic acid synthetase (ASS1), associated with citrullinemia; lecithin-cholesterol acyltransferase (LCAT) deficiency; amethylmalonic acidemia (MMA); NPC1 associated with Niemann-Pick disease, type Cl); propionic academia (PA); TTR associated with Transthyretin (TTR)-related Hereditary Amyloidosis; low density lipoprotein receptor (LDLR) protein, associated with familial hypercholesterolemia (FH), LDLR variant, such as those described in WO 2015 / 164778; PCSK9; ApoE and ApoC proteins, associated with dementia; UDP- glucouronosyltransferase, associated with Crigler-Najjar disease; adenosine deaminase, associated with severe combined immunodeficiency disease; hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch-Nyan syndrome; biotimidase, associated with biotimidase deficiency; alpha-galactosidase A (a-Gal A) associated with Fabry disease); beta-galactosidase (GLB1) associated with GM1 gangliosidosis; ATP7B associated with Wilson’s Disease; beta-glucocerebrosidase, associated with Gaucher disease type 2 and 3; peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; arylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease; sphingomyelinase (SMPD1) gene associated with Nieman Pick disease type A; argininosuccsinate synthase associated with adult onset type II citrullinemia (CTLN2); carbamoyl-phosphate synthase 1 (CPS1) associated with urea cycle disorders; survival motor neuron (SMN) protein, associated with spinal muscular atrophy; ceramidase associated with Farber lipogranulomatosis; b-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff diseases; aspartylglucosaminidase associated with aspartyl-glucosaminuria; a-fucosidase associated with fucosidosis; a-mannosidase associated with alpha-mannosidosis; porphobilinogen deaminase, associated with acute intermittent porphyria (AIP); alpha- 1 antitrypsin for treatment of alpha-1 antitrypsin deficiency (emphysema); erythropoietin for treatment of anemia due to thalassemia or to renal failure; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease. In certain embodiments, the protein is encoded by a transgene sequence including hormones and growth and differentiation factors including, without limitation, insulin, glucagon, glucagon-like peptide 1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor P superfamily, including TGF P, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), lysosomal acid lipase (LIPA or LAL), neurturin, agrin, any one of the family of semaphorins / collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. Other useful transgene encode lysosomal enzymes that cause mucopolysaccharidoses (MPS), including a-L-iduronidase (MPSI), iduronate sulfatase (MPSII), heparan N-sulfatase (sulfaminidase) (MPS IIIA, Sanfdippo A), a-N-acetyl- glucosaminidase (MPS IIIB, Sanfdippo B), acetyl-CoA:a-glucosaminide acetyltransferase (MPS IIIC, Sanfdippo C), N-acetylglucosamine 6-sulfatase (MPS IIID, Sanfdippo D), galactose 6-sulfatase (MPS IVA, Morquio A), P-Galactosidase (MPS IVB, Morquio B), N- acetyl-galactosamine 4-sulfatase (MPS VI, Maroteaux-Lamy), P-Glucuronidase (MPS VII, Sly), and hyaluronidase (MPS IX).

[0178] In certain embodiments, the protein is encoded by a transgene sequence including a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding P-lactamase, P- galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.

[0179] In certain embodiments, the protein is selected from a disease associated the indicated neurological diseases, disorders, syndrome and / or conditions include, but not limited to, spinal muscular atrophy (SMA) (associated with survival motor neuron protein (SMN2) gene), SMN1, amyotrophic lateral sclerosis (ALS) (superoxide dismutase type 1 (SOD1), FUS RNA binding protein (FUS), microRNA-155, chromosome 9 open reading frame 72 (C9orf72), or ataxin-2 (ATXN2) genes), Huntington disease (associated with huntingtin (HTT) gene), hATTR polyneuropathy (associated with transthyretin (TTR) gene), Alzheimer's disease (associated with MAP-tau (MAPT) gene), Multiple System Atrophy (associated with alpha-synuclein (SNCA)), Parkinson's disease (associated with alpha-synuclein (SNCA), leucine rich repeat kinase 2 (LRRK2) genes), centronuclear myopathy (associated with dynamin 2 (DNM2) gene), Angelman syndrome (associated with ubiquitin protein ligase E3A (UBE3A) gene), epilepsy (associated with glycogen synthase 1 (GYSI) gene), Dravet Syndrome (associated with sodium voltage-gated channel alpha subunit 1 (SNC1A) gene), Leukodystrophy (associated with glial fibrillary acidic protein (GFAP) gene), prion disease (associated with prion protein (PRNP) gene), and Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) (associated with amyloid beta precursor protein (APP) gene).

[0180] An rAAV having a mutant rAAV capsid as provided herein has a vector genome which comprises nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi or miRNA), antisense oligonucleotides etc. These may be in additional to or in alternative to a protein to be delivered. Compositions and Uses

[0181] Provided herein are compositions containing at least one rAAV stock (e.g., an rAAV9 engineered stock or rAAVhu68 engineered stock, wherein engineered capsid comprises an exogenous targeting motif as described herein) and an optional carrier, excipient and / or preservative.

[0182] In one aspect, provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is formulated at about 1 x 109genome copies (GC) / mL to about 1 x 1014GC / mL. In a further embodiment, the rAAV is formulated at about 3 x 109GC / mL to about 3 x 1013GC / mL. In yet a further embodiment, the rAAV is formulated at about 1 x 109GC / mL to about 1 x 1013GC / mL. In one embodiment, the rAAV is formulated at least about 1 x 1011GC / mL.

[0183] Provided herein, also, are compositions containing at least one therapeutic protein, polypeptide, nanoparticles and / or delivery system comprising the targeting motif as provided herein, and an optional carrier, excipient and / or preservative.

[0184] Provided herein, also, are methods of use of compositions as described herein. In certain embodiments, a method for targeted therapy to brain cells comprising administering to a patient in need thereof a stock of the rAAV (i.e., comprising modified AAV capsid) as described herein, wherein a therapeutic is targeted for delivery to cells in brain and / or spinal cord. In certain embodiments, a method for targeted therapy to heart cells comprising administering to a patient in need thereof a stock of the rAAV (i.e., comprising modified AAV capsid) as described herein, wherein a therapeutic is targeted for delivery to cells in heart. In certain embodiments, a method for targeted therapy to kidney cells comprising administering to a patient in need thereof a stock of the rAAV (i.e., comprising modified AAV capsid) as described herein, wherein a therapeutic is targeted for delivery to cells in kidney.

