Recombinant AAV mutant vectors with cardiac and skeletal muscle-specific targeting motifs and compositions containing same

Abstract

Provided herein are compositions comprising a muscle (cardiac and / or skeletal) cell targeting peptide linked to or inserted into a targeting protein of a recombinant vector having at least one exogenous peptide comprising Xn-RGD-n-mer-Xm. Compositions providing such conjugates, targeting peptides, or recombinant vectors with engineered capsid or envelope proteins are provided, along with uses thereof.

Description

[Technical Field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing ("UPN-22-10068PCT.xml", size: 183,452 bytes, and creation date: December 5, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] Adeno-associated virus (AAV) is currently the gene therapy vector of choice. AAV can deliver transgenes that are not associated with any human disease and are stably expressed for long periods from a non-integrated genome. However, AAV-mediated gene therapy is currently limited to the treatment of a small number of diseases due to challenges in delivery and tropism.

[0003] AAV vector-based therapeutic approaches have been approved by the U.S. Food and Drug Administration and other regulatory agencies worldwide for the treatment of Leber congenital amaurosis, lipoprotein lipase deficiency, and spinal muscular atrophy. A central challenge in gene therapy is the difficulty of regulating and targeting the expression of therapeutic transgenes in vivo, more specifically, targeting specific tissues, such as the heart and skeletal muscle.

[0004] There is a high and unmet need in adult and early-onset myocyte-related pathologies (cardiac and skeletal). In such cases, the lack of effective treatment can lead to high rates of transplantation and / or death. While a variety of cardiac and skeletal myocyte-based diseases are amenable to AAV gene replacement therapy, there is a need for specific and effective targeting of muscle tissue.

[0005] There remains a need for vectors that can specifically target selected tissues and cell types. Summary of the Invention

[0006] In one aspect, a recombinant adeno-associated particle (rAAV) comprises: (a) an adeno-associated virus (AAV) capsid comprising a VP1 protein, a VP2 protein, and a VP3 protein, wherein the capsid protein has an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises an "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGD (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 the control of a sequence that directs expression of the nucleic acid sequence encoding the gene product. Provided herein is a recombinant adeno-associated particle (rAAV) comprising: In certain embodiments, the rAAV comprises an exogenous targeting peptide comprising an n-mer that is (a) IIRGDPA (SEQ ID NO: 1) or (b) AVIRGDV (SEQ ID NO: 2). In certain embodiments, the exogenous targeting peptide is inserted between any two consecutive amino acids in the hypervariable region VIII (HVRVIII) or hypervariable region IV (HVRIV) at a suitable position in the parent AAV capsid. In certain embodiments, the parent AAV capsid is an AAV9, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 capsid. In certain embodiments, the exogenous targeting peptide is inserted into the hypervariable region between amino acids 588 and 589 in the AAV9 parent capsid, as determined based on the numbering of the VP1 amino acid sequence of SEQ ID NO: 25, or at an analogous position in an AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 parent AAV capsid. In certain embodiments, the exogenous targeting peptide is immediately preceded by a native AAV residue, e.g., "AQ." In certain embodiments, the rAAV capsid comprises AAV VP1, AAV VP2, and AAV VP3 proteins having a variant AAV VP3 region from amino acids 204 to 743 of any one of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, wherein the VP1, VP2, and VP3 proteins each have a heterogeneous population that further comprises highly deamidated residues (e.g., independently, from about 50% to about 100% deamidated) at positions N57, N329, N452, and N512, where the deamidated position numbering is based on the residue positions of SEQ ID NO:25 or SEQ ID NO:26.In certain embodiments, the rAAV capsid comprises an AAV VP1 protein, an AAV VP2 protein, and an AAV VP3 protein, wherein VP1 has a variant sequence of amino acids 1-743 of any one of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, and the AAV VP1 protein, AAV VP2 protein, and AAV VP3 protein further comprise highly deamidated residues at positions N57, N329, N452, and N512, where the deamidated position numbers are based on the residue positions of SEQ ID NO:25 or SEQ ID NO:26 (e.g., independently, about 50% to about 100% deamidated). In certain embodiments, the n-mer is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 108-125, or a sequence at least 95% identical to any one of SEQ ID NOs: 108-125.

[0007]

[0013] In another aspect, provided herein is a recombinant muscle cell targeting peptide, the recombinant muscle cell targeting peptide comprising a "Xn-n-mer-Xm", where (a) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (b) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), and (iii) Xm is 0, 1, 2, or 3 amino acids independently selected from any amino acid, and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the recombinant muscle cell targeting peptide targets cardiac myocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells.

[0008] In another aspect, provided herein is a nucleic acid molecule comprising a mutant AAV capsid VP1 gene comprising a nucleic acid sequence encoding the following n-mer: IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), RGDYAQV (SEQ ID NO: 20), RGDLHGY (SEQ ID NO: 22), PYQRGDH (SEQ ID NO: 24), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), IGRGDPN (SEQ ID NO: 21), or RGDYSTM (SEQ ID NO: 23).In certain embodiments, the nucleic acid sequence encoding the n-mer is selected from the group consisting of: (a) SEQ ID NO: 108, or a sequence at least 99% identical thereto (encoding IIRGDPA); (b) SEQ ID NO: 109, or a sequence at least 99% identical thereto (encoding AVIRGDV); (c) SEQ ID NO: 110, or a sequence at least 99% identical thereto (encoding IVRGDPA); (d) SEQ ID NO: 111, or a sequence at least 99% identical thereto (encoding MIRGDVK); (e) SEQ ID NO: 112, or a sequence at least 99% identical thereto (encoding AQHRGDV); (f) SEQ ID NO: 113, or a sequence at least 99% identical thereto (encoding VSRGDPN); (g) SEQ ID NO: 114, or a sequence at least 99% identical thereto (encoding VSRGDPA); (h) SEQ ID NO: 115, or a sequence at least 99% identical thereto (encoding PLVRGDI); (i) SEQ ID NO: 116, or a sequence at least 99% identical thereto (encoding PYV (j) SEQ ID NO: 117 or a sequence at least 99% identical thereto (encodes VVRGDPQ); (k) SEQ ID NO: 118 or a sequence at least 99% identical thereto (encodes VVQRGDV); (l) SEQ ID NO: 119 or a sequence at least 99% identical thereto (encodes QHRGDTQ); (m) SEQ ID NO: 120 or a sequence at least 99% identical thereto (encodes QIRGDLR); (n) SEQ ID NO: 121 or a sequence at least 99% identical thereto (encodes RGDYAQV); (o) SEQ ID NO: 122 or a sequence at least 99% identical thereto (encodes IGRGDPN); (p) SEQ ID NO: 123 or a sequence at least 99% identical thereto (encodes RGDLHGY); (q) SEQ ID NO: 124 or a sequence at least 99% identical thereto (encodes RGDYSTM); or (r) SEQ ID NO: 125 or a sequence at least 99% identical thereto (encodes PYQRGDH). In certain embodiments, the nucleic acid sequence encoding the AAV capsid protein comprises SEQ ID NO: 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106.

[0009] In certain embodiments, provided herein are fusion polypeptides or proteins comprising a muscle cell targeting peptide and a fusion partner comprising at least one polypeptide or protein. In certain embodiments, compositions comprising a fusion polypeptide or protein provided herein and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

[0010] Also provided herein are compositions and methods for using the rAAV, muscle cell targeting peptides, fusion polypeptides or proteins, and / or compositions described herein to deliver a therapeutic agent to a patient in need thereof. In certain embodiments, the therapeutic agent is targeted to a muscle cell, and optionally the muscle cell is a cardiac muscle cell or a skeletal muscle cell, optionally a gastrocnemius muscle cell, a deltoid muscle cell, a soleus muscle cell, a biceps brachii muscle cell, or a diaphragm muscle cell.

[0011] In certain embodiments, methods are provided for targeting therapy to muscle cells in a patient in need thereof, the methods comprising administering to the patient an rAAV as described herein. In certain embodiments, methods for treating cardiac and / or skeletal muscle disorders and / or diseases are provided.

[0013] Methods for treating cardiac and / or skeletal muscle disorders and / or diseases in a subject in need thereof are provided, the methods comprising delivering to the subject a stock of rAAV described herein. In certain embodiments, methods for treating one or more cardiac and / or skeletal (e.g., gastrocnemius, deltoid, soleus, biceps) muscle-based disorders and / or diseases in a subject in need thereof are provided, the methods comprising delivering to the subject a stock of rAAV described herein, wherein the encoded gene product is a protein, optionally an antibody.

[0012] These and other embodiments and advantages of the present invention will become apparent from this specification, including but not limited to the detailed description of the invention. [Brief explanation of the drawings]

[0013] [Figure 1A] Plotted cardiac enrichment scores from RGD screening round 2 for top performing cardiac candidates compared to top literature capsids are shown. [Figure 1B] Shown are muscle enrichment scores from RGD screening round 2 for top performing heart and muscle candidates compared to top heart candidates. [Figure 1C] Shown are gastrocnemius enrichment scores from RGD screening round 2 for top performing muscle candidates compared to top literature cardiac capsids. [Figure 2A] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the cardiac left ventricle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. DNA levels are plotted as GC / diploid cells. [Figure 2B] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the cardiac left ventricle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. RNA levels are plotted as copy number (#) / 100 ng of RNA. [Figure 2C] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the cardiac left ventricle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. Protein expression levels are plotted as picograms (pg) of GFP / micrograms (µg) of protein. [Figure 3A] RNA, DNA, and protein expression levels in tissue samples collected from the right ventricle of the heart after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors are shown. DNA levels are plotted as GC / diploid cells. [Figure 3B] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the cardiac right ventricle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. RNA levels are plotted as copy number (#) / 100 ng of RNA. [Figure 3C] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the cardiac right ventricle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. Protein expression levels are plotted as pg GFP / µg protein. [Figure 4A] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the gastrocnemius muscle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. DNA levels are plotted as GC / diploid cells. [Figure 4B] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the gastrocnemius muscle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. RNA levels are plotted as copy number (#) / 100 ng of RNA. [Figure 4C] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the gastrocnemius muscle after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. Protein expression levels are plotted as pg GFP / µg protein. [Figure 5A] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the liver after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. DNA levels are plotted as GC / diploid cells. [Figure 5B]Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the liver after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. RNA levels are plotted as copy number (#) / 100 ng of RNA. [Figure 5C] Figure 1 shows RNA, DNA, and protein expression levels in tissue samples collected from the liver after administration of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors. Protein expression levels are plotted as pg GFP / µg protein. [Figure 6] The RNA / DNA ratios (normalized to AAV9) for the biodistribution of AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors are shown. [Figure 7] Alignments of specific regions of the amino acid sequences of various AAV capsid proteins are shown: AAV9 (amino acids 566-615 of the AAV9 capsid; SEQ ID NO:27), AAV8 (amino acids 565-614 of the AAV8 capsid; SEQ ID NO:28), AAV7 (amino acids 567-616 of AAV7; SEQ ID NO:29), AAV6 (amino acids 550-599 of the AAV6 capsid; SEQ ID NO:30), AAV5 (amino acids 556-605 of AAV5; SEQ ID NO:31), AAV4 (amino acids 558-607 of the AAV4 capsid; SEQ ID NO:32), AAV3B (amino acids 564-613 of the AAV3B capsid; SEQ ID NO:33), AAV2 (amino acids 566-615 of the AAV2 capsid; SEQ ID NO:34), and AAV1 (amino acids 566-615 of the AAV1 capsid; SEQ ID NO:35). The HVRVIII regions (based on structural analysis) into which targeting peptides can be inserted are shown. [Figure 8] Percent GFP-positive area quantified from IHC analysis of cardiac (longitudinal) tissue samples is shown. [Figure 9] Percent GFP-positive area quantified from IHC analysis of cardiac (transverse) tissue samples is shown. [Figure 10]The RNA / DNA ratios (normalized to AAV9) for various vectors, AAV9, AAV9-IIRGDPA, and AAV9-X, analyzed in cardiac tissue are shown. [Figure 11] The RNA / DNA ratios (normalized to AAV9) for various vectors, AAV9, AAV9-IIRGDPA, and AAV9-X, analyzed in liver tissue are shown. [Figure 12A] RNA levels in cardiac tissue after rAAV transduction plotted as RNA transcript / 100 ng are shown. [Figure 12B] DNA levels in cardiac tissue after rAAV transduction plotted as GC per diploid genome are shown. [Figure 12C] Shown are RNA levels in liver tissue after rAAV transduction, plotted as RNA transcript / 100 ng. [Figure 12D] DNA levels in liver tissue after rAAV transduction plotted as GC per diploid genome are shown. [Figure 13A] The RNA / DNA ratios (normalized to AAV9) for various vectors, AAV9, AAV9-AVIRGDV, and AAV9-X, analyzed in cardiac tissue are shown. [Figure 13B] The RNA / DNA ratios (normalized to AAV9) for various vectors, AAV9, AAV9-AVIRGDV, and AAV9-X, analyzed in liver tissue are shown. [Figure 13C] RNA levels in cardiac tissue after rAAV transduction plotted as RNA transcript / 100 ng are shown. [Figure 13D] DNA levels in cardiac tissue after rAAV transduction plotted as GC per diploid genome are shown. [Figure 13E] Shown are RNA levels in liver tissue after rAAV transduction, plotted as RNA transcript / 100 ng. [Figure 13F]DNA levels in liver tissue after rAAV transduction plotted as GC per diploid genome are shown. [Figure 14A] DNA levels in cardiac tissue after rAAV transduction plotted as GC / diploid cells are shown. [Figure 14B] RNA levels in cardiac tissue after rAAV transduction plotted as copy number (#) / 100 ng of RNA are shown. [Figure 14C] Protein expression levels in cardiac tissue after rAAV transduction plotted as pg GFP / μg protein are shown. [Figure 15A] DNA levels in liver tissue after rAAV transduction plotted as GC / diploid cells are shown. [Figure 15B] RNA levels in liver tissue after rAAV transduction plotted as copy number (#) / 100 ng of RNA are shown. [Figure 15C] Protein expression levels in liver tissue after rAAV transduction plotted as pg GFP / μg protein are shown. [Figure 16A] DNA levels in gastrocnemius muscle tissue after rAAV transduction plotted as GC / diploid cells are shown. [Figure 16B] RNA levels in gastrocnemius muscle tissue after rAAV transduction plotted as copy number (#) / 100 ng of RNA are shown. [Figure 16C] Protein expression levels in gastrocnemius muscle tissue after rAAV transduction plotted as pg GFP / μg protein are shown. [Figure 17] Shown are GFP expression levels in iPSCM and C2C12 cells after rAAV transduction, plotted as relative light units (RLU, from microplate readings). [Figure 18A]Shown are rAAV (AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vector) titers measured in PBS or PBS containing 0.001% Pluronic formulation and plotted as GC / mL obtained from large-scale and small-scale rAAV preparations. [Figure 18B] Shown are pooled titers of rAAV (AAV9, AAV9-IIRGDPA, and AAV9-AVIRGDV vectors) measured in PBS or PBS containing 0.001% Pluronic formulation and plotted as GC / mL obtained from large-scale and small-scale rAAV preparations. [Figure 19A] Results of an ongoing survival study, plotted as survival probability, are shown. These results demonstrate similar and / or better survival observed in mice when administered AAV9-IIRGDPA or AAV9-AVIRGDV compared to the AAVhu68 vector, all containing TT1. [Figure 19B] Body weight plotted as grams (g) is shown. [Figure 20A] Representative ISH microscopic images of cardiac tissue are shown. [Figure 20B] Quantified percentage of ISH-positive cardiomyocytes in various treatment groups is shown. [Figure 21A] RNA levels plotted as normalized RNA transcript copy number (#) / 100 ng of RNA in cardiac tissue are shown. [Figure 21B] RNA levels in liver tissue plotted as normalized RNA transcript copy number (#) / 100 ng of RNA are shown. [Figure 21C] DNA levels plotted as GC per normalized diploid cell are shown. [Figure 21D] DNA levels in heart tissue plotted as GC per normalized diploid cell in liver tissue are shown. [Figure 21E]Protein (GFP) expression levels in thermal tissues plotted as pg of GFP per normalized µg of total protein are shown. [Figure 21F] Protein (GFP) expression levels in liver tissue plotted as pg of GFP per μg of total protein normalized are shown. [Figure 22] 1 shows the results of RNA biodistribution in various muscle tissue subtypes. [Figure 23] 1 shows the results of DNA biodistribution in various muscle tissue subtypes. [Figure 24] 1 shows the results of protein (GFP) biodistribution in various muscle tissue subtypes. [Figure 25A] Shown are the results of in situ hybridization (ISH) analysis in gastrocnemius muscle tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25B] Shown are the results of ISH analysis in diaphragm tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25C] 1 shows the results of ISH analysis in biceps femoris tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25D] Shown are the results of ISH analysis in gluteus maximus tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25E] Shown are the results of ISH analysis in deltoid muscle tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25F] 1 shows the results of ISH analysis in soleus muscle tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 25G] Shown are the results of ISH analysis in vastus lateralis muscle tissue plotted as percent GFP positivity normalized to AAV9 control. [Figure 26A]Shown are TT2 DNA levels in the left ventricle after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2, plotted as GC / diploid cells. [Figure 26B] TT2 RNA levels in the left ventricle after administration of AAVhu68.TT2, or AAV9-IIRGDPA.TT2, AAV9-AVIRGDV.TT2, plotted as vector GC / 100 ng of total RNA (normalized to U6) are shown. [Figure 26C] Shown are TT2 DNA levels in the septum after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.hTT2, plotted as GC / diploid cells. [Figure 26D] Shown are TT2 RNA levels in the septum after AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 administration, plotted as vector GC / 100 ng of total RNA (normalized to U6). [Figure 26E] Shown are TT2 DNA levels in the left atrium after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2, plotted as GC / diploid cells. [Figure 26F] Shown are TT2 RNA levels in the left atrium after AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 administration, plotted as vector GC / 100 ng of total RNA (normalized to U6). [Figure 26G] Shown are TT2 DNA levels in the liver following administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2, plotted as GC / diploid cells. [Figure 26H]Shown are TT2 RNA levels in the liver following AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 administration, plotted as vector GC / 100 ng of total RNA (normalized to U6). [Figure 26I] Shown are TT2 DNA levels in the diaphragm following administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2, plotted as GC / diploid cells. [Figure 26J] Shown are TT2 RNA levels in the diaphragm after AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 administration, plotted as vector GC / 100 ng of total RNA (normalized to U6). [Figure 26K] Shown are TT2 DNA levels in quadriceps muscle following administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2, plotted as GC / diploid cells. [Figure 26L] Shown are TT2 RNA levels in quadriceps muscle following AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 administration, plotted as vector GC / 100 ng of total RNA (normalized to U6). [Figure 27A] Shown are the results of the ISH analysis plotted as the percentage of ISH-positive cells in tissue from the left ventricle. [Figure 27B] Shown are the results of the ISH analysis plotted as the percentage of ISH-positive cells in tissue from the interventricular septum of the heart. [Figure 27C] Results of the ISH analysis plotted as percent ISH-positive cells in right ventricular tissue are shown. [Figure 27D] 1 shows the results of the ISH analysis plotted as the percentage of ISH-positive cells in the diaphragm tissue. [Figure 27E] 1 shows the results of the ISH analysis plotted as the percentage of ISH-positive cells in quadriceps tissue. [Figure 28A] Measured levels of aspartate aminotransferase (AST) in blood samples from D0 to D90 after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 are shown. [Figure 28B] Measured levels of alanine aminotransferase (ALT) in blood samples from D0 to D90 after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 are shown. [Figure 28C] Measured levels of platelet counts in blood samples from D0 to D90 after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 are shown. [Figure 28D] Measured levels of d-dimer in blood samples from DO to D90 after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 are shown. [Figure 28E] Measured levels of troponin I in blood samples from D0 to D90 after administration of AAVhu68.TT2, AAV9-IIRGDPA.TT2, or AAV9-AVIRGDV.TT2 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] In certain embodiments, recombinant muscle cell targeting peptides and nucleic acid sequences encoding them are provided herein. Also provided herein are fusion proteins, modified proteins, engineered viral capsids (e.g., recombinant adeno-associated virus (rAAV) capsids), and other moieties comprising the targeting peptide, wherein the targeting peptide is "Xn-n-mer-Xm," where Xn is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 4 or 3 amino acid residues, and the n-mers are IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDT and / or a full-length n-mer sequence of at least 6, at least 7, or a full-length n-mer of any one of Q (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), where Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Nucleic acid sequences encoding fusion proteins, modified proteins, and engineered viral capsids are also provided herein. In certain embodiments, the exogenous targeting motif modifies the native tissue specificity of the source (parent) protein, viral vector, viral capsid, or other moiety. In certain embodiments, compositions having one or more of the exogenous targeting peptides have enhanced or altered muscle cell targeting. In certain embodiments, compositions having one or more of the targeting peptides have enhanced or altered cardiac and / or skeletal muscle cell targeting (e.g., gastrocnemius muscle cell targeting). In certain embodiments, viral vectors having modified capsids containing targeting motifs exhibit increased transduction of AAV-producing cells in vitro.

