Compositions and methods for improved adeno-associated viral gene therapy

Abstract

Provided herein are recombinant adeno-associated virus (AAV) capsid proteins that contain an artificial peptide insert, as well as AAV particles containing such capsid proteins and methods of administering such AAV particles to a subject, e.g., so as to express a transgene of interest.

Description

[0001] ATTORNEY DOCKET NO.: 51772-007WO2

[0002] PATENT COMPOSITIONS AND METHODS FOR IMPROVED ADENO-ASSOCIATED VIRAL GENE THERAPY

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated in its entirety. Said XML copy, created on December 5, 2025, is named “51772-007WO2_Sequence_Listing_12_5_25.xml” and is 575,510 bytes in size.

[0005] BACKGROUND

[0006] Recombinant vectors derived from adeno-associated viruses (AAVs) have become a prevalent modality in gene therapy due, at least in part, to the remarkable safety profile of this non-pathogenic virus, as well as its potential to achieve transgene expression in a variety of tissues. Intramuscularly or intravenously administered AAV vectors have been explored as a vehicle for the delivery of various transgenes, including therapeutic and prophylactic proteins. Despite early successes in proof-of-concept studies, the clinical development of intramuscularly or intravenously administered AAV vectors has largely been limited as a result of insufficient potency, as the transduction efficiency that natural AAV serotypes exhibit - particularly for muscle cells - is often too low to achieve adequate levels of therapeutic or prophylactic protein secretion to the serum. Accordingly, there remains a need for AAV vectors that effectuate enhanced transduction activity, whether provided intramuscularly, intravenously, or byway of another route of administration.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention addresses the limitations of adeno associated viruses (AAVs) used in gene therapy. Historically, these vectors have been hindered by insufficient transduction efficiency, leading to inadequate levels of transgene expression. To address this, the current disclosure describes the discovery and optimization of peptide inserts that, upon incorporation into an AAV capsid protein, enhance transduction efficiency, tropism, and biodistribution in targeted tissue. Also described herein are exemplary processes for incorporating peptide inserts of the invention into an AAV capsid protein, producing recombinant AAV particles containing peptide inserts disclosed herein, and administering recombinant AAV particles to a patient in need thereof through various routes described herein.

[0009] In a first aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (I):

[0010] (X1)(X2)(X3)(X4)(X5)(X6)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X3), (X5), and (X6), independently, represents any naturally occurring amino acid.

[0011] In some embodiments, (X1) represents Thr, Ser, Asn, Gly, Leu, or Met. In some embodiments, (X1) represents Thr or Ser.

[0012] In some embodiments, (X3) represents Thr, lie, Ser, Asn, Ala, Arg, Met or Lys. In some embodiments, (X3) represents Thr. lie, Ser, Asn, or Ala.

[0013] In some embodiments, (X5) represents Pro, Lys, Vai, or lie. In some embodiments, (X5) represents Pro, Lys, or Vai. In some embodiments, (X5) represents Pro. ATTORNEY DOCKET NO.: 51772-007WO2

[0014] PATENT

[0015] In some embodiments, (X6) represents Ala, Gly, lie, Met, Gin, Ser, or Vai. In some embodiments, (X6) represents Ala, lie, Gin, or Vai.

[0016] In some embodiments, (X2) represents Lys.

[0017] In some embodiments, (X2) represents Arg.

[0018] In some embodiments, (X4) represents Lys.

[0019] In some embodiments, (X4) represents Arg.

[0020] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (II): (X1)(X2)(X3)(X4)(X5)(X6)G, wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X5), and (X6), independently, represents any naturally occurring amino acid.

[0021] In some embodiments, (X1) represents Thr, Ser, Met, His, Gly, or Trp. In some embodiments, (X1) represents Thr or Ser.

[0022] In some embodiments, (X2) represents lie, Phe, Leu, Lys, Arg, Ala, His, Met, Asn, Gin, Ser, Thr, or Vai. In some embodiments, (X2) represents lie, Phe, or Leu. In some embodiments, (X2) represents lie.

[0023] In some embodiments, (X5) represents Pro, lie, Vai, Ala, or Thr. In some embodiments, (X5) represents Pro or lie. In some embodiments, (X5) represents Pro.

[0024] In some embodiments, (X6) represents Ala, Vai, Gin, lie, Asn, Pro, or Ser. In some embodiments, (X6) represents Ala or Vai.

[0025] In some embodiments, (X3) represents Lys.

[0026] In some embodiments, (X3) represents Arg.

[0027] In some embodiments, (X4) represents Lys.

[0028] In some embodiments, (X4) represents Arg.

[0029] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (III): (X1)(X2)(X3)(X4)(X5)(X6)G, wherein each of (X4) and (X5), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid.

[0030] In some embodiments, (X1) represents Thr or Ser. In some embodiments, (X1) represents Thr. In some embodiments, (X2) represents lie, Lys, or Leu. In some embodiments, (X2) represents lie.

[0031] In some embodiments, (X3) represents Thr, Arg, Ala, or lie. In some embodiments, (X3) represents Thr or Arg.

[0032] In some embodiments, (X6) represents Vai, lie, Ala, Pro, or Gin. In some embodiments, (X6) represents Vai or lie.

[0033] In some embodiments, (X4) represents Lys.

[0034] In some embodiments, (X4) represents Arg.

[0035] In some embodiments, (X5) represents Lys.

[0036] In some embodiments, (X5) represents Arg. ATTORNEY DOCKET NO.: 51772-007WO2

[0037] PATENT

[0038] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (IV): (X1)(X2)(X3)(X4)P(X6)(X7), wherein (X4) represents an amino acid selected from the group consisting of Lys and Arg, (X7) represents an amino acid selected from the group consisting of Gly and Pro, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid.

[0039] In some embodiments, (X1) represents Thr, Ser, Met, or Asn. In some embodiments, (X1) represents Thr, Ser, or Met.

[0040] In some embodiments, (X2) represents lie, Leu, Phe, Arg, or Met. In some embodiments, (X2) represents lie, Leu, or Phe.

[0041] In some embodiments, (X3) represents lie, Thr, Ala, Met, Asn, Arg, Ser, or Tyr. In some embodiments, (X3) represents lie or Thr.

[0042] In some embodiments, (X6) represents Ala, Vai, lie, or Ser. In some embodiments, (X6) represents Ala or Vai.

[0043] In some embodiments, (X4) represents Lys.

[0044] In some embodiments, (X4) represents Arg.

[0045] In some embodiments, (X7) represents Gly.

[0046] In some embodiments, (X7) represents Pro.

[0047] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (V): (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Vai, and lie, (Polar) represents any amino acid listed in Table 1, below, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, lie, or Met. In some embodiments, (X3) represents Ala, lie, Met, Asn, Arg, Ser, or Thr.

[0048] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (VI): (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Vai, (Polar) represents any amino acid listed in Table 1, below, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, lie, or Met. In some embodiments, (X3) represents Ala, lie, Met, Asn, Arg, Ser, or Thr.

[0049] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (VII): (X1)(X2)(X3)(X4)(X5)(X6)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Asn, and Gin, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Arg, Vai, and lie, (X6) represents an amino acid selected from the group consisting ATTORNEY DOCKET NO.: 51772-007WO2

[0050] PATENT

[0051] of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, orThr.

[0052] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (VIII): (X1)(X2)(X3)(X4)P(Nonpolar)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Met, and Asn, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Nonpolar) represents Ala, Gly, lie, Met, or Vai. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0053] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (IX): (X1)(X2)(X3)(X4)P(X6)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Met, and Asn, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0054] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (X): (Polar)(X2)(X3)(X4)P(Nonpolar)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (Polar) represents any amino acid listed in Table 1, below, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, Gly, lie, Met, or Vai. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0055] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (XI): (X1)(X2)(X3)(X4)P(X6)G, wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Asn, and Gin, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, orThr.

[0056] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (XII): (X1)(X2)(X3)(X4)(X5)(Nonpolar)G, wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro and lie, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and each of (X1) and (X2), independently, represents any naturally occurring amino acid. In some embodiments, (X1) and (X2) represent amino acids that are not anionic. In some embodiments, (Nonpolar) represents Ala, lie, Pro, or Vai. In some embodiments, (X1) represents ATTORNEY DOCKET NO.: 51772-007WO2

[0057] PATENT

[0058] Gly, His, Met, Ser, Thr, or Trp. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0059] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (XIII): (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G, wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (Polar) represents any amino acid listed in Table 1, below, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain, and (X2) represents any naturally occurring amino acid. In some embodiments, (Polar) represents His, Ser, or Thr. In some embodiments, (Nonpolar) represents Ala, lie, Pro, or Vai. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0060] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (XIV): (X1)(X2)(X3)(X4)(X5)(X6)G, wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, and Met, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X2) represents any naturally occurring amino acid. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0061] In another aspect, the disclosure features a recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (XV): (X1)(Nonpolar)(X3)(X4)(X5)(X6)G, wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, and Met, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (Nonpolar) represents any amino acid listed in Table 1, below, as having a nonpolar, neutral side chain. In some embodiments, (Nonpolar) represents Ala, Phe, lie, Leu, Met, or Vai.

[0062] In some embodiments of the foregoing aspects, the peptide insert is incorporated into a wild-type AAV capsid protein. In some embodiments, the wild-type AAV capsid protein is a capsid protein that occurs naturally in an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, or AAVrh74. In some embodiments, the wild-type AAV capsid protein is a capsid protein that occurs naturally in AAV9.

[0063] In some embodiments, the wild-type AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the wild-type AAV capsid protein is a VP1 capsid protein.

[0064] In some embodiments, the peptide insert is incorporated into the wild-type AAV capsid protein between a pair of consecutive amino acid residues that are located within a variable region (VR) selected from the group consisting of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX. In some embodiments, the peptide insert is incorporated into the wild-type AAV capsid protein between a pair of consecutive amino acid residues that are located within VR-VIII. In some embodiments, the pair of consecutive amino acid residues is selected from the group consisting of (a) amino acids 581 and 582, ATTORNEY DOCKET NO.: 51772-007WO2

[0065] PATENT

[0066] (b) amino acids 582 and 583, (c) amino acids 583 and 584, (d) amino acids 584 and 585, (e) amino acids 585 and 586, (f) amino acids 586 and 587, (g) amino acids 587 and 588, (h) amino acids 588 and 589, (i) amino acids 589 and 590, (j) amino acids 590 and 591, (k) amino acids 591 and 592, or (I) amino acids 592 and 593, wherein the amino acids are numbered relative to the amino acid sequence of wild-type AAV9 VP1 (SEQ ID NO: 11).

[0067] In a further aspect, the disclosure features an AAV particle comprising any of the AAV capsid proteins of any one of the foregoing aspects or embodiments of the disclosure.

[0068] In some embodiments, the AAV capsid protein encapsulates an AAV genome comprising, in the 5’-to-3’ direction:

[0069] a) a first inverted terminal repeat (ITR);

[0070] b) a transgene of interest; and

[0071] c) a second ITR.

[0072] In some embodiments, the AAV genome further comprises, between the first ITR and the transgene, one or more transcription regulatory elements that modulate expression of the transgene.

[0073] In a further aspect, the disclosure features a method of expressing a transgene in a subject, the method comprising administering to the subject the AAV particle of any of the foregoing aspects or embodiments of the disclosure.

[0074] In some embodiments, the AAV particle is administered to the subject by way of intramuscular, intrahepatic, intravenous, intrathecal, intracerebroventricular, intracisternal, intrastriatal, intracerebral, intracerebrospinal, intracranial, intracortical, intradermal, transdermal, parenteral, intranasal, subcutaneous, percutaneous, intratracheal, intraocular, or intravascular administration. In some embodiments, the AAV particle is administered to the subject by way of intramuscular administration.

[0075] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In a further aspect, the disclosure features a kit comprising the AAV particle of any of the foregoing aspects or embodiments of the disclosure and a package insert, wherein the package insert instructs a user of the kit to administer the AAV particle to a subject in accordance with the method of any one of the above aspects or embodiments of the disclosure.

[0076] BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a graph showing the sequence logo of the peptide motif discovered in the initial stringent data analysis (as is described in the working examples, below). In brief, 506 unique peptides were subjected to clustering analysis using available web tools and this distinct peptide family motif was discovered. The experiments were performed as described in Example 1, below.

[0077] FIG.2 shows sequence logos for subgroups of the peptide family of the invention.

[0078] FIGS.3A-3C are a series of graphs showing the comparison of AAV9 and SIRKPAG (SEQ ID NO: 18) capsids for intramuscularly administered AAV in mice measuring the following in vivo: (FIG.3A) serum [glucagon-like peptide-1 (GLP-1 )] pM, (FIG. 3B) relative mRNA level from muscle, and (FIG.3C) relative mRNA level from liver. The SIRKPAG (SEQ ID NO: 18) capsid enhanced serum production of the reporter protein across a wide range of doses (Figure 3A). Local enhancement at the muscle injection site was pronounced, particularly at higher doses, for SIRKPAG (SEQ ID NO: 18) (Figure 3B). Rag mice ATTORNEY DOCKET NO.: 51772-007WO2

[0079] PATENT

[0080] were dosed intramuscularly with SIRKPAG (SEQ ID NO: 18) and AAV9 (each encapsulating identical cytomegalovirus-GLP-1 -fragment crystallizable (CMV-GLP-1-Fc) reporter constructs). Fourteen days later, serum was collected from a terminal bleed for GLP-1 -Fc reporter protein determination, and from the muscle and liver for reporter mRNA quantification. The experiments were performed as described in Example 4, below. FIG.3A abbreviations: GLP-1; glucagon-like peptide- 1.

[0081] FIGS.4A-4C are a series of bar graphs showing the comparison of AAV9 and TIRKPAG (SEQ ID NO: 57) capsids for intramuscular AAV in mice at 1 E9 GC / mouse measuring the following: (FIG. 4A) serum GLP-1 -Fc, (FIG.4B) GLP-1 -Fc mRNA from muscle, and (FIG.4C) GLP-1 -Fc mRNA from liver. The TIRKPAG (SEQ ID NO: 57) capsid enhanced serum production of the reporter protein 24-fold (FIG.

[0082] 4A). Local enhancement at the muscle injection site for TIRKPAG (SEQ ID NO: 57) was 71 -fold (vs AAV9, FIG. 4B). TIRKPAG (SEQ ID NO: 57) transduction of liver was 2% of AAV9 (FIG.4C), demonstrating superior muscle versus liver specificity. Rag mice were dosed at 1 E9 GC / mouse intramuscularly with TIRKPAG (SEQ ID NO: 57) and AAV9 (each encapsulating identical cytomegalovirus-GLP-1 -fragment crystallizable (CMV-GLP-1-Fc) reporter constructs). Fourteen days later, serum was collected from a terminal bleed for GLP-1 -Fc reporter protein determination, and from the muscle and liver for reporter mRNA quantification. The experiments were performed as described in Example 7, below. FIG.4A abbreviations: GLP-1 -Fc; glucagon-like peptide-1 -fragment crystallizable.

[0083] Definitions

[0084] As used herein, the term “about” refers to a value that is within 10% above or below the value being described. For example, “about 100 pounds” as used in the context of weight described herein includes quantities that are within 10% above or below 100 lbs. Additionally, when used in the context of a list of numerical quantities, it is to be understood that the term “about,” when preceding a list of numerical quantities, applies to each individual quantity recited in the list.

[0085] Throughout the specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0086] As used herein, “activity” refers to the functionality or effectiveness of a molecule or system. For example, “transduction activity” refers to the effectiveness of transferring genetic material into a cell with the aid of a viral vector, such as using the methods described herein.

[0087] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).

[0088] As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom.