[0185] Additionally, provided herein is a method of delivering of a transgene to one or more target cells of central nervous system (CNS) of a subject comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising engineered capsid comprising exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and rAAV further comprising a vector genome comprising the transgene operably linked to regulatory sequences that direct expression of the transgene in cell of CNS (e.g., brain cell). In certain embodiments, provided herein is a method of delivering of a transgene to one or more target cells of central nervous system (CNS) of a subject comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising engineered capsid comprising exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and rAAV further comprising a vector genome comprising the transgene operably linked to regulatory sequences that direct expression of the transgene in cell of CNS (e.g., brain cell). Provided herein, also, is a method of delivering of a transgene to one or more target cells of central nervous system (CNS) of a subject comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising engineered capsid comprising exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n- mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”- X”’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and rAAV further comprising a vector genome comprising the transgene operably linked to regulatory sequences that direct expression of the transgene in target cell of CNS. In certain embodiments, the target cells of the CNS are brain cells, brain endothelial cells, parenchymal cells, cells of the choroid plexus, ependymal cells, astrocytes, and / or and neurons, optionally neurons of the cortex, hippocampus, and / or striatum. In certain embodiments, the transgene encodes a secreted gene product. In certain embodiments, the AAV vector is delivered intrathecally, optionally via intra-ci sterna magna (ICM) injection. In certain embodiments, the AAV vector is delivered via intracerebroventricular injection. In certain embodiments, the AAV vector is delivered via intraparenchymal administration. In certain embodiments, the AAV vector is delivered via Ommaya Reservoir delivery system.

[0186] In certain embodiments, the target cells are heart cells. In certain embodiments, the target cells are kidney cells. In certain embodiments, the rAAV is delivered via renal venogram. In certain embodiments, the rAAV is delivered via renal arterial injection. In certain embodiments, the rAAV is delivered via ureteral injection. In certain embodiments, the rAAV is delivered via anterograde administration into renal artery. In certain embodiments, the rAAV is delivered via retrograde infusion into ureter. In certain embodiments, the rAAV is delivered via intraparenchymal injection (i.e., direct injection into a kidney). In certain embodiments, the rAAV is delivered using pressure controlling device.

[0187] Provided herein are also uses of an rAAV having a engineered capsid with one or more of exogenous targeting peptides, to target brain cells at higher levels of transduction than achieved using an AAV9 vector, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the rAAV achieves higher levels of transduction, for example relative to AAV9, of neurons in the cortex, hippocampus, and / or striatum. In certain embodiments, the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), or TAMRDPR (SEQ ID NO: 105). In certain embodiments, the rAAV having an engineered capsid with one or more of exogenous targeting peptides, target brain cells at higher levels of transduction than achieved using an AAV9 vector. In certain embodiments, the rAAV having an engineered capsid with one or more of exogenous targeting peptide, target heart cells at higher levels of transduction than achieved using an AAV9 vector. In certain embodiments, the rAAV having an engineered capsid with one core one or more of exogenous targeting peptide, target kidney cells at higher levels of transduction than achieved using an AAV9 vector.

[0188] In certain embodiments, a composition may contain at least a second, different rAAV stock. This second vector stock may vary from the first by having a different AAV capsid and / or a different vector genome. In certain embodiments, a composition as described herein may contain a different vector expressing an expression cassette as described herein, or another active component (e g., an antibody construct, another biologic, and / or a small molecule drug).

[0189] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0190] In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Suitably, the final formulation is adjusted to a physiologically acceptable pH, e.g., the pH may be in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other routes of delivery. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

[0191] In another embodiment, the composition includes a carrier, diluent, excipient and / or adjuvant. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

[0192] In certain embodiments, the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer. In one embodiment, the buffer is phosphate-buffered saline (PBS). In one embodiment, the formulation buffer PBS with total salt concentration of 200 mM, 0.001% (w / v) pluronic F68 (Final Formulation Buffer, FFB).

[0193] In certain embodiments, the compositions described herein are used in preparing medicaments for treating central nervous system disorders and diseases. Optionally, the compositions described herein are administered in the absence of an additional extrinsic pharmacological or chemical agent, or other physical disruption of the blood brain barrier.

[0194] Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and / or admixed with suitable excipients designed for delivery to the subject via injection, or for delivery by another route and / or device.

[0195] In certain embodiments, the composition comprises a viral vector (i.e., rAAV vector). The vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. In certain embodiments, the vectors are formulated for delivery via intranasal delivery devices. In certain embodiments, vectors are formulated for aerosol delivery devices, e.g., via a nebulizer or through other suitable devices. In certain embodiment, vectors are formulated for intrathecal delivery. In some embodiments, intrathecal delivery encompasses an injection into the spinal canal, e.g., the subarachnoid space. In some embodiments, other delivery route may be selected, e g., intracranial, intranasal, intracisternal, intracerebrospinal fluid delivery, among other suitable direct or systemic routes, i.e., Ommaya reservoir. In certain embodiments, vectors are formulated for intravenous delivery. Other conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e g., lung), oral inhalation, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In one embodiment, the vector is administered intranasally using intranasal mucosal atomization device (LMA® MAD Nasal™- MAD 110). In another embodiment the vector is administered intrapul monary in nebulized form using Vibrating Mesh Nebulizer (Aerogen® Solo) or MADgic™ Laryngeal Mucosal Atomizer. Routes of administration may be combined, if desired. Routes of administration and utilization of which for delivering rAAV vectors are also described in the following published US Patent Applications, the contents of each of which is incorporated herein by reference in its entirety: US 2018 / 0155412A1, US 2018 / 0243416 Al, US 2014 / 0031418 Al, and US

[0196] 2019 / 0216841A1.

[0197] As used herein, the term “dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration. Dosages of the recombinant vector (e.g., rAAV) will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of the recombinant vector (e.g., rAAV) is generally in the range of from about 25 to about 1000 microliters to about 5 mb of aqueous suspending liquid containing doses of from about 109to 4x1014GC of AAV vector. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in recombinant vectors, preferably AAV vectors comprising transgene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.

[0198] In certain embodiments, the compositions can be formulated in dosage units to contain an amount of rAAV that is in the range of about 1 x 109GC per gram of brain mass to about 1 x 1013genome copies (GC) per gram (g) of brain mass, including all integers or fractional amounts within the range and the endpoints. In another embodiment, the dosage is 1 x 1010GC per gram of brain mass to about 1 x 1013GC per gram of brain mass. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 109GC / g, about 1.5 x 109GC / g, about 2.0 x 109GC / g, about 2.5 x 109GC / g, about 3.0 x 109GC / g, about 3.5 x 109GC / g, about 4.0 x 109GC / g, about 4.5 x 109GC / g, about 5.0 x 109GC / g, about 5.5 x 109GC / g, about 6.0 x 109GC / g, about 6.5 x 109GC / g, about 7.0 x 109GC / g, about 7.5 x 109GC / g, about 8.0 x 109GC / g, about 8.5 x 109GC / g, about 9.0 x 109GC / g, about 9.5 x 109GC / g, about 1.0 x 1010GC / g, about 1.5 x 1010GC / g, about 2.0 x 1010GC / g, about 2.5 x 1010GC / g, about 3.0 x 1010GC / g, about 3.5 x 1010 GC / g, about 4.0 x IO10GC / g, about 4.5 x IO10GC / g, about 5.0 x IO10GC / g, about 5.5 x