[0015] Advantageously, in certain embodiments, provided herein is a recombinant muscle cell targeting peptide (also referred to as a "targeting peptide," "exogenous targeting peptide"), wherein the recombinant muscle cell targeting peptide comprises an "Xn-n-mer-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVR GDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length of any one of the n-mers, wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0016] In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide, wherein the exogenous targeting peptide is an "Xn-n-mer-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDV (SEQ ID NO: 14), and the like. P (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length of any one of the n-mers, wherein Xm is independently selected from any 0 to 10 amino acids. , 1, 2, or 3 amino acid residues. In certain embodiments, the exogenous targeting peptides provided herein provide a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells, compared to the parent capsid (e.g., AAV9, or another clade F capsid, or another clade capsid).

[0017] In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-IIRGDPA (SEQ ID NO: 1)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-AVIRGDV (SEQ ID NO: 2)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-IVRGDPA (SEQ ID NO: 8)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-MIRGDVK (SEQ ID NO: 9)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and optionally Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid.In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-AQHRGDV (SEQ ID NO: 10)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-VSRGDPN (SEQ ID NO: 11)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-VSRGDPA (SEQ ID NO: 12)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-PLVRGDI (SEQ ID NO: 13)-Xm." wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, and optionally, gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein is an engineered rAAV capsid comprising an exogenous targeting peptide comprising "Xn-PYVRGDP(SEQ ID NO: 14)-Xm," wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, and optionally, gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-VVRGDPQ (SEQ ID NO: 15)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-PTRGDVK (SEQ ID NO: 16)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid.In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-VVQRGDV (SEQ ID NO: 17)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-QHRGDTQ (SEQ ID NO: 18)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-QIRGDLR (SEQ ID NO: 19)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-RGDYAQV (SEQ ID NO: 20)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid.In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-IGRGDPN (SEQ ID NO: 21)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0 independently selected from any amino acid. , 1, 2, or 3 amino acid residues, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally, gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-RGDLHGY (SEQ ID NO: 22)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally, gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-RGDYSTM (SEQ ID NO: 23)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide comprising "Xn-PYQRGDH (SEQ ID NO: 24)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides a significant transduction advantage in muscle cells, including cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, compared to the parent capsid.

[0018] In certain embodiments, provided herein is an rAAV comprising a mutant AAV capsid having an exogenous targeting peptide identified herein. In certain embodiments, the mutant AAV capsid comprises an exogenous targeting peptide immediately preceded by mutated flanking amino acids compared to the parent AAV capsid. In certain embodiments, the mutated flanking amino acids comprise the exogenous targeting peptide along with 1, 2, 3, 4, 5, or 6 inserted amino acids. In certain embodiments, the entire exogenous targeting peptide is inserted into the parent AAV capsid. In still other embodiments, the amino acid sequence inserted into the capsid protein can include all or a fragment of the exogenous targeting peptide at the carboxy (COO-) or amino (N-) terminus (i.e., via insertion of 5' or 3' coding sequence therefor), further comprising 0 to 3 flanking amino acid residues as provided in the formula above. In certain embodiments, engineered rAAV capsids comprising targeting peptides provided herein exhibit reduced transduction to the liver (i.e., detargeted / detargeted) compared to their parent capsids (e.g., AAV9 or another clade F capsid (e.g., AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96) or other modifications thereof). In certain embodiments, engineered rAAV capsids comprising targeting peptides provided herein exhibit reduced transduction to the spleen (i.e., detargeted / detargeted) compared to their parent capsids (e.g., AAV9 or another clade F capsid or other modifications thereof).

[0019] Targeting peptides can be linked to recombinant proteins (e.g., for enzyme replacement therapy) or polypeptides (e.g., immunoglobulins) to form fusion proteins or conjugates to target desired tissues (e.g., muscle cells, cardiac myocytes, skeletal muscle cells, gastrocnemius muscle cells). In addition, targeting peptides can be linked to liposomes and / or nanoparticles (lipid nanoparticles, LNPs) to form peptide-coated liposomes and / or LNPs to target desired tissues. A sequence encoding at least one copy of the targeting peptide and an optional linking sequence can be fused in-frame with the coding sequence of the recombinant protein and co-expressed with the protein or polypeptide to provide a fusion protein or conjugate. Alternatively, other synthetic methods can be used to synthesize proteins, polypeptides, or other targeting peptides. The targeting peptide may form a conjugate with a peptide or another moiety (e.g., DNA, RNA, or small molecule). In certain embodiments, multiple copies of the targeting peptide are in the fusion protein / conjugate. A suitable method for conjugating a targeting peptide to a recombinant protein includes using a first crosslinker to modify the amino (N)-terminus and one or more residues on a recombinant human protein (e.g., an enzyme) to produce a first crosslinker-modified recombinant human protein, and using a second crosslinker to modify the amino (N)-terminus of a short extension linker region in front of the targeting peptide to produce a second crosslinker-modified variant targeting peptide, and then conjugating the first crosslinker-modified recombinant human protein to the second crosslinker-modified variant targeting peptide containing the short extension linker. Other suitable methods for conjugating targeting peptides to recombinant proteins include conjugating a first crosslinker-modified recombinant human protein to one or more second crosslinker-modified variant targeting peptides, where the first crosslinker-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 crosslinker-modified variant targeting peptides comprise one or more variant targeting peptides that include a modified N-terminal amino acid of a short extension linker preceding the targeting peptide. Still other suitable methods for conjugating targeting peptides to proteins, polypeptides, nanoparticles, or other biologically useful chemical moieties can be selected. For example, see U.S. Patent No. 9,545,450 B2 (NHS-phosphine crosslinkers, NHS-azide crosslinkers) and U.S. Published Patent Application No. 2018 / 0185503 A1 (aldehyde-hydrazide crosslinking).

[0020] In certain embodiments, an exogenous targeting peptide is engineered (i.e., inserted) at a suitable site within a protein or polypeptide (e.g., a viral capsid protein). In certain embodiments, the targeting peptide is flanked at its amino (N-) (e.g., optional Xn) and / or carboxy (COO-) (e.g., optional Xm) termini by short extension linkers. Such linkers can be about 1 to about 20 amino acid residues in length, or about 2 to about 20 amino acid 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 the N-terminal linker is selected independently from the carboxy-terminal linker, and the presence and length of the carboxy-terminal linker is selected independently from the N-terminal linker. Suitable short extension linkers can be provided using an approximately 5 amino acid flexible GS extension linker (glycine-glycine-glycine-glycine-serine), an approximately 10 amino acid extension linker comprising two flexible GS linkers, an approximately 15 amino acid extension linker comprising three flexible GS linkers, an approximately 20 amino acid extension linker comprising four flexible GS linkers, or any combination thereof.

[0021] In certain embodiments, compositions are provided that are useful for targeting muscle cells. In certain embodiments, the compositions include an engineered capsid (e.g., rAAV capsid), fusion protein, or another conjugate, comprising at least one exogenous targeting peptide comprising the core amino acid sequence (e.g., "n-mer") of IIRGDPA (SEQ ID NO: 1), flanked at the amino and / or carboxy termini 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 the core amino acid sequence (e.g., an "n-mer") of AVIRGDV (SEQ ID NO: 2), flanked at the amino and / or carboxy termini 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. The compositions are IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), and RGDYSTM (SEQ ID NO: 24). No. 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6 consecutive amino acids of any one of the n-mers, flanked at the amino and / or carboxy termini 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.

[0022] In certain embodiments, the targeting peptide core amino acid sequence is encoded by a nucleic acid sequence.

[0023] In certain embodiments, the targeting peptide core (e.g., "n-mer") is IIRGDPA (SEQ ID NO: 1). In certain embodiments, the targeting peptide core is AVIRGDV (SEQ ID NO: 2). In certain embodiments, the targeting peptide core is IVRGDPA (SEQ ID NO: 8). In certain embodiments, the targeting peptide core is MIRGDVK (SEQ ID NO: 9). In certain embodiments, the targeting peptide core is AQHRGDV (SEQ ID NO: 10). In certain embodiments, the targeting peptide core is VSRGDPN (SEQ ID NO: 11). In certain embodiments, the targeting peptide core is VSRGDPA (SEQ ID NO: 12). In certain embodiments, the targeting peptide core is PLVRGDI (SEQ ID NO: 13). In certain embodiments, the targeting peptide core is PYVRGDP (SEQ ID NO: 14). In certain embodiments, the targeting peptide core is VVRGDPQ (SEQ ID NO: 15). In certain embodiments, the targeting peptide core is PTRGDVK (SEQ ID NO: 16). In certain embodiments, the targeting peptide core is VVQRGDV (SEQ ID NO: 17). In certain embodiments, the targeting peptide core is QHRGDTQ (SEQ ID NO: 18). In certain embodiments, the targeting peptide core is QIRGDLR (SEQ ID NO: 19). In certain embodiments, the targeting peptide core is RGDYAQV (SEQ ID NO: 20). In certain embodiments, the targeting peptide core is IGRGDPN (SEQ ID NO: 21). In certain embodiments, the targeting peptide core is RGDLHGY (SEQ ID NO: 22). In certain embodiments, the targeting peptide core is RGDYSTM (SEQ ID NO: 23). In certain embodiments, the targeting peptide core is PYQRGDH (SEQ ID NO: 24). In certain embodiments, two or more copies of the targeting peptide are present in a conjugated or modified protein (e.g., a parvovirus capsid, a rAAV capsid). In certain embodiments, two or more different targeting peptide cores are present.

[0024] In certain embodiments, compositions are provided that are useful for targeting muscle cells. In certain embodiments, compositions are provided that are useful for targeting cardiomyocytes (i.e., cardiac tissue). In certain embodiments, compositions are provided that are useful for targeting skeletal muscle cells. In certain embodiments, compositions are provided that are useful for targeting gastrocnemius muscle cells. In certain embodiments, compositions are provided that target muscle cells but are also useful for detargeting cells in the liver. In certain embodiments, compositions are provided that target muscle cells but are also useful for detargeting cells in the spleen.

[0025] Amino acid core sequences IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDP (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN ( Also provided herein are compositions comprising an engineered rAAV capsid, fusion protein, or another conjugate comprising at least one exogenous targeting peptide comprising SEQ ID NO:21), RGDLHGY (SEQ ID NO:22), RGDYSTM (SEQ ID NO:23), and / or PYQRGDH (SEQ ID NO:24), wherein the inserted targeting peptide is flanked by 0, 1, 2, or 3 amino acids at the amino and / or carboxy terminus of the motif, and is optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

[0026] 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 biological and chemical moieties are suitable for use with the targeting peptide(s) provided herein.

[0027] In certain embodiments, provided herein are compositions comprising a nucleic acid molecule, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule, e.g., naked DNA, naked plasmid DNA, messenger RNA (mRNA), linked to a targeting peptide sequence motif described herein. In some embodiments, the nucleic acid molecule is further associated with various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions, and other polymer, lipid and / or cholesterol-based-nucleic acid conjugates, and other constructs as described herein. See, for example, WO2014 / 089486, US2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085A1, US9670152B2, and US8,853,377B2, X. Su, et al., Mol. Pharmaceutics, 2011, 8(3), pp774-787, web publication: March 21, 2011, WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930, all of which are incorporated herein by reference. In certain embodiments, targeting peptide motifs are chemically linked to the nanoparticle surface, and the nanoparticles encapsulate nucleic acid molecules. In some embodiments, nanoparticles comprising surface-linked targeting peptides are designed for targeted, tissue-specific delivery. In some embodiments, two or more different targeting peptides are linked to the surface of the nanoparticle. Suitable chemical linkages or crosslinks include those known to those of skill in the art.

[0028] Capsid In certain embodiments, a recombinant parvovirus having a modified parvovirus capsid is provided, wherein the capsid comprises an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises an "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Such recombinant parvoviruses can be hybrid bocavirus / AAV or recombinant AAV vectors (rAAV). In other embodiments, other viral vectors can be generated with one or more exogenous targeting peptides in exposed capsid proteins to modulate and / or alter the targeting specificity of the viral vector compared to the parent vector, wherein the one or more exogenous targeting peptides comprise "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PL VRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length 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.

[0029] An exogenous targeting peptide can be inserted into hypervariable loop (HVR) VIII (also referred to as HVR8) at any suitable position (and / or engineered via mutation of the sequence encoding the adjacent amino acid residue(s)). For example, based on the numbering of the AAV9 capsid, a peptide is inserted between amino acids 588 and 589 (QA) of the AAV9 capsid protein, with linkers of various lengths, based on the numbering of the AAV9 VP1 (also referred to as Vp1 or vp1) amino acid sequence: SEQ ID NO: 25. See also WO2019 / 168961, published September 6, 2019, and WO2020 / 160582, filed September 7, 2018, which contain Table G providing the deamidation pattern of AAV9. The amino acid residue positions (see amino acid numbering) are identical in AAVhu68 (SEQ ID NO: 26). However, other sites within HVRVIII can also be selected. Alternatively, another exposed loop HVR (e.g., HVRIV) can be selected for insertion. Equivalent HVR regions can be selected in other capsids. In certain embodiments, the positions of HVRVIII and HVRIV are determined using the algorithms and / or alignment techniques described in U.S. Patent No. 9,737,618 B2 (column 15, lines 3-23) and U.S. Patent No. 10,308,958 B2 (column 15, line 46 to column 16, line 6), which are incorporated herein by reference in their entireties. In certain embodiments, the targeting peptide can be inserted (and / or engineered) into the hypervariable loop HVRVIII described in U.S. Provisional Patent Application No. 63 / 119,863, filed December 1, 2020, and International Patent Application No. PCT / US2021 / 061312, filed December 1, 2022, which are incorporated herein by reference in their entireties.In certain embodiments, the AAV1 capsid protein is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted into a suitable position in the HVRVIII region at amino acids 582-585 or the HVRIV region at amino acids 456-459, based on vp1 numbering (Gurda, B.L., et al., Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions, 2012, Journal of Virology, June 12, 2013, 87(16):). In certain embodiments, AAV8 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted into the HVRVIII region at amino acids 586-591 or the HVRIV region at amino acids 456-460, based on the VP1 numbering (Gurda, B.L., 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).

[0030] In certain embodiments, the parent AAV capsid is an AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, or AAVrh91 capsid.

[0031] In certain embodiments, the exogenous targeting peptide is engineered and / or inserted into the hypervariable region between amino acids 588 and 589 in the AAV9 parent capsid, as determined based on the numbering of the VP1 amino acid sequence of SEQ ID NO:25, or into an analogous position in an AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parent AAV capsid.

[0032] In certain embodiments, the exogenous targeting peptide has an amino acid sequence at its carboxy terminus and at its amino terminus that is immediately preceded by "AQ" at position 588 of a parent capsid that is a clade F capsid, e.g., an AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95, or AAVhu96 capsid.

[0033] In certain embodiments, residues of the parent AAV capsid sequence are conserved (i.e., there are no substitutions and / or deletions at 1, 2, and / or 3 amino acid residues at the N-terminus and / or C-terminus immediately preceding the target peptide insert, compared to the parent AAV capsid amino acid sequence). In certain embodiments, there are no deletions at 1, 2, and / or 3 amino acid residues at the N-terminus and / or C-terminus immediately preceding the target peptide insert, compared to the parent AAV capsid amino acid sequence. In certain embodiments, one or more amino acid residues are modified at the N-terminus and / or C-terminus immediately preceding the n-mer, and / or one or more residues of an n-mer sequence are provided in the mutant AAV capsid. In certain embodiments, there are one or more amino acid residues modified at positions immediately adjacent to the N-terminus and / or C-terminus of the n-mer, and / or one or more residues of an n-mer sequence are provided in the mutant AAV capsid.

[0034] In certain embodiments, AAV9 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 588 and 589 (QA) based on VP1 numbering. In other embodiments, AAVhu68 or another clade F capsid is selected as the parent capsid. In certain embodiments, AAV8 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 590 and 591 (NT) based on VP1 numbering. In certain embodiments, AAV7 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 589 and 590 (NT) based on VP1 numbering. In certain embodiments, AAV6 is selected as the parent capsid, and targeting peptides with linkers of various lengths are In certain embodiments, AAV5 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 588 and 589 (ST) based on VP1 numbering. In certain embodiments, AAV5 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 577 and 578 (TT) based on VP1 numbering. In certain embodiments, AAV4 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 586 and 587 (SN) based on VP1 numbering. In certain embodiments, AAV3 / 3B is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 588 and 589 (NT) based on VP1 numbering. In certain embodiments, AAV2 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 587 and 589 (NR) based on VP1 numbering. In certain embodiments, AAV1 is selected as the parent capsid, and targeting peptides with linkers of various lengths are inserted (and / or engineered) into suitable positions in the HVRVIII region at amino acids 588 and 589 (ST) based on VP1 numbering.Also, see FIG. 7, which shows a sequence diagram of AAV9 (amino acids 566-615 of the AAV9 capsid, SEQ ID NO: 27), AAV8 (amino acids 565-614 of the AAV8 capsid, SEQ ID NO: 28), AAV7 (amino acids 567-616 of the AAV7 capsid, SEQ ID NO: 29), AAV6 (amino acids 550-599 of the AAV6 capsid, SEQ ID NO: 30), AAV5 (amino acids 556-605 of the AAV5 capsid, SEQ ID NO: 31), AAV4 (amino acids 558-607 of the AAV4 capsid, SEQ ID NO: 32), AAV5 (amino acids 559-609 of the AAV5 capsid, SEQ ID NO: 33), AAV6 (amino acids 560-561 of the AAV6 capsid, SEQ ID NO: 34), AAV5 (amino acids 561-562 of the AAV5 capsid, SEQ ID NO: 35), AAV6 (amino acids 562-563 of the AAV6 capsid, SEQ ID NO: 36), AAV6 (amino acids 563-564 of the AAV6 capsid, SEQ ID NO: 37), AAV6 (amino acids 564-565 of the AAV6 capsid, SEQ ID NO: 38), AAV6 (amino acids 565-566 of the AAV6 capsid, SEQ ID NO: 39), AAV6 (amino acids 566-567 of the AAV6 capsid, SEQ ID NO: 40), AAV6 (amino acids 567-568 of the AAV6 capsid, SEQ ID NO: 41), AAV6 (amino acids 568-569 of the A FIG. 1 shows an alignment of specific regions of the amino acid sequences of various AAV capsid proteins: AAV3B (amino acids 564-613 of the AAV3B capsid; SEQ ID NO: 32), AAV3B (amino acids 564-613 of the AAV3B capsid; SEQ ID NO: 33), AAV2 (amino acids 566-615 of the AAV2 capsid; SEQ ID NO: 34), and AAV1 (amino acids 566-615 of the AAV1 capsid; SEQ ID NO: 35), which focuses on the region HVRVIII into which (based on structural analysis) a targeting peptide may be inserted.

[0035] In certain embodiments, the parent capsid is modified to comprise an "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the parent capsid is a clade F or any one of the following: (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer of at least 6, at least 7, or a full-length n-mer of any one of the following: AAV (e.g., AAVhu68, AAV9, AAVhu31, AAVhu32, AAVhu95, AAVhu96), clade E (e.g., AAV8), or clade A AAV (e.g., AAV1, AAVrh91) capsid, or a non-parvovirus capsid (e.g., herpes simplex virus, etc.), tailored to enhance expression and / or otherwise modulate targeting to muscle cells (e.g., cardiac (cardiac cells) or skeletal (gastrocnemius) cells). See, for example, WO2020 / 223231 (rh91, including a table with deamidation patterns), published November 5, 2020; U.S. Provisional Patent Application No. 63 / 065,616, filed August 14, 2020; See U.S. Provisional Patent Application No. 63 / 109,734, filed on May 4, 2020, and International Patent Application No. PCT / US21 / 45945, filed on August 13, 2021, and now published as WO2022 / 036220, all of which are incorporated by reference in their entireties. In certain embodiments, AAV capsids with reduced capsid deamidation may be selected. See, e.g., PCT / US19 / 19804 and PCT / US18 / 19861, both filed on February 27, 2019, and incorporated by reference in their entireties.

[0036] In certain embodiments, the mutant capsids described herein are characterized by having deamidation patterns similar to their parent AAV, as described, for example, in US2020 / 0056159 published February 20, 2020 (AAVhu68, highly deamidated at N57, N329, N452, and N512), with small, optional amounts of deamidation), and US2020 / 0407750 published December 31, 2020 (AAV9, highly deamidated at N57, N329, N452, and N512), each of which is incorporated by reference in its entirety.

[0037] In certain embodiments, a recombinant adeno-associated virus particle (rAAV) comprises: (a) an adeno-associated virus (AAV) capsid comprising a VP1 protein, a VP2 protein, and a VP3 protein, wherein the capsid protein has an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, the exogenous targeting peptide comprising "Xn-n-mer-Xm"; (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGD A recombinant adeno-associated virus particle (rAAV) is provided, comprising: (i) an adeno-associated virus (AAV) capsid, wherein the AAV capsid is an n-mer sequence of at least 6, at least 7, or a full-length n-mer of any one of LHGY (SEQ ID NO: 22), RGDYS™ (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer; and (ii) 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 the control of a sequence that directs expression of the nucleic acid sequence encoding the gene product.In certain embodiments, the rAAV comprises a capsid protein having an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), and / or any combination thereof. In certain embodiments, rAAVs comprising capsid proteins comprising exogenous targeting peptides described herein (i.e., engineered rAAV capsids) have greater muscle specificity, targeting, and / or efficacy compared to the parent rAAV capsid.