[0089] As used herein, the term "genetically modified" or "transformed" or "transfected" or "transduced" by exogenous DNA (e.g., via a recombinant virus) refers to when such DNA has been introduced inside ATTORNEY DOCKET NO.: 51772-007WO2

[0090] PATENT

[0091] the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0092] As used herein, the term “level” refers to a level of a molecule, for example, a protein or nucleic acid, as compared to a reference. The reference may be any useful reference, as defined herein. By a “decreased level” and an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference. A level of a protein may be expressed in mass / vol (e.g., g / dL, mg / mL, pg / mL, or ng / mL) or percentage relative to total protein in a sample.

[0093] As used herein, the “length” of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the quantity of nucleotides from the 5’ to the 3’ end of the nucleic acid. Exemplary molecular biology techniques that may be used to determine the length of a nucleic acid of interest are known in the art.

[0094] As used herein, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides of any length. Examples of polynucleotides are DNA polynucleotides and RNA polynucleotides. All nucleic acid sequences herein are written in the 5’-to-3’ direction and are to be construed accordingly. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non- nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single- stranded molecules. Unless otherwise specified or required, any embodiment herein that comprises a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0095] A nucleic acid molecule, e.g., a nucleic acid molecule in a vector (e.g., an expression vector, a viral vector) may be introduced into a host cell. As used herein, the term “host cell” refers to not only to the particular cell(s) into which the nucleic acid molecule has been introduced, but also to the progeny or potential progeny of such a cell. A host cell may be a “producer cell,” which refers to cells involved in ex vivo amplifying and manufacturing of a viral product. Many suitable producer cells are known to those skilled in the art; producer cells may be prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells, insect cells, plant cells, mammalian cells). Representative producer cells include, without limitation, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. A host cell may be a “target cell,” which refers to cells in a human or animal, for example muscle cells, that may be targeted for a therapeutic purpose, such as treating a genetic disorder described herein.

[0096] Methods for introducing nucleic acid molecules into host cells are well known in the art and include, without limitation, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral -mediated nucleic acid transfer (e.g., transduction). ATTORNEY DOCKET NO.: 51772-007WO2

[0097] PATENT

[0098] As used herein, the term “codon” refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule, or coding strand of DNA, that specifies a particular amino acid or a starting or stopping signal for translation. The term codon also refers to base triplets in a DNA strand.

[0099] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as therapeutic proteins, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding therapeutic proteins, and other such nucleic acids for use in delivery of a gene product to a mammalian subject (which may be referred to as "transgenes" to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

[0100] As used herein, the term “gene” refers to a region of DNA that encodes a protein. A gene may include regulatory regions and a protein-coding region. In some embodiments, a gene may include two or more introns and three or more exons, wherein each intron forms an intervening sequence between two exons. The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene may contain an open reading frame that is capable of encoding a gene product. One example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, e.g., a functional RNA product, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for nucleases, etc., which is transcribed but not translated.

[0101] As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product. The gene product may be an RNA, peptide, or protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and / or other functional elements. Embodiments may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0102] As used herein, the term “gene expression” or “transgene expression” refers to the process by which a nucleic acid is transcribed from a nucleic acid molecule, and often, translated into a peptide or protein. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof. Reference to a measurement of “gene expression” may refer to measurement of the product of transcription (e.g., RNA or mRNA), the product of translation (e.g., peptides or proteins). The term "gene expression product" or "gene product" is a molecule resulting from expression of a particular gene, as defined above. Gene expression products include, e.g., a polypeptide, an aptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, a non-coding RNA (ncRNA), and the like. In some ATTORNEY DOCKET NO.: 51772-007WO2

[0103] PATENT

[0104] embodiments, gene products include active polypeptide product or inhibitory RNA from a transcribed gene. In some embodiments, the transgene is operably linked to expression control sequences.

[0105] As used herein, the term “wild-type” or “non-mutant” form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation). In some embodiments, the wild-type gene may serve as a reference to compare a variant gene that is associated with a genetic disorder, e.g., as described in Table 4 herein.

[0106] As used herein, the term “variant” or “mutant” refers to any gene with a change in sequence, such that the sequence is not identical to that of the wild-type gene and results in an altered form of the gene. A mutation may be selected from the group including a single nucleotide point mutation that results in a premature termination codon, a single nucleotide insertion, a single nucleotide deletion, the insertion of two or more contiguous nucleotides, the deletion of two or more contiguous nucleotides, the duplication of a contiguous region within a gene (e.g., an exon), or the deletion of a contiguous region within a gene. A mutated gene may include a single mutation, or multiple mutations. A mutation may occur in any region of the gene. Gene mutations include the substitution, insertion, or deletion of a single base in DNA or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.

[0107] As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1, below.

[0108] Table 1. Representative physicochemical properties of naturally-occurring amino acids Electrostatic

[0109] Side3 Letter 1 Letter character at Steric Amino Acid chain

[0110] Code Code physiological pH Volume1

[0111] Polarity

[0112] (7.4)

[0113] Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic large Asparagine Asn N polar neutral intermediate Aspartic acid Asp D polar anionic intermediate Cysteine Cys C nonpolar neutral intermediate Glutamic acid Glu E polar anionic intermediate Glutamine Gin Q polar neutral intermediate Glycine Gly G nonpolar neutral small

[0114] Both neutral and

[0115] Histidine His H polar cationic forms in large equilibrium at pH 7.4

[0116] Isoleucine lie I nonpolar neutral large Leucine Leu L nonpolar neutral large

[0117]

[0118] ATTORNEY DOCKET NO.: 51772-007WO2

[0119] PATENT

[0120] Electrostatic

[0121] Side3 Letter 1 Letter character at Steric Amino Acid chain

[0122] Code Code physiological pH Volume1

[0123] Polarity

[0124] (7.4)

[0125] Lysine Lys K polar cationic large Methionine Met M nonpolar neutral large Phenylalanine Phe F nonpolar neutral large

[0126] nonProline Pro P neutral intermediate polar

[0127] Serine Ser S polar neutral small Threonine Thr T polar neutral intermediate Tryptophan Trp W nonpolar neutral bulky Tyrosine Tyr Y polar neutral large Valine Vai V nonpolar neutral intermediate

[0128]

[0129] Abased on volume in A3: 50-100 is small, 100-150 is intermediate,

[0130] 150-200 is large, and >200 is bulky

[0131] From this table it is appreciated that the conservative amino acid families include (i) G, A, V, L and I; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).

[0132] “Percent (%) sequence identity,” with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity may be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art may determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which may alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0133] 100 multiplied by (the fraction X / Y)

[0134] where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A ATTORNEY DOCKET NO.: 51772-007WO2

[0135] PATENT

[0136] is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0137] “Percent (%) sequence complementarity” with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity may be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art may determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared. As an illustration, the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, (which may alternatively be phrased as a given nucleic acid sequence, A that has a certain percent complementarity to a given nucleic acid sequence, B) is calculated as follows:

[0138] 100 multiplied by (the fraction X / Y)

[0139] where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0140] As used herein, the term “functional,” with respect to a gene, refers to a functional gene product, for example, a functional protein. A gene product is functional if it fulfills its normal (wild-type) functions. Disruption of the gene prevents expression of a functional factor encoded by the gene and contains an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and / or a promoter and / or an operator that is necessary for expression of the gene in the animal.

[0141] As used herein, the term “plasmid” refers to an extrachromosomal circular double stranded DNA molecule into which additional DNA segments may be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked. ATTORNEY DOCKET NO.: 51772-007WO2

[0142] PATENT

[0143] As used herein, the term “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which is incorporated herein by reference as it pertains to nucleic acid regulatory elements. Additionally, the term “promoter” may refer to a synthetic promoter, which are regulatory DNA sequences that do not occur naturally in biological systems.

[0144] Synthetic promoters contain parts of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and may be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types. The term “promoter,” as used herein, refers to a region within the regulatory region of a gene that enables initiation of the transcription of a gene into a messenger RNA, wherein transcription is initiated with the binding of an RNA polymerase on or nearby the promoter.

[0145] As used herein, the term “operably linked” in the context of a nucleic acid refers to a nucleic acid that is placed into a structural or functional relationship with another nucleic acid. For example, one segment of DNA may be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding region so as to facilitate transcription of the coding region. In other examples, the operably linked nucleic acids are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.

[0146] Additionally, two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present. The term “operably linked” may also refer to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. A vector containing a nucleic acid molecule may have one or more elements for expression operably linked to such a nucleic acid molecule, and further may include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene), and / or those that may be used in purification of a polypeptide (e.g., 6xHis tag). Elements for expression include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an expression element is a promoter sequence. Expression elements also may include one or more introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid molecule. Expression elements may be of bacterial, yeast, insect, mammalian, or viral origin and vectors may contain a combination of expression elements from different origins. As used herein, operably linked means that elements for expression are positioned in a vector relative to a coding sequence in such a way as to direct or regulate expression of the coding sequence. ATTORNEY DOCKET NO.: 51772-007WO2

[0147] PATENT

[0148] Vectors, including expression vectors, are commercially available or may be produced by recombinant technology.

[0149] As used herein, the term “regulatory element” or “regulatory sequence” refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).

[0150] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus, or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest. Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that may be used for the expression of transgenes as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of a transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0151] As used herein, the terms " AAV viral particle" refer to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide. The term “particle" as used herein, refers to any suitable sized particles for delivery of the engineered AAV capsid system components described herein. Suitable sizes include macro-, micro-, and nano-sized particles. In some embodiments, any of the of the engineered AAV capsid system components (e.g., polypeptides such as peptide inserts, polynucleotides, vectors and combinations thereof described herein) may be attached to, coupled to, integrated with, otherwise associated with one or more particles or component thereof as described herein. The particles described herein may then be administered to a cell or organism by an appropriate route and / or technique. In some embodiments, particle delivery may be selected and be advantageous for delivery of the polynucleotide or vector components. It will be appreciated that in embodiments, particle delivery may also be advantageous for other engineered capsid system molecules and formulations described elsewhere herein.

[0152] The term "rAAV" is an abbreviation that refers to recombinant AAV vectors. " Recombinant," as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a ATTORNEY DOCKET NO.: 51772-007WO2

[0153] PATENT

[0154] polynucleotide found in nature. The term "rAAV vector” refers to a polynucleotide, such as a nucleic acid genome, within an AAV viral particle. The terms include replicates of the original polynucleotide construct and progeny of the original virus construct. If an AAV particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, e.g., a transgene to be delivered to a target cell, an RNAi agent or CRISPR agent to be delivered to a target cell, etc.), it is typically referred to as a "recombinant AAV (rAAV) virion" or an "rAAV viral particle" or an “rAAV particle.” In general, the heterologous polynucleotide is flanked by at least one, and generally by two, AAV ITRs. Recombinant AAV polynucleotides also encompass polynucleotides encoding rAAV (e.g., a single stranded polynucleotide encoding rAAV (ss-rAAV); a double stranded polynucleotide encoding rAAV (ds-rAAV), e.g., plasmids encoding rAAV; and the like). Unless otherwise indicated, the viral vectors of the invention are recombinant, as they contain heterologous nucleic acids.

[0155] As used herein, the terms “adeno-associated virus” and “AAV” include, but are not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., Fields et al. Virology, 4thed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been identified recently. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Moris et al. Virol. 33:375 (2004). The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NO — 002077, NO — 001401, NO — 001729, NO— 001863, NO— 001829, NO— 001862, NO— 000883, NO— 001701, NO— 001510, NO— 006152, NO— 006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NO— 001358, NO — 001540, AF513851, AF513852 and AY530579; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Bantel-Schaal et al. J. Virol. 73:939 (1999); Chiorini et al. J. Virol. 71:6823 (1997); Chiorini et al. J. Virol. 73:1309 (1999); Gao et al. Proc. Nat. Acad. Sci. USA 99:11854 (2002); Moris et al. Virol. 33:375 (2004); Muramatsu et al. Virol. 221:208 (1996); Ruffing et al. J. Gen. Virol. 75:3385 (1994); Rutledge et al. J. Virol. 72:309 (1998); Schmidt et al. J. Virol. 82:8911 (2008); Shade et al. J. Virol. 58:921 (1986); Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and US 6,156,303; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences.

[0156] From a structural perspective, AAV is a nonpathogenic parvovirus composed of a 4.7 kb singlestranded DNA genome within a non-enveloped, icosahedral capsid. The genome contains three open reading frames (ORF) flanked by inverted terminal repeats (ITR) that function as the viral origin of replication and packaging signal. The Rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The Cap ORF encodes three structural proteins (VP 1-3) that assemble to form a 60-mer viral capsid. Finally, an ORF present as an alternate reading frame within the Cap gene produces the assembly-activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid ATTORNEY DOCKET NO.: 51772-007WO2

[0157] PATENT

[0158] assembly process. There are several naturally occurring ("wild-type") serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0159] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC.001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC 006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC 006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrhIO); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U. S. Pat. No. 6,156,303.

[0160] The sequences of naturally existing Cap (capsid) proteins associated with AAV serotypes are known in the art and include those disclosed herein as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, and AAVrh74. The terms "variant AAV capsid protein" or " AAV variant” refer to an AAV capsid protein comprising an amino acid sequence that includes at least one modification or substitution (including deletion, insertion, point mutation, etc.) relative to a naturally existing or "wildtype" AAV capsid protein sequence, e.g., as set forth in Table 3 herein. A variant AAV capsid protein may have about 80% identity or more to the amino acid sequence of a wild-type capsid protein, for example, 85% identity or more, 90% identity or more, or 95% identity or more to the amino acid sequence of the wild-type capsid protein, for example, 98% or 99% identity to the wild-type capsid protein. A variant AAV capsid protein may not be a wild-type capsid protein.

[0161] As used herein, the term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. AAV “Rep” and “Cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV Rep and Cap are referred to herein as AAV “packaging genes”.

[0162] “Rep proteins” are proteins which fill capsids with a polynucleotide molecule (e.g., viral DNA) into a capsid. In addition, a Rep protein may enable nucleic acid molecule replication, transcriptional regulation of a nucleic acid molecule, and / or site-specific integration (e.g., chromosomal integration) of a nucleic acid molecule. The Rep protein may be from any parvovirus. As one of skill in the art will appreciate, in some parvovirus family virus species, a Rep protein is referred to as a “non-structural (NS) protein”. As used herein, a Rep protein may refer to a NS protein. As used herein, a Rep protein may refer to an AAV Rep protein, which have been found in all AAV serotypes examined to date, and a synthetic Rep protein. For example, in some embodiments, an AAV Rep protein (e.g., AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78) is a capsid protein having an amino acid sequence derived from a ATTORNEY DOCKET NO.: 51772-007WO2

[0163] PATENT

[0164] particular AAV serotype, for example AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, and ovine AAV. Alternatively, for example, as used herein, a Rep protein may include a synthetic Rep protein. Any suitable Rep protein may be used.

[0165] As used herein, the term “viral capsid protein” refers to a capsid protein composing a proteinaceous shell. Such a proteinaceous shell is generally composed of one or more viral capsid proteins and when assembled is capable of being loaded with one or more polynucleotide molecules. A viral capsid protein described herein may, for example, be a viral protein VP1, VP2, or VP3. Further, a viral capsid protein described herein may refer to a synthetic protein or a viral capsid protein from Parvoviridae (e.g., an AAV). A “viral capsid protein” as used herein refers to any of the AAV capsid proteins that are components of AAV viral particles.

[0166] As used herein, the term “same capsid species” refers to a population of capsids having the same defined stoichiometry of viral capsid protein components, which may include one or more of VP1, VP2, and VP3.

[0167] As used herein, the terms “viral protein 1” and “VP1” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytic entry and receptor-mediated fusion). As used herein, a VP1 may self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP1 may exhibit selfbinding properties and self-assemble around the exterior of a respective VP1 -containing capsid. As used herein, a VP1 may be synthetic or a VP1 derived from Parvoviridae (e.g., an AAV). For example, a VP1 derived from an AAV may be a VP1 derived from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered.