[0199] 1010GC / g, about 6.0 x IO10GC / g, about 6.5 x IO10GC / g, about 7.0 x IO10GC / g, about 7.5 x IO10GC / g, about 8.0 x 1010GC / g, about 8.5 x 1010GC / g, about 9.0 x IO10GC / g, about

[0200] 9.5 x 1010GC / g, about 1.0 x 1011GC / g, about 1.5 x 1011GC / g, about 2.0 x 1011GC / g, about 2.5 x 1011GC / g, about 3.0 x 1011GC / g, about 3.5 x 1011GC / g, about 4.0 x 1011GC / g, about 4.5 x 1011GC / g, about 5.0 x 1011GC / g, about 5.5 x 1011GC / g, about 6.0 x

[0201] 1011GC / g, about 6.5 x 1011GC / g, about 7.0 x 1011GC / g, about 7.5 x 1011GC / g, about 8.0 x 1011GC / g, about 8.5 x 1011GC / g, about 9.0 x 1011GC / g, about 9.5 x 1011GC / g, about 1.0 x 1012GC / g, about 1.5 x 1012GC / g, about 2.0 x 1012GC / g, about 2.5 x 1012GC / g, about 3.0 x 1012GC / g, about 3.5 x 1012GC / g, about 4.0 x 1012GC / g, about 4.5 x 1012GC / g, about 5.0 x 1012GC / g, about 5.5 x 1012GC / g, about 6.0 x 1012GC / g, about 6.5 x IO’2GC / g, about 7.0 x 1012GC / g, about 7.5 x 1012GC / g, about 8.0 x IO12GC / g, about 8.5 x 1012GC / g, about 9.0 x 1012GC / g, about 9.5 x 1012GC / g, about 1.0 x 1013GC / g, about

[0202] 1.5 x 1013GC / g, about 2.0 x 1013GC / g, about 2.5 x 1013GC / g, about 3.0 x 1013GC / g, about 3.5 x 1013GC / g, about 4.0 x 1013GC / g, about 4.5 x 1013GC / g, about 5.0 x 1013GC / g, about 5.5 x 1013GC / g, about 6.0 x 1013GC / g, about 6.5 x 1013GC / g, about 7.0 x 1013GC / g, about 7.5 x 1013GC / g, about 8.0 x 1013GC / g, about 8.5 x 1013GC / g, about 9.0 x 1013GC / g, about 9.5 x 1013GC / g, or about 1.0 x 1014GC / g brain mass. In certain embodiments, the dose may be in the range of about 1 x 109GC / g brain mass to about 1 x

[0203] 1012GC / g brain mass. In certain embodiments, the dose may be in the range of about 3 x 1010GC / g brain mass to about 3 x 1011GC / g brain mass. In certain embodiments, the dose may be in the range of about 5 x 1010GC / g brain mass to about 1.85 x 1011GC / g brain mass.

[0204] In certain embodiments, the replication-defective virus (e.g., rAAV) compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109GC to about 1.0 x 1016GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 1012GC to 1.0 x 1014GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xlO9, or 9xl09GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10, 3xlO10, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xlO10, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO11, 2xlOn, 3xlOn, 4xlOu, SxlO11, 6xlOn, 7xlOu, 8xlOn, or 9xlOnGC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO14, 2xl014, 3xl014, 4x1014, 5xl014, 6xl014, 7xl014, 8xl014, or 9xl014GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6x1015, 7x1015, 8x1015, or 9x1015GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from IxlO10to about IxlO12GC per dose including all integers or fractional amounts within the range.

[0205] In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 1.0 x 109GC to about 1.0 x 1014GC, including all integers or fractional amounts within the range. In certain embodiments, the rAAV compositions is fonnulated in dosage units to contain about I x lO12GC / kg. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 1 x 1013GC / kg. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 2.5 x 1013GC / kg. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 5 x 1013GC / kg.

[0206] In one embodiment, for human application the dose can range from 109to about 7xl013GC per dose including all integers or fractional amounts within the range.

[0207] These above doses may be administered in a variety of volumes of carrier, excipient or buffer formulation, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of carrier, excipient or buffer is at least about 25 pL. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 75 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL. In yet another embodiment, the volume is about 200 pL. In another embodiment, the volume is about 225 pL. In yet another embodiment, the volume is about 250 pL. In yet another embodiment, the volume is about 275 pL. In yet another embodiment, the volume is about 300 pL. In yet another embodiment, the volume is about 325 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 375 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 pL. In another embodiment, the volume is between about 700 and 1000 pL.

[0208] In one embodiment, the viral constructs may be delivered in doses of from at least about least IxlO9GCs to about 1 x 1015, or about 1 x I011to 5 x 1013GC. Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in the art. For example, volumes of about 1 pL to 150 mb may be selected, with the higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mb to about 10 mL, for older infants, about 0.5 mb to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected. For pre-teens and teens, volumes up to about 50 mL may be selected. Other suitable volumes and dosages may be determined. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and / or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, Ommaya reservoir device, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery.

[0209] The composition, the suspension or the pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes. In one embodiment, the pharmaceutical composition comprises a formulation buffer suitable for delivery via intracerebroventricular (ICV), intrathecal (IT), intracisternal, intracisternal magna (IDM), administration by direct injection into the substantia nigra and / or ventral tegmental area, or intravenous (IV) routes of administration. In certain embodiments, the rAAV or the pharmaceutical composition comprises a formulation buffer suitable for intravenous, intraparenchymal (dentate nucleus) and / or intrathecal administration to a patient in the need thereof. In certain embodiments, the rAAV or the pharmaceutical composition comprises a formulation buffer suitable for infusion into the renal artery, via catheter or injection, retrograde infusion into the renal vein, retrograde infusion into the ureter, or subcapsular injection into the parenchyma of the kidney.

[0210] As used herein, the terms “intrathecal delivery” or “intrathecal administration” refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular, intracerebroventricular (ICV) suboccipital / intracisternal, and / or Cl-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cisterna magna (intracisternal magna; ICM). Intracisternal delivery may increase vector diffusion and / or reduce toxicity and inflammation caused by the administration. See, e.g., Christian Hinderer et al, Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna, Mol Ther Methods Clin Dev. 2014; 1 : 14051. Published online 2014 Dec 10. doi: 10.1038 / mtm.2014.51.

[0211] As used herein, the terms “intraci sternal delivery” or “intraci sternal administration” refer to a route of administration for drugs directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cisterna magna or via permanently positioned tube.

[0212] As used herein, the term “intraparenchymal (dentate nucleus)” or IDN refers to a route of administration of a composition directly into dentate nuclei. IDN allows for targeting of dentate nuclei and / or cerebellum. In certain embodiments, the IDN administration is performed using ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and ventricular cannula, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0213] In one embodiment, a frozen composition is provided which contains an rAAV in a buffer solution as described herein, in frozen form. Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers or preservatives is present in this composition. Suitably, for use, a composition is thawed and titrated to the desired dose with a suitable diluent, e.g., sterile saline or a buffered saline.