[0038] In certain embodiments, provided herein is a recombinant adeno-associated virus 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 the NDVRAVS (SEQ ID NO: 36) sequence. In certain embodiments, the rAAV comprises an AAV2 capsid, and the AAV2 capsid protein comprises at least one or more of an exogenous targeting peptide comprising "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), PYVRGDP (SEQ ID NO: 15), PYVRGDP (SEQ ID NO: 16), PYVRGDP (SEQ ID NO: 17), PYVRGDP (SEQ ID NO: 18), PYVRGDP (SEQ ID NO: 19), PYVRGDP (SEQ ID NO: 20), PYVRGDP (SEQ ID NO: 21), PYVRGDP (SEQ ID NO: 22), PYVRGDP (SEQ ID NO: 23), PYVRGDP (SEQ ID NO: 24), PYVRGDP (SEQ ID NO: 25), PYVRGDP (SEQ ID NO: 26), PYVRGDP (SEQ ID NO: 27), PYVRGDP (SEQ ID NO: 28), PYVRGDP (SEQ ID NO: 29), PYVRGDP (SEQ ID NO: 30), PYVRGDP (SEQ ID NO: 31), PYVRGDP (SEQ ID NO: 32), PYVRGDP (SEQ ID NO: 33), PYVRGDP (SEQ ID NO: 34), PYVRGDP (SEQ (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid;

[0039] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous targeting peptides comprising "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), PYVRGDP (SEQ ID NO: 15), PYVRGDP (SEQ ID NO: 16), PYVRGDP (SEQ ID NO: 17), PYVRGDP (SEQ ID NO: 18), PYVRGDP (SEQ ID NO: 19), PYVRGDP (SEQ ID NO: 20), PYVRGDP (SEQ ID NO: 21), PYVRGDP (SEQ ID NO: 22), PYVRGDP (SEQ ID NO: 23), PYVRGDP (SEQ ID NO: 24), PYVRGDP (SEQ ID NO: 25), PYVRGDP (SEQ ID NO: 26), PYVRGDP (SEQ ID NO: 27), PYVRGDP (SEQ ID NO: 28), PYVRGDP (SEQ ID NO: 29), PYVRGDP (SEQ ID NO: 30), PYVRGDP (SEQ ID NO: 31), PYVRGDP (SEQ ID NO: 32), PYVRGDP (SEQ ID NO: 33), PYVRGDP (SEQ ID NO: 34), PYVRGDP (SEQ ID NO: ), VVRGDPQ (SEQ ID NO:15), PTRGDVK (SEQ ID NO:16), VVQRGDV (SEQ ID NO:17), QHRGDTQ (SEQ ID NO:18), QIRGDLR (SEQ ID NO:19), RGDYAQV (SEQ ID NO:20), IGRGDPN (SEQ ID NO:21), RGDLHGY (SEQ ID NO:22), RGDYSTM (SEQ ID NO:23), or PYQRGDH (SEQ ID NO:24), or an n-mer sequence of at least 6, at least 7, or the full length 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 AAV9 capsid, wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, and the exogenous targeting peptide is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13). , PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), PYQRGDH (SEQ ID NO: 24), and / or any combination thereof.In certain embodiments, the rAAV comprises an AAV9 capsid, the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, and the exogenous targeting peptide comprises IIRGDPA (SEQ ID NO: 1). In certain embodiments, the rAAV comprises an AAV9 capsid, the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, and the exogenous targeting peptide comprises AVIRGDV (SEQ ID NO: 2).

[0040] In certain embodiments, the rAAV comprises an AAVhu68 capsid having an AAVhu68 capsid protein comprising one or more of an exogenous targeting peptide comprising "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), PYVRGDP (SEQ ID NO: 15), PYVRGDP (SEQ ID NO: 16), PYVRGDP (SEQ ID NO: 17), PYVRGDP (SEQ ID NO: 18), PYVRGDP (SEQ ID NO: 19), PYVRGDP (SEQ ID NO: 20), PYVRGDP (SEQ ID NO: 21), PYVRGDP (SEQ ID NO: 22), PYVRGDP (SEQ ID NO: 23), PYVRGDP (SEQ ID NO: 24), PYVRGDP (SEQ ID NO: 25), PYVRGDP (SEQ ID NO: 26), PYVRGDP (SEQ ID NO: 27), PYVRGDP (SEQ ID NO: 28), PYVRGDP (SEQ ID NO: 29), PYVRGDP (SEQ ID NO: 30), PYVRGDP (SEQ ID NO: 31), PYVRGDP (SEQ ID NO: 32), PYVRGDP (SEQ ID NO: 33), PYVRGDP (SEQ ID NO: 34), PYVRGDP (S (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 protein comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), PYQRGDH (SEQ ID NO: 24), and / or any combination thereof.

[0041] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-IIRGDPA (SEQ ID NO: 1)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising IIRGDPA (SEQ ID NO: 1).

[0042] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-AVIRGDV (SEQ ID NO: 2)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising AVIRGDV (SEQ ID NO: 2).

[0043] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-IVRGDPA (SEQ ID NO: 8)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising IVRGDPA (SEQ ID NO: 8).

[0044] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-MIRGDVK (SEQ ID NO: 9)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising MIRGDVK (SEQ ID NO: 9).

[0045] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising at least one or more of an exogenous peptide comprising "Xn-AQHRGDV (SEQ ID NO: 10)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising at least one or more of an exogenous peptide comprising AQHRGDV (SEQ ID NO: 10).

[0046] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-VSRGDPN (SEQ ID NO: 11)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising VSRGDPN (SEQ ID NO: 11).

[0047] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-VSRGDPA (SEQ ID NO: 12)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising VSRGDPA (SEQ ID NO: 12).

[0048] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-PLVRGDI (SEQ ID NO: 13)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising PLVRGDI (SEQ ID NO: 13).

[0049] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-PYVRGDP (SEQ ID NO: 14)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising PYVRGDP (SEQ ID NO: 14).

[0050] In certain embodiments, the rAAV has an AAV9 capsid protein that includes one or more exogenous peptides that include "Xn-VVRGDPQ (SEQ ID NO: 15)-Xm." wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising VVRGDPQ (SEQ ID NO: 15).

[0051] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-PTRGDVK (SEQ ID NO: 16)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising PTRGDVK (SEQ ID NO: 16).

[0052] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-VVQRGDV (SEQ ID NO: 17)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising VVQRGDV (SEQ ID NO: 17).

[0053] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-QHRGDTQ (SEQ ID NO: 18)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising QHRGDTQ (SEQ ID NO: 18).

[0054] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-QIRGDLR (SEQ ID NO: 19)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising QIRGDLR (SEQ ID NO: 19).

[0055] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-RGDYAQV (SEQ ID NO: 20)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising RGDYAQV (SEQ ID NO: 20).

[0056] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-IGRGDPN (SEQ ID NO: 21)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. or three amino acid residues. In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising IGRGDPN (SEQ ID NO: 21).

[0057] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-RGDLHGY (SEQ ID NO: 22)-Xm", where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising RGDLHGY (SEQ ID NO: 22).

[0058] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-RGDYSTM (SEQ ID NO: 23)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising RGDYSTM (SEQ ID NO: 23).

[0059] In certain embodiments, the rAAV comprises an AAV9 capsid having an AAV9 capsid protein comprising one or more exogenous peptides comprising "Xn-PYQRGDH (SEQ ID NO: 24)-Xm," where Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and 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 an AAV9 capsid protein comprising one or more exogenous peptides comprising PYQRGDH (SEQ ID NO: 24).

[0060] In certain embodiments, the rAAV comprises a mutant AAV9-IIRGDPA capsid or a mutant AAVhu68-IIRGDPA capsid, each comprising mutant VP1, VP2, and VP3 proteins, each comprising a heterogeneous population of proteins containing an IIRGDPA (SEQ ID NO: 1) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-IIRGDPA (or AAVhu68-IIRGDPA), wherein each AAV VP1, AAV VP2, and AAV VP3 protein is a heterogeneous population and comprises the AAV VP3 region of SEQ ID NO: 73 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises a heterogeneous population of each of capsid proteins having deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the percentage of deamidation at one or more of these positions is greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0061] The percentage of deamidation at each highly deamidated position may differ from one another within a rAAV capsid.For example, the percentage of deamidation at N57 of an AAV capsid may differ from the percentage of deamidation at each of the other highly deamidated residues, which may have a higher (or lower) percentage of deamidation.Similarly, the percentage of deamidation at N329, N452, and / or N512 may differ from one another. Each may differ from the others. In certain embodiments, the percentage of deamidation for each highly deamidated position is determined for all VP proteins within a single rAAV capsid or stock of rAAV capsids. In certain embodiments, the parent clade F capsid contains a VP protein that is highly deamidated at all four of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween. In certain embodiments, there may be little deamidation at other positions within the capsid. In certain embodiments, there are no post-translational modifications within the peptide insert of the mutant capsid (e.g., AVIRGDV). In certain embodiments, the rAAV comprises a mutant AAV9-AVIRGDV capsid or a mutant AAVhu68-AVIRGDV capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an AVIRGDV (SEQ ID NO: 2) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-AVIRGDV (or AAVhu68-AVIRGDV)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 75 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). A protein that further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions.In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0062] In certain embodiments, the rAAV comprises a mutant AAV9-IVRGDPA capsid or a mutant AAVhu68-IVRGDPA capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an IVRGDPA (SEQ ID NO: 8) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-IVRGDPA (or AAVhu68-IVRGDPA)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 77 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween. In certain embodiments, the rAAV comprises a mutant AAV9-MIRGDVK capsid or a mutant AAVhu68-MIRGDVK capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing the MIRGDVK (SEQ ID NO: 9) peptide insert. In certain embodiments, the proteins contain three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512. The rAAV is further characterized by having deamidation at positions N57 (VP1 only), N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has a capsid comprising AAV9-MIRGDVK (or AAVhu68-MIRGDVK)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins, comprising the VP3 region of SEQ ID NO:79 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO:25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the residue positions of the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0063] In certain embodiments, the rAAV comprises a mutant AAV9-AQHRGDV capsid or a mutant AAVhu68-AQHRGDV capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, with a heterogeneous population of proteins comprising the AQHRGDV (SEQ ID NO: 10) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-AQHRGDV (or AAVhu68-AQHRGDV)-VP proteins, with each VP1, VP2, and VP3 protein being a heterogeneous population and comprising the VP3 region of SEQ ID NO: 81 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0064] In certain embodiments, the rAAV comprises a mutant AAV9-VSRGDPN capsid or a mutant AAVhu68-VSRGDPN capsid, each comprising mutant VP1, VP2, and VP3 proteins, each comprising a heterogeneous population of proteins containing a VSRGDPN (SEQ ID NO: 11) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-VSRGDPN (or AAVhu68-VSRGDPN)-VP proteins, each comprising a heterogeneous population of VP1, VP2, and VP3 proteins, each comprising the VP3 region of SEQ ID NO: 83 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0065] In certain embodiments, the rAAV comprises mutant VP1, mutant VP2, and mutant V The rAAVs comprise mutant AAV9-VSRGDPA capsids or mutant AAVhu68-VSRGDPA capsids containing the P3 protein, each having a heterogeneous population of proteins containing a VSRGDPA (SEQ ID NO: 12) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAVs have capsids containing AAV9-VSRGDPA (or AAVhu68-VSRGDPA)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins, and which comprise the VP3 region of SEQ ID NO: 85 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0066] In certain embodiments, the rAAV comprises a mutant AAV9-PLVRGDI capsid or a mutant AAVhu68-PLVRGDI capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a PLVRGDI (SEQ ID NO: 13) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-PLVRGDI (or AAVhu68-PLVRGDI)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 87 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0067] In certain embodiments, rAAVs comprise mutant AAV9-PYVRGDP capsids or mutant AAVhu68-PYVRGDP capsids, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each comprising a heterogeneous population of proteins containing a PYVRGDP (SEQ ID NO: 14) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, rAAVs comprise capsids comprising AAV9-PYVRGDP (or AAVhu68-PYVRGDP)-VP proteins, each comprising a heterogeneous population of VP1, VP2, and VP3 proteins, each comprising the VP3 region of SEQ ID NO: 89 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, More than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or between about 70% and about 100%, or any value therebetween.

[0068] In certain embodiments, the rAAV comprises a mutant AAV9-VVRGDPQ capsid or a mutant AAVhu68-VVRGDPQ capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a VVRGDPQ (SEQ ID NO: 15) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-VVRGDPQ (or AAVhu68-VVRGDPQ)-VP proteins, wherein each VP1, VP2, and VP3 protein is a heterogeneous population and comprises the VP3 region of SEQ ID NO: 91 (approximately amino acid 203 to approximately amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0069] In certain embodiments, the rAAV comprises a mutant AAV9-VVQRGDV capsid or a mutant AAVhu68-VVQRGDV capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a VVQRGDV (SEQ ID NO: 17) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-VVQRGDV (or AAVhu68-VVQRGDV)-VP proteins, wherein each VP1, VP2, and VP3 protein is a heterogeneous population and comprises the VP3 region of SEQ ID NO: 93 (approximately amino acid 203 to approximately amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0070] In certain embodiments, the rAAV comprises a mutant AAV9-QHRGDTQ capsid or a mutant AAVhu68-QHRGDTQ capsid, each comprising mutant VP1, VP2, and VP3 proteins, each comprising a heterogeneous population of proteins containing a QHRGDTQ (SEQ ID NO: 18) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and / or N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-QHRGDTQ (or AAVhu68-QHRGDTQ)-VP proteins, each comprising a heterogeneous population of VP1, VP2, and VP3 proteins, each comprising the VP3 region of SEQ ID NO: 95 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). Based on the position of the residues in the parent capsid, N57 (VP1 only), N32 and / or N512. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0071] In certain embodiments, the rAAV comprises a mutant AAV9-QIRGDLR capsid or a mutant AAVhu68-QIRGDLR capsid, each comprising mutant VP1, VP2, and VP3 proteins, each having a heterogeneous population of proteins containing a QIRGDLR (SEQ ID NO: 19) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-QIRGDLR (or AAVhu68-QIRGDLR)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins, and which comprises the VP3 region of SEQ ID NO: 97 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0072] In certain embodiments, the rAAV comprises a mutant AAV9-RGDYAQV capsid or a mutant AAVhu68-RGDYAQV capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an RGDYAQV (SEQ ID NO: 20) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-RGDYAQV (or AAVhu68-RGDYAQV)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 99 (approximately amino acid 203 to approximately amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0073] In certain embodiments, the rAAV comprises a mutant AAV9-IGRGDPN capsid or a mutant AAVhu68-IGRGDPN capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, with a heterogeneous population of proteins comprising an IGRGDPN (SEQ ID NO: 21) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV comprises an AAV9-IGRGGDPN capsid or a mutant AAVhu68-IGRGDPN capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, with a heterogeneous population of proteins comprising an IGRGDPN (SEQ ID NO: 21) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. The capsid comprises a capsid comprising DPN (or AAVhu68-IGRGDPN)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins, and which comprises the VP3 region of SEQ ID NO:101 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO:25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the residue positions of the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0074] In certain embodiments, the rAAV comprises a mutant AAV9-RGDLHGY capsid or a mutant AAVhu68-RGDLHGY capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an RGDLHGY (SEQ ID NO: 22) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-RGDLHGY (or AAVhu68-RGDLHGY)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 103 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0075] In certain embodiments, the rAAV comprises a mutant AAV9-RGDYS™ capsid or a mutant AAVhu68-RGDYS™ capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an RGDYS™ (SEQ ID NO: 23) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids comprising AAV9-RGDYS™ (or AAVhu68-RGDYS™)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins and comprises the VP3 region of SEQ ID NO: 105 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The protein further comprises deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0076] In certain embodiments, the rAAV comprises a mutant AAV9-PYQRGDH capsid or a mutant AAVhu68-PYQRGDH capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each comprising the PYQRGDH (SEQ ID NO: 24) peptide. The rAAV has a heterogeneous population of proteins containing deamidated asparagines at positions N57, N329, N452, and N512. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the rAAV has capsids containing AAV9-PYQRGDH (or AAVhu68-PYQRGDH)-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins, comprising the VP3 region of SEQ ID NO: 107 (from about amino acid 203 to about amino acid 736, based on the residue positions of SEQ ID NO: 25 (AAV9)). The proteins further comprise deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the residue positions of the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions, hi certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0077] In certain embodiments, the rAAV comprises a mutant AAV9-GQVRVGV capsid or a mutant AAVhu68-GQVRVGV capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a GQVRVGV (SEQ ID NO: 3) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has a mutant capsid comprising AAV-GQVRVGV-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid contains VP proteins that are highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0078] In certain embodiments, rAAV comprises mutant AAV9-MDAHYVR capsids or mutant AAVhu68-MDAHYVR capsids comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing the MDAHYVR (SEQ ID NO: 4) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has mutant capsids comprising AAV-MDAHYVR-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residues in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions, hi certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0079] In certain embodiments, the rAAV comprises mutant VP1, mutant VP2, and mutant V The present invention also includes mutant AAV9-TQAVPLK capsids or mutant AAVhu68-TQAVPLK capsids containing the P3 protein, each having a heterogeneous population of proteins containing the TQAVPLK (SEQ ID NO: 5) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 have mutant capsids containing AAV-TQAVPLK-VP proteins, each a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residues in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions, hi certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0080] In certain embodiments, the rAAV comprises a mutant AAV9-VVHQTGL capsid or a mutant AAVhu68-VVHQTGL capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a VVHQTGL (SEQ ID NO: 6) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has a mutant capsid comprising AAV-VVHQTGL-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the residue positions in the parent capsid. In certain embodiments, the capsid contains VP proteins that are highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0081] In certain embodiments, rAAV comprises a mutant AAV9-MAISRER capsid or a mutant AAVhu68-MAISRER capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each comprising a heterogeneous population of proteins containing a MAISRER (SEQ ID NO: 7) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 comprises a mutant capsid comprising an AAV-MAISRER-VP protein, each comprising a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residues in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions, hi certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0082] In certain embodiments, the rAAV comprises a mutant AAV9-IVRGDPA capsid or a mutant AAVhu68-IVRGDPA capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an IVRGDPA (SEQ ID NO: 8) peptide insert. In certain embodiments, the proteins are further characterized by three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, the mutant rAAV9 or mutant rAAVhu68 comprises a mutant capsid comprising AAV-IVRGDPA-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins having deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residues in the parent capsid. In certain embodiments, the capsid comprises a VP protein that is highly deamidated at all of these positions, hi certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0083] In certain embodiments, the rAAV comprises a mutant AAV9-MIRGDVK capsid or a mutant AAVhu68-MIRGDVK capsid comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a MIRGDVK (SEQ ID NO: 9) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has a mutant capsid comprising AAV-MIRGDVK-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid contains VP proteins that are highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0084] In certain embodiments, the rAAV comprises a mutant AAV9-PTRGDVK capsid or a mutant AAVhu68-PTRGDVK capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing a PTRGDVK (SEQ ID NO: 16) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has a mutant capsid comprising AAV-PTRGDVK-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid contains VP proteins that are highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0085] In certain embodiments, the rAAV comprises a mutant AAV9-RGDYREL capsid or a mutant AAVhu68-RGDYREL capsid, each comprising mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogeneous population of proteins containing an RGDYREL (SEQ ID NO: 128) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines at positions N57, N329, N452, and N512, as well as optional deamidation at other positions within the parent capsid sequence. In certain embodiments, mutant rAAV9 or mutant rAAVhu68 has a mutant capsid comprising AAV-RGDYREL-VP proteins, each of which is a heterogeneous population of VP1, VP2, and VP3 proteins with deamidation at about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the position of the residue in the parent capsid. In certain embodiments, the capsid contains VP proteins that are highly deamidated at all of these positions. In certain embodiments, the percentage of deamidation at one or more of these highly deamidated positions is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or between about 70% and about 100%, or any value therebetween.