[0168] As used herein, the terms “viral protein 2” and “VP2” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP2 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus. As used herein, a VP2 may self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP2 may self-assemble within the interior of a respective VP2-containing capsid. As used herein, a VP2 may be synthetic or a VP2 derived from Parvoviridae (e.g., an AAV). For example, a VP2 derived from an AAV may be a VP2 derived from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered.

[0169] As used herein, the terms “viral protein 3” and “VP3” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP3 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell ATTORNEY DOCKET NO.: 51772-007WO2

[0170] PATENT

[0171] nucleus. As used herein, a VP3 may self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP3 may self-assemble within the interior of a respective VP3-containing capsid. As used herein, a VP3 may be synthetic or a VP3 derived from Parvoviridae (e.g., an AAV). For example, a VP3 derived from an AAV may be a VP3 derived from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered.

[0172] As used herein, the terms “encapsidation,” “encapsulating,” “encapsidate,” and the like refer to non-enzymatically driven encasing of nucleic acid molecules in a capsid shell. Thus, when a suitable population of viral capsid proteins (e.g., VP1, VP2, and / or VP3) and conditions are combined with a nucleic acid molecule, the nucleic acid molecule may be encapsidated by the capsid to form an assembled capsid particle packaged with one or more nucleic acid molecules. The encapsidation process may occur simultaneously with capsid assembly or after capsid assembly is complete.

[0173] An “ITR” is a palindromic nucleic acid, e.g., an inverted terminal repeat, that is about 120 nucleotides to about 250 nucleotides in length and capable of forming a hairpin. The term “ITR” includes the site of the viral genome replication that may be recognized and bound by a parvoviral protein (e.g., Rep78 / 68). An ITR may be from any AAV, with serotype 2 being preferred. An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term “ITR” does not require a wild-type parvoviral ITR (e.g., a wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutations), as long as the ITR functions to mediate virus packaging, replication, integration, and / or provirus rescue, and the like. The “5’ ITR” is intended to mean the parvoviral ITR located at the 5’ boundary of the nucleic acid molecule; and the term “3’ ITR” is intended to mean the parvoviral ITR located at the 3’ boundary of the nucleic acid molecule.

[0174] As used herein, the term "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0175] As used herein, the term "helper virus functions" refers to functions encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral proteins is transfected into a producer cell along with an rAAV vector. The terminology "infectious" virus or viral particle is one that comprises a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this ATTORNEY DOCKET NO.: 51772-007WO2

[0176] PATENT

[0177] disclosure and in the art. Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11: S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0178] As used herein, the term "tropism" as used herein refers to the preferential targeting by a virus (e.g., an AAV) of cells of a particular host species or of particular cell types within a host species. For example, a virus that may infect cells of the heart, lung, liver, and muscle has a broader (i.e., increased) tropism relative to a virus that may infect only lung and muscle cells. Changes in tropism can also manifest as more subtle shift in cell-type preference generally or in cell-type preference in the context of a particular route of administration. Tropism may also include the dependence of a virus on particular types of cell surface molecules of the host. For example, some viruses may infect only cells with surface glycosaminoglycans, while other viruses may infect only cells with sialic acid (such dependencies may be tested using various cells lines deficient in particular classes of molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the virus's relative preferences. For example, a first virus may be able to infect all cell types but is much more successful in infecting those cells with surface glycosaminoglycans. A second virus may be considered to have a similar (or identical) tropism as the first virus if the second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), even if the absolute transduction efficiencies are not similar. For example, the second virus might be more efficient than the first virus at infecting every given cell type tested, but if the relative preferences are similar (or identical), the second virus may still be considered to have a similar (or identical) tropism as the first virus. In some embodiments, the tropism of a viral particle comprising a subject variant AAV capsid protein is not altered relative to a naturally occurring viral particle. In some embodiments, the tropism of a viral particle comprising a subject variant AAV capsid protein is expanded (i.e., broadened) relative to a naturally occurring viral particle. In some embodiments, the tropism of a viral particle comprising a subject variant AAV capsid protein is reduced relative to a naturally occurring viral particle.

[0179] As used herein, the term "heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV that includes a polynucleotide not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.

[0180] As used herein, the term “hybridize” refers to the formation of a stable duplex of nucleic acids by way of annealing mediated by inter-strand hydrogen bonding, for example, according to Watson-Crick base pairing. The nucleic acids of the duplex may be, for example, at least 50% complementary to one another. The “stable duplex” formed upon the hybridization of one nucleic acid to another is a duplex structure that is not denatured by a stringent wash. Exemplary stringent wash conditions are known in the art and include temperatures of about 5° C less than the melting temperature of an individual strand of the ATTORNEY DOCKET NO.: 51772-007WO2

[0181] PATENT

[0182] duplex and low concentrations of monovalent salts, such as monovalent salt concentrations. The complementarity of the nucleic acids of the duplex may be low overall but there may be segments of the nucleic acid that are contiguous and fully complementary to an equal-length segment of the target that, in the duplex form, allow for hybridizing across the target’s length.

[0183] As used herein, the phrases “specifically binds” and “binds” refer to a binding reaction which is determinative of the presence of a particular molecule, in a heterogeneous population of ions, salts, small molecules, and / or proteins that is recognized. A variety of assay formats may be used to determine the affinity of a ligand for a specific protein. For example, solid-phase ELISA assays are routinely used to identify ligands that specifically bind a target protein. See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of assay formats and conditions that may be used to determine specific protein binding.

[0184] As used herein, the terms “subject” and “patient” refer to an animal (e.g., a mammal, such as a human).

[0185] As used herein, the terms “administering,” “administration,” and the like refer to directly giving a patient an agent, for example, a therapeutic agent (e.g., a pharmaceutical composition including a viral vector including a nucleic acid sequence encoding a transgene operably linked to a promoter), by any effective route. Exemplary routes of administration are described herein and include systemic administration routes, such as intravenous injection, as well as routes of administration directly to muscle cells.

[0186] As used herein, the term “dose” refers to the quantity of a therapeutic agent, such as a viral vector described herein, that is administered to a subject at a particular instant for the treatment of a disorder, such as to treat or ameliorate one or more symptoms of a disorder described herein. A therapeutic agent as described herein may be administered in a single dose or in multiple doses over the course of a treatment period, as defined herein. In each case, therapeutic agent may be administered using one or more unit dosage forms of therapeutic agent, a term that refers to a one or more discrete compositions containing a therapeutic agent that collectively constitute a single dose of the agent.

[0187] As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of composition, vector construct, or viral vector described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a disease, it is an amount of the composition, vector construct, or viral vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but may nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition, vector construct, or viral vector of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition, vector ATTORNEY DOCKET NO.: 51772-007WO2

[0188] PATENT

[0189] construct, or viral vector of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regime may be adjusted to provide the optimum therapeutic response. In the practice of the methods of the present invention, an “effective amount” of any one of the compounds or a combination of any of the compounds or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination.

[0190] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0191] As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, such as a nucleic acid or vector described herein, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.

[0192] As used herein, the term “sample” refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental, or dermal), pancreatic fluid, chorionic villus sample, and cells) isolated from a subject.

[0193] As used herein, “treat”, “treatment”, or “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” may also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0194] As used herein, the term “therapeutic protein” refers to (i) a protein whose deficiency or lack of activity is associated with a disorder (e.g., a genetic disorder, for example, a loss-of-function disorder recited in Table 4), as well as (ii) a protein that is not necessarily deficient in a patient, but whose supplementation would nonetheless have a beneficial effect on the patient.

[0195] DETAILED DESCRIPTION

[0196] Recombinant vectors derived from adeno-associated viruses (AAVs) are widely used in gene therapy, but have historically been hindered by insufficient transduction efficiency, resulting in levels of transgene expression that fall short of that required for robust therapeutic or prophylactic activity. This problem is directly addressed by the present invention, which provides peptide inserts that, upon ATTORNEY DOCKET NO.: 51772-007WO2

[0197] PATENT

[0198] incorporation into an AAV capsid protein, engender unexpected improvements in various important parameters, including transduction efficiency, tropism, and residence time in desired tissue.

[0199] Using the compositions and methods disclosed herein, one may incorporate a peptide insert of the invention into AAV capsid protein, such as an existing, wild-type AAV capsid protein (e.g., VP1, VP2, VP3) associated with any known AAV serotype (e.g., AAV, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAVrh74). AAV capsids of the invention may, in turn, be used to encapsulate an AAV nucleic acid vector (e.g., encoding a therapeutic or prophylactic transgene of interest), thereby producing a viral particle. Viral particles described herein may be administered to patients by any of a variety of routes of administration, including intramuscular delivery, among an array of others described herein.

[0200] Described in more detail below is the discovery and optimization (including in vivo AAV activity, muscle versus liver specificity, and vector manufacturability) of a number of capsid peptide inserts. Also described below are exemplary processes for incorporating peptide inserts of the invention into an AAV capsid protein, as well as methods of producing recombinant AAV particles containing peptide inserts disclosed herein and methods of administering the same to a patient in need thereof.

[0201] Peptide Inserts

[0202] The peptide inserts of the invention are amino acid sequences that may be inserted into any one or more AAV capsid proteins, such as a wild-type AAV capsid protein known in the art. In some embodiments, the peptide inserts may be of various length, e.g., 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, or 40 amino acids, or more.

[0203] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (I):

[0204] (X1)(X2)(X3)(X4)(X5)(X6)G

[0205] Formula (I)

[0206] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X3), (X5), and (X6), independently, represents any naturally occurring amino acid. In some embodiments, (X1) represents Thr, Ser, Asn, Gly, Leu, or Met. In some embodiments, (X1) represents Thr or Ser. In some embodiments, (X3) represents Thr, lie, Ser, Asn, Ala, Arg, Met or Lys. In some embodiments, (X3) represents Thr. lie, Ser, Asn, or Ala. In some embodiments, (X5) represents Pro, Lys, Vai, or lie. In some embodiments, (X5) represents Pro, Lys, or Vai. In some embodiments, (X5) represents Pro. In some embodiments, (X6) represents Ala, Gly, lie, Met, Gin, Ser, or Vai. In some embodiments, (X6) represents Ala, lie, Gin, or Vai. In some embodiments, (X2) represents Lys. In some embodiments, (X2) represents Arg. In some embodiments, (X4) represents Lys. In some embodiments, (X4) represents Arg. ATTORNEY DOCKET NO.: 51772-007WO2

[0207] PATENT

[0208] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (II):

[0209] (X1)(X2)(X3)(X4)(X5)(X6)G

[0210] Formula (II)

[0211] wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X5), and (X6), independently, represents any naturally occurring amino acid. In some embodiments, (X1) represents Thr, Ser, Met, His, Gly, or Trp. In some embodiments, (X1) represents Thr or Ser. In some embodiments, (X2) represents lie, Phe, Leu, Lys, Arg, Ala, His, Met, Asn, Gin, Ser, Thr, or Vai. In some embodiments, (X2) represents lie, Phe, or Leu. In some embodiments, (X2) represents lie. In some embodiments, (X5) represents Pro, lie, Vai, Ala, or Thr. In some embodiments, (X5) represents Pro or lie. In some embodiments, (X5) represents Pro. In some embodiments, (X6) represents Ala, Vai, Gin, lie, Asn, Pro, or Ser. In some embodiments, (X6) represents Ala or Vai. In some embodiments, (X3) represents Lys. In some embodiments, (X3) represents Arg. In some embodiments, (X4) represents Lys. In some embodiments, (X4) represents Arg.

[0212] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (III):

[0213] (X1)(X2)(X3)(X4)(X5)(X6)G

[0214] Formula (III)

[0215] wherein each of (X4) and (X5), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid. In some embodiments, (X1) represents Thr or Ser. In some embodiments, (X1) represents Thr. In some embodiments, (X2) represents lie, Lys, or Leu. In some embodiments, (X2) represents lie. In some embodiments, (X3) represents Thr, Arg, Ala, or lie. In some embodiments, (X3) represents Thr or Arg. In some embodiments, (X6) represents Vai, lie, Ala, Pro, or Gin. In some embodiments, (X6) represents Vai or lie. In some embodiments, (X4) represents Lys. In some embodiments, (X4) represents Arg. In some embodiments, (X5) represents Lys. In some embodiments, (X5) represents Arg.

[0216] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (IV):

[0217] (X1)(X2)(X3)(X4)P(X6)(X7)

[0218] Formula (IV)

[0219] wherein (X4) represents an amino acid selected from the group consisting of Lys and Arg, (X7) represents an amino acid selected from the group consisting of Gly and Pro, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid. In some embodiments, (X1) represents Thr, Ser, Met, or Asn. In some embodiments, (X1) represents Thr, Ser, or Met. In some embodiments, (X2) represents lie, Leu, Phe, Arg, or Met. In some embodiments, (X2) represents lie, Leu, or Phe. In some embodiments, (X3) represents lie, Thr, Ala, Met, Asn, Arg, Ser, or Tyr. In some embodiments, (X3) represents lie or Thr. In some embodiments, (X6) represents Ala, Vai, lie, or Ser. In some embodiments, (X6) represents Ala or Vai. In some embodiments, (X4) represents Lys. In some embodiments, (X4) represents Arg. In some embodiments, (X7) represents Gly. In some embodiments, (X7) represents Pro. ATTORNEY DOCKET NO.: 51772-007WO2

[0220] PATENT

[0221] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (V):

[0222] (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G

[0223] Formula (V)

[0224] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Vai, and lie, (Polar) represents any amino acid listed in Table 1, above, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, lie, or Met. In some embodiments, (X3) represents Ala, lie, Met, Asn, Arg, Ser, or Thr.

[0225] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (VI):

[0226] (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G

[0227] Formula (VI)

[0228] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Vai, (Polar) represents any amino acid listed in Table 1, above, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, lie, or Met. In some embodiments, (X3) represents Ala, lie, Met, Asn, Arg, Ser, or Thr.

[0229] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (VII):

[0230] (X1)(X2)(X3)(X4)(X5)(X6)G

[0231] Formula (VII)

[0232] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Asn, and Gin, (X5) represents an amino acid selected from the group consisting of Pro, Lys, Arg, Vai, and lie, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0233] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (VIII):

[0234] (X1)(X2)(X3)(X4)P(Nonpolar)G

[0235] Formula (VIII)

[0236] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Met, and Asn, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, ATTORNEY DOCKET NO.: 51772-007WO2

[0237] PATENT

[0238] (Nonpolar) represents Ala, Gly, lie, Met, or Vai. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0239] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (IX):

[0240] (X1)(X2)(X3)(X4)P(X6)G

[0241] Formula (IX)

[0242] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Met, and Asn, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0243] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (X):

[0244] (Polar)(X2)(X3)(X4)P(Nonpolar)G

[0245] Formula (X)

[0246] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (Polar) represents any polar amino acid, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and (X3) represents any naturally occurring amino acid. In some embodiments, (Polar) represents Thr, Ser, or Asn. In some embodiments, (Nonpolar) represents Ala, Gly, lie, Met, or Vai. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0247] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (XI):

[0248] (X1)(X2)(X3)(X4)P(X6)G

[0249] Formula (XI)

[0250] wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, Asn, and Gin, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X3) represents any naturally occurring amino acid. In some embodiments, (X3) represents Ala, lie, Lys, Met, Asn, Arg, Ser, or Thr.