[0214] In certain embodiments, provided herein is a composition comprising one or more exogenous brain cell-targeting peptide(s) comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n- mer comprises exogenous targeting motif X’-G-X”-X”’ DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, together with one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base. In certain embodiments, provided herein is a composition comprising one or more exogenous brain cell-targeting peptide(s) comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, together with one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base. In certain embodiments, provided herein is a composition comprising one or more exogenous brain cell-targeting peptide(s) comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n- mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, together with one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base. Further provided are compositions comprising nucleic acid sequences encoding same.

[0215] In certain embodiments, a composition comprising a fusion polypeptide or protein, or a nucleic acid sequence encoding the fusion polypeptide or protein, or a nanoparticle containing same are provided. The composition may further comprise one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

[0216] In certain embodiments, a nucleic acid sequence encoding the fusion polypeptide protein is encapsulated in a lipid nanoparticle (LNP). As used herein, the phrase “lipid nanoparticle” or “nanoparticle” refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more nucleic acid sequences to one or more target cells (e.g., cell in CNS, brain cell, heart cell, kidney cell). Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactidepolyglycolide copolymers, poly caprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine. In one embodiment, the transfer vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid sequence encapsulated therein to a target cell. Useful lipid nanoparticles for nucleic acid sequence comprise a cationic lipid to encapsulate and / or enhance the delivery of such nucleic acid sequence into the target cell that will act as a depot for protein production. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. The contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG- modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available. See, e g., WO2014 / 089486, US 2018 / 0353616A1 , and US 8,853,377B2, which are incorporated by reference. In certain embodiments, LNP formulation is performed using routine procedures comprising cholesterol, ionizable lipid, helper lipid, PEG-lipid and polymer forming a lipid bilayer around encapsulated nucleic acid sequence (Kowalski et al., 2019, Mol. Ther. 27(4):710-728). In some embodiments, LNP comprises a cationic lipids (i.e. N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), or l,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) with helper lipid DOPE. In some embodiments, LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine-based ionizable lipids (cKK- E12). In some embodiments, polymer comprises a polyethyleneimine (PEI), or a poly(P- amino)esters (PBAEs). See, e.g., WO2014 / 089486, US 2018 / 0353616A1, US2013 / 0037977A1, W02015 / 074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”-X” ’DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, i.e., decorated surface with targeting peptide. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, i.e., decorated surface with targeting peptide. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102), YGVKDPR (SEQ ID NO: 103), TGMIDPR (SEQ ID NO: 104), TAMRDPR (SEQ ID NO: 105), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n- mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, i.e., decorated surface with targeting peptide. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45), and / or combination thereof.

[0217] In certain embodiments, provided herein is a composition, e.g., an rAAV having an engineered capsid with at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer comprises exogenous targeting peptide motif X’-G-X”- X’ ”DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, is useful for delivering a therapeutic to a patient in need thereof. In certain embodiments, provided herein is a composition, e.g., an rAAV having a modified (i.e., engineered) capsid with at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is QGVKDPR (SEQ ID NO: 39), NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), TGMRDPN (SEQ ID NO: 57), TVMRDPR (SEQ ID NO: 54), RGVQDPR (SEQ ID NO: 44), DGMRDPR (SEQ ID NO: 43), or QGVRDPR (SEQ ID NO: 45); and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, is useful for delivering a therapeutic to a patient in need thereof. In certain embodiments, provided herein is a composition, e.g., an rAAV having an engineered capsid with at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer TGVLDPR (SEQ ID NO: 98), YGGPDPR (SEQ ID NO: 99), QGIQDPR (SEQ ID NO: 100), TDMRDPR (SEQ ID NO: 101), TGMRDPK (SEQ ID NO: 102). YGVKDPR (SEQ ID NO: 103). TGMIDPR (SEQ ID NO: 104). or TAMRDPR (SEQ ID NO: 105): and (hi) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, is useful for delivering a therapeutic to a patient in need thereof.

[0218] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one QGVKDPR (SEQ ID NO: 39) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0219] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one NGLKDPR (SEQ ID NO: 33) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0220] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TGVKDPR (SEQ ID NO: 42) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0221] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TGMRDPN (SEQ ID NO: 57) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0222] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TVMRDPR (SEQ ID NO: 54) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0223] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one RGVQDPR (SEQ ID NO: 44) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0224] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one DGMRDPR (SEQ ID NO: 43) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0225] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one QGVRDPR (SEQ ID NO: 45) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0226] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TGVLDPR (SEQ ID NO: 98) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0227] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one YGGPDPR (SEQ ID NO: 99) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0228] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one QGIQDPR (SEQ ID NO: 100) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0229] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TDMRDPR (SEQ ID NO: 101) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0230] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TGMRDPK (SEQ ID NO: 102) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0231] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one YGVKDPR (SEQ ID NO: 103) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0232] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TGMIDPR (SEQ ID NO: 104) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0233] In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one TAMRDPR (SEQ ID NO: 105) peptide and optional linker sequences, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in CNS (brain and / or spinal cord).

[0234] In certain embodiments, a composition comprising an rAAV having a modified capsid comprising exogenous targeting peptide as described herein, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in kidney.

[0235] In certain embodiments, a composition comprising an rAAV having a modified capsid comprising exogenous targeting peptide as described herein, is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery in heart.