[0086] In certain embodiments, a rAAV comprises an AAV capsid, wherein the AAV capsid protein comprises an exogenous peptide immediately preceded by "AQ". In certain embodiments, a rAAV comprises an AAV capsid, wherein the AAV capsid protein comprises an exogenous peptide, where the exogenous peptide is adjacent to "AQ" (e.g., "AQ-IIRGDPA (SEQ ID NO: 1)", "AQ-IIRGDPA (SEQ ID NO: 1)-AQ", or "IIRGDPA (SEQ ID NO: 1)-AQ"). In certain embodiments, a rAAV comprises an AAV capsid, wherein the AAV capsid protein comprises an exogenous peptide immediately preceded by native residues of the parent AAV, which may be unmodified at the amino (N-) terminus and / or carboxy (COO-) terminus. In certain embodiments, the rAAV comprises an AAV capsid, wherein the AAV capsid protein comprises an exogenous peptide immediately preceded by a native residue of the parent AAV, which may be mutated at the amino (N-) and / or carboxy (COO-) terminus. In certain embodiments, where the parent capsid is an AAV9 capsid or other clade F capsid, the AAV9 parent capsid or other clade F parent capsid is unmodified at the residues adjacent to 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 adjacent to "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 an exogenous peptide, wherein the exogenous peptide is adjacent to "AQA" at the carboxy (COO-) terminus. In certain embodiments, where 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 residues adjacent to the inserted exogenous targeting peptide. In certain embodiments, the rAAV comprises an AAV capsid, wherein the AAV capsid protein comprises an exogenous peptide, and the exogenous peptide is adjacent to a mutated trimer "ENT" at the amino (N-) terminus of the exogenous peptide.In certain embodiments, the rAAV comprises an AAV capsid, the AAV capsid protein comprising an exogenous peptide, the exogenous peptide flanked at the amino (N-) terminus of the exogenous peptide by mutant trimers "SHQ", "SWQ", "SAI", "GAQ", "FAQ", "QAQ", "AAQ", "SGQ", "SGM". In certain embodiments, the rAAV comprises an AAV capsid, the AAV capsid protein comprising an exogenous peptide, the exogenous peptide flanked at the carboxy (COO-) terminus by mutant trimers "QQA", "NQA", "AMA", "AQC", "GQA", "ARA", "GRA". In other embodiments, for example, when a non-clade F parent AAV is selected and / or to reduce the number of AAV residues inserted, flanking residues may be modified.

[0087] In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SHQIIRGDPAQQA" (SEQ ID NO: 39). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SHQIIRGDPAQQA" (SEQ ID NO: 39). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQIIRGDMQAQA" (SEQ ID NO: 40). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIIRGDMQAQA" (SEQ ID NO: 40). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SWQITRGDPAAQA" (SEQ ID NO: 41). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SWQITRGDPAAQA" (SEQ ID NO: 41). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SWQITRGDPAAQA" (SEQ ID NO: 41). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAIIIRGDPHAQA" (SEQ ID NO: 42). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAIIIRGDPHAQA" (SEQ ID NO: 42).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "GAQIIRGDPQAQA" (SEQ ID NO: 43). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "GAQIIRGDPQAQA" (SEQ ID NO: 43). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "GAQIIRGDPAQQA" (SEQ ID NO: 44). In certain embodiments, the rAAV comprises an AAV capsid having a capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "GAQIIRGDPAQQA" (SEQ ID NO: 44). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence of "FAQIIRGDQAAQA" (SEQ ID NO: 45). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "FAQIIRGDQAAQA" (SEQ ID NO: 45). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQIIRGDNANQA" (SEQ ID NO: 46). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIIRGDNANQA" (SEQ ID NO: 46). In certain embodiments, the rAAV is modified.In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "QAQIIRGDPQAQA" (SEQ ID NO: 47). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "QAQIIRGDPQAQA" (SEQ ID NO: 47). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "AAQYIRGDPAAQA" (SEQ ID NO: 48). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "AAQYIRGDPAAQA" (SEQ ID NO: 48). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence of "SGQVIRGDPAAQA" (SEQ ID NO: 49). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SGQVIRGDPAAQA" (SEQ ID NO: 49). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQIGRGDPQAQA" (SEQ ID NO: 50). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIGRGDPQAQA" (SEQ ID NO: 50).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQIIRGDGAAMA" (SEQ ID NO: 51). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIIRGDGAAMA" (SEQ ID NO: 51). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SGMIIRGDPAAQA" (SEQ ID NO: 52). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SGMIIRGDPAAQA" (SEQ ID NO: 52). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence of "SAQIIRGDNAAQC" (SEQ ID NO: 53). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIIRGDNAAQC" (SEQ ID NO: 53). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "QAQIIRGDPAAQA" (SEQ ID NO: 54). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "QAQIIRGDPAAQA" (SEQ ID NO: 54).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide adjacent to modified amino acid residues, wherein the capsid protein is , "SAQIIRGDPAAQA" (SEQ ID NO: 55). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQIIRGDPAAQA" (SEQ ID NO: 55). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAYAVARGDVAQA" (SEQ ID NO: 56). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAYAVARGDVAQA" (SEQ ID NO: 56). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQQVIRGDVGQA" (SEQ ID NO: 57). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQQVIRGDVGQA" (SEQ ID NO: 57). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQAGIRGDVARA" (SEQ ID NO: 58). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQAGIRGDVARA" (SEQ ID NO: 58).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "GAQAAIRGDVAQA" (SEQ ID NO: 59). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "GAQAAIRGDVAQA" (SEQ ID NO: 59). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SWQAVVRGDVAQA" (SEQ ID NO: 60). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SWQAVVRGDVAQA" (SEQ ID NO: 60). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence of "QAQAVIRGDVAQA" (SEQ ID NO: 61). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "QAQAVIRGDVAQA" (SEQ ID NO: 61). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQAVIRGDVGRA" (SEQ ID NO: 62). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQAVIRGDVGRA" (SEQ ID NO: 62).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide adjacent to modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SGQAVIRGDVARA" (SEQ ID NO: 63). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SGQAVIRGDVARA" (SEQ ID NO: 63). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAYAVIRGDVAQA" (SEQ ID NO: 64). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAYAVIRGDVAQA" (SEQ ID NO: 64). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SWQAAIRGDVAQA" (SEQ ID NO: 65). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SWQAAIRGDVAQA" (SEQ ID NO: 65). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQAYIRGDVAQA" (SEQ ID NO: 66). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQAYIRGDVAQA" (SEQ ID NO: 66). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide adjacent to modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAMAVIRGDVAQA" (SEQ ID NO: 67).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAMAVIRGDVAQA" (SEQ ID NO: 67). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence of "SAHAVIRGDVAQA" (SEQ ID NO: 68). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAHAVIRGDVAQA" (SEQ ID NO: 68). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQAVVRGDVAQA" (SEQ ID NO: 69). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SAQAVVRGDVAQA" (SEQ ID NO: 69). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SHQAVIRGDVAQA" (SEQ ID NO: 70). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises an amino acid sequence at least 99% identical to "SHQAVIRGDVAQA" (SEQ ID NO: 70). In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, wherein the capsid protein comprises the amino acid sequence of "SAQAVIRGDVAQA" (SEQ ID NO: 81).In certain embodiments, the rAAV comprises an AAV capsid having an AAV capsid protein comprising an exogenous peptide flanked by modified amino acid residues, the capsid protein comprising the exogenous peptide. The protein comprises an amino acid sequence that is at least 99% identical to "SAQAVIRGDVAQA" (SEQ ID NO: 81).

[0088] In certain embodiments, capsids from the AAV of the F-class, such as AAVhu68 or AAV9, are selected as parent capsids.Methods for generating vectors with AAV9 capsids or AAVhu68 capsids, and / or chimeric capsids derived from AAV9 are described.See, for example, US 7,906,111, which is incorporated herein by reference.See also US Provisional Patent Application No. 63 / 093,275, filed October 18, 2020, which is incorporated herein by reference. Other AAV serotypes that transduce nasal cells or another suitable target (e.g., muscle or lung) can be selected as the source of capsid for AAV viral vectors, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, rh8, AAVrh32.33 (see, e.g., U.S. Published Patent Application No. 2007-0036760-A1, U.S. Published Patent Application No. 2009-0197338-A1, and EP 1310571). Also, recombinant AAVs such as those described in WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent Nos. 7,790,449 and 7,282,199 (AAV8), WO2005 / 033321 (AAV9), and WO2006 / 110689, or those yet to be discovered, or based thereon, can be used as a source of AAV capsids. See, for example, WO2020 / 223232A1 (AAV rh90), WO2020 / 223231A1, International Application No. PCT / US21 / 45945 filed August 13, 2021 (AAV rh91), and WO2020 / 223236A1 (AAV rh92, AAV rh93, AAV rh91.93), which are incorporated by reference in their entireties. These documents also describe other AAVs that can be selected to generate AAVs and are incorporated by reference. In some embodiments, AAV capsids (caps) for use in viral vectors can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV caps or their encoding nucleic acids.In some embodiments, the AAV capsid is a chimera comprising two, three, four, or more domains of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vpl, Vp2, and Vp3 (also referred to as vp1, vp2, vp3, or VP1, VP2, VP3) monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned caps.

[0089] In certain embodiments, mutant AAV capsids are produced by manipulating a nucleic acid sequence encoding an exogenous targeting peptide insert into the AAV VP1 coding sequence. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:108, or a sequence encoding SEQ ID NO:1 (IIRGDPA) that is 95% to 100% or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:109, or a sequence encoding SEQ ID NO:2 (AVIRGDV) that is 95% to 100% or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:110, or a sequence encoding SEQ ID NO:8 (IVRGDPA) that is 95% to 100% or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:111, or a sequence encoding SEQ ID NO:9 (MIRGDVK) that is 95% to 100% or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 112, or a sequence that encodes or is 95% to 100% or at least 99% identical to SEQ ID NO: 10 (AQHRGDV). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is the sequence SEQ ID NO:113, or a sequence that encodes SEQ ID NO:11 (VSRGDPN), and is 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:114, or a sequence that encodes SEQ ID NO:12 (VSRGDPA), and is 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:115, or a sequence that encodes SEQ ID NO:13 (PLVRGDI), and is 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:116, or a sequence that encodes SEQ ID NO:14 (PYVRGDP), and is 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:117, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO:15 (VVRGDPQ). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:118, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO:17 (VVQRGDV). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:119, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO:18 (QHRGDTQ). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO:120, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO:19 (QIRGDLR). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 121, or a sequence that encodes and is 95% to 100% identical or at least 99% identical to SEQ ID NO: 20 (RGDYAQV). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 122, or a sequence that encodes and is 95% to 100% identical or at least 99% identical to SEQ ID NO: 21 (IGRGDPN).In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 123, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO: 22 (RGDLHGY). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 124, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO: 23 (RGDYSTM). In certain embodiments, the coding sequence of the exogenous targeting peptide insert is SEQ ID NO: 125, or a sequence that encodes and is 95% to 100% or at least 99% identical to SEQ ID NO: 24 (PYQRGDH).

[0090] In certain embodiments, the exogenous peptide is inserted between amino acids 588 and 589 in the AAVhu68 capsid (based on the amino acid sequence of SEQ ID NO: 26). In other embodiments, the exogenous peptide is inserted between amino acids 588 and 589 in the AAV9 capsid (based on the amino acid sequence of SEQ ID NO: 25). Still other suitable locations for these insertions may be determined. In still other embodiments, these peptides may be used in other vectors or compositions for targeting.

[0091] In certain embodiments, the coding sequence of a mutant AAV9 capsid having an exogenous targeting peptide inserted into the hypervariable region between amino acids 588 and 589 in the AAV9 parent capsid is SEQ ID NO:72 (IIRGDPA), SEQ ID NO:74 (AVIRGDV), SEQ ID NO:76 (IVRGDPA), SEQ ID NO:78 (MIRGDVK), SEQ ID NO:80 (AQHRGDV), SEQ ID NO:82 (VSRGDPN), SEQ ID NO:84 (VSRGDPA), SEQ ID NO:86 (PLVRGDI), SEQ ID NO:88 (PYVRGDP), SEQ ID NO:90 (VVRGDPQ), SEQ ID NO:92 (VVQRGDV), SEQ ID NO:94 (QHRGDTQ), SEQ ID NO:96 (QIRGDLR), SEQ ID NO:98 (RGDYAQV), SEQ ID NO:100 (IGRGDPN), SEQ ID NO:102 (RGDLHGY), SEQ ID NO:104 (RGDYSTM), or SEQ ID NO:106 (PYQRGDH).

[0092] In other embodiments, the mutant AAV9 is any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:73 (IIRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:75 (AVIRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:77 (IVRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:79 (MIRGDVK mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:81 (AQHRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:83 (VSRGDPN mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:85 (VSRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:87 (PLVRGDI mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:89 (PYVRGDP mutant VP1). , any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:91 (VVRGDPQ mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:93 (VVQRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:95 (QHRGDTQ mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:97 (QIRGDLR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:99 (RGDYAQV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:101 (IGRGDPN mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:103 (RGDLHGY mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:105 (RGDYSTM mutant VP1), or any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:407 (PYQRGDH mutant VP1).

[0093] In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:72, or a sequence encoding SEQ ID NO:73 (IIRGDPA mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:74, or a sequence encoding SEQ ID NO:75 (AVIRGDV mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:76, or a sequence encoding SEQ ID NO:77 (IVRGDPA mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:78, or a sequence encoding SEQ ID NO:79 (MIRGDVK mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:80, or a sequence encoding SEQ ID NO:81 (AQHRGDV mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:82, or a sequence encoding SEQ ID NO:83 (VSRGDPN mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:84, or a sequence encoding SEQ ID NO:85 (VSRGDPA mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:86, or a sequence encoding SEQ ID NO:87 (PLVRGDI mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO: 88, or a sequence that is 95% to 100% identical or at least 99% identical to SEQ ID NO: 89 (PYVRGDP mutant VP1).In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:90, or a sequence that is 95% to 100% identical or at least 99% identical to SEQ ID NO:91 (VVRGDPQ mutant VP1). In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO:92, or a sequence that encodes SEQ ID NO:93 (VVQRGDV mutant VP1). The coding sequence of the mutant AAV9 capsid is 95% to 100% identical or at least 99% identical to SEQ ID NO:94, or a sequence encoding SEQ ID NO:95 (QHRGDTQ mutant VP1). In certain embodiments, the coding sequence of the mutant AAV9 capsid is 95% to 100% identical or at least 99% identical to SEQ ID NO:96, or a sequence encoding SEQ ID NO:97 (QIRGDLR mutant VP1). In certain embodiments, the coding sequence of the mutant AAV9 capsid is 95% to 100% identical or at least 99% identical to SEQ ID NO:98, or a sequence encoding SEQ ID NO:99 (RGDYAQV mutant VP1). In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO: 100, or a sequence encoding SEQ ID NO: 101 (IGRGDPN mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO: 102, or a sequence encoding SEQ ID NO: 103 (RGDLHGY mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO: 104, or a sequence encoding SEQ ID NO: 105 (RGDYSTM mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto. In certain embodiments, the coding sequence of the mutant AAV9 capsid is SEQ ID NO: 106, or a sequence encoding SEQ ID NO: 107 (PYQRGDH mutant VP1) that is 95% to 100% identical thereto, or at least 99% identical thereto.

[0094] As used herein, the term "clade" in relation to a group of AAVs refers to a group of AAVs that are phylogenetically related to each other, as determined using a neighbor-joining algorithm based on an alignment of AAV vp1 amino acid sequences with a bootstrap value of at least 75% (out of at least 1000 replicates) and a Poisson-corrected distance measure of 0.05 or less. Neighbor-joining algorithms are described in the literature, 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 a modified Nei-Gojobori method. Using these techniques and computer programs and the sequence of the AAV vp1 capsid protein, one of skill in the art can readily determine whether a selected AAV falls within one of the clades identified herein, another clade, or is outside these clades. See, for example, G Gao, et al., J Virol, 2004 Jun;78(12):6381-6388, which identifies clades A, B, C, D, E, and F and provides the nucleic acid sequences of novel AAVs, GenBank accession numbers AY530553 to AY530629. See also WO2005 / 033321.

[0095] As used herein, an "AAV9 capsid" refers to a self-assembling AAV capsid composed of multiple AAV9 vp proteins. AAV9 vp proteins are typically expressed as alternative splice variants encoded by nucleic acid sequences, which encode the vp1 amino acid sequence of GenBank Accession No. AAS99264. These splice variants result in proteins of different lengths. In certain embodiments, "AAV9 capsid" includes AAVs having an amino acid sequence 99% identical to or 99% identical to AAS99264. See also WO2019 / 168961, published September 6, 2019, which includes Table G, which provides deamidation patterns of AAV9. See also US7906111 and WO2005 / 033321. When specified, "AAV9 variant" refers to any AAV variant described, for example, in WO2016 / 049230, US8 ,927,514, US2015 / 0344911, and US8,734,809.

[0096] rAAVhu68 consists of an AAVhu68 capsid and a vector genome. The AAVhu68 capsid is a collection of a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term "heterogeneous" or any grammatical variant thereof refers to a collection of non-identical components, for example, having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. Also, "Adeno-Associated Virus (AAV) Clade F Vector and See PCT / US2018 / 019992, WO2018 / 160582, entitled "Uses Therefor," which are incorporated herein by reference in their entireties.

[0097] For other recombinant viral vectors, the suitable exposed part of the viral capsid or envelope protein responsible for targeting specificity is selected for the insertion of targeting peptide.For example, in adenovirus, it may be desirable to modify the hexon protein.In lentivirus, the envelope fusion protein can be modified to include one or more copies of targeting motif.For vaccinia virus, the major glycoprotein can be modified to include one or more copies of targeting motif.Preferably, these recombinant viral vectors are replication-defective for safety purposes.

[0098] Expression cassettes and vectors The vector genome sequence packaged in AAV capsid and delivered to host cells typically consists of at least a transgene and its regulatory sequence, and AAV inverted terminal repeats (ITRs). A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a desired polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows the transcription, translation, and / or expression of the transgene in the cells of the target tissue.

[0099] The AAV sequences of the vector typically include cis-acting AAV 5' and AAV 3' inverted terminal repeat (ITR) sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). The ITR sequences are approximately 145 base pairs (bp) in length. Preferably, substantially the entire ITR-encoding sequences are used in the molecule, although some minor modifications of these sequences are tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520-532 (1996)). One example of such a molecule for use in the present invention is a “cis-acting” plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory elements are located within the 5′ and 3′ AAV The capsid is flanked by ITR sequences (also referred to as "AAV 5' ITR", "5' ITR", "AAV 5' ITR", or "5' ITR", "AAV 3' ITR", "3' ITR", "AAV 3' ITR", or "3' ITR"). In one embodiment, the ITRs are from an AAV different from the one supplying the 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 ΔITR, in which the D sequence and terminal separation sites (trs) are deleted, is A specific embodiment has been described. In a specific embodiment, the vector genome contains a 130-base-pair shortened AAV2 ITR, lacking the external A element. Without wishing to be bound by theory, it is believed that the shortened ITR is restored 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. If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0100] In certain embodiments, provided herein is an rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5'-end and / or extreme 3'-end of the nucleic acid molecule, which is a vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule comprising a 5'-AAV ITR, an expression cassette, and a 3'-AAV ITR.

[0101] In certain embodiments, rAAVs comprise a vector genome that includes, from 5' to 3', a nucleic acid molecule comprising: AAV-5'ITR-optional enhancer-promoter-optional intron-coding sequence (e.g., test transgene)-polyadenylation (polyA) signal sequence-AAV3'-ITR. In other embodiments, the orientation of the ITRs may vary from the orientation presented in the vector genome of the nucleic acid (e.g., plasmid) used for production. Thus, in certain embodiments, rAAVs may comprise a vector genome flanked by 3' and 5'AAV ITRs, respectively. In certain embodiments, rAAVs may comprise a vector genome flanked by two 5'AAV ITRs. In certain embodiments, rAAVs may comprise a vector genome flanked by two 3'AAV ITRs. In other embodiments, the rAAVs provided herein may be partially truncated such that the 5'AAV ITR and / or 3'AAV ITR are not detectable in the vector genome packaged in the final rAAV product.

[0102] In addition to the key elements identified above for a recombinant AAV vector, the vector also contains the necessary conventional control elements operably linked to the transgene in a manner that allows for its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that flank the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0103] Regulatory control elements typically include promoter sequences located as part of expression control sequences, for example, between a selected 5' ITR sequence and a coding sequence. Constitutive promoters, regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), tissue-specific promoters, or promoters that respond to physiological cues can be used in the vectors described herein.

[0104] Examples of constitutive promoters suitable for controlling the expression of therapeutic products include, but are not limited to, chicken beta (β)-actin (CB) promoter, CB7 promoter (a promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer and a chicken β-actin promoter, optionally with a spacer sequence, optionally with a chimeric intron comprising a chicken beta-actin intron, and further comprising a chicken beta-actin splicing donor (exon sequence, chicken beta-actin intron) and a rabbit beta-globin splicing acceptor), human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), Simian virus 40 (SV40) early and late promoters, U6 promoter, metallothionein promoter, EFlα promoter, ubiquitin promoter, hypoxanthine phosphoribosyltransferase (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, and β-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)), the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus, and other constitutive promoters known to those skilled in the art. Examples of tissue-specific or cell-specific 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, FoxJ1 (which targets ciliated cells). In other embodiments, the selection of a cardiac-specific promoter may be desirable. See, for example, R.M. Devatiirov, et al., "Human library of cardiac promoters" in and enhancers”, bioRxiv, pp. 1-27, bioRxiv preprint; posted June 15, 2020, which is incorporated herein by reference in its entirety. Preferably, such promoters are of human origin. In other embodiments, the selection of muscle-specific promoters may be desirable, such as the muscle creatine kinase promoter, the human skeletal α-actin promoter, the desmin gene promoter, etc. See, e.g., Skopenkova, VV, Muscle Specific Promoters for Gene Therapy, Acta See Naturae, 2021, Jan-Mar;13(1):47-58, which is incorporated herein by reference in its entirety.