[0251] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (XII):

[0252] (X1 )(X2)(X3)(X4)(X5)(Nonpolar)G

[0253] Formula (XII)

[0254] wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro and lie, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and each of (X1) and (X2), independently, represents any naturally occurring amino acid. In some embodiments, (X1) and (X2) represent amino acids that are not anionic. In some embodiments, (Nonpolar) represents Ala, lie, Pro, or Vai. In some embodiments, (X1) represents Gly, His, Met, Ser, Thr, ATTORNEY DOCKET NO.: 51772-007WO2

[0255] PATENT

[0256] or Trp. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0257] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (XIII):

[0258] (Polar)(X2)(X3)(X4)(X5)(Nonpolar)G

[0259] Formula (XIII)

[0260] wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (Polar) represents any amino acid listed in Table 1, above, as having a polar, neutral side chain, (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain, and (X2) represents any naturally occurring amino acid. In some embodiments, (Polar) represents His, Ser, or Thr. In some embodiments, (Nonpolar) represents Ala, lie, Pro, or Vai. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0261] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (XIV):

[0262] (X1)(X2)(X3)(X4)(X5)(X6)G

[0263] Formula (XIV)

[0264] wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, and Met, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (X2) represents any naturally occurring amino acid. In some embodiments, (X2) represents Ala, Phe, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, or Vai.

[0265] In some embodiments, a peptide insert of the invention has an amino acid sequence represented by Formula (XV):

[0266] (X1 )(Nonpolar)(X3)(X4)(X5)(X6)G

[0267] Formula (XV)

[0268] wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, (X1) represents an amino acid selected from the group consisting of Thr, Ser, and Met, (X5) represents an amino acid selected from the group consisting of Pro, Vai, and lie, (X6) represents an amino acid selected from the group consisting of Ala, Vai, lie, Met, and Leu, and (Nonpolar) represents any amino acid listed in Table 1, above, as having a nonpolar, neutral side chain. In some embodiments, (Nonpolar) represents Ala, Phe, lie, Leu, Met, or Vai.

[0269] An AAV capsid protein of the invention may, in turn, encapsulate a viral vector (e.g., an AAV vector), thereby furnishing a recombinant AAV particle. In some embodiments, an engineered AAV capsid of the invention containing one or more of the peptide inserts may direct or redirect AAVs to novel cellular receptors, resulting in enhanced transduction activity from intramuscular or other direct tissue routes of administration. Peptide inserts described herein may enhance muscle transduction and muscle-specificity when delivered intramuscularly or intravenously in vivo.

[0270] Table 2 illustrates exemplary peptide inserts of the invention. ATTORNEY DOCKET NO.: 51772-007WO2

[0271] PATENT

[0272] Table 2. Exemplary Peptide Inserts Peptide sequence SEQ ID NO.

[0273] TKSRPAG 17 SIRKPAG 18 SIRQKVG 19 SRNKPIG 20 TFKKPAG 21 LRNKPAG 22 TLRKPAG 23 SKTRPAG 24 TRIKPVG 25 MIKKPVG 26 NRIKPAG 27 TIKRPVG 28 TITKKPG 29 SIRRPAG 30 TKNKPVG 31 MKTKPVG 32 TKAKPAG 33 TRAKPVG 34 MMKKPVG 35 TKIKPSG 36 GKTKPAG 37 SKMRPVG 38 TKIRPMG 39 SRIKPVP 40 TKIRPQG 41 TRTKVAG 42 SKIRPGG 43 SKTKPQG 44 TLRKIAG 45 SKSKPIG 46 TRTKVVG 47 TIIKKIG 48 TIKFKVG 49 TKIKPAG 50 TKIKPVG 51 TKTKPAG 52 TKTKPVG 53

[0274]

[0275] ATTORNEY DOCKET NO.: 51772-007WO2

[0276] PATENT

[0277] Peptide sequence SEQ ID NO.

[0278] SKI KPAG 54 SKIKPVG 55 SKTKPAG 56 TIRKPAG 57 TIRKPQG 58 TRSKPQG 59 TRNKPAG 60 SRKKPVG 61 SRSRPAG 62 TIRRPIG 63 TKRKKVG 64 SIRRPVG 65 TRTKPQG 66 TIRRPAG 67 TRSKPVG 68 SRSKPVG 69 TRNKPVG 70 SRSKPAG 71 TIIKPAG 72 TRTRPIG 73 TIRKIVG 74 SRTKPQG 75 MRTRPAG 76 TRIRPIG 77 GRRKPAG 78 SRTKPAG 79 TRSKPIG 80 MISKPAG 81 TRTRIVG 82 TRAKPQG 83 TRAKPGG 84 SRIKPQG 85 SRTRPAG 86 TIRRPQG 87 TRNRPAG 88 TRAKPAG 89 TRIRPVG 90 NFIKPAG 91

[0279]

[0280] ATTORNEY DOCKET NO.: 51772-007WO2

[0281] PATENT

[0282] Peptide sequence SEQ ID NO.

[0283] TFTKIAG 92 NRTRPIG 93 TIRKKVG 94 NRSKPAG 95 SITKKQG 96 TRNRPQG 97 TLIRPAG 98 SRIRPAG 99 TMTRPVG 100 SLIRPAG 101 MLTKPVG 102 MLIRPAG 103 SRIRIQG 104 TIIRPAG 105 TIIRPVG 106 TRTKPIG 107 SRIKPAG 108 TLIKPAG 109 TIRKVVG 110 TIRRPSP 111 TIMRPAG 112 TRIKPAG 113 MFAKPAG 114 SIKRPAG 115 TRTKPVG 116 TRTMVVG 117 SLIRPVG 118 SIRKPIG 119 TITKPAG 120 TFTKPAG 121 MITRPAG 122 MITRPVG 123 SIIRVAG 124 TLAKKIG 125 SLIKPVG 126 SIM KPAG 127 TIRMPAP 128 SFTKPVG 129

[0284]

[0285] ATTORNEY DOCKET NO.: 51772-007WO2

[0286] PATENT

[0287] Peptide sequence SEQ ID NO.

[0288] TKTKKAG 130 QMPRTPG 131 MRIRPAG 132 TRMKPAG 133 TKSRPSG 134 TITRPIG 135 NFARPIG 136 TLTKPAG 137 SIIRPVG 138 TLIKPVG 139 TLTKPVG 140 NIIRPIG 141 MLNRPAG 142 SRIRVAG 143 SIIKPVG 144 TLAKPMG 145 TITRPAG 146 NLRRPVG 147 MRNMPAG 148 NRTKPAP 149 SKIRPAG 150 SRIKPVG 151 TRIKPIG 152 TKSKPSG 153 GKIKPIG 154 SLSKPAG 155

[0289] M LI KPAG 156 SIIKPAG 157 SIARPSG 158 TKNRPAG 159 TKSKPAG 160 TLIKKAG 161 LIRKPIG 162 SITKPSG 163 TKNKPAG 164 TLKRPAG 165 LIRKPAG 166 TMIRPMG 167

[0290]

[0291] ATTORNEY DOCKET NO.: 51772-007WO2

[0292] PATENT

[0293] Peptide sequence SEQ ID NO.

[0294] TITKPVG 168 TRIRVAG 169 TIAKPAG 170 TRMKVAG 171 TRMKPVG 172 SKIRPVG 173 TIIKPVG 174 TITKPSG 175 TITKPIG 176 SLKKPAG 177 TLKKPAG 178 NLRRPIG 179 TIKRPAG 180 SI KM PAG 181 TISRPAG 182 MRAKPVG 183 GRNKPAG 184 LIRKPVG 185 SIIKVAG 186 MITKPSG 187 TKTRIAG 188 SKSKPAG 189 SIIQKVG 190 SINKPVG 191 SMIKPVG 192 TLIKPIG 193 TLNKPVG 194 MITKPVG 195 TISKPVG 196 SMKKPSG 197 TIIKPIG 198 SITKPVG 199 TKIKPAP 200 SIKKPAG 201 SKIRPIG 202 LRNRPVP 203 SINRPVG 204 SITKPAG 205

[0295]

[0296] ATTORNEY DOCKET NO.: 51772-007WO2

[0297] PATENT

[0298] Peptide sequence SEQ ID NO.

[0299] SKAKVVG 206 TLNKPAG 207 SRMKPAG 208 TKTKPMG 209 TIKKPMG 210 TRAQPAG 211 TRRKPAG 212 TRRRPVG 213 TKTRPVG 214 NIKKPAG 215 SKTRVAG 216 MLKKPQG 217 SLNRPAG 218 SRTKPVG 219 SLTRPIG 220 SIAKPAG 221 TIKKPAG 222 NIIKPIG 223 TMMKPVG 224 TMRKPVG 225 TIKKPQG 226 TIIRVVP 227 SKIRVVG 228 SKTKPSG 229 TKTRPIG 230 TKNRPSG 231 TIIKKVG 232 NKIKPVG 233 TFKKPVG 234 LITKPAG 235 SIMKPVG 236 MKTKPGG 237 SKMRVQG 238 TKIRPAG 239 SIIKKVG 240 SIKKPSG 241 MKIFPAG 242 TKTRPQG 243

[0300]

[0301] ATTORNEY DOCKET NO.: 51772-007WO2

[0302] PATENT

[0303] Peptide sequence SEQ ID NO.

[0304] SIN KPAG 244 NLRMKAG 245 TMRKPIG 246 GRIKPAG 247 TIKRPIG 248 TKARPAG 249 TKMRVIG 250 TINKPAG 251 MKTRPIP 252 TLAVPFK 253 MIAKPAG 254 TKIRPVG 255 TIMRKVG 256 SKTRPVG 257 GMTKPAG 258 TRTKKVG 259 TINKPVG 260 TKTKPQG 261 TIKKPVG 262 GLTKPIG 263 TLKQPAG 264 TKNKIVG 265 MIKRPAP 266 SIAKVVG 267 SKNKPAG 268 TLMFPGG 269 TIKKPIG 270 GIRKPAG 271 SKTKPVG 272 TLNKIAG 273 MINKPVG 274 SIKKPVG 275 NLTKPVG 276 MKTKPVP 277 TISKIAG 278 MKIRPVG 279 TKMKPAG 280 TIMKPVG 281

[0305]

[0306] ATTORNEY DOCKET NO.: 51772-007WO2

[0307] PATENT

[0308] Peptide sequence SEQ ID NO.

[0309] LITKVVG 282 SKTRKAG 283 TKIKPQG 284 TKIKVVG 285 GLKKPMG 286 TRTMKQG 287 MKIKPAG 288 TIIKKAG 289 TIIQPVG 290 TKNKPIG 291 NIIRKVG 292 TIKRPQP 293 TKSKPVG 294 TKTFIAG 295 SITQPAG 296 TKTKPSG 297 RMPVQKG 298 SMSRPIG 299 SKTKPIG 300 TRRKVAG 301 MKIKPSG 302 NKIKPGG 303 SRTQPAG 304 SKARPVG 305 LFIKPVG 306 LKIKPVG 307 TKIKPIG 308 MIIKPVG 309 SLKKVAG 310 SITQPVG 311 MKNKPVG 312 LKTRPVG 313 SINKIVG 314 TKARPGG 315 TLRKPVG 316 TIIFPAG 317 MKTKPQG 318 TMRKPQG 319

[0310]

[0311] ATTORNEY DOCKET NO.: 51772-007WO2

[0312] PATENT

[0313] Peptide sequence SEQ ID NO.

[0314] TIRRVPG 320 LINKPVG 321 TLRQPVG 322 MKIKPVG 323 SIRRPPG 324 TLRKPIG 325 SISKPVG 326 SKIKPVP 327 TKSKPIG 328 TKIMKSG 329 TFKKPIG 330 TKTKPIG 331 N I KF PAG 332 SKKKVAG 333 MKSMVAG 334 TKAKIMG 335 TLKQPQG 336 SRMRPVG 337 SISKKVG 338 TITKKVG 339 SRTKKAG 340 MKIKPVP 341 SKAKPVG 342 TKRQPAG 343 GKARPAG 344 NIAKPVG 345 MIIRPVG 346 MKTKPAG 347 SLAKPIG 348 TLKKPIG 349 TKTKVAP 350 SRTRPVG 351 NNSTRGG 352 TMSKIVG 353 TRRKPVG 354 SRAKPAG 355 SIKKPAP 356 SKKFPMG 357

[0315]

[0316] ATTORNEY DOCKET NO.: 51772-007WO2

[0317] PATENT

[0318] Peptide sequence SEQ ID NO.

[0319] TFTKKVG 358 NRTKPVG 359 TIKKVAG 360 NKTKPVG 361 GKIKPAG 362 SIAKPIG 363 GIAKPVG 364 TIKKPSP 365 SITFPIG 366 MKSRPVG 367 TKIMPVG 368 GKNKPAG 369 LNTTKPI 370 SLTRKQG 371 SLTKKAG 372 SITMPAG 373 TKRKPIG 374 TKKQPIG 375 TKTKKVG 376 MKSKVVG 377 LIKKPAG 378 SIIQPAG 379 SKRKPAG 380 LKIKPIG 381 KNPAKPP 382 TKIKPPG 383 SNYVKQT 384 TKIMPIG 385 KNPTKPA 386 LINKPIG 387 TKRRPAG 388 TLMQPIG 389 GLRKPVG 390 SIIMPVG 391 GIKQKPG 392 SRRKPQG 393 TKTMPQG 394 SISKKQG 395

[0320]

[0321] ATTORNEY DOCKET NO.: 51772-007WO2

[0322] PATENT

[0323] Peptide sequence SEQ ID NO.

[0324] GIKKPAG 396 TKKFPVG 397 TKRKPVG 398 SRTKPIG 399 TIKKPPG 400 NKKRPVG 401 SKRRPVP 402 TRRRPAG 403 SRIMKAG 404 SFRQPVG 405 TRKKPAG 406 MITMPVG 407 MKKKPIG 408 MLRKPSG 409 TMRRPQG 410 MINKPPG 411 TFIKPVG 412 IKVARPP 413 TIKKKVG 414 TKKRPAG 415 NRRKPVG 416 GRKKPIG 417 SLARPVP 418 SLRKPIG 419 SKRKPIG 420 SKKKPVG 421 SMAKPIG 422 SIRKKIG 423 TKKKPVG 424 TKKKPMG 425 SKRKPVG 426 NKRKPVG 427 NKKKPVG 428 TRKKVGG 429 TRKKPVG 430 NKNMKGG 431 TKKKPQG 432 TKKKPSG 433

[0325]

[0326] ATTORNEY DOCKET NO.: 51772-007WO2

[0327] PATENT

[0328] Peptide sequence SEQ ID NO.

[0329] TLSQPVG 434 SKKKPAG 435 MKIKPPG 436 SRKKPQG 437 TKKKPAG 438 SKKRPSG 439 LMTMPVG 440 TKRKPAG 441 NKIKKVG 442 SKIRPPG 443 TKIKKVG 444 SKIKKVG 445 SKRKPQG 446 TFTRKIG 447 SRMKPVG 448 SFSKPPG 449 SKRRPVG 450 TKRRPVG 451 SKKKVVG 452 MIIRKAG 453 SRKKPIG 454 SKIKKAG 455 TKKKPPG 456 SISKPPG 457 TKIKKAG 458 NRKRPVG 459 MKRKPVG 460 TKAKKVG 461 SKTKKAG 462 NKRMKVG 463 SRKKVVG 464 SLRRKVP 465 TIIRPPG 466 GKKKPQG 467 MKKKPGG 468 LLRKIPG 469 SIRKKVG 470 MKTKKAG 471

[0330]

[0331] ATTORNEY DOCKET NO.: 51772-007WO2

[0332] PATENT

[0333] Peptide sequence SEQ ID NO.