[0236] In certain embodiments, the methods and compositions may be useful for treatment of autosomal dominant form of long-QT syndrome caused by a loss-of-function and partial dominant negative mutations in KCNQ1 gene (i.e., amenable to gene replacement or knockdown / replace approach). The autosomal dominant form of long-QT syndrome is associated with syncope and sudden cardiac death usually occurring during exercise or emotional stress, and many patients remain at-risk despite standard of care (beta blockers, cardiac sympathetic denervation) and require Implantable Cardioverter Defibrillator (ICD). See also, Huang H., et al., Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations, Sci. Adv. 2018, 4: 1-12, epub March 7, 2018, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of hypertrophic cardiomyopathy. In certain embodiments, the methods and compositions may be useful for treatment of hypertrophic cardiomyopathy caused by loss-of-function mutations in the MYBPC3 gene (i.e., amenable to gene therapy). See also, Mearini G., et al., Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice, Nature Communication, 2014, 5:5515, epub December 2, 2014, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of long WT syndrome type 2 caused by a loss-of-function mutation in hERG (Kvl l. l; also, Kvl l.l voltage-gated potassium channel) gene. See also, Curran ME., et al., A Molecular Basis for Cardiac Arrhythmia: HERG Mutations Cause Long QT Syndrome, Cell, Voi. 80, 795-803, March 10, 1995, and Hylten-Cavallius, L., et al., Patients With Long-QT Syndrome Caused by Impaired hERG-Encoded Kvl l.l Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated With Reactive Hypoglycemia, Circulation, 2017;135: 1705-1719, which are incorporated herein by reference in their entirety. In certain embodiments, the methods and compositions may be useful for treatment of transthyretin amyloid cardiomyopathy (ATTR-CM). In certain embodiments, the methods and compositions may be used in treatment of ATTR-CM caused by mutations in the transthyretin (TTR) gene (i.e., amenable to gene therapy). See also, Yamamoto H, Yokochi T. Transthyretin cardiac amyloidosis: an update on diagnosis and treatment. ESC Heart Fail. 2019 De c;6(6): 1128-1139; and Jain A, Zahra F. Transthyretin Amyloid Cardiomyopathy (ATTR-CM) [Updated 2023 Apr 27], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 Jan, ncbi.nlm.nih.gov / books / NBK574531 / , which are incorporated herein by reference in their entirety. In certain embodiments, the methods and compositions may be useful for treatment of LMNA cardiomyopathy or a disease caused by loss-of-function mutation in the LMNA gene. See also, Kang, S., et al., Laminopathies; Mutations on single gene and various human genetic diseases, BMB Rep. 2018; 51(7): 327-337, and US Provisional Patent application No. 63 / 293,680, filed December 24, 2021, International Patent Application No. PCT / US2022 / 082383, filed December 24, 2022, now Publication No. WO 2023 / 122803 Al, published June 29, 2023, which are incorporated herein by reference in their entirety. In certain embodiments, the methods and compositions may be useful for treatment of heart failure, ischemia-reperfusion injury, myocardial infarction, ventricular remodeling, or a disease associated with extracellular superoxide dismutase 3 (SOD3 or EcSOD). See also, US Patent Application Publication No. US 2013 / 0136729A1, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of myocardial infarction, reduced ejection fraction of the heart or a disease associated with myc transcription factor, cyclin T 1 and cyclin-dependent kinase 9 (CDK9). See also, International Patent Application Publication No. W02020 / 165603A1, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of heart failure, or heart tissue damage, or degeneration, or a disease associated with cyclin A2 protein. See also, International Patent Application Publication No. W02020 / 051296A1, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of dilated cardiomyopathy (DCM), a heart failure, a cardiac fibrosis, a heart inflammation, an ischemic heart disease, a myocardial infarction, an ischemic / reperfusion (I / R) related injuries, a transverse aortic constriction, or a disease associated with YY1 or BMP7 protein. See also, International Patent Application Publication No. W02021 / 021021A1, which is incorporated herein by reference in its entirety. In certain embodiments, the methods and compositions may be useful for treatment of dilated cardiomyopathy or a disease associated with cardiac Apoptosis Repressor with Caspase Recruitment Domain (cARC). See also, International Patent Application Publication No. W02021 / 016126A1, which is incorporated herein by reference in its entirety. Symptoms of cardiomyopathy or a disease associated with a mutation in a LMNA gene, KCNQ1 gene, MYBPC3 gene, TAZ gene, or hERG gene include atrioventricular (AV) conduction block, atrial fibrillation, arrhythmia including atrial arrhythmia such as atrial flutter and atrial tachycardia, and ventricular arrhythmias including sustained ventricular tachycardias, and ventricular fibrillation (VF) and / or heart failure. In certain embodiments, the methods and compositions described herein may be useful to ameliorate one or more symptoms of cardiomyopathy including increased average life span, and / or reduction in progression towards heart failure.

[0237] In certain embodiments, provided herein is a method for targeted therapy to Central Nervous System (CNS) comprising administering to a patient or subject in need thereof a stock of the rAAV as described herein. In certain embodiments, provided herein is a method for treating of one or more of cognitive disorders, neurodegenerative disorders, and / or a disease of the central nervous system by administering to a patient or a subject in need thereof a stock of the rAAV as described herein, wherein the encoded gene product is a protein, or an antibody.

[0238] In certain embodiments, a rAAV having a modified capsid as described herein may be delivered in a co-therapeutic regimen which further comprises one or more other active components. In certain embodiments, the regimen may involve co-administration of an immunomodulatory component. In certain embodiments, provided herein is a rAAV having a modified capsid as described herein for use in delivery with immunosuppressive co-therapeutic regimen, and methods thereof. Without wishing to be bound by theory, immune suppression co-therapy does one or more of the following: induces anergy or immunologic tolerance to the rAAV and / or Transgene; blocks an immune response to optimize efficacy; minimize de novo immune response against transgene; minimize impact of pre-existing immune response to transgene; minimize impact of pre-existing immune response to AAV; prevent immune medicated toxicity; minimize destruction of TG expressing cells; reduce axonopathy / DRG neurotoxicity in NHPs.

[0239] In certain embodiments, provided herein is rAAV having modified capsid as described herein for use in a method further comprising a combination therapy, such as transient co-treatment with an immunosuppressant before and / or during treatment with rAAV. Optionally, immunosuppressive co-therapy may be used as a precautionary measure without prior assessment of neutralizing antibodies to the AAV vector capsid and / or other components of the formulation. Prior immunosuppression therapy may be desirable to prevent potential adverse immune reaction to the transgene product (gene of interest) i.e., where the transgene product may be seen as “foreign.”

[0240] Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids or corticosteroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mycophenolate mofetil , methotrexate, leflunomide (Arava), cyclophosphamide, chlorambucil (Leukeran), a chloroquine (e.g., hydroxychloroquine), quinine sulfate, mefloquine, a combination of atovaquone and proguanil, sulfasalazine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporinabatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz), cyclosporine, tacrolimus, sirolimus, IFN-P, IFN-y, an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent, and combinations of these drugs .

[0241] In certain embodiments, the immunosuppressive therapy may be started 0, 1, 2, 7, or more days prior to the gene therapy administration. Such therapy may involve co-administration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected. In one embodiment, the two or more drugs may be, e.g., one or more corticosteroids (e.g., a prednelisone or prednisone) and optionally, MMF and / or a calcinuerin inhibitor (e.g., tacrolimus or sirolimus (i.e., rapamycin)). In one embodiment, the two or more drugs are micophenolate mofetil (MMF) and / or sirolimus. In another embodiment, the two or more drugs may be, e.g., methylprednisolone, prednisone, tacrolimus, and / or sirolimus. In certain embodiments, the drugs are MMF and tacrolimus for 0 to 15 days pre-vector delivery and maintaining for about 8 weeks with MMF and / or throughout follow-up appointments with tacrolimus. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. In certain embodiments, patients are dosed initially with an IV steroid (e.g., methylprednisolone) to load the dose, followed by with an oral steroid (e.g., prednisolone) that is gradually tapered down so that the patient is off steroids by week 12. The corticosteroid treatment is supplemented by tacrolimus (for 24 weeks) and / or sirolimus (for 12 weeks), and can be further supplemented with MMF. When using both tacrolimus and sirolimus, the dose of each should be a low dose adjusted to maintain a blood trough level of about 4 ng / mL to about 8 ng / ml, or a total of about 8 ng / mL to about 16 ng / mL. In certain embodiments, when only one of these agents is used, the total dose for tacrolimus and / or sirolimus may be in the range of about 16 ng / mL to about 24 ng / mL. If only one of the agents is used, the label dose (higher dose) should be employed; e.g., tacrolimus at 0.15-0.20 mg / kg / day given as two divided doses every 12 hours; and sirolimus at 1 mg / m2 / day; the loading dose should be 3 mg / m2. If MMF is added to the regimen, the dose for tacrolimus and / or sirolimus can be maintained since the mechanisms of action differ. These and other therapies may be started at about day - 14 to day -1 (e.g., day -2, day 0, etc.), and continue to about to up to about 1 week (7 days), or up to about 60 days, or up to about 12 weeks, or up to about 16 weeks, or up to about 24 weeks, or up to about 48 weeks, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0242] In certain embodiments, patients will receive immune suppression (IS) as follows: corticosteroids: methylprednisolone 10 mg / kg IV once on Day 1 predose and oral prednisone starting at 0.5 mg / kg / day on Day 2 with gradual tapering and discontinuation by Week 12; Tacrolimus: 1 mg BID by mouth Day 2 to Week 24 with tapering over 8 weeks between Week 24 and 32; Sirolimus: (a loading dose on Day -2 and then sirolimus 0.5 mg / m2 / day divided in BID dosing until Week 48. In certain embodiments, IS therapy is discontinued at Week 48 post dosing with the rAAV.