[0105] In certain embodiments, the regulatory sequence comprises one or more of the following: a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signal such as splicing and polyadenylation signal (polyA), a sequence that stabilizes cytoplasmic mRNA, such as woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), and a sequence that enhances 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. For example, such promoters include, but are not limited to, chicken beta-actin (CB) promoter, a hybrid of a cytomegalovirus immediate-early enhancer and a chicken beta-actin promoter (CB7 promoter), a human cytomegalovirus (CMV) promoter, a ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), a U6 promoter, a metallothionein promoter, an EFlα promoter, a ubiquitin promoter, a hypoxanthine phosphoribosyltransferase (HPRT) promoter, a dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991)), an adenosine deaminase promoter, a phosphoglycerol kinase (PGK) promoter, a pyruvate kinase promoter, a phosphoglycerol mutase promoter, and a beta-actin promoter (Lai et al., al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)), the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus, and other constitutive promoters known to those skilled in the art.

[0106] In certain embodiments, the promoter is a tissue- or cell-specific promoter. In certain embodiments, the promoter is a cardiac-specific promoter, such as the cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (α-MHC), or myosin light chain 2 (MLC-2) promoter. See also Pacak, CA, et al., Tissue-specific promoters improve specificity of AAV9-mediated transgene expression following intravascular gene delivery in neonatal mice, Genetic Vaccines and Therapy 2008, 6:13. In certain embodiments, the expression cassette comprises a promoter that is the chicken cardiac troponin T promoter (also referred to as chicken TnT or chTnT). In certain embodiments, the promoter is a hybrid cardiac promoter comprising the cytomegalovirus immediate-early (CMV IE) enhancer and the chicken cardiac troponin T (chicken cTnT or chTnT) promoter. See also International Patent Application No. PCT / US2022 / 082384, filed December 24, 2022, now published WO2023 / 122804, which is incorporated herein by reference in its entirety. In certain embodiments, the enhancer is an alpha-myosin heavy chain enhancer.

[0107] Suitable inducible promoters for controlling the expression of therapeutic products include promoters that respond to exogenous agents (such as pharmacological agents) or physiological cues.These response elements include but are not limited to the hypoxia response element (HRE) that binds to HIF-Iα and β, such as the metal ion response element 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 the heat shock response element described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., pp167-220, 1991).

[0108] 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 a transcription factor activated by a pharmacological agent or, in alternative embodiments, a physiological cue). A promoter system that is not leaky and can be tightly controlled is preferred. Examples of regulatable promoters that are ligand-dependent transcription factor complexes that can be used in the present invention include, but are not limited to, members of the nuclear receptor superfamily that are activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysone, and analogs and mimetics thereof), and rTTA, which is activated by tetracycline. In one aspect of the present invention, the gene switch is an EcR-based gene switch. Examples of such systems include, but are not limited to, those described in U.S. Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006 / 0014711, 2007 / 0161086, and International Published Application No. 01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Patent No. 7,091,038, U.S. Published Patent Applications Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, and International Published Applications Nos. 01 / 70816, 02 / 066612, 02 / 066613, 02 / 066614, 02 / 066615, 02 / 29075, and 2005 / 108617 (each of which is incorporated by reference in its entirety). An example of a nonsteroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression system (New England Biolabs, Ipswich, MA).

[0109] Still other promoter systems include those that utilize tetracycline (tet) response elements (e.g., those described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551) or those described by Lee et al. Response elements may include, but are not limited to, hormone response elements such as those described by Hynes 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), as well as other inducible promoters known in the art. Using such promoters, expression of soluble hACE2 constructs can be regulated, 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)-regulated system (Geneswitch; Wang Y et al., 1994, Proc. Natl. Acad. Sci. USA., 91(17):8180-4; Schillinger et al. al., 2005, Proc. Natl. Acad. Sci. US A. 102(39):13789-94), and the humanized tamoxifen-dep regulatory system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

[0110] In another embodiment, the gene switch is based on heterodimerization of FK506-binding protein (FKBP) with FKBP rapamycin-related protein (FRAP), and is regulated through rapamycin or its non-immunosuppressive analogs. Examples of such systems include, but are not limited to, ARGENT™ transcription technology (ARIAD Pharmaceuticals, Cambridge, Mass.), and those described in U.S. Patent 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. 2009 / 10100535, 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 ... Patent Nos. 5,869,337, 5,871,753, 6,011,018, 6,043,082, 6,046,047, 6,063,625, 6,140,120, 6,165,787, 6,972,193, 6,326,166, 7,008,780, 6,133,456, 6,150,527, 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. Pat. No. 7,196,192, U.S. Pat. No. 6,476,200, U.S. Pat. No. 6,492,106, WO94 / 18347, WO96 / 20951, WO96 / 06097, WO97 / 31898, WO96 / 41865, WO98 / 02441, WO95 / 33052, WO99110508, WO99110510, WO99 / 36553, WO99 / 41258, WO01114387, the ARGENT™ regulated transcription system described in 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 its analogs, called "rapalogs." Examples of suitable rapamycins are provided in the references listed above in connection with the description of the ARGENT™ system. In one embodiment, the molecule is rapamycin (e.g., marketed by Pfizer as Rapamune™). 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), and rapamycin analogs ("rapalogs") that are readily prepared by chemical modification 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, AP26113 (Ariad), AP1510 (Amara, JF, et al., 1997, Proc Natl Acad Sci USA, 94(20):10618-23), AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692, and AP1889, which have a "bump" designed to minimize interaction with endogenous FKBP. Other rapalogs, such as AP23573 [Merck], may also be selected. In certain embodiments, rapamycin or a suitable analog may be delivered locally to AAV-transfected cells in the nasopharynx. This local delivery may be achieved by intranasal injection, via a bolus, cream, or gel, or locally to cells. See U.S. Patent Application No. 2019 / 0216841A1, which is incorporated herein by reference.

[0111] Other suitable enhancers include those appropriate for the desired target tissue indication. In one embodiment, the expression cassette contains one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or different from each other. For example, the enhancer may include a CMV immediate-early enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the duplicated enhancer copies may be separated by one or more sequences. In yet another embodiment, the expression cassette further contains an intron, such as a chicken beta-actin intron. Other suitable introns include those known in the art and described, for example, in WO2011 / 126808. Examples of suitable polyadenylation (polyA) sequences include, for example, rabbit beta globin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. One 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. al, Gene Therapy (2009) 16:605-619).

[0112] In certain embodiments, the expression cassette may include one or more expression enhancers, such as post-transcriptional regulatory elements from woodchuck (WPRE), human (HPRE), ground squirrel (GPRE), or arctic ground squirrel (AGSPRE) hepatitis virus, or synthetic post-transcriptional regulatory elements. These expression-enhancing elements are particularly advantageous when placed in the 3'UTR and can significantly increase mRNA stability and / or protein yield. In certain embodiments, the provided expression cassette includes a regulatory sequence that is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided within the vector genomes described herein and are well known in the art.

[0003] It is well known that WPREs are used in various cell lines (e.g., those described in U.S. 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 mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, e.g., containing a mutation in the start codon of the WHX gene. In certain embodiments, a modified WPRE element is engineered to eliminate expression of the WHX protein, and the modified WPRE is a mutant version containing 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 appears to be sufficient to eliminate expression of a truncated WHX protein, based on sensitive flow cytometry analysis of various human cell lines transduced with lentivirus containing a WPRE-GFP fusion construct (Zanta-Boussif et al., 2009). See also Kingsman SM, Mitrophanous K., & Olsen See JC (2005), Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (WPRE). Gene Ther. 12(1):3-4, and Zanta-Boussif MA, Charrier S., Brice-Ouzet A., Martin S., Opolon P., Thrasher AJ, Hope TJ, & 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 their entireties. In other embodiments, the enhancer is selected from a non-viral source. In certain embodiments, the WPRE sequence is absent.

[0113] An AAV vector genome may contain sequences encoding multiple gene products (e.g., encoding one or more proteins, peptides, miRs, miR seeds, or targets). In certain embodiments, transgenes may be used to correct or ameliorate genetic defects, including those in which a normal gene is expressed at less than normal levels or in which a functional gene product is not expressed. Alternatively, a transgene may provide a cell with a product that is not naturally expressed in the cell type or host. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide to be expressed in the host cell. The present invention further encompasses the use of multiple transgenes. In certain situations, different transgenes 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 subunits is large (e.g., immunoglobulin, platelet-derived growth factor, or dystrophin protein). In certain situations, different transgenes may be used to encode each subunit of a protein (e.g., immunoglobulin domain, immunoglobulin heavy chain, immunoglobulin light chain). In one embodiment, cells produce a multi-subunit protein after infection / transfection with a virus containing each of the different subunits. In another embodiment, the different subunits of the 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 5 kilobases). As an alternative to an IRES, the DNA may be separated by a sequence encoding a 2A peptide that self-cleaves in a post-translational event. For example, ML Donnelly, et al, (Jan 1997) J. Gen. Virol., 78 (Pt1): 13-21, S. Furler, S et al, (June 2001) Gene Ther., 8 (11): 864-873, H. Klump, et al. See, (May 2001) Gene Ther., 8(10):811-817. This 2A peptide is significantly smaller than an IRES, making it well suited for use when space is a limiting factor. More often, when a transgene is large, consists of multiple subunits, or two transgenes are delivered simultaneously, coadministration of rAAVs carrying the desired transgene(s) or subunits allows them to concatenate in vivo to form a single vector genome. In such embodiments, for coexpression in host cells, a first AAV may carry an expression cassette expressing a single transgene, and a second AAV may carry an expression cassette expressing a different transgene. However, the selected transgene may encode any biologically active product or other product (e.g., a product desired for research).

[0114] In addition to the elements identified above for the expression cassette, the vector also contains conventional control elements operably linked to the coding sequence in a manner that allows for the transcription, translation, and / or expression of the encoded product in cells transfected with the plasmid vector or infected with the virus produced by the present invention. Examples of other suitable transgenes are provided herein. As used herein, "operably linked" sequences include both expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0115] Expression control sequences include appropriate enhancers, transcription factors, transcription terminators, promoters, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, e.g., the woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE), sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance secretion of the encoded product.

[0116] In one embodiment, 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 approximates the size of a native AAV genome. Thus, in one embodiment, 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 than about 5.2 kb in size. The size of the vector genome can be manipulated based on the size of regulatory sequences, including promoters, enhancers, introns, polyA, etc. See Wu et al., Mol Ther, Jan 2010, 18(1):80-6 (incorporated herein by reference).

[0117] Thus, in one embodiment, an intron is included in the vector. Suitable introns include chicken beta-actin intron, human beta-globin IVS2 (Kelly et al, Nucleic Acids Research, 43(9):4721-32(2015)), Promega chimeric intron (Almond, B. and Schenborn, ETA Comparison of pCI-neo-Vector and pcDNA4 / HisMax Vector), and hFIX intron. Various introns suitable for use herein are known in the art and include, but are not limited to, those found at bpg.utoledo.edu / ~afedorov / lab / eid.html (incorporated herein by reference). See also Shepelev V., Fedorov A. Advances in See the Exon-Intron Database. Briefings in Bioinformatics 2006, 7:178-185 (hereby incorporated by reference).

[0118] In certain embodiments, the mutant rAAV comprises an expression cassette further comprising at least one miRNA target sequence 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 (including the expression cassette) further comprising the seed, binding site, or complete sequence of at least one miRNA. MicroRNAs (or miRNAs or miRs) are 19-25 nucleotide non-coding RNAs that downregulate gene expression by binding to a nucleic acid target site and either reducing the stability or inhibiting translation of the nucleic acid molecule. In some embodiments, the microRNA sequence comprises a seed region, e.g., a sequence of the region between positions 2 and 8 of the mature microRNA, that has a Watson-Crick sequence that is fully or partially complementary to the miRNA target sequence of the nucleic acid. Such at least one miRNA may also contain a coding sequence for a therapeutic protein, enzyme, or other moiety and be used in combination, including in an expression cassette or vector genome operably linked to the coding sequence. In certain embodiments, the vector genome may contain one to eight miRNA sequences, which may be the same or different. Optionally, the vector genome does not contain any therapeutic transgenes other than the miRNA sequences.

[0119] In some embodiments, the miRNA binding site, e.g., miR183 and / or miR182 binding site, is complementary to an miRNA expressed in DRG (dorsal root ganglion) neurons. In some embodiments, the miR binding site complementary to an miR expressed in DRG neurons comprises, for example, a nucleotide sequence disclosed in WO2020 / 132455 and WO2023 / 087019 (the contents of which are incorporated herein by reference in their entirety).

[0120] As another non-limiting example, the vector genome (expression cassette) may include miR-122 miRNA to regulate, e.g., reduce, expression of a gene product in the liver. In some embodiments, the vector genome (expression cassette) may include miRNA, e.g., miR-142-3p, to regulate, e.g., reduce expression of a gene product in cells or tissues of the hematopoietic lineage, including, for example, immune cells (e.g., antigen-presenting cells or APCs, including dendritic cells (DCs), macrophages, and B lymphocytes). Other suitable miRNAs may include, for example, miR-206 (skeletal muscle), miR-018b, and 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, WO2019 / 035690A1, WO2019 / 035690A1, WO2022 / 147181A1, and Brazilian Patent Publication No. BR102018067702A2, all of which are incorporated herein by reference.

[0121] Several different vector genomes were generated in the work described herein, however, it will be understood by those skilled in the art that other genome configurations may be selected, including promoters, enhancers, and other regulatory sequences that may be substituted for other coding sequences.

[0122] rAAV vector production For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector, e.g., a plasmid, that is delivered to a production (packaging) host cell. Plasmids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic, insect, or mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art. In certain embodiments, the production host cell is a human or insect cell. In certain embodiments, the production host cells are HEK293 cells, HuH-7 cells, BHK cells, or Vero cells. In certain embodiments, the production host cells are in suspension cell culture.

[0123] In certain embodiments, provided herein are production host cells comprising a recombinant nucleic acid molecule described herein, a nucleic acid sequence encoding an AAV capsid protein, and sufficient AAV rep and helper functions to allow packaging of the vector genome into an AAV capsid.

[0124] In certain embodiments, the insertion of an exogenous targeting peptide, the exogenous targeting peptide comprises an "Xn-n-mer-Xm", where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) is selected from IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDP (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), Q an n-mer selected from IRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length of any one of the n-mers; (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; wherein inclusion of an exogenous targeting peptide in the AAV capsid confers a production advantage compared to methods that do not include at least one copy of the motif in the AAV capsid; and the production cells are 293 cells.

[0125] In certain embodiments, host cells are stably or transiently transfected with genetic elements (e.g., plasmids or other nucleic acid molecules) that express the mutant AAV capsids provided herein. In certain embodiments, such genetic elements comprise a nucleic acid sequence encoding a mutant AAV VP1 coding sequence with mutant peptide(s) inserted therein, operably linked to expression control sequences that enable expression of the AAV capsid proteins in the packaging host cell. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:73 (IIRGDPA), which optionally is the nucleic acid sequence of SEQ ID NO:72, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:75 (AVIRGDV), which optionally is the nucleic acid sequence of SEQ ID NO:74, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:77 (IVRGDPA), which optionally is the nucleic acid sequence of SEQ ID NO:76, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:79 (MIRGDVK), which optionally is the nucleic acid sequence of SEQ ID NO:78, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:81 (AQHRGDV), which optionally is the nucleic acid sequence of SEQ ID NO:80, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO:83 (VSRGDPN), which optionally is the nucleic acid sequence of SEQ ID NO:82, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO: 85 (VSRGDPA), Optionally, the peptide insert is a nucleic acid sequence of SEQ ID NO: 84, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the peptide insert coding sequence is a sequence encoding SEQ ID NO: 87 (PLVRGDI), which is optionally a nucleic acid sequence of SEQ ID NO: 86, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the peptide insert coding sequence is a sequence encoding SEQ ID NO: 89 (PYVRGDP), which is optionally a nucleic acid sequence of SEQ ID NO: 88, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the peptide insert coding sequence is a sequence encoding SEQ ID NO: 91 (VVRGDPQ), 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 of the peptide insert is a sequence encoding SEQ ID NO:93 (VVQRGDV), which optionally is the 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 of the peptide insert is a sequence encoding SEQ ID NO:95 (QHRGDTQ), which optionally is the 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 of the peptide insert is a sequence encoding SEQ ID NO:97 (QIRGDLR), which optionally is the 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 of the peptide insert is a sequence encoding SEQ ID NO:99 (RGDYAQV), which optionally is the nucleic acid sequence of SEQ ID NO:98, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO: 101 (IGRGDPN), which is optionally the nucleic acid sequence of SEQ ID NO: 100, or a sequence that is 95% to 100% identical or at least 99% identical thereto.In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO: 103 (RGDLHGY), which optionally is the nucleic acid sequence of SEQ ID NO: 102, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO: 105 (RGDYS™), which optionally is the nucleic acid sequence of SEQ ID NO: 104, or a sequence 95% to 100% identical or at least 99% identical thereto. In certain embodiments, the coding sequence of the peptide insert is a sequence encoding SEQ ID NO: 107 (PYQRGDH), which optionally is the nucleic acid sequence of SEQ ID NO: 106, or a sequence 95% to 100% identical or at least 99% identical thereto.

[0126] In certain embodiments, host cells are stably or transiently transfected with genetic elements (e.g., plasmids or other nucleic acid molecules) that express the mutant AAV capsids provided herein. In certain embodiments, such genetic elements comprise a nucleic acid sequence encoding a mutant AAV VP1 coding sequence with mutant peptide(s) (i.e., exogenous targeting peptide(s)) inserted therein, operably linked to expression control sequences that 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 of the AAVhu68 capsid. In other embodiments, these peptides are inserted between amino acids 588 and 589 of 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 sequences of mutant AAV9 capsids having an exogenous targeting peptide inserted into the hypervariable region between amino acids 588 and 589 in the AAV9 parent capsid are selected from the group consisting of SEQ ID NO: 72 (IIRGDPA), SEQ ID NO: 74 (AVIRGDV), SEQ ID NO: 76 (IVRGDPA), SEQ ID NO: 78 (MIRGDVK), SEQ ID NO: 80 (AQHRGDV), SEQ ID NO: 82 (VSRGDV), SEQ ID NO: 83 (VIRGDPA), SEQ ID NO: 84 (VIRGDPA), SEQ ID NO: 85 (VIRGDPA), SEQ ID NO: 86 (VIRGDPA), SEQ ID NO: 87 (VIRGDPA), SEQ ID NO: 88 (VIRGDPA), SEQ ID NO: 89 (VIRGDPA), SEQ ID NO: 90 (V PN), SEQ ID NO: 84 (VSRGDPA), SEQ ID NO: 86 (PLVRGDI), SEQ ID NO: 88 (PYVRGDP), SEQ ID NO: 90 (VVRGDPQ), SEQ ID NO: 92 (VVQRGDV), SEQ ID NO: 94 (QHRGDTQ), SEQ ID NO: 96 (QIRGDLR), SEQ ID NO: 98 (RGDYAQV), SEQ ID NO: 100 (IGRGDPN), SEQ ID NO: 102 (RGDLHGY), SEQ ID NO: 104 (RGDYSTM), or SEQ ID NO: 106 (PYQRGDH). In other embodiments, the mutant AAV9 is any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:73 (IIRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:75 (AVIRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:77 (IVRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:79 (MIRGDVK mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:81 (AQHRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:83 (VSRGDPN mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:85 (VSRGDPA mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:87 (PLVRGDI mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:89 (PYVRGDP mutant VP1). , any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:91 (VVRGDPQ mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:93 (VVQRGDV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:95 (QHRGDTQ mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:97 (QIRGDLR mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:99 (RGDYAQV mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:101 (IGRGDPN mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:103 (RGDLHGY mutant VP1), any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:105 (RGDYSTM mutant VP1), or any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:407 (PYQRGDH mutant VP1).

[0127] Methods for preparing AAV-based vectors (e.g., having AAV9 or other AAV capsids) are known. See, for example, U.S. Published Patent Application No. 2007 / 0036760 (February 15, 2007), which is incorporated herein by reference. The present invention is not limited to the use of AAV9 or other Clade F AAV amino acid sequences, but encompasses peptides and / or proteins containing terminal β-galactose linkages produced by other methods known in the art, including, for example, chemical synthesis, other synthetic techniques, or other methods. Any of the AAV capsid sequences provided herein can be readily produced using a variety of techniques. Suitable production techniques are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY). Alternatively, peptides can also be synthesized using well-known solid-phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc., 85:2149; Stewart and Young, Solid Phase AAV capsid proteins can be synthesized by Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62. These methods may involve culturing host cells that contain a minigene consisting of a nucleic acid sequence encoding the AAV capsid, a functional rep gene, at a minimum the AAV inverted terminal repeats (ITRs) and a transgene, and sufficient helper functions to allow packaging of the minigene into AAV capsid proteins. These and other suitable production methods are within the knowledge of those skilled in the art and are not a limitation of the present invention.

[0128] The components required to be cultured in a host cell for packaging an AAV minigene into an AAV capsid can be provided to the host cell in trans. Alternatively, the necessary components (e.g., minigene, rep sequence, cap sequence, and / or helper functions) can be packaged in a host cell. Any one or more of these may be provided by a stable host cell that has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most preferably, 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 transgenes. As yet another alternative, the 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, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by those of skill in the art.