[0334] SKSKKAG 472 TKNKKVG 473 MRARPVG 474 TKSRKPG 475 TLKKKPG 476 SKARKVG 477 TKTRKAG 478 TRIKPPG 479 TLRKKAG 480 SRRKPVG 481 GKRKPIG 482 SKTQPAG 483 GRKRPVG 484 LKIMPPG 485 TRIKKQG 486 MKRRPVG 487 LRKKPVG 488 LRKRPAG 489 TRNKKAG 490 NIRKKAG 491 TIRKKAG 492 TRIKKAG 493 SKNKKAG 494 MKRKPAG 495 TKRKVAG 496 TRRMKVG 497 LIRKKVG 498 MRIKPPG 499 TRRRPVP 500 TKRKKAG 501 TRARKIG 502 SRIKKAG 503 SRAKKAG 504 TKRKIAG 505 TRRKVVG 506 MRRMVQG 507 MKRKPGG 508 TRKRKVG 509

[0335]

[0336] ATTORNEY DOCKET NO.: 51772-007WO2

[0337] PATENT

[0338] Peptide sequence SEQ ID NO.

[0339] LKRRPAG 510 SKKRKAG 511 NIKRKGG 512 TRRKKQG 513 TRKKKAG 514 TKIRKAG 515 SKKKKVG 516 GKSRKIG 517 MKRRPPG 518 TKRRKVG 519 MRRKPVG 520 TKKKKAG 521 TRRKKVG 522 TKRRKAG 523 TMRKKAG 524 LKRKKAG 525 SQRKPAG 526 SFRKPAG 527 SIRKTAG 528 WIRKPAG 529 STRKPAG 530 SVRKPAG 531 SIRKPNG 532 SIRKPPG 533 SIYKPAG 534 SARKPAG 535 SSRKPAG 536 SNRKPAG 537 SHRKPAG 538 SIRKPSG 539 HIRKPAG 540 SIRYPAG 541 SIRHPAG 542 SIRKPQG 543 SIRKAAG 544 SIRKPGG 545 SIFKPAG 546 SIGKPAG 547

[0340]

[0341] ATTORNEY DOCKET NO.: 51772-007WO2

[0342] PATENT

[0343] Peptide sequence SEQ ID NO.

[0344] SIRKPMG 548 SIRKPTG 549 SGRKPAG 550 EIRKPAG 551 SIRLPAG 552 SIRKPVG 553 DIRKPAG 554 SIRSPAG 555 SICKPAG 556 SILKPAG 557 SIPKPAG 558 NIRKPAG 559 SIRKPFG 560 SIRKPAS 561 PIRKPAG 562 SIRQPAG 563 SIRNPAG 564 SIVKPAG 565 SIRKPEG 566 SIRKPXG 567 SIRVPAG 568 SLRKPAG 569 SIQKPAG 570 FIRKPAG 571 SISKPAG 572 SIRTPAG 573 SXRKPAG 574 SIRKPLG 575 SIRKPAV 576 SIRKPHG 577 SI RM PAG 578 SIHKPAG 579

[0345] S IRC PAG 580 SIRKRAG 581 SIRAPAG 582 SIRFPAG 583 SIRIPAG 584 SIRKPDG 585

[0346]

[0347] ATTORNEY DOCKET NO.: 51772-007WO2

[0348] PATENT

[0349] Peptide sequence SEQ ID NO.

[0350] SIRKEAG 586 SIRKLAG 587 SRRKPAG 588 SIRKPYG 589 SIRKPAW 590 SIRDPAG 591 SIRPPAG 592 SIRKPRG 593 SIRKDAG 594 SMRKPAG 595 SIRGPAG 596 RIRKPAG 597 KIRKPAG 598 SIRKPAA 599 SERKPAG 600 YIRKPAG 601 SIRKSAG 602 AIRKPAG 603 MIRKPAG 604 SPRKPAG 605 SIRKQAG 606 SIWKPAG 607 SIRKPAD 608 IIRKPAG 609 SIRKMAG 610 SIRKNAG 611 SIRWPAG 612 SI EKPAG 613 SIREPAG 614 SIDKPAG 615 SDRKPAG 616 QIRKPAG 617 XIRKPAG 618 VIRKPAG 619 CIRKPAG 620 SWRKPAG 621 SYRKPAG 622 SCRKPAG 623

[0351]

[0352] ATTORNEY DOCKET NO.: 51772-007WO2

[0353] PATENT

[0354] Peptide sequence SEQ ID NO.

[0355] SIXKPAG 624

[0356] SIRXPAG 625

[0357] SIRKKAG 626

[0358] SIRKXAG 627

[0359] SIRKGAG 628

[0360] SIRKWAG 629

[0361] SIRKVAG 630

[0362] SIRKHAG 631

[0363] SIRKYAG 632

[0364] SIRKCAG 633

[0365] SIRKIAG 634

[0366] SIRKFAG 635

[0367] SIRKPKG 636

[0368] SIRKPWG 637

[0369] SIRKPCG 638

[0370] SIRKPAK 639

[0371] SIRKPAQ 640

[0372] SIRKPAE 641

[0373] SIRKPAX 642

[0374] SIRKPAT 643

[0375] SIRKPAP 644

[0376] SIRKPAR 645

[0377] SIRKPAM 646

[0378] SIRKPAL 647

[0379] SIRKPAN 648

[0380] SIRKPAH 649

[0381] SIRKPAY 650

[0382] SIRKPAC 651

[0383] SIRKPAI 652

[0384] SIRKPAF 653

[0385]

[0386] AAV Capsid Proteins

[0387] The peptide inserts of the invention may be incorporated into any one or more AAV capsid proteins that are known in the art. AAV capsid proteins compose the exterior, non-nucleic acid portion of the viral particle and are encoded by the AAV Cap gene. The Cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for particle assembly. The sequences of naturally existing capsid ATTORNEY DOCKET NO.: 51772-007WO2

[0388] PATENT

[0389] proteins associated with AAV serotypes are known in the art and include those disclosed herein as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhl 0, and AAVrh74.

[0390] Table 3 illustrates several non-limiting examples of naturally occurring AAV capsid proteins.

[0391] Table 3. Capsid Proteins of AAV Serotypes

[0392] Serotype Capsid Sequence SEQ ID Protein NO. AAV1 VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDD 1 GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKR LNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMA DNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNN HLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTST VQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYA HSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPA GMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYN LNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTA LDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHA MGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEW ELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIG TRYLTRPL AAV2 VP1 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS 2 RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDS GDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEP LGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRL NFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMAD NNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNN HLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNG SQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDI RDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLN GRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDI EKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQ GVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWEL

[0393]

[0394] ATTORNEY DOCKET NO.: 51772-007WO2

[0395] PATENT

[0396] Serotype Capsid Sequence SEQ ID Protein NO.

[0397] QKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL AAV3 VP1 MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDN 3

[0398] RRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLK AGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILE PLGLVEEAAKTAPGKKGAVDQSPQEPDSSSGVGKSGKQPARK RLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPM ADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYN NHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKKLSFKLFNIQVRGVTQNDGTTTIANNLTST VQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYA HSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGP QSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKY HLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASN AELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTGTV NHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGF GLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEI EWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRP IGTRYLTRNL AAV4 VP1 MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNA 4

[0399] RGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKA GDNPYLKYNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLE PLGLVEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKQPAKKKL VFEDETGAGDGPPEGSTSGAMSDDSEMRAAAGGAAVEGGQG ADG VG N ASG D WHO DSTWS EG H VTTTSTRTWVLPTYN N H LYKR LGESLQSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNW GM RPKAM RVKI FN IQVKEVTTSNG ETTVAN N LTSTVQI FADSSY ELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGNTSQQQT DRNAFYCLEYFPSQMLRTGNNFEITYSFEKVPFHSMYAHSQSL DRLMNPLIDQYLWGLQSTTTGTTLNAGTATTNFTKLRPTNFSNF KKNWLPGPSIKQQGFSKTANQNYKIPATGSDSLIKYETHSTLDG RWSALTPGPPMATAGPADSKFSNSQLIFAGPKQNGNTATVPGT LIFTSEEELAATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGA VPGMVWQNRDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKHPP PQIFIKNTPVPANPATTFSSTPVNSFITQYSTGQVSVQIDWEIQKE RSKRWNPEVQFTSNYGQQNSLLWAPDAAGKYTEPRAIGTRYLT HHL

[0400]

[0401] ATTORNEY DOCKET NO.: 51772-007WO2

[0402] PATENT

[0403] Serotype Capsid Sequence SEQ ID Protein NO.

[0404] AAV5 VP1 MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQAR 5

[0405] GLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAG DNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPF GLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAG PSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGN ASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSV DGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWG FRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQL PYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSS FFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLAN PLVDQYLYRFVSTN NTGG VQFN KN LAG RYANTYKN WFPG PMG RTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMT NNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQ PVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWME RDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTP VPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNP EIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL AAV6 VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDD 6

[0406] GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKR LNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMA DNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNN HLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTST VQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYA HSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPA GMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYN LNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTA LDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHV MGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEW ELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIG TRYLTRPL AAV7 VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDN 7

[0407] GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARK

[0408]

[0409] ATTORNEY DOCKET NO.: 51772-007WO2

[0410] PATENT

[0411] Serotype Capsid Sequence SEQ ID Protein NO.

[0412] RLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGTVAAGGGAPM ADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYN NHLYKQISSETAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTI QVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG SQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPS TMAEQAKNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHL NGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTGATNKTTL ENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQ GALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLK HPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTR YLTRNL AAV8 VP1 MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDG 8

[0413] RGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQA GDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEP LGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMA DNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNN HLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTI QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG SQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNT M ANQAKN WLPG PCYRQQRVSTTTGQN N N SN FAWTAGTKYHL NGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADY SDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQ GALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLK HPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTR YLTRNL AAV8 VP2 TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGD 9

[0414] SESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGAD GVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISN GTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINN NWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRL

[0415]

[0416] ATTORNEY DOCKET NO.: 51772-007WO2

[0417] PATENT

[0418] Serotype Capsid Sequence SEQ ID Protein NO.

[0419] MNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKN WLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLAN PGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSE EEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVW QNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKN TPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL AAV8 VP3 TMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT 10

[0420] STRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFD FNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQN EGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVF MIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTY TFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANT QTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNS NFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIF GKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQ QNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQY STGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNT EGVYSEPRPIGTRYLTRNL AAV9 VP1 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 11

[0421] ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLE PLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKR LNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVAD NNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNH LYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTST VQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLND GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYA HSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNM AVQG RN Yl PG PSYRQQR VSTTVTQN N NSEFAWPG ASSWALNG RNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDA DKVM ITN EEEI KTTN PVATESYGQVATN HQSAQAQAQTG WVQN QGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGM KHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGT RYLTRNL

[0422]

[0423] ATTORNEY DOCKET NO.: 51772-007WO2

[0424] PATENT

[0425] Serotype Capsid Sequence SEQ ID Protein NO.

[0426] AAV9 VP2 TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 12

[0427] SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVG SSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTS GGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNW GFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSS FYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLM NPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPG PAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEE EIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMV WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILI KNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL AAV9 VP3 MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTS 13

[0428] TRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNN GVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFM IPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYE FENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQT LKFS VAG PSN M AVQG RN Yl PG PSYRQQ R VSTTVTQN N N S E FA WPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFG KQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSA QAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVN TEGVYSEPRPIGTRYLTRNL AAV10 VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDD 14

[0429] GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEAAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKK RLNFGQTGESESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPM ADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYN NHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTS TIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYA HSQSLDRLMNPLIDQYLYYLSRTQSTGGTQGTQQLLFSQAGPA N MSAQAKN WLPG PCYRQQRVSTTLSQN N NSN FAWTG ATKYHL

[0430]

[0431] ATTORNEY DOCKET NO.: 51772-007WO2

[0432] PATENT

[0433] Serotype Capsid Sequence SEQ ID Protein NO.

[0434] NGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGRDNV DYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQANTGPIVGNVN SQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFG LKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGT RYLTRNL AAVrhIO VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDD 15

[0435] GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLK AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKK RLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPM ADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYN NHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTS TIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYA HSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPN NMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHL NGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNV DYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVN SQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFG LKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPIGT RYLTRNL AAV10rh74 VP1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDN 16

[0436] GRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQ AGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKK RLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPM ADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYN NHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTS TIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYA HSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPN N MSAQAKN WLPG PCYRQQRVSTTLSQN N NSN FAWTG ATKYHL NGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNV DYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVN SQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFG

[0437]

[0438] ATTORNEY DOCKET NO.: 51772-007WO2

[0439] PATENT

[0440] Serotype Capsid Sequence SEQ ID Protein NO.

[0441] LKHPPPQILIKNTPVPADPPTTFTKAKLASFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGT RYLTRNL

[0442]

[0443] In some embodiments, a peptide insert of the invention may be incorporated into a variant AAV capsid protein, such as a variant of any of the capsid protein sequence recited in Table 3, above. For example, an AAV peptide insert of the invention may be incorporated into an amino acid sequence having at least, e.g., 85% identity to the amino acid sequence of any one of SEQ ID NOs: 3501 -3516 (e.g., at least 85% identity, 87% identity, 88% identity, 89% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity to any one of SEQ ID NOs: 1-16).

[0444] Peptide inserts of the invention may be incorporated at one of several different sites within an AAV Cap gene. Peptide inserts may be incorporated in any one or more of nine variable loop regions on the surface of the AAV capsid, for example, VR-I, VR-II, VR-II I, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX. In preferred embodiments, peptides of the invention may be inserted between any amino acids of VR-VIII (e.g., of AAV9), for example, between amino acids 581 and 582, amino acids 582 and 583, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, amino acids 589 and 590, amino acids 590 and 591, amino acids 591 and 592, or amino acids 592 and 593, with numbering of amino acids with respect to VP1 (DiMattia et al., Journal of Virology 86:12 (2012), the disclosure of which is incorporated herein by reference).

[0445] Methods of Screening for Optimized AAV Capsid Variants

[0446] Various processes may be used to characterize and further refine AAV capsid variants containing peptide inserts. For example, such variants may be screened to identify optimized AAV capsid variants with the use of library-based approaches. Generally, an AAV capsid library may be generated by expressing engineered capsid variants, each containing a peptide insert of the invention, in an appropriate AAV producer cell line.

[0447] After first-round administration, one or more engineered AAV viral particles having an engineered capsid variant may then be used to form a filtered AAV capsid library. Desirable AAV viral particles may be identified by measuring the mRNA expression of the capsid variants and determining which variants are highly expressed in the desired cell type(s) as compared to non-desired cells type(s). Those that are highly expressed in the desired cell, tissue, and / or organ type are the desired AAV capsid variant particles.

[0448] The recombinant AAV capsid variant particles identified from the first round may then be administered to various non-human animals. In some embodiments, the animals used in the second round of selection and identification are not the same as those animals used for first round selection and identification. Similar to round 1, after administration the top expressing variants in the desired cell, tissue, and / or organ type(s) may be identified by measuring viral mRNA expression in the cells. Administration at ATTORNEY DOCKET NO.: 51772-007WO2

[0449] PATENT

[0450] each round may be systemic. The top variants, or optimized capsid variants, identified after round two may then be optionally barcoded and optionally pooled. In some embodiments, optimized capsid variants from the second round may then be administered to a non-human primate to identify the top cell-specific variant(s), particularly if the end use for the top variant is in humans.

[0451] In some embodiments, the method of generating an AAV capsid variant may include the steps of: (a) expressing a vector system described herein that contains an engineered AAV capsid polynucleotide in a ceil to produce engineered AAV virus particle capsid variants; (b) harvesting the engineered AAV virus particle capsid variants produced in step (a); (c) administering engineered AAV virus particle capsid variants to one or more first subjects, wherein the engineered AAV virus particle capsid variants are produced by expressing an engineered AAV capsid variant vector or system thereof in a cell and harvesting the engineered AAV virus particle capsid variants produced by the cell; and (d) identifying one or more engineered AAV capsid variants produced at a significantly high level by one or more specific cells or specific cell types in the one or more first subjects.