[0243] In certain embodiments, the method further comprises administering subject with intramuscular steroid or corticosteroid prior to and / or post administration of rAAV. In certain embodiments, the method further comprises administering subject with oral steroid or corticosteroid prior to and / or post administration with rAAV.

[0244] In certain embodiments, the immunosuppressive therapy regimen is as follows: Corticosteroids:

[0245] In the morning of vector administration (Day 1 predose), patients receive methylprednisolone lOmg / kg IV (maximum of 500 mg) over at least 30 minutes. The methylprednisolone is administered before the lumbar puncture and intrathecal (IC) injection of rAAV. Premedication with acetaminophen and an antihistamine is optional.

[0246] On Day 2, oral prednisone is started with the goal to discontinue prednisone by Week 12. The dose of prednisone is as follows: Day 2 to the end of Week 2: 0.5 mg / kg / day. Week 3 and 4: 0.35 mg / kg / day. Week 5-8: 0.2 mg / kg / day. Week 9-12: 0.1 mg / kg.

[0247] Prednisone is discontinued after Week 12. The exact dose of prednisone can be adjusted to the next higher clinically practical dose. Sirolimus: 2 days prior to vector administration (Day -2): a loading dose of sirolimus 1 mg / m2 every 4 hours x 3 doses is administered. From Day -1 : sirolimus 0.5 mg / m2 / day divided in twice a day dosing with target blood level of 4-8 ng / ml. Sirolimus is discontinued after the Week 48 visit.

[0248] Tacrolimus: Tacrolimus is started on Day 2 (the day following rAAV) at a dose of 1 mg twice daily and adjusted to achieve a blood level 4-8 ng / mL for 24 Weeks. Starting at Week 24 visit, tacrolimus is tapered off over 8 weeks. At week 24 the dose is decreased by approximately 50%. At Week 28 the dose is further decreased by approximately 50%. Tacrolimus is discontinued at Week 32.

[0249] In one embodiment, the method further comprises administering to a subject anti-AAV neutralizing antibodies (NAb) to reduce peripheral transduction, and mitigate the potential risk of transgene-induced toxicity. In certain embodiments, the method further comprises detect the presence of systemic AAV NAb prior to treating with anti-AAV NAb, wherein patients with levels of anti-AAV NAb in excess of a predetermined level against the rAAV capsid (or a sero-crossreactive capsid) do not require pretreatment. Such levels may be, e.g., in excess of about 1 : 10, about 1 :20, about 1 :50, about 1 : 100, about 1 :250, or higher or lower levels. In certain embodiments, the method further comprises intravenously administering human anti-AAV polyclonal antibodies (e.g., plasma-derived, pooled human immunoglobulin (IVIG)), an anti-AAV monoclonal antibody, or a cocktail of anti-AAV antibodies, to a patient about 1 day to about 2 hours before treatment with a rAAV as described herein.

[0250] In certain embodiments, a combination regimen is provided for preventing off- target delivery rAAV, the regimen comprising (a) pretreating the patient by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid in a recombinant AAV vector, and (b) administering to the central nervous system (CNS) rAAV as described herein (e.g., rAAV). See also, US Provisional Patent Application No. 63 / 328,227, filed April 6, 2022, and International Patent Application No. PCT / US2023 / 065422, filed April 6, 2023, now Publication No. WO2023 / 196892A1 which are incorporated herein by reference in their entirety. As used herein, the term “NAb titer” a measurement of how much neutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno- Associated Viruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, which is incorporated by reference herein.

[0251] In certain embodiments, a combination regimen is provided including an anti-IgG enzymes, which have been described as being useful for depleting anti-AAV antibodies (and thus may permit administration to patients testing above a threshold level of antibody for the selected AAV capsid), and / or delivery of anti-FcRN antibodies and / or one or more of a) a steroid or combination of steroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and / or (f) gamma interferon. anti-FcRN antibodies include, e.g., rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all form HanAll BioPhrma Co. Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx S.E.), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd). In certain embodiments, a combinations of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti- FcRn ligand (i.e., a peptide or protein construct binding human FcRn so as to inhibit IgG binding).

[0252] In one embodiment, a combination regimen for treating a patient with MPSIIIB is provided, wherein the regiment includes administering a vector describe herein in combination with a ligand which inhibits binding of human FcRn and pre-existing patient neutralizing antibodies (e.g., IgG). In certain embodiments, the patient may be naive to any therapeutic treatment with a vector and may have pre-existing immunity due to prior infections with a wild-type virus. In other embodiments, the patient may have neutralizing antibodies as a result of a prior treatment or vaccination. In certain embodiments, the patient may have neutralizing antibodies 1 : 1 to 1 :20, or in excess of 1 :2, in excess of 1 :5, in excess of 1 : 10, in excess of 1 :20, in excess of 1 :50, in excess of 1 : 100, in excess of 1 :200, in excess of 1 OO or higher. In certain embodiments, a patient has neutralizing antibodies in the range of 1 : 1 to 1 :200, or 1 :5 to 1 :100, or 1 :2 to 1 : 20, or 1 :5 to 1 : 50, or 1 :5 to 1 :20. In certain embodiments, a patient receives a single anti-FcRn ligand (e.g., anti- FcRn antibody) as the sole agent to modulate FcRn-IgG binding and to permit effective vector delivery. In other embodiments, a patient may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-regulator (e.g., interferon gamma), an IgG enzyme, or another suitable component). Such combinations may be particularly desirable for patients having particularly high neutralizing antibody levels (e.g., in excess of 1 :200).