[0129] These rAAVs are particularly suitable for gene delivery for therapeutic purposes and infection prevention. Furthermore, the compositions of the present invention can also be used to produce a desired gene product in vitro. For in vitro production, the desired product (e.g., protein) may be obtained from the desired culture after transfecting host cells with rAAV containing a molecule encoding the desired product and culturing the cell culture under conditions that allow expression. The expressed product can then be purified and isolated, if desired. Suitable techniques for transfection, cell culture, purification, and isolation are known to those skilled in the art. Methods for generating and isolating AAVs suitable for use as vectors are known in the art. Generally, see, for example, Grieger & Samulski, 2005, "Adeno-associated See, "A 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 of 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 cassette. The cap and rep genes can be supplied in trans.

[0130] In one embodiment, the expression cassettes described herein are engineered into genetic elements (e.g., shuttle plasmids) that transfer the immunoglobulin construct sequences carried thereon to packaging host cells for production viral vectors. In one embodiment, the selected genetic elements can be delivered to AAV packaging cells by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be generated. Alternatively, the expression cassettes can be used to generate viral vectors other than AAV or for the in vitro production of antibody mixtures. Methods used to generate such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0131] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid that lacks the desired genomic sequence to be packaged in it. These may also be referred to as "empty" capsids. Such capsids lack a detectable expression cassette. These empty capsids may contain no functional genomic sequence or only partially packaged genomic sequence that is insufficient to achieve expression of a gene product. These empty capsids are non-functional for delivering a gene of interest to a host cell.

[0132] The recombinant AAV described herein can be produced using known techniques. For example, see WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2. Such methods involve culturing host cells containing an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid, a functional rep gene, at least an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper functions to allow the expression cassette to be packaged into AAV capsid proteins. Accordingly, methods for producing capsids, coding sequences, and methods for producing rAAV vectors have been described. For example, see Gao, et al., Proc. Natl. Acad. Sci. USA 100 (10), 6081-6086 (2003) and US2013 / 0045186A1.

[0133] In certain embodiments, the rAAV is produced (manufactured) using triple transfection technology. In certain embodiments, the rAAV is produced using stable mammalian cell lines. In certain embodiments, the stable cell line comprises one or more of the following: (a) a first plurality of polynucleotide molecules comprising a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for producing replication-deficient rAAV vectors (Rep52 and Rep78), wherein the rep protein coding sequence is operably linked to a doxycycline-inducible promoter that directs expression of the rep protein in the cell line; (b) at least a second plurality of polynucleotide molecules each encoding an adenovirus (Ad) helper protein necessary for producing replication-deficient rAAV vectors, wherein the at least second plurality of polynucleotide molecules each encode ... Ad E2A DNA-binding protein (DBP) coding sequence and an Ad E4ORF6 coding sequence, wherein the Ad E2A DBP coding sequence and the Ad E4ORF6 coding sequence are operably linked to a doxycycline-inducible promoter that directs expression of the Ad helper protein in the cell line; and (c) at least a second plurality of polynucleotide molecules each encoding an adenovirus (Ad) helper protein necessary for producing replication-deficient rAAV vectors, wherein the at least second plurality of polynucleotide molecules each encode an Ad E2A DNA-binding protein (DBP) coding sequence and an Ad E4ORF6 coding sequence. (d) at least a third plurality of nucleic acid molecules, each of which comprises an AAV VP1 coding sequence encoding AAV VP1, VP2, and VP3 proteins that self-assemble to form an AAV capsid after expression in a cell, wherein the AAV VP1 coding sequence is operably linked to a promoter that directs expression of the VP1 coding sequence in a cell line. See also U.S. Provisional Patent Application No. 63 / 490,222, filed March 14, 2023, which is incorporated herein by reference.

[0134] In one embodiment, the cells are produced in a suitable cell culture (e.g., HEK293 cells). Methods for producing gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA used to produce the gene therapy vector, generating the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are an AAV cis-plasmid encoding the AAV genome and the gene of interest for packaging into the capsid, an AAV trans-plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process can include method steps such as initiating cell culture, passaging the cells, seeding the cells, transfecting the cells with plasmid DNA, changing the medium to serum-free medium after transfection, and harvesting the vector-containing cells and culture medium. Harvested vector-containing cells and culture medium are referred to herein as crude cell harvests. In yet another system, gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For a review of these production systems generally, see, 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, 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 are incorporated herein by reference in their 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.

[0135] The crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the 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.

[0136] The vector drug product is purified and empty capsids are removed using a two-step affinity chromatography purification at high salt concentrations, followed by anion exchange resin chromatography. These methods are described in more detail in International Patent Application Nos. PCT / US2016 / 065970, entitled "Scalable Purification Method for AAV9," filed December 9, 2016, and US 11,098,286 B2, both of which are incorporated by reference. AAV8 purification methods are described in International Patent Application Nos. PCT / US2016 / 065976, entitled "Scalable Purification Method for AAV8," filed December 9, 2016, and US 11,015,174 B2, both of which are incorporated by reference. Purification methods for rh10 include International Patent Application Nos. PCT / US16 / 066013, entitled "Scalable Purification Method for AAVrh10," filed December 9, 2016, and US 11,028,372 B2, which are incorporated herein by reference. Purification methods for AAV1 include International Patent Application Nos. PCT / US2016 / 065974, entitled "Scalable Purification Method for AAV1," filed December 9, 2016, and US 11,015,173 B2, which are incorporated herein by reference.

[0137] To calculate empty and filled particle content, the vp3 band volume for a selected sample (e.g., in the present example, a preparation purified on an iodixanol gradient, where GC number = particle number) is plotted against the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the peak for the test sample. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL - GC / mL yields empty pt / mL. Dividing empty pt / mL by pt / mL and multiplying by 100 yields the percentage of empty particles.

[0138] In general, methods for assaying AAV vector particles with empty capsids and packaged genomes are 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. See, for example, (2000) 74:9281-9293). To test for denatured capsids, the method involves subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% Tris-acetate in buffer, running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as a primary antibody to bind to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semiquantitatively determine the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method that can detect radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescent detection kit. For example, in SDS-PAGE, samples from column fractions can be heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins resolved in a precast gradient polyacrylamide gel (e.g., Novex). Silver staining can be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA.After inactivation of the nuclease, the sample is further diluted and amplified using a TaqMan™ fluorogenic probe specific to the primers and the DNA sequence between them. The number of cycles required to reach a predetermined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0139] In addition, other examples of measuring empty-to-filled particle ratios are also known in the art. Sedimentation velocity measured in an analytical ultracentrifuge (AUC) can detect aggregates and other trace components and provide excellent quantification of the relative amounts of different particle species based on their different sedimentation coefficients. This is an absolute method based on the fundamental units of length and time and does not require a standard molecule as a reference. The vector sample is loaded into a two-channel cell with a 12 mm path length and a charcoal-epon centerpiece. The supplied dilution buffer is loaded into the reference channel of each cell. The loaded cells are then placed in an AN-60Ti analytical rotor and analyzed by a Beckman-Coulter centrifuge equipped with both absorbance and RI detectors. The samples are loaded into a ProteomeLab XL-I analytical ultracentrifuge. After full temperature equilibration at 20°C, the rotor is brought to a final running speed of 12,000 rpm. A280 scans are recorded approximately every 3 minutes for approximately 5.5 hours (a total of 110 scans for each sample). The raw data are analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resulting size distribution is graphed and the peaks are integrated. The percentage value associated with each peak represents the peak area fraction of the total area under all peaks. Based on raw data generated at 280 nm, many laboratories use these values ​​to calculate the empty:filled particle ratio. However, empty and filled particles have different extinction coefficients at this wavelength, so the raw data can be adjusted accordingly. Before and after extinction coefficient adjustment, The ratio of the empty particle and filled monomer peak values ​​at both ends is used to determine the empty particle:filled particle ratio.

[0140] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serine protease, e.g., proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Preferably, the sample is diluted with a volume of proteinase K buffer equal to the sample size. The proteinase K buffer can be concentrated two-fold or more. Typically, proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally performed at about 55°C for about 15 minutes, but may also be performed at lower temperatures (e.g., about 37°C to about 50°C) for longer periods (e.g., about 20 minutes to about 30 minutes) or at higher temperatures (e.g., up to about 60°C) for shorter periods (e.g., about 5-10 minutes). Similarly, heat inactivation is typically performed at about 95°C for about 15 minutes, although the temperature may be lowered (e.g., about 70 to about 90°C) and the time extended (e.g., about 20 to about 30 minutes). The sample is then diluted (e.g., 1:1000) and subjected to TaqMan analysis as described for standard assays. Quantification can also be performed using ViroCyt or flow cytometry.

[0141] Additionally or alternatively, droplet digital PCR (ddPCR) can be used.For example, the method for determining single-stranded and self-complementary AAV vector genome titer by ddPCR is described.For example, see 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.

[0142] In certain embodiments, the manufacturing process for the rAAV (e.g., including engineered rAAV) described herein involves the methods described in U.S. Provisional Patent Application No. 63 / 371,597, filed August 16, 2022, and U.S. Provisional Patent Application No. 63 / 371,592, filed August 16, 2022, which are incorporated by reference in their entireties.

[0143] Therapeutic Proteins and Delivery Systems In certain embodiments, provided herein are fusion partners, conjugate partners, and recombinant vectors containing the muscle-targeting peptides provided herein comprising a "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15). , PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length of any one of these n-mers; and (iii) Xm are 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, which are useful for a variety of therapeutic proteins, polypeptides, nanoparticles, and delivery systems. Examples of proteins and compounds useful for the compositions and targeted delivery provided herein are described below. Viral vectors, nanoparticles It will be understood that these and other delivery systems contain a sequence encoding a selected protein (or conjugate) for expression in vivo.

[0144] In some embodiments, provided herein is an rAAV having a modified capsid bearing one or more exogenous targeting peptides, wherein the exogenous targeting peptide comprises an "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDP (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ ( (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV comprises a vector genome comprising a desired transgene and a promoter for use in a target cell, as detailed above, optionally assessed for contamination by conventional methods, and then formulated into a pharmaceutical composition for administration to a subject in need thereof. Such formulations include a pharmaceutically and / or physiologically acceptable vehicle or carrier (such as buffered saline or other buffers (e.g., HEPES) to maintain pH at an appropriate physiological level), and optionally other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is typically a liquid.

[0145] In certain embodiments, the protein, polypeptide, nanoparticle, and / or delivery system, including viral vectors (rAAV) and nanoparticles comprising exogenous targeting peptides provided herein, is useful for treating one or more cardiac and / or skeletal (e.g., gastrocnemius) muscle-based disorders. Such diseases and / or disorders may include, but are not limited to, autoimmune diseases, cancer, muscular dystrophies, neuromuscular diseases, sugar or glycogen storage diseases, cardiomyopathies, and infections affecting muscle cells. More specifically, such diseases and / or disorders may include, but are not limited to, Huntington's disease, myotonic dystrophy (type 1 or type 2), facioscapular dystrophy (FSHD), Duchene muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, oculopharyngeal muscular dystrophy, Barth syndrome, MPS III disease, Pompe disease, Fabry disease, Charcot-Marie-Tooth disease, Friedreich's ataxia, dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-related cardiomyopathy, myotubular myopathy, primary merosin deficiency, Dannon disease, idiopathic dilated cardiomyopathy (DCM), or diseases associated with mutations in the LMNA gene. Examples of genes and proteins associated with diseases and / or disorders, e.g., spinal muscular atrophy (SMA, SMN1), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., frataxin), cardiomyopathies (LMNA), Charcot-Marie-Tooth disease (MFN2). Also, U.S. Provisional Patent Application No. 63 / 293,680, filed December 24, 2021; International Patent Application No. PCT / US2021 / 041406, filed July 13, 2021, now published No. WO2022 / 015715A1; International Patent Application No. PCT / US2022 / 076939, filed September 23, 2022; International Patent Application No. PCT / US2020 / 066167, filed December 18, 2020, now published No. WO2021 / 127533A1; International Patent Application No. PCT / US2022 / 025879, filed April 22, 2022, now published No. WO2022 / 226263A1; and International Patent Application No. PCT / US2022 / 030163A1, filed October 8, 2021. See International Patent Application No. PCT / US2021 / 054145, now published as WO2022 / 076803, which are incorporated by reference in their entireties.

[0146] In certain embodiments, proteins useful in the compositions provided herein include 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), angiopoietin, 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 alpha (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any of the transforming growth factor beta superfamily. The transgene sequences include hormones and growth and differentiation factors, including, but not limited to, one of the following: TGFβ, activin, inhibin, or any of the bone morphogenetic proteins (BMPs) BMP1-15, 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 semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, tyrosine hydroxylase.Other useful transgenes encode lysosomal enzymes that cause mucopolysaccharidoses (MPS), including α-L-iduronidase (MPS I), iduronate sulfatase (MPS II), heparan N-sulfatase (sulfaminidase) (MPS IIIA, Sanfilippo A), α-N-acetyl-glucosaminidase (MPS IIIB, Sanfilippo B), acetyl-CoA:α-glucosaminide acetyltransferase (MPS IIIC, Sanfilippo C), N-acetylglucosamine 6-sulfatase (MPS IIID, Sanfilippo D), galactose 6-sulfatase (MPS IVA, Morquio A), β-galactosidase (MPS IVB, Morquio B), N-acetyl-galactosamine 4-sulfatase (MPS VI, Maroteaux-Lamy), and β-glucuronidase (MPS VII, Sly), and hyaluronidase (MPS IX).

[0147] In certain embodiments, proteins useful in the compositions provided herein are encoded by transgene sequences containing reporter sequences that generate a detectable signal upon expression. Such reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-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), influenza hemagglutinin protein, and others known in the art (for which high-affinity antibodies exist or can be produced by conventional means), as well as fusion proteins (including, among others, membrane-bound proteins appropriately fused to antigen tag domains derived from hemagglutinin or Myc).

[0148] The rAAVs having the mutant rAAV capsids provided herein have vector genomes that include nucleic acid sequences encoding proteins that act, for example, as transcriptional repressors, antisense molecules, ribozymes, and low-inhibitory nucleic acid sequences, including, but not limited to, RNAi, siRNA, microRNAi (mRNAi or miRNA), and antisense oligonucleotides, in addition to or in place of the delivered protein. It's okay to have it.

[0149] Compositions and Uses Provided herein are compositions containing at least one rAAV stock (e.g., an engineered stock of rAAV9 or an engineered stock of rAAVhu68, wherein the engineered capsid comprises an exogenous targeting motif described herein) and optional carriers, excipients, and / or preservatives.

[0150] In one aspect, a pharmaceutical composition is provided comprising a rAAV described herein in a formulation buffer. In one embodiment, the rAAV is present in a concentration of about 1 x 10 9 Genome copies (GC) / mL ~ approx. 1 x 10 14 In a further embodiment, the rAAV is formulated at about 3 x 10 GC / mL. 9 GC / mL ~ approx. 3×10 13 In yet a further embodiment, the rAAV is formulated at about 1 x 10 GC / mL. 9 GC / mL ~ approx. 1×10 13 In one embodiment, the rAAV is formulated at a concentration of at least about 1 x 10 GC / mL. 11 It is formulated in GC / mL.

[0151] Also provided herein are compositions comprising at least one therapeutic protein, polypeptide, nanoparticle, and / or delivery system comprising a targeting motif provided herein, and optional carriers, excipients, and / or preservatives.

[0152] Also provided herein are methods of using the compositions described herein. In certain embodiments, a method for targeted therapy to muscle cells comprises administering to a patient in need thereof a stock of rAAV described herein, wherein the therapeutic agent is targeted for delivery to muscle cells (e.g., cardiac cells (heart), skeletal muscle cells (e.g., gastrocnemius muscle)) and detargeted to liver cells.

[0153] Additionally, provided herein is a method of delivering a transgene to one or more muscle cells of a subject, comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising an engineered capsid protein comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises an "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PVRGDP (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVRGDPQ (SEQ ID NO: 17), VVRGDPQ (SEQ ID NO: 18), VVRGDPQ (SEQ ID NO: 19), VVRGDPQ (SEQ ID NO: 20), VVRGDPQ (SEQ ID NO: 21), VVRGDPQ (SEQ ID NO: 22), VVRGDPQ (SEQ ID NO: 23), VVRGDPQ (SEQ ID NO: 24), VVRGDPQ (SEQ ID NO: 25), VVRGDPQ (SEQ ID NO: 26), VVRGDPQ (SEQ ID NO: 27), VVRGDPQ (SEQ ID NO: 28), VVRGDPQ (SEQ ID NO: 29), VVRGDPQ (SEQ ID NO: 30), VVRGDPQ (SEQ ID NO: 31), VVRGDPQ (SEQ ID NO: 32), VVRGDPQ (S (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, which are useful for a variety of therapeutic proteins, polypeptides, nanoparticles, and delivery systems, and the rAAV further comprises a vector genome comprising a transgene operably linked to a regulatory sequence that directs expression of the transgene in muscle cells.

[0154] In certain embodiments, the target muscle cells are cardiac muscle cells, smooth muscle cells, and / or skeletal muscle cells. In certain embodiments, the transgene encodes a secreted gene product. In certain embodiments, the AAV vector is delivered intravenously.

[0155] rAA with engineered capsids carrying one or more exogenous targeting peptides to target muscle cells with higher levels of transduction than can be achieved using AAV9 vectors Also provided herein is the use of V, wherein the exogenous targeting peptide comprises an "Xn-n-mer-Xm", where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTRGDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or an n-mer sequence of at least 6, at least 7, or the full length 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.

[0156] In certain embodiments, the composition may contain at least a second, different rAAV stock. This second vector stock may differ from the first vector stock by having a different AAV capsid and / or a different vector genome. In certain embodiments, the composition described herein may contain a different vector expressing the expression cassette described herein, or another active ingredient (e.g., an antibody construct, another biological agent, and / or a small molecule drug).

[0157] 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, etc. 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 composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present invention into suitable host cells. In particular, rAAV vector-delivered transgenes can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0158] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, e.g., an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Preferably, the final formulation is adjusted to a physiologically acceptable pH (e.g., pH may range from 6 to 9, or 6.5 to 7.5, 7.0 to 7.7, or 7.2 to 7.8). For intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pH values ​​within the broadest ranges, and subranges thereof, may be selected for other delivery routes. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be shipped as a concentrate that is diluted for administration to a subject. In another embodiment, the composition may be lyophilized and reconstituted at the time of administration.

[0159] A suitable surfactant or combination of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), such as SOLUTOL HS15 (macrogol (polyethylene glycol) The following surfactants may be selected: 15-hydroxystearate, LABRASOL® (polyoxycaprylic acid glyceride), polyoxy10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, with the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0160] In another embodiment, the composition comprises a carrier, diluent, excipient, and / or adjuvant. Suitable carriers can be readily selected by those skilled in the art based on the indication for which the transport virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer 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 contain components that prevent rAAV from adhering to the injection tubing but do not interfere with rAAV binding activity in vivo. A suitable surfactant, or surfactant combination, can be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl-terminated bifunctional block copolymer surfactant is selected, such as Poloxamer 188 (also known under the trade names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, and Kolliphor® P188), which has a neutral pH and an average molecular weight of 8400. Other surfactants and poloxamers may be selected, i.e., nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, where the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core and the last digit x 10 gives the percentage of 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.

[0161] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such formulations may contain a buffered saline solution, such as Harvard buffer, which contains one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water. In one embodiment, the buffer is phosphate buffered saline (PBS). In one embodiment, the formulation buffer PBS (final formulation buffer, FFB) contains a total salt concentration of 200 mM and 0.001% (w / v) Pluronic F68.

[0162] Optionally, in addition to the rAAV and carrier(s), the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0163] The compositions according to the invention may comprise a pharmaceutically acceptable carrier as defined above. Preferably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with suitable excipients designed for delivery to a subject via injection or by another route or device.

[0164] In certain embodiments, the composition comprises a viral vector (i.e., an rAAV vector). The vector is administered in an amount sufficient to transfect cells and provide a sufficient level of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with a medically acceptable physiological effect, which can be determined by one skilled in the art. In certain embodiments, the vector is formulated for systemic or direct delivery to the desired organ (e.g., lung), oral inhalation, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes.

[0165] As used herein, the term "dosage" or "amount" can refer to the total dosage or amount delivered to a subject over the course of treatment, or the dosage or amount delivered in a single unit (or multiple units or divided doses) administration. The dosage of a viral vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore may vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 25 to about 1000 microliters to about 5 mL, or about 1000 microliters to about 5 mL. 9 ~4×10 14 The range of the aqueous suspension containing the dose of AAV vector of GC.Dosage is adjusted to balance the therapeutic benefit against any side effects, and such dosage can vary depending on the therapeutic application that recombinant vector is used for.The expression level of transgene can be monitored to determine the dosage frequency that results in viral vector, preferably AAV vector containing minigene.Optionally, the dosage regimen similar to that described for therapeutic purposes can be used for immunization using the composition of the present invention.