[0452] The method may further include the steps of: (e) administering some or all engineered AAV capsid variant particles identified in step (d) to one or more second subjects; and (f) identifying one or more optimized AAV capsid variant particles produced at a significantly high level in one or more specific cells or specific ceil types in the one or more second subjects. The ceil in step (a) may be a prokaryotic cell or a eukaryotic ceil. In some embodiments, the administration in step (c), step (e), or both is systemic. In some embodiments, one or more first subjects, one or more second subjects, or both, are non-human mammals. In some embodiments, one or more first subjects, one or more second subjects, or both, are each independently selected from the group consisting of: a wild-type non-human mammal, a humanized non-human mammal, a disease-specific non-human mammal model, and a non-human primate.

[0453] AAV Vectors for Nucleic Acid Delivery

[0454] Nucleic acids encoding optimized capsid variants containing the peptide inserts of the invention may be incorporated into an AAV particle in order to facilitate introduction into a target cell of interest. AAV particles useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh74, and others. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0455] Also useful in conjunction with the compositions and methods described herein are pseudotyped AAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9. For example, a representative pseudotyped vector is an AAV8 vector encoding a ATTORNEY DOCKET NO.: 51772-007WO2

[0456] PATENT

[0457] therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 2. Techniques involving the construction and use of pseudotyped AAV particles are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0458] AAV Particle Production

[0459] AAV particles of the invention, containing one or more of the optimized AAV capsid proteins described herein, may be manufactured from producer cells. There are various approaches to producing AAV particles, often differing based on how the adenovirus helper factors are provided to a producer cell.

[0460] As non-limiting examples, in some embodiments, a method of producing AAV particles may include introducing, into cell lines that stably harbor AAV replication- and capsid-encoding polynucleotides, adenovirus helper factors and the target AAV genome (containing, e.g., a transgene of interest). In some embodiments, a method of producing AAV particles includes co-transfection of an appropriate producing cell line with three vectors (e.g., plasmid vectors): (1) an AAV genome that contains a transgene of interest between two ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotides; and (3) a vector containing helper polynucleotides.

[0461] The construction of AAV particles has been described, e.g., in US Patent Nos. 5,173,414;

[0462] 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery. The engineered AAV vectors and systems thereof described herein may be produced by any of these methods.

[0463] Cell-based Vector Amplification and Expression

[0464] Vectors may be designed for expression of one or more elements of the optimized AAV capsid system in a suitable producer cell. In some embodiments, the suitable producer cell is a eukaryotic cell. Suitable producer cells include, but are not limited to, insect cells and mammalian cells. The vectors may be viral-based or non-viral based.

[0465] In some embodiments, the vector is a baculovirus vector or expression vector and may be suitable for expression of polynucleotides and / or proteins in insect cells. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Ceil. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). rAAV (recombinant adeno-associated viral) vectors are preferably produced in insect cells, e.g., Spodoptera frugiperda S19 insect cells, grown in serum-free suspension culture. Serum-free insect cells may be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405).

[0466] In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). The mammalian expression vector may include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and / or proteins in the mammalian cell. For ATTORNEY DOCKET NO.: 51772-007WO2

[0467] PATENT

[0468] example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More detail on suitable regulatory elements is described elsewhere herein. For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.

[0469] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277) and muscle-specific promoters (Skopenkova, et al, 2021. Acta Naturae 13:47-58). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regards to these eukaryotic vectors, mention is made of U. S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments may utilize viral vectors, with regards to which mention is made of U. S. Patent application 13 / 092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U. S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. In some embodiments, a regulatory element may be operably linked to one or more elements of an optimized AAV capsid system so as to drive expression of the one or more elements of the optimized AAV capsid system described herein.

[0470] Methods for the Delivery of Exogenous Nucleic Acids to Host Cells

[0471] The vector or vector components described herein may be delivered into a host cell, such as a producer cell or a target cell, by any suitable method. Suitable methods of the invention include, but are not limited to, those described below.

[0472] Techniques that may be used to introduce a polynucleotide, such as codon-optimized DNA or RNA (e.g., mRNA, tRNA, siRNA, miRNA, shRNA, chemically modified RNA) into a mammalian cell are well known in the art. For example, electroporation may be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0473] Additional techniques useful for the transfection of target cells are the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the ATTORNEY DOCKET NO.: 51772-007WO2

[0474] PATENT

[0475] uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0476] Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids are contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that may be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0477] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

[0478] Impalefection is another technique that may be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires.

[0479] Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures may express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0480] Magnetofection may also be used to deliver nucleic acids to target cells. The magnetofection principle is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated ATTORNEY DOCKET NO.: 51772-007WO2

[0481] PATENT

[0482] by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0483] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0484] Microvesicles represent another potential vehicle that may be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, may be used to efficiently deliver proteins into a cell that subsequently catalyze the sitespecific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0485] Cell-free Vector and Polynucleotide Expression

[0486] The AAV capsid proteins of the invention may be incorporated into a vector or suitable polynucleotide in a cell-free in vitro system. In other words, AAV capsid proteins may be transcribed and optionally translated in vitro. In vitro transcription / translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription may include T7, SP6, T3, promoter regulatory sequences that may be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0487] In vitro translation may be stand-alone (e.g., translation of a purified polyribonucleotide) or linked / coupled to transcription. In some embodiments, the cell-free (or in vitro) translation system may include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. The extracts may include various macromolecular components that are needed for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). Other components may be included or added during the translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other co-factors (Mg2+, K+, etc.). As previously mentioned, in vitro translation may be based on RNA or DNA starting material. Some translation systems may utilize an RNA template as starting material (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems may utilize a DNA template as a starting material (e.g., E cob-based systems). In these systems transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available. ATTORNEY DOCKET NO.: 51772-007WO2

[0488] PATENT

[0489] Selectable Markers and Tags

[0490] Selectable markers and tags may be used in conjunction with cell-based and cell-free vector amplification and expression. One or more of the engineered polynucleotides of the present invention (e.g., an optimized AAV capsid polynucleotide) may be operably linked, fused to, or otherwise modified to include a polynucleotide that encodes a selectable marker or tag, which may be a polynucleotide or polypeptide. In some embodiments, the polypeptide encoding a polypeptide selectable marker may be incorporated in the engineered polynucleotide of the present invention (e.g., an optimized AAV capsid polynucleotide) such that the selectable marker polypeptide, when translated, is inserted between two amino acids between the N- and C- terminus of an engineered polypeptide (e.g., an optimized AAV capsid polypeptide).

[0491] It will be appreciated that the polynucleotide encoding such selectable markers or tags may be incorporated into a polynucleotide encoding one or more components of the engineered AAV capsid system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure.

[0492] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that may allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FIAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which may be readily identified (e.g., phenotypic markers such as b-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); polynucleotides that may generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0493] Selectable markers and tags may be operably linked to one or more components of the optimized AAV capsid system or other compositions and / or systems described herein via suitable linker.

[0494] The vector or vector system may include one or more polynucleotides encoding one or more targeting moieties. In some embodiments, the targeting moiety encoding polynucleotides may be included ATTORNEY DOCKET NO.: 51772-007WO2

[0495] PATENT

[0496] in the vector or vector system, such as a viral vector system, such that they are expressed within and / or on the virus particle(s) produced such that the virus particles may be targeted to specific cells, tissues, organs, etc. In some embodiments, the targeting moiety encoding polynucleotides may be included in the vector or vector system such that the engineered polynucleotide(s) of the present invention (e.g., an optimized AAV capsid polynucleotide(s)) and / or products expressed therefrom include the targeting moiety and may be targeted to specific ceils, tissues, organs, etc. In some embodiments, such as non-viral carriers, the targeting moiety may be attached to the carrier (e.g., polymer, lipid, inorganic molecule etc.) and may be capable of targeting the carrier and any attached or associated engineered polypeptides of the present invention, or other compositions of the present invention described herein, to specific cells, tissues, organs, etc. In some embodiments, the specific cells are muscle cells.

[0497] Methods of Treatment

[0498] The methods and compositions of the disclosure may be used to treat any disease with a genetic association. In some embodiments, an AAV particle of the invention containing an AAV vector and one or more of the optimized AAV capsid proteins described herein may be delivered to a subject in need thereof, as a therapy for one or more diseases. In some embodiments, the disease to be treated is a genetic or epigenetic disease. In some embodiments, the therapy is gene therapy. In some embodiments, the disease to be treated is not a genetic or epigenetic disease. In some embodiments, compositions of the invention may be incorporated into a pharmaceutical composition or pharmaceutical formulation and may be administered to a subject via any of the routes of administration described herein. In some embodiments, an AAV particle or pharmaceutical composition described herein may be delivered to a subject in need thereof as a treatment or prevention (or as a part of a treatment or prevention) of a disease. The specific disease to be treated and / or prevented by delivery of an AAV particle or pharmaceutical composition described herein may be dependent on the cargo coupled to, attached to, contained in, or otherwise associated with the AAV particle or pharmaceutical composition.

[0499] Genetic diseases that may be treated by administering an AAV particle or pharmaceutical composition of the invention are discussed in greater detail below. Other diseases that may be treated by administering an AAV particle or pharmaceutical composition of the invention, include, but are not limited to, any of the following: Acinetobacter infections, actinomycosis, African sleeping sickness, AIDS / HIV, amoebiasis, Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacterial meningitis, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, balantidiasis, Bartonellosis, Baylisascaris infection, BK virus infection, Black Piedra, Biastocytosis, Blastomycosis, Bolivian hemorrhagic fever, Botulism, Brazilian hemorrhagic fever, brucellosis, Bubonic plague, Burkholderia infection, buruli ulcer, calicivirus invention, campylobacteriosis, cancer, Candidiasis, Capiilariasis, Carrion’s disease, Cat-scratch disease, cellulitis, Chagas Disease, Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydia pneumoniae, Cholera, Chromoblastomycosis, Chytridiomycosis, Clonorchiasis, Clostridium difficile colitis, Coccidioidomycosis, Colorado tick fever, rhinovirus / coronavirus infection (common cold), Creutzfeldt-Jakob disease, Crimean-congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans (CLM), cyclosporiasis, cysticercosis, cytomegalovirus infection, Dengue fever, Desmodesmus infection, Dientamoebiasis, Diphtheria, Diphyllobothriasis, ATTORNEY DOCKET NO.: 51772-007WO2

[0500] PATENT

[0501] Dracunculiasis, Ebola, Echinococcosis, Ehrlichiosis, Enterobiasis, Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema Infectiosum, Exanthem subitum, Fascioliasis, Fascioiopsiasis, fatal familial insomnia, filariasis, Clostridium perfringens infection, Fusobacterium infection, Gas gangrene (clostridial myonecrosis), geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale, Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot, and mouth disease, hantavirus pulmonary syndrome, heartland virus disease, helicobacter pylori infection, hemorrhagic fever with renal syndrome, Hendra virus infection, Hepatitis (all groups A, B, C, D, E), herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, Human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus, Hymenolepiasis, Epstein-Barr infection, mononucleosis, influenza, isosporiasis, Kawasaki disease, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires disease and Potomac Fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, measles, Middle East respiratory syndrome, Melioidosis, meningitis, Meningococcal disease, Metagonimiasis, Microsporidosis, Molluscum contagiosum, Monkeypox, Mumps, Murine typhus, Mycoplasma pneumonia, Mycoplasma genitalium infection, Mycetoma, Myiasis, Conjunctivitis, Nipah virus infection, Norovirus, Variant Creutzfeldt-Jakob disease, Nocardiosis, Onchocerciasis, Opisthorchiasis, Paracoccidioidomycosis, Paragonimiasis, Pasteurellosis, Pediculosisi capitis, Pediculosis corporis, Pediculosis pubis, pelvic inflammatory disease, pertussis, plague, pneumococcal infection, pneumocystis pneumonia, pneumonia, poliomyelitis, prevotella infection, primary amoebic meningoencephalitis, progressive multifocal leukoencephalopathy, Psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinovirus infection, rickettsial infection, Rickettsialpox, Rift Valley Fever, Rocky Mountain Spotted Fever, Rotavirus infection, Rubella, Salmonellosis, SARS, Scabies, Scarlet fever, Schistosomiasis, sepsis, Shigellosis, Shingles, Smallpox, Sporotrichosis, Staphylococcal infection (including methicillin-resistant Staphylococcus aureus or MRSA), strongyloidiasis, subacute sclerosing panencephalitis, Syphilis, Taeniasis, tetanus, Trichophyton species infection, Toxocariasis, Toxoplasmosis, Trachoma, Trichinosis, Trichiniasis, Tuberculosis, Tularemia, Typhoid Fever, Typhus Fever, Ureaplasma urealyticum infection, Valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio species infection, Viral pneumonia, West Nile Fever, White Piedra, Yersinia pseudotuberculosis, Yersiniosis, Yellow fever, Zeaspora, Zika fever, Zygomycosis and combinations thereof.

[0502] Some other diseases and disorders that may be treated using an AAV particle or pharmaceutical composition of the present disclosure include, but are not limited to, muscle diseases of infectious or non- infectious origin, endocrine diseases (e.g., Type I and Type II diabetes, gestational diabetes, hypoglycemia, glucagonoma, goiter, hyperthyroidism, hypothyroidism, thyroiditis, thyroid cancer, thyroid hormone resistance, parathyroid gland disorders, osteoporosis, osteitis deformans, rickets, osteomalacia, hypopituitarism, pituitary tumors, etc.), skin conditions of infections and non-infectious origin, eye diseases of infectious or non-infectious origin, gastrointestinal disorders of infectious or non-infectious origin, cardiovascular diseases of infectious or non-infectious origin, brain and neuron diseases of infectious or non-infectious origin, nervous system diseases of infectious or non-infectious origin, bone diseases of infectious or non-infectious origin, reproductive system diseases of infectious or non- ATTORNEY DOCKET NO.: 51772-007WO2

[0503] PATENT

[0504] infectious origin, renal system diseases of infectious or non-infectious origin, blood diseases of infectious or non-infectious origin, lymphatic system diseases of infectious or non-infectious origin, immune system diseases of infectious or non-infectious origin, and mental-illnesses of infectious or non-infectious origin, among others.

[0505] In some embodiments, the disease is a muscle disease or disorder, neuromuscular disease or disorder, a musculoskeletal disease or disorder, a muscular dystrophy, or a cardiomyopathy. In some embodiments, the disease or disorder may be selected from any one or more of the following categories: (a) a muscle disease;

[0506] (b) a muscular dystrophy;

[0507] (c) a neuromuscular disease;

[0508] (d) a musculoskeletal disease;

[0509] (e) a cardiomyopathy;

[0510] (f) an expanded repeat disease;

[0511] (g) a dominant negative disease;

[0512] (h) a sugar or glycogen storage disease;

[0513] (i) a viral, bacterial, or fungal disease;

[0514] (j) a progeroid disease;

[0515] (k) an autoimmune disease;

[0516] (l) a cancer;

[0517] (m) a neurodegenerative disease;

[0518] (n) a lysosomal storage disease; or

[0519] (o) any combination thereof.