[0253] In certain embodiments, an anti-FcRn ligand(s) (e.g., antibodies) is administered to a patient having neutralizing antibodies prior to and, optionally, concurrently with a selected viral vector. In certain embodiments, continued expression of an anti-FcRn ligand post administration of the gene therapy vector may desired on a short-term (transient basis), e.g., until such time as the viral vector clears from the patient. In certain embodiments, persistent expression of an anti-FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, including, e.g., in the viral vector expressing the therapeutic transgene. However, this embodiment is not desirable where the therapeutic gene being delivered is an antibody or antibody construct or another construct comprising an IgG chain. In such embodiments, where an antibody construct having an IgG chain is being delivered via a viral vector to a patient having pre-existing immunity, the anti-FcRn ligand is delivered or dosed transiently so that the amount of anti- FcRn ligand in the circulation is cleared from the sera before effective levels of vector- mediated transgene product are expressed.

[0254] In certain embodiments, the FcRn ligand is delivered one to seven days prior to administration of the vector (e.g., rAAV). In certain embodiments, the FcRn ligand is delivered daily. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day as the vector is administered. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least one day to four weeks post-rAAV administration. In certain embodiments, the ligand is delivered for four weeks to six months post-rAAV administration. In certain embodiments, the ligand is dosed via a different route of administration than the rAAV. In certain embodiments, the ligand is dosed orally, intravenously, or intraperitoneally. See also, International Patent Application No. PCT / US2021 / 037575, filed June 16, 2021, and now published WO 2021 / 257668 Al, which is incorporated herein by reference in its entirety.

[0255] In certain embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises administering at least one or more of immunosuppressive agents comprising corticosteroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent. In certain embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises administering one or more of: (a) a corticosteroid or combination of corticosteroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and / or (f) gamma interferon. In certain embodiments, the one or more of immunosuppressive agents is administered (i) prior to rAAV administration, (ii) post-rAAV administration, or (iii) prior to and post-rAAV administration. In certain embodiments, the rAAV is for use in a co- therapeutic regimen which further comprises co-administering a ligand which specifically prevents binding between human neonatal Fc receptor (FcRn) and the neutralizing antibodies without interfering with albumin binding to FcRn. In certain embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises pretreating the patient or the subject by systemically administering a composition comprising anti -AAV capsid neutralizing antibodies directed against an AAV capsid of the recombinant AAV vector.

[0256] It should be understood that the compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0257] Kit In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1 : 1 to a 1 :5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device are included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol / PBS.

[0258] It should be understood that the compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0259] Apparatus and Method for Delivery of a Pharmaceutical Composition

[0260] In one aspect, the vectors, rAAV or compositions thereof provided herein may be administered intrathecally via the method and / or the device provided in this section and described in WO 2017 / 136500 and WO 2018 / 160582, which are incorporated by reference herein. Alternatively, other devices and methods may be selected. In certain embodiments, the method comprises the steps of CT-guided sub-occipital injection via spinal needle into the cisterna magna of a patient. As used herein, the term Computed Tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by computer from a series of plane cross-sectional images made along an axis. In certain embodiments, the apparatus is described in US Patent Publication No. 2018-0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the vectors, rAAV or compositions thereof provided herein may be administered using Ommay a Reservoir. It should be understood that the compositions in the device described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0261] An “immunoglobulin molecule” is a protein containing the immunologically-active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The terms “antibody” and “immunoglobulin” may be used interchangeably herein.

[0262] “Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno- Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390, which is incorporated by reference herein.

[0263] As used herein when used to refer to vp capsid proteins, the term “heterogenous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 61 provides the encoded amino acid sequence of the AAVhu68 vp l protein. The term “heterogenous” as used in connection with vpl, vp2 and vp3 proteins (alternatively termed isoforms), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine (N - G) pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0264] As used herein, a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified. For example, a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherwise specified. A “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidated, 50% to 100% deamidated, 70% to 100% deamidated, 75% to 100% deamidated, or 70% to 90% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at the reference amino acid position. Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.

[0265] As used herein, a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected. See, e.g., WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, filed September 7, 2018. See, also, e.g., WO 2020 / 223231, published November 5, 2020 (rh91, including table with deamidation pattern), US Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and US Provisional Patent Application No. 63 / 109,734, filed November 4, 2020 and International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, which are all incorporated herein by reference in its entirety.

[0266] The compositions described herein may be used in a regimen involving coadministration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

[0267] The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0268] The term “heterologous” when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0269] A “replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.

[0270] A “recombinant AAV” or “rAAV” is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non- AAV coding sequences packaged within the AAV capsid. In certain embodiments, the capsid contains about 60 proteins composed of vpl proteins, vp2 proteins, and vp3 proteins, which selfassemble to form the capsid. Unless otherwise specified, “recombinant AAV” or “rAAV” may be used interchangeably with the phrase “rAAV vector.” The rAAV is a “replicationdefective virus” or “viral vector”, as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5’ and 3’ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0271] The term “nuclease-resistant” indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process. As used herein, the term “host cell” may refer to the packaging cell line in which the rAAV is produced from the plasmid. In the alternative, the term “host cell” may refer to the target cell in which expression of the transgene is desired.

[0272] As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, expression cassette comprising coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR. In certain embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a trans-complementing AAV. Further, other ITRs, e.g., self-complementary (scAAV) ITRs, may be used. Both single- stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0273] As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. In certain embodiments, non-viral genetic elements used in manufacture of a rAAV, will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, but the use of other suitable genetic elements is contemplated. Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV. Alternatively, such a production plasmid may carry the vector genome which is packaged into the rAAV.

[0274] As used herein, a “parental capsid” refers to a non-mutated, non-engineered or a non-modified capsid selected from parvovirus or other viruses (e.g., AAV, adenovirus, HSV, RSV, etc.). In certain embodiments, the parental capsid includes any naturally occurring AAV capsids comprising a wild-type genome encoding for capsid proteins (i.e., vp proteins), wherein the capsid proteins direct the AAV transduction and / or tissuespecific tropism, n some embodiments, the parent capsid is selected from AAV which natively targets CNS. In other embodiments, the parental capsid is selected from AAV which do not natively target CNS.

[0275] As used herein, the terms “target cell” and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector. The term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart. In one embodiment, the target tissue is brain. In certain embodiments, the target cell is one or more cell type of the CNS (e.g., brain cell and / or brain endothelial cell), including but not limited to astrocytes, neurons, glial cells, ependymal cells, and cells of the choroid plexus. In certain embodiments, the targ...

Claims

WHAT IS CLAIMED IS:

1. A recombinant adeno-associated virus particle (rAAV) comprising:(a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein:(i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid;(ii) n-mer is NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), or an n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and(iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and(b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences.

2. A recombinant adeno-associated virus particle (rAAV) comprising:(a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein:(i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid;(ii) n-mer comprises exogenous targeting peptide motif X’-G-X”- X’”DPR (SEQ ID NO: 88), wherein X’ is W, Y, T, Q, R, N, E, D, X” is L, I, M, K, V, R, T, and X’” is K, P, R, Q, L, K, S; and(iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and(b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences.