[0166] The replication-deficient virus composition may be administered in a dose of about 1.0 x 10 for a human patient (to treat an average subject weighing 70 kg), including all whole numbers and decimals within the range. 9 GC~approx. 1.0×10 16 GC range, preferably 1.0 x 10 12 GC~1.0×10 14 Dosage units can be formulated to contain an amount of replication-deficient virus that is within the GC range. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers and decimals within the range. 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9×10 9In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9×10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9×10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 14 , 2 × 10 14 , 3×1014 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers and decimals within the range. 15 , 2 × 10 15 , 3×10 15 , 4×1 0 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9×10 15 In one embodiment, for human applications, the dose is 1 x 10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 In certain embodiments, the rAAV composition can range from about 1 x 10 13 In certain embodiments, the rAAV composition is formulated in a dosage unit containing about 2.5 x 10 GC / kg. 13 In certain embodiments, the rAAV composition is formulated in a dosage unit containing about 5×10 GC / kg. 13 It is formulated in dosage units containing GC / kg.

[0167] In one embodiment, for human applications, the dose is 10 per dose, including all integers or fractions within the range. 9 GC~approx. 7×10 13 It can be a range of GC.

[0168] These above-mentioned doses may be administered in various volumes of the carrier, excipient, or buffer formulation, ranging from about 25 to about 1000 microliters, or greater, or any number within that 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 the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is about 700-1000 μL.

[0169] In one embodiment, the rAAV construct is about 1 x 10 9 GC~approx. 1×10 15 , or approximately 1 × 10 11 ~5×10 13The recombinant vector may be delivered in a dose of GC. Suitable volumes for delivery of these doses and concentrations can be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL can be selected, with higher volumes being selected for adults. Typically, for newborns, a suitable volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For pre-teens and teenagers, a volume of up to about 50 mL may be selected. Other suitable volumes and dosages can be determined. The dosage is adjusted to balance the therapeutic benefit against any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is used.

[0170] The compositions according to the invention may comprise a pharmaceutically acceptable carrier as defined above. Preferably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with suitable excipients designed for delivery to a subject via injection.

[0171] The compositions, suspensions, or pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. In certain embodiments, the rAAV or pharmaceutical composition is administered intravenously to a patient in need thereof. The formulation may include a formulation buffer suitable for administering the formulation.

[0172] In certain embodiments, provided herein are compositions comprising one or more exogenous muscle cell targeting peptides comprising "Xn-n-mer-Xm," together with one or more physiologically compatible carriers, excipients, and / or aqueous suspension bases, wherein (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the selected n-mer is selected from IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13). , PYVRGDP (SEQ ID NO:14), VVRGDPQ (SEQ ID NO:15), PTRGDVK (SEQ ID NO:16), VVQRGDV (SEQ ID NO:17), QHRGDTQ (SEQ ID NO:18), QIRGDLR (SEQ ID NO:19), RGDYAQV (SEQ ID NO:20), IGRGDPN (SEQ ID NO:21), RGDLHGY (SEQ ID NO:22), RGDYSTM (SEQ ID NO:23), or PYQRGDH (SEQ ID NO:24), or an n-mer sequence of at least 6, at least 7, or the full length 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. Further provided are compositions comprising nucleic acid sequences encoding same.

[0173] In certain embodiments, compositions are provided comprising a fusion polypeptide or protein, or a nucleic acid sequence encoding the fusion polypeptide or protein, or nanoparticles containing the same. The compositions may further comprise one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

[0174] In certain embodiments, the nucleic acid sequence encoding the fusion polypeptide protein is encapsulated in lipid nanoparticles (LNPs). As used herein, the term "lipid nanoparticles" refers to a transfer vehicle that comprises 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., muscle cells (cardiac, skeletal, smooth)). Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylate, polyalkylcyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, dendrimers, and polyethyleneimine. In one embodiment, the transfer vehicle is selected based on its ability to promote transfection of the nucleic acid sequence encapsulated therein into target cells. Lipid nanoparticles useful for nucleic acid sequences contain cationic lipids to encapsulate and / or enhance the delivery of such nucleic acid sequences to target cells, which function as a depot for protein production. As used herein, the term "cationic lipid" refers to any of several lipid species that carry a net positive charge at a selected pH, e.g., physiological pH. Contemplated lipid nanoparticles can be prepared by including a multi-component lipid mixture in varying ratios using 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, US2018 / 0353616A1, and US8,853,377B2, which are incorporated by reference.In certain embodiments, LNP formulations are made using routine procedures that include cholesterol, ionizable lipids, helper lipids, PEG-lipids, and polymers that form a lipid bilayer around the encapsulated nucleic acid sequence. (Kowalski et al., 2019, Mol. Ther. 27(4):710-728). In some embodiments, the LNPs comprise a cationic lipid (i.e., N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) with the helper lipid DOPE. In some embodiments, the LNPs comprise the ionizable lipid Dlin-MC3-DMA or a diketopiperazine-based ionizable lipid (cKK-E12). In some embodiments, the polymer comprises polyethyleneimine (PEI) or poly(β-amino)ester (PBAE). See, e.g., WO2014 / 089486, US2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085A1, US9670152B2, and US8,853,377B2, which are incorporated by reference.In certain embodiments, a lipid nanoparticle (LNP) comprises at least one exogenous targeting peptide comprising "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTR GDVK (SEQ ID NO: 16), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), RGDYAQV (SEQ ID NO: 20), IGRGDPN (SEQ ID NO: 21), RGDLHGY (SEQ ID NO: 22), RGDYSTM (SEQ ID NO: 23), or PYQRGDH (SEQ ID NO: 24), or any one of the n-mers, at least 6, at least 7, or full-length n-mer sequences of consecutive amino acids, and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, i.e., the surface is decorated with a targeting peptide. In certain embodiments, the lipid nanoparticle (LNP) comprises at least one IIRGDPA (SEQ ID NO: 1) peptide. In certain embodiments, the lipid nanoparticle (LNP) comprises at least one AVIRGDV (SEQ ID NO: 2) peptide.

[0175] In certain embodiments, for example, a composition comprising an rAAV having an engineered capsid with at least one exogenous targeting peptide comprising "Xn-n-mer-Xm," where (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and (ii) the n-mer is selected from the group consisting of IIRGDPA (SEQ ID NO: 1), AVIRGDV (SEQ ID NO: 2), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), PTR and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Provided herein are compositions useful for delivering therapeutic agents to patients in need thereof, wherein the rAAV has a modified capsid bearing at least one IIRGDPA (SEQ ID NO: 1) peptide, and the therapeutic agent is targeted for delivery to muscle cells. In certain embodiments, compositions comprising an rAAV having a modified capsid having at least one core AVIRGDV (SEQ ID NO: 2) are useful for delivering a therapeutic agent to a patient in need thereof, wherein the therapeutic agent is targeted for delivery into muscle cells. In embodiments, compositions comprising rAAVs having modified capsids with at least one IIRGDPA (SEQ ID NO: 1) peptide are useful for delivering therapeutic agents to patients in need thereof, where the therapeutic agents are targeted for delivery to muscle cells and detargeted to the liver. In certain embodiments, compositions comprising rAAVs having modified capsids with at least one core AVIRGDV (SEQ ID NO: 2) are useful for delivering therapeutic agents to patients in need thereof, where the therapeutic agents are targeted for delivery into muscle cells and detargeted to the liver.

[0176] In certain embodiments, the methods and compositions are useful for treating mitochondrial cardiomyopathy associated with Barth syndrome. Barth syndrome is a rare X-linked recessive disorder characterized by loss-of-function mutations in the TAZ gene (i.e., amenable to gene therapy). Barth syndrome is associated with childhood-onset cardiomyopathy (i.e., by age 5 years) accompanied by neutropenia, mild mitochondrial myopathy (skeletal muscle weakness), and mild intellectual disability. See also Sabbah, HN, Barth syndrome cardiomyopathy: targeting the mitochondria with elamipretide, Heart Failure Reviews (2021) 26:237-253, incorporated herein by reference in its entirety.

[0177] In certain embodiments, the methods and compositions are useful for treating autosomal dominant forms of long QT syndrome (i.e., amenable to gene replacement or knockdown / replacement approaches), which are caused by loss-of-function and partial dominant-negative mutations in the KCNQ1 gene. Autosomal dominant forms of long QT syndrome are associated with syncope and sudden cardiac death, typically occurring during exercise or mental stress, and many patients remain at risk despite standard treatments (beta-blockers, cardiac sympathetic denervation) and require implantable cardioverter-defibrillators (ICDs). Also see Huang H., et al., Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensors. See "Domain Mutations," Sci. Adv. 2018, 4:1-12, epub March 7, 2018, which is incorporated by reference in its entirety.

[0178] In certain embodiments, the present method and composition are useful for treating hypertrophic cardiomyopathy.In certain embodiments, the present method and composition can be used for treating hypertrophic cardiomyopathy caused by loss-of-function mutation of MYBPC3 gene (i.e., suitable for 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 (the entirety of which is incorporated herein by reference).

[0179] In certain embodiments, the methods and compositions are useful for treating transthyretin amyloid cardiomyopathy (ATTR-CM). In certain embodiments, the methods and compositions can be used to treat ATTR-CM caused by mutations in the transthyretin (TTR) gene (i.e., suitable for gene therapy). Also, Yamamoto H, Yokochi T.Transthyretin cardiac amyloidosis:an update on diagnosis and treatment.ESC Heart Fail.2019 Dec;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 entireties.

[0180] In certain embodiments, the methods and compositions are useful for treating long QT syndrome type 2, which is caused by loss-of-function mutations in the hERG (Kv11.1, also known as Kv11.1 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 Kv11.1 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 entireties.

[0181] In certain embodiments, the methods and compositions are useful for treating LMNA cardiomyopathy or diseases caused by loss-of-function mutations in the LMNA gene. See also Kang, S., et al., Laminopathies; Mutations on single genes and various human genetic diseases, BMB Rep. 2018; 51(7):327-337, U.S. Provisional Patent Application No. 63 / 293,680, filed December 24, 2021, International Patent Application No. PCT / US2022 / 082383, filed December 24, 2022, and current publication No. WO2023 / 122803A1, published June 29, 2023, which are incorporated herein by reference in their entireties.

[0182] In certain embodiments, the methods and compositions are useful for treating heart failure, ischemia-reperfusion injury, myocardial infarction, ventricular remodeling, or diseases associated with extracellular superoxide dismutase 3 (SOD3 or EcSOD). See also U.S. Patent Application Publication No. 20130136729A1, which is incorporated herein by reference in its entirety.

[0183] In certain embodiments, the methods and compositions are useful for treating myocardial infarction, heart failure with reduced ejection fraction, or diseases associated with myc transcription factors, cyclin T1, and cyclin-dependent kinase 9 (CDK9). See also International Patent Application Publication No. WO 2020 / 165603 A1, which is incorporated herein by reference in its entirety.

[0184] In certain embodiments, the methods and compositions are useful for treating heart failure, or cardiac tissue damage or degeneration, or diseases associated with cyclin A2 protein. See also International Patent Application Publication No. 2020 / 051296A1, the entire contents of which are incorporated herein by reference.

[0185] In certain embodiments, the methods and compositions are useful for treating dilated cardiomyopathy (DCM), heart failure, cardiac fibrosis, carditis, ischemic heart disease, myocardial infarction, ischemia / reperfusion (I / R)-related injury, aortic coarctation, or diseases associated with YY1 or BMP7 proteins. See also International Patent Application Publication No. 2021 / 021021A1, which is incorporated herein by reference in its entirety.

[0186] In certain embodiments, the methods and compositions are directed to treating dilated cardiomyopathy or caspase recruitment. These compounds are useful for treating diseases associated with cardiac apoptosis inhibitors having the cardiac apoptosis inhibitor (cARC) domain. See also International Patent Application Publication No. WO 2021 / 016126A1, which is incorporated herein by reference in its entirety.

[0187] Symptoms of cardiomyopathy or diseases associated with mutations in the LMNA, KCNQ1, MYBPC3, TAZ, or hERG genes include atrioventricular (AV) conduction block, arrhythmias including atrial arrhythmias such as atrial fibrillation, atrial flutter, and atrial tachycardia, and ventricular arrhythmias including sustained ventricular tachycardia, and ventricular fibrillation (VF), and / or heart failure.

[0188] In certain embodiments, the methods and compositions described herein are useful for alleviating one or more symptoms of cardiomyopathy, including increasing life expectancy and / or reducing progression to heart failure.

[0189] In certain embodiments, the methods and compositions are useful for treating or alleviating one or more symptoms of muscular dystrophy.

[0190] In certain embodiments, the methods and compositions are useful for treating or alleviating one or more symptoms of Duchenne muscular dystrophy (dystrophin, DMD), Becker muscular dystrophy (dystrophin, DMD), Danon disease (LAMP2), myotubular myopathy (myotubularin, MRM1), primary merosin deficiency (merosin, LAMA2), Pompe disease (alpha-1,4-glucosidase, GAA), limb-girdle muscular dystrophy (calpain 3, CAPN3), oculopharyngeal muscular dystrophy (PABPN1), or muscular dystrophies associated with dysferlin (DYSF), alpha-sarcoglycan (LGMD2D), beta-sarcoglycan (SGCB), or fukutin-related protein (FKRP) proteins.

[0191] In certain embodiments, rAAVs having modified capsids described herein can be delivered in a co-therapeutic regimen further comprising one or more other active components. In certain embodiments, the regimen can include co-administration of an immunomodulatory component. Such immunomodulatory regimens can include, for example, immunosuppressants such as, but not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or cytostatic agents, including agents active against immunophilins. Immunosuppressants may include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25)-specific or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-alpha) binders. In certain embodiments, immunosuppressive therapy may be initiated prior to gene therapy administration. Such therapy may involve co-administration of two or more agents (e.g., prednelisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin) on the same day. One or more of these agents may be continued at the same dose or at adjusted doses after gene therapy administration. Such therapy may be for about 1 week, about 15 days, about 30 days, about 45 days, 60 days, or longer, as needed. Still other co-therapeutic agents may be used, for example, anti-IgG enzymes, which have been described as useful for depleting anti-AAV antibodies (thus allowing administration of antibodies above a threshold level of a selected AAV capsid to the patient being tested), and / or other agents described, for example, in WO2021 / 257668, filed December 23, 2021, entitled "Compositions and Methods for Treatment of Gene Therapy Patients" (U.S. Provisional Patent Application No. 63 / 044, filed June 17, 2020). No. 62 / 135,998, filed January 11, 2021, and U.S. Provisional Patent Application No. 63 / 152,085, filed February 22, 2021), 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.

[0192] kit In certain embodiments, a kit is provided that includes a concentrated vector suspended in a formulation (optionally frozen), an optional dilution buffer, and a device and components required for intravenous administration. In another embodiment, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow injection. Such a buffer may allow for approximately a 1:1 to 1:5 dilution of the concentrated vector, or greater. In other embodiments, larger or smaller volumes of buffer or sterile water are included to allow for dose titration and other adjustments by the treating physician. In yet other embodiments, the kit includes one or more components of the device. Suitable dilution buffers, such as saline, phosphate-buffered saline (PBS), or glycerol / PBS, are available.

[0193] It is to be understood that the compositions in the kits described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0194] An "immunoglobulin molecule" is a protein containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently bound to each other and capable of specifically combining with an antigen. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The terms "antibody" and "immunoglobulin" may be used interchangeably herein.

[0195] The term "neutralizing antibody titer" (NAb titer) is a measure of the production of neutralizing antibodies (e.g., anti-AAV NAbs) that neutralize the physiological effects of the targeted epitope (e.g., AAV). Anti-AAV NAb titers can be measured, for example, as described in Calcedo, R., et al., "Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses." Journal of Infectious Diseases, 2009, 199(3): pp. 381-390, which is incorporated herein by reference.

[0196] As used herein, when used to refer to vp capsid proteins, the term "heterologous" or any grammatical variation thereof refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 26 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The term "heterologous" used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1, vp2, and vp3 proteins that have modifications from predicted amino acid residues. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. For example, specific subpopulations may contain asparagine -Contains at least one, two, three, or four highly deamidated asparagine (N) positions in glycine (NG) pairs, and optionally further contains other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications.

[0197] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that share at least one defined common characteristic and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins, unless otherwise specified, can be at least one vp1 protein and less than all vp1 proteins in an assembled AAV capsid. A "subpopulation" of vp3 proteins, unless otherwise specified, can be one vp3 protein to less than all vp3 proteins in an assembled AAV capsid. For example, in an assembled AAV capsid, vp1 protein can be a subpopulation of vp proteins, vp2 protein can be another subpopulation of vp proteins, and vp3 can be yet a further subpopulation of vp proteins. In another example, the vp1, vp2, and vp3 proteins can include subpopulations with at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidation, 50%-100% deamidation, 70%-100% deamidation, 75%-100% deamidation, or 70%-90% deamidation at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position. Such percentages can be determined using 2D gels, mass spectrometry, or other suitable techniques.

[0198] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite heterogeneity in capsid proteins due to deamidation, rAAVs within a stock are expected to share the same vector genome. A stock can contain, for example, rAAVs with capsids that have a heterogeneous deamidation pattern characteristic of a selected AAV capsid protein and a selected production system. A stock can be produced from a single production system or pooled from multiple runs of a production system. Various production systems can be selected, including, but not limited to, those described herein. See, for example, WO2019 / 168961, published September 6, 2019, and WO2020 / 160582, filed September 7, 2018, which includes Table G, which provides the deamidation pattern of AAV9. See also, for example, WO2020 / 223231 published on November 5, 2020 (rh91, including a table with deamidation patterns), U.S. Provisional Patent Application No. 63 / 065,616 filed on August 14, 2020, U.S. Provisional Patent Application No. 63 / 109,734 filed on November 4, 2020, and International Patent Application No. PCT / US21 / 45945 filed on August 13, 2021, all of which are incorporated by reference in their entirety into this specification.

[0199] The compositions described herein can be used in a regimen involving the simultaneous administration of other active agents.Any suitable method or route can be used to administer such other agents.Administration routes include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.Optionally, the AAV compositions described herein can also be administered by one of these routes.

[0200] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. During infection, the first Rather than waiting for cell-mediated synthesis of two strands, the two complementary halves of scAAV will assemble to form a single double-stranded DNA (dsDNA) unit 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, for example, in 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.

[0201] The term "heterologous," when used with reference to a protein or nucleic acid, indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids having two or more sequences from unrelated genes arranged to create a new functional nucleic acid are typically produced recombinantly. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0202] A "replication-deficient virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged within a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-deficient, i.e., they are unable to produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy because replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0203] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle containing two elements: an AAV capsid and a vector genome containing at least a non-AAV coding sequence packaged within the AAV capsid. In certain embodiments, the capsid contains approximately 60 proteins composed of vp1, vp2, and vp3 proteins, which self-assemble to form the capsid. Unless otherwise specified, "recombinant AAV" or "rAAV" can be used interchangeably with the phrase "rAAV vector." Because rAAV lacks any functional AAV rep or cap genes and cannot generate progeny, it is a "replication-deficient virus" or "viral vector." In certain embodiments, the only AAV sequences are AAV inverted terminal repeats (ITRs), typically located at the extreme 5' and 3' ends of the vector genome, allowing genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0204] The term "nuclease-resistant" indicates that the AAV capsid is assembled around an expression cassette designed to deliver a transgene into a host cell, and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

[0205] As used herein, the term "host cell" can refer to a packaging cell line in which an rAAV is produced from a plasmid. Alternatively, the term "host cell" can refer to a target cell in which expression of a transgene is desired.

[0206] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside the rAAV capsid that forms the viral particle. Such nucleic acid sequences include AAV inverted terminal repeats (ITRs). In the example herein, the vector genome includes, from 5' to 3', at least the AAV 5' ITR, an expression cassette containing coding sequence(s) (i.e., transgene(s)), and the AAV 3' ITR. In certain embodiments, the ITRs are from AAV2 (a different AAV source from the capsid), or other full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV or trans-complementing AAV that provides the rep function during production. Additionally, other ITRs, such as self-complementary (scAAV) ITRs, can be used. Both single-stranded and self-complementary (sc) AAVs are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transcription, translation, and / or expression of the transgene in cells of the 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 and a nucleic acid sequence encoding a protein of interest operably linked to a regulatory control sequence (that directs its expression in the target cell), where the vector-specific sequence may be a terminal repeat sequence that 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.

[0207] As used herein, "operably linked" sequences include both expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0208] In certain embodiments, non-viral genetic elements used in the production of rAAV will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, although the use of other suitable genetic elements is contemplated. Such production plasmids may encode sequences expressed during rAAV production, such as AAV capsid or rep proteins necessary for the production of rAAV, that are not packaged into the rAAV. Alternatively, such production plasmids may carry vector genomes that are packaged into the rAAV.

[0209] As used herein, "parent capsid" refers to a non-mutated, non-engineered, or non-modified capsid selected from parvoviruses or other viruses (e.g., AAV, adenovirus, HSV, RSV, etc.). In certain embodiments, the parent capsid includes any naturally occurring AAV capsid comprising a wild-type genome encoding capsid proteins (i.e., vp proteins), which direct AAV transduction and / or tissue-specific tropism. In some embodiments, the parent capsid is selected from an AAV that naturally targets muscle cells. In other embodiments, the parent capsid is selected from an AAV that does not naturally target muscle cells.