[0520] Using an AAV particle or pharmaceutical composition of the invention, a variety of disorders of the central nervous system (CNS) may be treated. The following is a non-limiting list of genes associated with CNS-associated disorders that may be expressed using an AAV vector of the invention: neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase type 1 (SOD1), and amino acid decorboxylase (AADC), among others. For example, a useful transgene in the treatment of Parkinson’s disease (PD) encodes TH, which is a rate limiting enzyme in the synthesis of dopamine. A transgene encoding GTPCH, which generates the TH cofactor tetrahydrobiopterin, may also be used in the treatment of PD. A transgene encoding GDNF, BDNF, or AADC, which facilitates conversion of L-Dopa to dopamine (DA), may also be used for the treatment of PD. For the treatment of ALS, a useful transgene may encode: GDNF, BDNF, or CNTF. Also, for the treatment of ALS, a useful transgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibits the expression of SOD1. For the treatment of ischemia, a useful transgene may encode NAIP or NGF. A transgene encoding Beta-glucuronidase (GUS) may be useful for the treatment of certain lysosomal storage diseases (e.g., Mucopolysaccharidosis type VII (MPS VII)). A transgene encoding a prodrug activation gene, e.g., HSV-Thymidine kinase which converts ganciclovir to a toxic nucleotide which disrupts DNA synthesis and leads to cell death, may be useful for treating certain cancers, e.g., when administered in combination with the prodrug. A transgene encoding an endogenous opioid, such a p-endorphin may be useful for treating pain. Other examples of transgenes ATTORNEY DOCKET NO.: 51772-007WO2

[0521] PATENT

[0522] that may be expressed using the AAV vectors of the invention will be apparent to the skilled artisan (See, e.g., Costantini L C, et al., Gene Therapy (2000) 7, 93-109).

[0523] The AAV vectors described herein may include a polynucleotide encoding a therapeutic protein useful for the treatment of a genetic disorder. In some embodiments, the therapeutic transgene (e.g., a transgene encoding a therapeutic protein) may encode a polypeptide, a short peptide fragment, or a full-length protein. In some embodiments of the disclosure, the transgene contained within the AAV vector encodes a therapeutic RNA molecule that inhibits the expression of an endogenous gene of interest. Such inhibitory nucleic acids may specifically bind (e.g., hybridizes to) at least a portion of a target nucleic acid, such as a target RNA, pre-mRNA, or mRNA, and inhibit its expression or activity. In some embodiments, the inhibitory nucleic acid is complementary to a protein coding region or non-coding region (e.g., 5’UTR, 3’UTR, intron, etc.). In some embodiments, the inhibitory nucleic acid is complementary to a wild-type nucleic acid or a naturally occurring variant thereof. In some embodiments, the inhibitory nucleic acid is single stranded or double-stranded. In some embodiments, the inhibitory nucleic acid is an interfering RNA molecule, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or double-stranded RNA(dsRNA).

[0524] The following table provides a list of exemplary transgenes of the invention and their corresponding associated diseases which may be treated or prevented by administering an AAV particle or pharmaceutical composition of the invention.

[0525] Table 4. Exemplary transgenes of the invention and associated diseases Diseases Genes

[0526] Achondroplasia FGFR3

[0527] Achromatopsia CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, ACHM2, ACHM3

[0528] Acute Renal Injury NFkappaB, AATF, p85alpha, FAS, Apoptosis cascade elements (e.g., FASR, Caspase 2, 3, 4, 6, 7, 8, 9, 10, AKT, TNF alpha, IGF1, IGF1R, RIPK1), p53

[0529] Age Related Macular Aber, CCL2, CC2, CP, Timp3, cathepsinD, Degeneration VLDLR, CCR2, sFLTOI (VEGF / PIGF (placental growth factor) binding domain of human VEGFRI / Flt-1 (hVEGFRI) fused to the Fe portion of human IgG(l) through a polyglycine linker) Acquired Immune Deficiency Syndrome (AIDS) KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1

[0530] Albinism (including oculocutaneous albinism TYR, OCA2, TYRP1, SLC45A2, SLC24A5, (Types 1-7) and ocular albinism) C10orf11

[0531] Alkaptonuria HGD

[0532] Alpha-1 Antitrypsin Deficiency (AATD or AIAD) AAT, SERPINA1, those set forth in

[0533] or Hereditary Emphysema WO2017165862, PiZ allele

[0534]

[0535] ATTORNEY DOCKET NO.: 51772-007WO2

[0536] PATENT

[0537] Diseases Genes

[0538] Amyotrophic lateral sclerosis (ALS) SOD1, ALS2, ALS3, ALS5,

[0539] ALS7, STEX, FUS, TARDBP, VEGF (VEGF-a; VEGF-b; VEGF-c), DPP6, NEFH, PTGS1, SLC1A2, TNFRSF10B, PRPH, HSP90AA1, CRIA2, IFNG, AMPA2 SWOB, FGF2, AOX1, OS, TXN, RAPHJ1, MAP3K5, NBEAL1, GPX1, ICA1L, RAC1, MAPT, ITPR2, ALS2CR4, GLS, ALS2CR8, CNTFR, ALS2CR11, FOLH1, FAM117B, P4HB, CNTF, SQSTM1, STRADB, NAIP, NLR, YWHAQ, SLC33A1, TRAK2, SCA1, NIF3L1, NIF3, PARD3B, COX8A, CDK15, HECW1, HECT, 02, WW 15, N0S1, MET, S0D2, HSPB1, NEFL, CTSB, ANG, HSPA8, RNase A, VAPB, VAMP, SNCA, alpha HGF, CAT, ACTB, NEFM, TH, BCL2, FAS, CASP3, CLU, SMN1, G6PD, BAX, HSF1, RNF19A, JUN, ALS2CR12, HSPA5, MAPK14, APEX1, TXNRD1, N0S2, TIMP1, CASP9, XIAP, GLG1, EPO, VEGFA, ELN, GDNF, NFE2L2, SLC6A3, HSPA4, APOE, PSMB8, DCTN2, TIMP3, KIFAP3, SLC1A1, SMN2, CCNC, STUB1, ALS2, PRDX6, SYP, CABIN1, CASP1, GART, CDK5, ATXN3, RTN4, C1QB, VEGFC, HTT, PARK7, XDH, GFAP, MAP2, CYCS, FCGR3B, CCS, UBL5, MMP9m SLC18A3, TRPM7, HSPB2, AKT1, DEERL1, CCL2, NGRN, GSR, TPPP3, APAF1, BTBD10, GLUD1, CXCR4, SLC1A3, FLT1, P0N1, AR, LIF, ERBB3, GAS1, CD44, TP53, TLR3, GRIA1, GAPDH, AMPA, GRIK1, DES, CHAT, FLT4, CHMP2B, BAG1, CHRNA4, GSS, BAK1, KDR, GSTP1, OGG1, IL6

[0540] Alzheimer's Disease E1, CHIP, UCH, UBB, Tau, LRP, PICALM, CLU,

[0541] PS1, S0RL1, CR1, VLDLR, UBA1, UBA3, CHIP28, AQP1, UCHL1, UCHL3, APP, AAA, CVAP, AD1, APOE, AD2, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BDNF, BLMH, BMH, PSEN1, AD3, ALAS2, ABCA1, BIN1, BDNF, BTNL8, C1ORF49, CDH4, CHRNB2, CKLFSF2, CLEC4E, CR1L, CSF3R, CST3,

[0542]

[0543] ATTORNEY DOCKET NO.: 51772-007WO2

[0544] PATENT

[0545] Diseases Genes

[0546] CYP2C, DAPK1, ESR1, FCAR, FCGR3B, FFA2, FGA, GAB2, GALP, GAPDHS, GDNF, GMPB, HP, HTR7, IDE, IF127, IFI6, IFIT2, IL1RN, IL-1RA, IL8RA, IL8RB, JAG1, KCNJ15, LRP6, MAPT, MARK4, MPHOSPH1, MTHFR, NBN, NCSTN, NIACR2, NGF, NMNAT3, NTM, ORM1, P2RY13, PBEF1, PCK1, PICALM, PLAU, PLXNC1, PRNP, PSEN1, PSEN2, PTPRA, RALGPS2, RGSL2, SELENBP1, SLC25A37, SORL1, Mitoferrin-1, TF, TFAM, TNF, TNFRSF10C, UBE1C Amyloidosis APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ,

[0547] TTR, PALB

[0548] Amyloid Neuropathy TTR, PALB

[0549] Anemia CDAN1, CDA1, RPS19, DBA, PKLR, PK1,

[0550] NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT

[0551] Angelman Syndrome UBE3A

[0552] Anxiety BDNF

[0553] Arthritis, Rheumatoid Arthritis TNFR: Fc

[0554] Attention Deficit Hyperactivity Disorder (ADHD) PTCHD1

[0555] Autoimmune Lymphoproliferative Syndrome TNFRSF6, APT1, FAS, CD95, ALPS1A Autism, Autism spectrum disorders (ASDs), PTCHD1, Mecp2, BZRAP1, MDGA2, Sema5A, including Asperger's and a general diagnostic Neurexin 1, GLO1, RTT, PPMX, MRX16, RX79, category called Pervasive Developmental NLGN3, NLGN4, KIAA1260, AUTSX2, FMR1, Disorders (PDDs) FMR2, FXR1, FXR2, MGLUR5, ATP1 OC, CDH10,

[0556] GRM6, MGLUR6, CDH9, CNTN4, NLGN2, CNTNAP2, SEMA5A, DHCR7, NLGN4X, NLGN4Y, DPP6, NLGN5, EN2, NRCAM, MDGA2, NRXN1, FMR2, AFF2, FOXP2, OR4M2, OXTR, FXR1, FXR2, PAH, GABRA1, PTEN, GABRA5, PTPRZ1, GABRB3, GABRG1, HIRIP3, SEZ6L2, HOXA1, SHANK3, IL6, SHBZRAP1, LAMB1, SLC6A4, SERT, MAPK3, TAS2R1, MAZ, TSC1, MDGA2, TSC2, MECP2, UBE3A, WNT2, see also 20110023145

[0557] Autosomal Dominant Polycystic Kidney Disease PKD1, PKD2

[0558] (ADPKD) - (includes diseases such as von Rippel- Lindau disease and Tuberous Sclerosis Complex

[0559]

[0560] ATTORNEY DOCKET NO.: 51772-007WO2

[0561] PATENT

[0562] Diseases Genes

[0563] disease)

[0564] Autosomal Recessive Polycystic Kidney Disease PKDH1

[0565] (ARPKD)

[0566] Ataxia-Telangiectasia (aka Louis Bar syndrome) ATM

[0567] B-Cell Non-Hodgkin Lymphoma BCL7A, BCL7

[0568] Bardet-Biedl Syndrome ARL6, BBS1, BBS2, BBS4, BBS5, BBS7, BBS9,

[0569] BBS10, BBS12, CEP290, INPP5E, LZTFL1, MKKS, MKS1, SDCCAGS, TRIM32, TTC8 Bare Lymphocyte Syndrome TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11,

[0570] MHC2TA, C2TA, RFX5, RFXAP, RFX5 Bartter's Syndrome (Types I, II, III, IVA and B, and SLC12A1 (type I), KCNJ1 (type II), CLCNKB (type V) III), BSND (type IV A), or both the CLCNKA CLCNKB genes (type IV B), CASR (type V) Batten's Disease, Late Infantile Neuronal Ceroid CLN2

[0571] Lipofuscinosis

[0572] Becker Muscular Dystrophy DMD, BMD, MYF6

[0573] Best Disease (Vitelliform VMD2

[0574] Macular Dystrophy Type 2)

[0575] Bipolar Disorder BDNF

[0576] Bleeding Disorders TBXA2R, P2RX1, P2XI

[0577] Blue Cone Monochromacy OPN1LW, 0PN1MW, LCR

[0578] Breast Cancer BRCA1, BRCA2, COX-2

[0579] Bruton's Disease (aka X-linked BTK

[0580] Agammglobulinemia)

[0581] Mayavan's disease AS PA

[0582] Cancers (e.g., Lymphoma, Chronic Lymphocytic FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC, Leukemia (CLL), B Cell Acute Lymphocytic TRBC, those described in WO2015048577 Leukemia (B-ALL), Acute Lymphoblastic

[0583] Leukemia, Acute Myeloid Leukemia, NonHodgkin's Lymphoma (NHL), Diffuse Large Cell

[0584] Lymphoma (DLCL), Multiple Myeloma, Renal Cell

[0585] Carcinoma (RCC), Neuroblastoma, Colorectal

[0586] Cancer, Breast Cancer, Ovarian Cancer,

[0587] Melanoma, Sarcoma, Lung Cancer, Esophageal

[0588] Cancer, Hepatocellular Carcinoma, Pancreatic

[0589] Cancer, Astrocytoma, Mesothelioma, Head and

[0590] Neck Cancer, and Medulloblastoma

[0591] Cardiovascular Diseases IL1 B, XDH, TP53, PTGS, MB, IL4, ANGPT1, ABCGu8, CTSK, PTGIR, KCNJ11, INS, CRP,

[0592]

[0593] ATTORNEY DOCKET NO.: 51772-007WO2

[0594] PATENT

[0595] Diseases Genes

[0596] PDGFRB, CCNA2, PDGFB, KCNJ5, KCNN3, CAPN10, ADRA2B, ABCG5, PRDX2, CPAN5, PARP14, MEX3C, ACE, RNF, IL6, TNF, STN, SERPINE1, ALB, ADIPOQ, APOB, APOE, LEP, MTHFR, APOA1, EDN1, NPPB, NOS3, PPARG, PLAT, PTGS2, CETP, AGTR1, HMGCR, IGF1, SELE, REN, PPARA, PON1, KNG1, CCL2, LPL, VWF, F2, ICAM1, TGFB, NPPA, IL10, EPO, SOD1, VCAM1, IFNG, LPA, MPO, ESR1, MAPK, HP, F3, CST3, COG2, MMP9, SERPINC1, F8, HMOX1, APOC3, IL8, PROL1, CBS, NOS2, TLR4, SELP, ABCA1, AGT, LDLR, GPT, VEGFA, NR3C2, IL18, NOS1, NR3C1, FGB, HGF, IL1 A, AKT1, LIPC, HSPD1, MAPK14, SPP1, ITGB3, CAT, UTS2, THBD, F10, CP, TNFRSF11 B, EGFR, MMP2, PLG, NPY, RHOD, MAPK8, MYC, FN1, CMA1, PLAU, GNB3, ADRB2, SOD2, F5, VDR, ALOX5, HLA- DRB1, PARP1, CD40LG, PON2, AGER, IRS1, PTGS1, ECE1, F7, IRMN, EPHX2, IGFBP1, MAPK10, FAS, ABCB1, JUN, IGFBP3, CD14, PDE5A, AGTR2, CD40, LCAT, CCR5, MMP1, TIMP1, ADM, DYT10, STAT3, MMP3, ELN, USF1, CFH, HSPA4, MMP12, MME, F2R, SELL, CTSB, ANXA5, ADRB1, CYBA, FGA, GGT1, LIPG, HIF1A, CXCR4, PROC, SCARB1, CD79A, PLTP, ADD1, FGG, SAA1, KCNH2, DPP4, NPR1, VTN, KIAA0101, FOS, TLR2, PPIG, IL1R1, AR, CYP1A1, SERPINA1, MTR, RBP4, APOA4, CDKN2A, FGF2, EDNRB, ITGA2, VLA-2, CABIN1, SHBG, HMGB1, HSP90B2P, CYP3A4, GJA1, CAV1, ESR2, LTA, GDF15, BDNF, CYP2D6, NGF, SP1, TGIF1, SRC, EGF, PIK3CG, HLA-A, KCNQ1, CNR1, FBN1, CHKA, BEST1, CTNNB1, IL2, CD36, PRKAB1, TPO, ALDH7A1, CX3CR1, TH, F9, CH1, TF, HFE, IL17A, PTEN, GSTM1, DMD, GATA4, F13A1, TTR, FABP4, PON3, APOC1, INSR, TNFRSF1 B, HTR2A, CSF3, CYP2C9, TXN, CYP11 B2, PTH, CSF2, KDR, PLA2G2A, THBS1, GCG, RHOA, ALDH2, TCF7L2, NFE2L2, NOTCH1, UGT1 A1, IFNA1,

[0597]