3. The rAAV of claim 1 or claim 2, wherein the exogenous targeting peptide is inserted between any two contiguous amino acids in the hypervariable region VIII (HVRVIII) or IV (HVRIV) at a suitable location of a parental AAV capsid.

4. The rAAV of claim 3, wherein the parental AAV capsid is AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, or AAVrh91 .

5. The rAAV of any one of claims 1 to 3, wherein the exogenous targeting peptide is inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 parental capsid as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 60, or an analogous position in a AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parental AAV capsid.

6. The rAAV of any one of claims 1 to 3 or 5, wherein the exogenous targeting peptide is immediately preceded by amino acids “AQ”.

7. The rAAV of any one of claims 1 to 5, wherein the rAAV capsid comprises AAV VP3 proteins having a mutant VP3 region of: amino acids 204 to 743 of SEQ IDNO: 64, 66, 68, 70, 72, 74, 76, or 78, further comprising highly deamidated residues at positions N57, N329, N452, and N512, wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 60 or SEQ ID NO: 61.

8. The rAAV of any one of claims 1 to 5, wherein the rAAV capsid comprises AAV VP1 proteins, AAV VP2 proteins and AAV VP3 proteins having a mutant VP1 of: amino acids 1 to 743 of SEQ ID NO: 64, 66, 68, 70, 72, 74, 76, or 78, further comprising highly deamidated residues at positions N57, N329, N452 and N512, wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 60 or SEQ ID NO: 61.

9. The rAAV of any one of claims 1 to 4, wherein the n-mer is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 90-97, or a sequence at least 95% identical to any one of SEQ ID NOs: 90-97.

10. A composition comprising a stock of the rAAV of any one of claims 1 to 9, and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

11. A recombinant brain cell-targeting peptide, wherein the recombinant brain cell-targeting peptide comprises “Xn - n-mer - Xm”, wherein:(a) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid;(b) n-mer selected from NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), or a n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and(c) Xm is 0, 1, 2, or 3 amino acid / s independently selected from any amino acid; and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

12. A conjugate comprising:(a) a recombinant brain cell-targeting peptide, wherein the recombinant brain cell-targeting peptide comprises “Xn - n-mer - Xm”, wherein:(i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid;(ii) an n-mer selected from NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), or a n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and(iii) Xm is 0, 1, 2, or 3 amino acid / s independently selected from any amino acid; and(b) a nanoparticle, a second molecule, or a viral capsid protein.

13. The recombinant brain cell targeting peptide of claim 11 or the conjugate of claim 12, wherein the recombinant brain cell-targeting peptide targets brain endothelial cell.

14. A composition comprising the recombinant brain cell-targeting peptide of any one of claims 11 to 13, and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

15. A recombinant nucleic acid molecule encoding the recombinant brain celltargeting peptide of any of claims 11 to 13.

16. A nucleic acid molecule comprising a mutant AAV capsid VP1 gene comprising a nucleic acid sequence encoding an n-mer of: NGLKDPR (SEQ ID NO: 33), TGVKDPR (SEQ ID NO: 42), DGMRDPR (SEQ ID NO: 43), TGMRDPN (SEQ ID NO: 57), QGVRDPR (SEQ ID NO: 45), RGVQDPR (SEQ ID NO: 44), TVMRDPR (SEQ ID NO: 54), QGVKDPR (SEQ ID NO: 39), or a n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers.

17. The nucleic acid molecule of claim 16, wherein the sequence encoding the n-mer is:(a) SEQ ID NO: 90 of a sequence at least 99% identical to SEQ ID NO: 90 (encoding NGLKDPR (SEQ ID NO: 33)),(b) SEQ ID NO: 91 of a sequence at least 99% identical to SEQ ID NO: 91 (encoding TGVKDPR (SEQ ID NO: 42),(c) SEQ ID NO: 92 of a sequence at least 99% identical to SEQ ID NO: 92 (encoding DGMRDPR (SEQ ID NO: 43),(d) SEQ ID NO: 93 of a sequence at least 99% identical to SEQ ID NO: 93 (encoding TGMRDPN (SEQ ID NO: 57),(e) SEQ ID NO: 94 of a sequence at least 99% identical to SEQ ID NO: 94 (encoding QGVRDPR (SEQ ID NO: 45),(!) SEQ ID NO: 95 of a sequence at least 99% identical to SEQ ID NO: 95 (encoding RGVQDPR (SEQ ID NO: 44),(g) SEQ ID NO: 96 of a sequence at least 99% identical to SEQ ID NO: 96 (encoding TVMRDPR (SEQ ID NO: 54), or(h) SEQ ID NO: 97 of a sequence at least 99% identical to SEQ ID NO: 97 (encoding QGVKDPR (SEQ ID NO: 39).

18. A fusion polypeptide or protein comprising the recombinant brain celltargeting peptide according to any of claims 8 to 10 and a fusion partner which comprises at least one polypeptide or protein.

19. A composition comprising a fusion polypeptide or protein of claim 14 and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

20. Use of a stock of the rAAV of any one of claims 1 to 6, the recombinant brain cell-targeting peptide of any one of claims 11 to 13, or a fusion polypeptide or protein of claim 14, or a composition of any one of claims 7, 14, or 19, for delivering a therapeutic to a patient or a subject in need thereof.

21. A method for targeted therapy to Central Nervous System (CNS) comprising administering to a patient or a subject in need thereof a stock of the rAAV according to claim 1 .

22. A method for targeted therapy to brain cell comprising administering to a patient or a subject in need thereof a stock of the rAAV according to claim 123. A method for targeted therapy to brain cell comprising administering to a patient or a subject in need thereof a stock of the rAAV according to claim 1, wherein a therapeutic is targeted for delivery brain and / or spinal cord.

24. Use of an rAAV according to any one of claims 1 to 13, use of a composition according to claim 14, or a method for treating of one or more of cognitive disorders, neurodegenerative disorders, and / or a disease of the central nervous system by administering to a patient or a subject in need thereof a stock of the rAAV according to claim 1, wherein the encoded gene product is a protein, or an antibody.

25. The method according to any one of claims 21 to 24, wherein the method is part of a co-therapeutic regimen which further comprises administering or more of immunosuppressive agents comprising corticosteroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent.

26. The method according to any one of claims 21 to 25, wherein the method is part of a co-therapeutic regimen which further comprises administering one or more of: (a) a corticosteroid or combination of corticosteroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc- IgA binding; and / or (f) gamma interferon.

27. The method according to claim 21 or claim 26, wherein the one or more of immunosuppressive agents is administered (i) prior to rAAV administration, (ii) post- rAAV administration, or (iii) prior to and post-rAAV administration.

29. The method according to any one of claims 21 to 24, wherein the method is part of a co-therapeutic regimen which further comprises co-administering a ligand which specifically prevents binding between human neonatal Fc receptor (FcRn) and the neutralizing antibodies without interfering with albumin binding to FcRn.

30. The method according to any one of claims 21 to 24, wherein the method is part of a co-therapeutic regimen which further comprises pretreating the patient or the subject by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid of the recombinant AAV vector.

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