[0210] As used herein, the terms "target cell" and "target tissue" can refer to any cell or tissue intended to be transduced by the subject AAV vector. This term applies to cells of muscle, liver, lung, respiratory epithelium, the central nervous system, neurons, eyes ( In one embodiment, the target tissue is muscle tissue. In certain embodiments, the target cells are one or more muscle cell types (e.g., cardiac muscle cells, gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells).

[0211] As used herein, "cardiac cells" refers to heart cells, cardiomyocytes, "Cardiac" refers to common cardiac tissue cells, including but not limited to cardiac muscle cells (cardiomyocytes), conductive cells, fibroblasts, endothelial cells, smooth muscle cells, and pericytes.

[0212] As used herein, "variant capsid" or "variant AAV" or "variant AAV capsid" refers to a modified, engineered, or mutated capsid, wherein the capsid protein includes an insertion of a tissue-specific targeting peptide, and the modified insertion is not a naturally occurring variant.

[0213] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and operably linked regulatory sequences that direct or regulate the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, "operably linked" sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of a promoter, enhancer, intron, Kozak sequence, polyadenylation sequence, and TATA signal. An expression cassette may include, among other elements, regulatory sequences upstream (from 5') of the gene sequence, e.g., one or more of a promoter, enhancer, intron, etc., and enhancer, or regulatory sequences downstream (to 3') of the gene sequence, e.g., a 3' untranslated region (3'UTR) containing a polyadenylation site. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, and the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5' untranslated region (5' UTR). In certain embodiments, the expression cassette comprises the nucleic acid sequence of one or more gene products. In some embodiments, the expression cassette can be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell. Typically, such expression cassettes can be used to generate viral vectors and comprise the coding sequence of a gene product described herein adjacent to a packaging signal in the vector genome, as well as other expression control sequences, such as those described herein. In certain embodiments, the vector genome can comprise two or more expression cassettes.

[0214] The term "translation" in the context of the present invention relates to the process at the ribosome, whereby an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.

[0215] The term "expression" is used herein in its broadest sense and includes the production of RNA, or the production of RNA and protein. Expression may be transient or stable.

[0216] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity over at least about 95-99% of the aligned sequences. Preferably, the homology is over the full-length sequence, or its open reading frame, or over at least 15 nucleotides. The homology is across another suitable fragment of length. Examples of suitable fragments are described herein.

[0217] In the context of nucleic acid sequences, the terms "percent identity," "sequence identity," "percent sequence identity," or "percent identical" refer to the residues in two sequences that are the same when aligned for correspondence. The length of sequence identity comparison can be, and is preferred, over the full length of a genome, the full length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides. However, identity between smaller fragments, e.g., at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, or at least about 36 or more nucleotides, can also be desired.

[0218] Percent identity can be readily determined for proteins, polypeptides, amino acid sequences spanning the full length of about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequences encoding the sequences. Suitable amino acid fragments can be at least about 7 amino acids in length and can be up to about 700 amino acids.

[0219] Examples of suitable fragments are described herein. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more than 97% identity. Identity can be easily determined by those skilled in the art by relying on algorithms and computer programs known to those skilled in the art.

[0220] Generally, when referring to "identity," "homology," or "similarity" between two different sequences, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence.

[0221] Identity can be determined by preparing an alignment of sequences using various algorithms and / or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm). Alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal Omega," "Clustal W," "MUSCLE," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME," all accessible through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can also be used. Additionally, several algorithms known in the art exist and can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 10.1. Fasta™ provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG version 10.1, which is incorporated herein by reference. For amino acid sequences, sequence alignment programs such as "Clustal Omega", "Clustal X", "MUSCLE", "MAP", "PIMA", "MSA", " Programs such as "BLOCKMAKER," "MEME," and "Match-Box" are available. Generally, one of these programs is used with default settings, but one of skill in the art can change these settings as needed. Alternatively, one of skill in the art can use another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, JD Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0222] In certain embodiments, the effective amount may be determined based on animal models rather than human patients.

[0223] As noted above, the term "about," when used to adjust a numerical value, means a variation of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or any value therebetween) from the given reference, unless otherwise specified.

[0224] In certain cases, the terms "E+#" or "e+#" are used to refer to the exponent. For example, "5E10" or "5e10" refers to 5 x 10 10 These terms may be used interchangeably.

[0225] As used throughout this specification and claims, the terms "comprise", "contain", and variations thereof, including "comprises", "comprising", "contain" and "containing", among other variations, are inclusive of other components, elements, integers, steps, etc. The terms "consists of" or "consisting of" exclude other components, elements, integers, steps, etc.

[0226] The terms "a" or "an" refer to one or more, e.g., "enhancer" Note that "a" (or "an"), "one or more," and "at least one" refer to one or more enhancers. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0227] With respect to these descriptions of the invention, it is contemplated that each of the compositions described herein is, in another embodiment, useful in the methods of the invention. Additionally, it is contemplated that each of the compositions described herein that are useful in the methods is, in another embodiment, itself an embodiment of the invention.

[0228] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide general guidance to those skilled in the art for many of the terms used in this application. [Example]

[0229] The following examples are merely illustrative and do not limit the invention described herein.

[0230] Currently, there are no approved AAV gene therapies for cardiac disorders. One reason for this is The inherent difficulty in transducing cardiomyocytes via systemic AAV injection is a key challenge. While significant cardiac transduction is achievable via the naturally occurring AAV variant AAV9, the specificity of this transduction is lacking. Indeed, the majority of injected vectors end up transducing the liver, and the high doses required for therapeutically relevant cardiac transduction therefore pose the risk of liver damage, adverse immune responses, and potential death. One potential solution to this problem is local injection of gene therapy drugs. However, this technique is invasive and difficult to control, thus posing another set of safety risks that require further innovation to overcome. Therefore, a major goal of the gene therapy field is to create gene transfer agents that can specifically and non-invasively transduce myocardial cells. Thus, there exists an unmet clinical need for AAV vectors that can transduce myocardium more efficiently than the current standard in the art.

[0231] Similarly, AAV9 has historically performed poorly at transducing skeletal muscle, posing a major obstacle to current efforts aimed at treating numerous genetic disorders, such as Duchenne muscular dystrophy. Therefore, AAV vectors with enhanced skeletal muscle tropism are another powerful target for viral vector engineering.

[0232] Small peptide insertions into flexible loops on the surface of the AAV capsid have been shown to be able to mediate interactions with novel cellular receptors. In one example discovered at CalTech (AAV9-PHP.B), a seven-amino acid peptide inserted into the HVR8 loop on AAV9 mediates interaction with Ly6a, a GPI-anchored receptor on the cerebrovasculature of several mouse strains. This interaction drives transport of AAV9-PHP.B across the blood-brain barrier, resulting in approximately 50-fold higher transduction of brain cells than AAV9.

[0233] Studies were conducted to identify peptide inserts that could improve AAV9 capsid transduction of both skeletal and cardiac muscle. To accomplish this, we leveraged previous work in AAV capsid engineering that showed that the well-studied RGD peptide motif (a targeting motif for numerous integrins) was able to target AAV vectors to skeletal muscle (and to a lesser extent cardiac muscle) in the context of the AAV9 HVR8 loop.

[0234] We generated a library of hundreds of thousands of AAV9 insertion variants, each containing an RGD-containing peptide, individually inserted at the HVR8 locus (between positions 588 and 589). Each variant was barcoded to identify the transgene it carried as well as the capsid protein it contained, allowing analysis of the relative abundance of each variant after RNA extraction from transduced tissue. This library was injected intravenously into high-dose rhesus macaques, and the animals were sacrificed at 2 weeks for RNA extraction and analysis.

[0235] Total RNA was then collected, mRNA was enriched from the total RNA, and the mRNA was converted to cDNA, followed by next-generation sequencing of the resulting cDNA library. Thus, by sequencing the entire expressed genome in both cardiac and skeletal muscle, we were able to select hits for validation in a second, less noisy, more targeted library experiment. Variants were selected for high levels of enrichment across the range of vector library representation (tissue rpm / injected vector library rpm). In addition, several vectors from a separate screening in NHPs (driven by a synaptic promoter but still expressed in the heart) were also included in the second round of screening. The second round of libraries also included several key controls. First, AAV9-PHP.B (insert TLAVPFK (SEQ ID NO: 37)) and a related insert (TLAGPFK (SEQ ID NO: 38)), both of which are part of the AAV9-PHP.B library. Despite having an insert at position 588, it functioned similarly to AAV9 in the heart. We also included several previously reported vectors with known skeletal muscle tropism as positive controls. Each variant was encoded by three different synonymous codons, providing better confidence in performance metrics based on clustering of synonymous variants. From this secondary screen, we were able to identify numerous variants with 2- to 22-fold increases in both cardiac and skeletal muscle transduction.

[0236] Example 1 Production of rAAV containing the gene (protein) of interest In this study, engineered rAAVs containing engineered AAV capsids containing exogenous targeting peptides were generated and comparative studies were performed. In some cases, rAAVs containing the aGFP gene were generated and used to evaluate rAAV transduction and transduction expression. In some cases, rAAVs containing test gene X (TGX, where X is 1, 2, 3, etc.) were generated and used to evaluate rAAV transduction and gene expression.

[0237] Triple transfection technology is used to generate rAAVs using (1) a cis plasmid encoding the AAV2 rep protein and the AAV9 VP1cap gene, (2) a cis plasmid containing adenovirus helper genes not provided by the packaging cell line expressing adenovirus E1a, and (3) a trans plasmid containing a vector genome for packaging into AAV capsids. See, for example, US2020 / 0056159. The trans plasmid is designed to contain a vector genome containing a transgene of interest (e.g., GFP). The vector genome contains an AAV 5' inverted terminal repeat (ITR) and an AAV 3' ITR at the extreme 5' and 3' ends, respectively. The ITRs flank the sequence of an expression cassette packaged into the AAV capsid, which contains a sequence encoding the protein of interest. The expression cassette further contains a regulatory sequence operably linked to the protein-coding sequence, and the regulatory control sequence includes at least one of a promoter, an enhancer, and a polyA sequence.

[0238] The nucleic acid molecule comprising the vector genome packaged in a capsid contains an AAV2-5' inverted terminal repeat (ITR) and an AAV2-3' ITR at the extreme 5' and 3' ends of the vector genome, respectively. The vector genome further comprises an expression cassette packaged in the AAV capsid, having a sequence encoding test transgene 2 (TT2) between the ITRs. The expression cassette further comprises regulatory sequences operably linked to the engineered coding sequence, the regulatory control sequences including an optional enhancer, a promoter, and an optional intron (e.g., a hybrid CB7 promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, a chicken beta-actin intron, and an optional spacer sequence), and the expression cassette further comprises a rabbit beta-globin (RBG) polyA.

[0239] A summary of the productivity and recovery of rAAV production containing TT2 (e.g., using iCellis 200m2 with downstream chromatographic purification) is summarized below in Table A (recovery summary), Table B (productivity), and Table C (enrichment summary). [Table 1] [Table 2] [Table 3]

[0240] These results indicate that in the yield and manufacturability assessments, productivity and enrichment values ​​were comparable among AAVhu68, AAV9-IIRGDPA, and AAV9-AVIRGDV capsids.

[0241] In addition, mass spectrometry (N=1) of deamidation of rAAV capsids containing the AAV9 mutant AVIRGDV was performed, and the results are shown in Table D. [Table 4]

[0242] As can be seen, the AAV9-AVIRGDV mutant retains the deamidation pattern of AAV9 in that it is highly deamidated (at least 50% deamidated) at positions N57, N329, N452, and N512 [see US2020 / 0407750A1, incorporated herein by reference].

[0243] Example 2 Improvement of AAV9 for cardiac transduction In this study, we designed and generated an AAV9 library containing a comprehensive collection of all possible RGD 7-mer peptides (inserted into the HVRVIII (HVR8) region of the AAV9 capsid). This was used to administer a dose of 5 × 10 RATs in heart and muscle-focused (i.e., different integrin configurations) NHPs (heart vs. muscle) collected at day 14 for heart and gastrocnemius (muscle) tissue. 13 The RGD-inserted variants were used for selection through two rounds of selection at 1000 ng / kg (GC / kg). In round 2 of NHP cardiac selection, a mini-library design with an embedded control sequence, an AAV9-like negative control vector, and the best-reported RGD variant from the literature as a positive control was used. Figure 1A shows the plotted cardiac enrichment scores from round 2 of RGD screening for top-performing cardiac candidates compared to top literature capsids. I...

Claims

1. A recombinant adeno-associated virus particle (rAAV), comprising: (a) an adeno-associated virus (AAV) capsid comprising a VP1 protein, a VP2 protein, and a VP3 protein, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region that comprises an exogenous targeting peptide, and the exogenous targeting peptide comprises an "Xn-n-mer-Xm"; (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is selected from the group consisting of AVIRGDV (SEQ ID NO: 2), IIRGDPA (SEQ ID NO: 1), RGDYAQV (SEQ ID NO: 20), RGDLHGY (SEQ ID NO: 22), PYQRGDH (SEQ ID NO: 24), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13) ), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), VVQRGDV (SEQ ID NO: 17), QHRGDTQ (SEQ ID NO: 18), QIRGDLR (SEQ ID NO: 19), IGRGDPN (SEQ ID NO: 21), or RGDYSTM (SEQ ID NO: 23), or an n-mer sequence of at least 6, at least 7, or the full length of any one of said n-mers; (iii) an adeno-associated virus (AAV) capsid, wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged within the AAV capsid, the vector genome comprising a nucleic acid sequence encoding a gene product under the control of a sequence that directs expression of the nucleic acid sequence encoding the gene product.

2. the exogenous targeting peptide is (a) AVIRGDV (SEQ ID NO: 2), or (b) the rAAV of claim 1, comprising IIRGDPA (SEQ ID NO: 1).

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

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

5. 4. The rAAV of any one of claims 1 to 3, wherein the exogenous targeting peptide is inserted into the hypervariable region between amino acids 588 and 589 in an AAV9 parent capsid as determined based on the numbering of the VP1 amino acid sequence of SEQ ID NO:25, or into an analogous position in an AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 parent capsid.

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

7. The rAAV capsid is an amino acid sequence of SEQ ID NO: 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107.

6. The rAAV of any one of claims 1 to 5, comprising an AAV VP3 protein having a mutant VP3 region from amino acids 204 to 743, and further comprising highly deamidated residues at positions N57, N329, N452, and N512, the deamidated position numbers being based on the residue positions of SEQ ID NO:25 or SEQ ID NO:

26.

8. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV capsid comprises an AAV VP1 protein, an AAV VP2 protein, and an AAV VP3 protein having a mutant VP1 at amino acids 1 to 743 of SEQ ID NO:73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, and further comprises 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:25 or SEQ ID NO:

26.

9. 5. 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: 108-125, or a sequence at least 95% identical to any one of SEQ ID NOs: 108-125.

10. A composition comprising a stock of rAAV according to 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 muscle cell targeting peptide comprising "Xn-n-mer-Xm", (a) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (b) the n-mer is selected from the group consisting of AVIRGDV (SEQ ID NO: 2), IIRGDPA (SEQ ID NO: 1), IVRGDPA (SEQ ID NO: 8), MIRGDVK (SEQ ID NO: 9), AQHRGDV (SEQ ID NO: 10), VSRGDPN (SEQ ID NO: 11), VSRGDPA (SEQ ID NO: 12), PLVRGDI (SEQ ID NO: 13), PYVRGDP (SEQ ID NO: 14), VVRGDPQ (SEQ ID NO: 15), VVQRGDV (SEQ ID NO: 17), ), QHRGDTQ (SEQ ID NO:18), QIRGDLR (SEQ ID NO:19), RGDYAQV (SEQ ID NO:20), IGRGDPN (SEQ ID NO:21), RGDLHGY (SEQ ID NO:22), RGDYSTM (SEQ ID NO:23), or PYQRGDH (SEQ ID NO:24), or an n-mer sequence of at least 6, at least 7, or the full length of any one of said n-mers; (c) Xm is 0, 1, 2, or 3 amino acids independently selected from any amino acid; Optionally, the recombinant muscle cell targeting peptide is conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

12. the recombinant muscle cell targeting peptide (a) AVIRGDV (SEQ ID NO: 2), or (b) the recombinant muscle cell targeting peptide of claim 11, comprising IIRGDPA (SEQ ID NO: 1).

13. 12. The recombinant muscle targeting peptide of claim 11, wherein the recombinant muscle cell targeting peptide targets cardiac myocytes.

14. 12. The recombinant muscle cell targeting peptide of claim 11, wherein the recombinant muscle cell targeting peptide targets skeletal muscle cells, optionally gastrocnemius muscle cells.

15. A recombinant muscle cell targeting peptide according to any one of claims 11 to 14, and one or more of a compatible carrier, excipient, and / or aqueous suspension base.

16. A nucleic acid molecule encoding the recombinant muscle cell targeting peptide of any one of claims 11 to 14.

17. A nucleic acid molecule comprising a mutant AAV capsid VP1 gene comprising a nucleic acid sequence encoding an n-mer of AVIRGDV (SEQ ID NO:2), IIRGDPA (SEQ ID NO:1), RGDYAQV (SEQ ID NO:20), RGDLHGY (SEQ ID NO:22), PYQRGDH (SEQ ID NO:24), IVRGDPA (SEQ ID NO:8), MIRGDVK (SEQ ID NO:9), AQHRGDV (SEQ ID NO:10), VSRGDPN (SEQ ID NO:11), VSRGDPA (SEQ ID NO:12), PLVRGDI (SEQ ID NO:13), PYVRGDP (SEQ ID NO:14), VVRGDPQ (SEQ ID NO:15), VVQRGDV (SEQ ID NO:17), QHRGDTQ (SEQ ID NO:18), QIRGDLR (SEQ ID NO:19), IGRGDPN (SEQ ID NO:21), or RGDYSTM (SEQ ID NO:23).

18. the sequence encoding the n-mer is (a) SEQ ID NO: 109, or a sequence at least 99% identical thereto (encoding AVIRGDV); (b) SEQ ID NO: 108, or a sequence at least 99% identical thereto (encoding IIRGDPA); (c) SEQ ID NO: 110 or a sequence at least 99% identical thereto (encoding IVRGDPA); (d) SEQ ID NO: 111 or a sequence at least 99% identical thereto (encoding MIRGDVK); (e) SEQ ID NO: 112 or a sequence at least 99% identical thereto (encoding AQHRGDV); (f) SEQ ID NO: 113 or a sequence at least 99% identical thereto (encoding VSRGDPN); (g) SEQ ID NO: 114 or a sequence at least 99% identical thereto (encoding VSRGDPA); (h) SEQ ID NO: 115 or a sequence at least 99% identical thereto (encoding PLVRGDI); (i) SEQ ID NO: 116 or a sequence at least 99% identical thereto (encoding PYVRGDP); (j) SEQ ID NO: 117 or a sequence at least 99% identical thereto (encoding VVRGDPQ); (k) SEQ ID NO: 118 or a sequence at least 99% identical thereto (encoding VVQRGDV); (l) SEQ ID NO: 119 or a sequence at least 99% identical thereto (encoding QHRGDTQ); (m) SEQ ID NO: 120 or a sequence at least 99% identical thereto (encoding QIRGDLR); (n) SEQ ID NO: 121 or a sequence at least 99% identical thereto (encoding RGDYAQV); (o) SEQ ID NO: 122 or a sequence at least 99% identical thereto (encoding IGRGDPN); (p) SEQ ID NO: 123 or a sequence at least 99% identical thereto (encoding RGDLHGY); (q) SEQ ID NO: 124 or a sequence at least 99% identical thereto (RGDYSTM ), or (r) The nucleic acid molecule of claim 17, which is SEQ ID NO: 125 or a sequence at least 99% identical thereto (encoding PYQRGDH).

19. 19. The nucleic acid molecule of claim 17 or 18, wherein the nucleic acid sequence encoding the AAV VP1 gene is SEQ ID NO: 74, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106.

20. A fusion polypeptide or protein comprising a recombinant muscle cell targeting peptide according to any one of claims 11 to 14 and a fusion partner comprising at least one polypeptide or protein.

21. 21. A composition comprising the fusion polypeptide or protein of claim 20 and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.

22. 22. Use of an rAAV stock according to any one of claims 1 to 9, a recombinant muscle cell targeting peptide according to any one of claims 11 to 14, or a fusion polypeptide or protein according to claim 20, or a composition according to any one of claims 10, 15, or 21, for the delivery of a therapeutic agent to a patient in need thereof.

23. A method for targeted therapy to muscle cells in a subject in need thereof, the method comprising administering to the subject a stock of rAAV described in claim 1.

24. 1. A method for targeted delivery of a gene product to muscle cells in a subject in need thereof, the method comprising administering to the subject a stock of rAAV of claim 1, optionally wherein the muscle cells are cardiomyocytes and / or skeletal muscle cells, optionally gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells.

25. 1. A method for treating muscle cell disorders and / or diseases in a subject in need thereof, the method comprising delivering to the subject a stock of rAAV described in claim 1, wherein the encoded gene product is a protein, optionally an antibody.

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