[0598] ATTORNEY DOCKET NO.: 51772-007WO2

[0599] PATENT

[0600] Diseases Genes

[0601] PPARD, SIRT1, GNHR1, PAPPA, ARR3, NPPC, AHSP, PTK2, IL13, MTOR, ITGB2, GSTT1, IL6ST, CPB2, CYP1A2, HNF4A, SLC64A, PLA2G6, TNFSF11, SLC8A1, F2RL1, AKR1 A1, ALDH9A1, BGLAP, MTTP, MTRR, SULT1A3, RAGE, C4B, P2RY12, RNLS, CREB1, POMC, RAC1, LMNA, CD59, SCM5A, CYP1 B1, MIF, MMP13, TIMP2, CYP19A1, CUP21A2, PTPN22, MYH14, MBL2, SELPLG, AOC3, CTSL1, PCNA, IGF2, ITGB1, CAST, CXCL12, IGHE, KCNE1, TFRC, COL1A1, COL1A2, IL2RB, PLA2G10, ANGPT2, PROCR, NOX4, HAMP, PTPN11, SLCA1, IL2RA, CCL5, IRF1, CFAR, CACA, EIF4E, GSTP1, JAK2, CYP3A5, HSPG2, CCL3, MYDSS, VIP, SOAT1, ADRBK1, NR4A2, MMPS, NPR2, GCH1, EPRS, PPARGC1A, F12, PECAM1, CCL4, CERPINA34, CASR, FABP2, TTF2, PROS1, CTF1, SGCB, YME1 L1, CAMP, ZC3H12A, AKR1 B1, MMP7, AHR, CSF1, HDAC9, CTGF, KCNMA1, UGT1A, PRKCA, COMT, SWOB, EGR1, PRL, IL15, DRD4, CAMK2G, SLC22A2, CCL11, PGF, THPO, GP6, TACR1, NTS, HNF1A, SST, KCDN1, LOC646627, TBXAS1, CUP2J2, TBXA2R, ADH1 C, ALOX12, AHSG, BHMT, GJA4, SLC25A4, ACLY, ALOX5AP, NUMA1, CYP27B1, CYSLTR2, SOD3, LTC4S, UCN, GHRL, APOC2, CLEC4A, KBTBD10, TNC, TYMS, SHC1, LRP1, SOCS3, ADH1 B, KLK3, HSD11 B1, VKORC1, SERPINB2, TNS1, RNF19A, EPOR, ITGAM, PITX2, MAPK7, FCGR3A, LEEPR, ENG, GPX1, GOT2, HRH1, NR112, CRH, HTR1 A, VDAC1, HPSE, SFTPD, TAP2, RMF123, PTK2Bm NTRK2, IL6R, ACHE, GLP1R, GHR, GSR, NQO1, NR5A1, GJB2, SLC9A1, MAOA, PCSK9, FCGR2A, SERPINF1, EDN3, UCP2, TFAP2A, C4BPA, SERPINF2, TYMP, ALPP, CXCR2, SLC3A3, ABCG2, ADA, JAK3, HSPA1A, FASN, FGF1, F11, ATP7A, CR1, GFPA, ROCK1, MECP2, MYLK, BCHE, LIPE, ADORA1, WRN, CXCR3, CD81, SMAD7, LAMC2,

[0602]

[0603] ATTORNEY DOCKET NO.: 51772-007WO2

[0604] PATENT

[0605] Diseases Genes

[0606] MAP3K5, CHGA, IAPP, RHO, ENPP1, PTHLH, NRG1, VEGFC, ENPEP, CEBPB, NAGLU, F2RL3, CX3CL1, BDKRB1, ADAMTS13, ELANE, ENPP2, CISH, GAST, MYOC, ATP1 A2, NF1, GJB1, MEF2A, VCL, BMPR2, TUBB, CDC42, KRT18, HSF1, MYB, PRKAA2, ROCK2, TFP1, PRKG1, BMP2, CTNND1, CTH, CTSS, VAV2, NPY2R, IGFBP2, CD28, GSTA1, PPIA, APOH, S100A8, IL11, ALOX15, FBLN1, NR1H3, SCD, GIP, CHGB, PRKCB, SRD5A1, HSD11 B2, CALCRL, GALNT2, ANGPTL4, KCNN4, PIK3C2A, HBEGF, CYP7A1, HLA-DRB5, BNIP3, GCKR, S100A12, PADI4, HSPA14, CXCR1, H19, KRTAP19-3, IDDM2, RAC2, YRY1, CLOCK, NGFR, DBH, CHRNA4, CACNA1C, PRKAG2, CHAT, PTGDS, NR1H2, TEK, VEGFB, MEF2C, MAPKAPK2, TNFRSF11 A, HSPA9, CYSLTR1, MAT1A, OPRL1, IMPA1, CLCN2,

[0607] DLD, PSMA6, PSMBS, CHI3L1, ALDH1 B1, PARP2, STAR, LBP, ABCC6, RGS2, EFNB2, GJB6, APOA2, AMPD1, DYSF, FDFT1, EMD2, CCR6, GJB3, IL1RL1, ENTPD1, BBS4, CELSR2, F11 R, RAPGEF3, HYAL1, ZNF259, ATOX1, ATF6, KHK, SAT1, GGH, TIMP4, SLC4A4, PDE2A, PDE3B, FADS1, FADS2, TMSB4X, TXNIP, LIMS1, RHOB, LY96, FOXO1, PNPLA2, TRH, GJC1, SLC7A5, FTO, GJD2, PRSC1, CASP12, GPBAR1, PXK, IL33, TRIB1, PBX4, NUPR1, 15-SEP, CILP2, TERC, GGT2, MTCO1, UOX, AVP, ANGPLT3

[0608] Cataract CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CCL, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1 Catecholaminergic Polymorphic Ventricular CASQ2

[0609] Tachycardia (CPVT)

[0610]

[0611] ATTORNEY DOCKET NO.: 51772-007WO2

[0612] PATENT

[0613] Diseases Genes

[0614] CDKL-5 Deficiencies or Mediated Diseases CDKL5

[0615] Charcot-Marie-Tooth (CMT) disease (Types 1, 2, PMP22 (CMT1 A and E), MPZ (CMT1 B), LITAF 3, and 4) (CMT1 C), EGR2 (CMT1 D), NEFL (CMT1 F), GJB1 (CMT1X), MFN2 (CMT2A), KIF1B (CMT2A2B), RAB7A (CMT2B), TRPV4 (CMT2C), GARS (CMT2D), NEFL (CMT2E), GAPD1 (CMT2K), HSPB8 (CMT2L), DYNC1H1 (CMT2O), LRSAM1 (CMT2P), IGHMBP2 (CMT2S), MORC2 (CMT2Z), GDAP1 (CMT4A), MTMR2 or SBF2 / MTMR13 (CMT4B), SH3TC2 (CMT4C), NDRG1 (CMT4D), PRX (CMT4F), FIG4 (CMT4J), NT-3

[0616] Chediak-Higashi Syndrome LYST

[0617] Choroideremia CHM, REP1

[0618] Chorioretinal Atrophy PRDM13, RGR, TEAD1

[0619] Chronic Granulomatous Disease CYBA, CYBB, NCF1, NCF2, NCF4 Chronic Heart Failure SERCA2

[0620] Chronic Mucocutaneous Candidiasis AIRE, CARD9, CLEC7A IL12B, IL12B1, IL1 F,

[0621] IL17RA, IL17RC, RORC, STAT1, STAT3, TRAF31P2

[0622] Cirrhosis KRT18, KRT8, CIRH1 A, NAIC, TEX292,

[0623] KIAA1988

[0624] Colon Cancer (Familial Adenomatous Polyposis FAP: APC, HNPCC: MSH2, MLH1, PMS2, SH6, (FAP) and Hereditary Nonpolyposis Colon Cancer PMS1

[0625] (HNPCC))

[0626] Combined Immunodeficiency IL2RG, SCIDX1, SCIDX, IMD4, HIV-1 (CCL5,

[0627] SCYA5, D17S136E, TCP228)

[0628] Cone(-rod) Dystrophy AIPL1, CRX, GUA1A, GUCY2D, PITPM3,

[0629] PROM1, PRPH2, RIMS1, SEMA4A, ABCA4, ADAM9, ATF6, C21ORF2, C8ORF37, CACNA2D4, CDHR1, CERKL, CNGA3, CNGB3, CNNM4, CNAT2, IFT81, KCNV2, PDE6C, PDE6H, POC1B, RAX2, RDH5, RPGRIP1, TTLL5, RetCGI, GUCY2E

[0630] Congenital Stationary Night Blindness CABP4, CACNA1 F, CACNA2D4, GNAT1,

[0631] CPR179, GRK1, GRM6, LRIT3, NYX, PDE6B, RDH5, RHO, RLBP1, RPE65, SAG, SLC24A1, TRPM1

[0632] Congenital Fructose Intolerance ALDOB

[0633]

[0634] ATTORNEY DOCKET NO.: 51772-007WO2

[0635] PATENT

[0636] Diseases Genes

[0637] Cori's Disease (Glycogen Storage Disease Type AGL

[0638] HI)

[0639] Corneal Clouding and Dystrophy APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3,

[0640] CDG2, TACSTD2, TROP2, M1 S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD

[0641] Cornea Plana Congenital KERA, CNA2

[0642] Cri Du Chat Syndrome, also Deletions involving only band 5p15.2 to the entire known as 5p Syndrome and Cat Cry Syndrome short arm of chromosome 5, e.g., CTNND2, TERT Crigler-Najjar syndrome UGT1A1

[0643] Cystic Fibrosis (CF) CTFR, ABCC7, CF, MRP7, SCNN1A, those described in WG2015157070

[0644] Diabetic Nephropathy Gremlin, 12 / 15- lipoxygenase, TIM44

[0645] Dent Disease (Types 1 and 2) Type 1: CLCN5, Type 2: ORCL Dentatorubral-Pallidoluysian Atrophy (DRPLA) Atrophin- 1 and ATN1

[0646] (aka Haw River and Naito-Oyanagi

[0647] Disease)

[0648] Down Syndrome Chromosome 21 trisomy

[0649] Drug Addiction PRKCE, DRD2, DRD4, ABAT, GRIA2, GRM5, GRIN1, HTR1b, GRIN2A, DRD3, PDYN, GRIA1 Duane Syndrome (Types 1, 2, and 3, including CHN1, indels on chromosomes 4 and 8 subgroups A, B, and C). Other names for this

[0650] condition include Duane's Retraction Syndrome

[0651] (or DR Syndrome), Eye Retraction Syndrome,

[0652] Retraction Syndrome, Congenital retraction

[0653] Syndrome, and Stilling-Turk-Duane Syndrome

[0654] Duchenne Muscular Dystrophy (DMD) DMD, BMD, dystrophin gene, intron flanking exon 51 of DMD gene, exon 51 mutations in DMD gene, see also WG2013163628 and US Pat. Pub.

[0655] 20130145487

[0656] Eating Disorders BDNF

[0657] Edward's Syndrome (Trisomy 18) Complete or partial trisomy of chromosome 18 Ehlers-Danlos Syndrome (Types l-VI) COL5A1, COL5A2, COL1 A1, COL3A1, TNXB, PLOD1, COL1A2, FKBP14, ADAMTS2 Emery-Dreifuss Muscular Dystrophy LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A Enhanced S-Cone Syndrome NR2E3, NRL

[0658] Epilepsy NPY

[0659] Fabry's Disease GLA, AGA

[0660]

[0661] ATTORNEY DOCKET NO.: 51772-007WO2

[0662] PATENT

[0663] Diseases Genes

[0664] Facioscapulohumeral Muscular Dystrophy FSHMD1A, FSHD1A, FRG1

[0665] Factor H and Factor H-like 1 HF1, CFH, HUS

[0666] Factor V Leiden Thrombophilia and Factor V Factor V (F5)

[0667] Deficiency

[0668] Factor V and Factor VII Deficiency MCFD2

[0669] Factor VII Deficiency F7

[0670] Factor X Deficiency F10

[0671] Factor XI Deficiency F11

[0672] Factor XII Deficiency F12, HAF

[0673] Factor XI HA Deficiency F13A1, F13A

[0674] Factor XIIIB Deficiency F13B

[0675] Familial Hypercholesterolemia APOB, LDLR, PCSK9

[0676] Familial Mediterranean Fever (FMF) also called MEFV

[0677] Recurrent Polyserositis or Familial Paroxysmal

[0678] Polyserositis

[0679] Fanconi Anemia FANCA, FACA, FA1, FA, FAA, FAAP95,

[0680] FAAP90, FLJ34064, FANCC, FANCG, RAD51, BRCA1, BRCA2, BRIP1, BACH1, FANCJ, FANCB, FANCD1, FANCD2, FANCD, FAD, FANCE, FACE, FANCF, FANCI, ERCC4, FANCL, FANCM, PALB2, RAD51C, SLX4, UBE2T, FANCB, XRCC9, PHF9, KIAA1596 Fanconi Syndrome Types I (Childhood onset) and FRTS1, GATM

[0681] II (Adult Onset)

[0682] Fragile X Syndrome and related disorders FMR1, FMR2, FXR1, FXR2, mGLUR5 Fragile XE Mental Retardation (aka Martin Bell FMR1

[0683] Syndrome)

[0684] Friedreich Ataxia (FRDA) FXN / X25

[0685] Fuchs Endothelial Corneal Dystrophy TCF4, COL8A2

[0686] Galactosemia GALT, GALK1, GALE

[0687] Gastrointestinal Epithelial CISH

[0688] Cancer, Gl cancer

[0689] Gaucher Disease (Types 1, 2, and 3, as well as GBA, GCase

[0690] other unusual forms that may not fit into these

[0691] types)

[0692] Glaucoma MYOC, TIGR, GLC1 A, JOAG, GPOA, OPTN,

[0693] GLC1E, FIP2, HYPL, NRP, CYP1 B1, GLC3A, OPA1, NTG, NPG, CYP1 B1, GLC3A, those described in WO2015153780

[0694]

[0695] ATTORNEY DOCKET NO.: 51772-007WO2

[0696] PATENT

[0697] Diseases Genes

[0698] Glomerulosclerosis CC chemokine ligand 2

[0699] Glycogen Storage Diseases (Types l-VI - See SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, also Cori's Disease, Pompe Disease, McArdle's LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, Disease, Hers Disease, and Von Gierke's PYGL, PFKM, see also Cori's Disease, Pompe's disease) Disease, McArdle's disease, Hers Disease, and Von Gierke' s disease

[0700] RBC Glycolytic Enzyme Deficiency any mutations in a gene for an enzyme in the glycolysis pathway including mutations in genes for hexokinases I and II, glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase Bm triosephosphate isomerase, glyceraldehyde-3- phosphate dehydrogenase, phosphoglycerokinase, phosphoglycerate mutase, enolase I, pyruvate kinase

[0701] GM2 Gangliosidoses (e.g., Sandhoff Disease and Hexosaminidase-alpha, hexosaminidase-beta, Tay-Sachs Disease) HEX-A

[0702] Glycogen Storage Disease Type la (GSDIa) G6Pase

[0703] Haemophilia A F8

[0704] Hartnup Disease SLC6A19

[0705] Hearing Loss NOX3, Hes5, BDNF

[0706] Hemochromatosis (HH) HFE, H63D

[0707] Hemophagocytic Lymphohistiocytosis Disorders PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,

[0708] HLH3, FHL3

[0709] Hemorrhagic Disorders PI, ATT, F5

[0710] Hers Disease (Glycogen Storage Disease Type PYGL

[0711] VI)

[0712] Hereditary Angioedema (HAE) kalikrein B1

[0713] Hereditary Hemorrhagic ACVRL1, ENG, SMAD4

[0714] Telangiectasia (Osler-Weber- Rendu Syndrome)

[0715] Hereditary Spherocytosis NK1, EPB42, SLC4A1, SPTA1, SPTB Hereditary Persistence of Fetal Hemoglobin HBG1, HBG2, BCL11 A, promoter region of HBG 1 and / or 2 (in the CCAAT box) Hemophilia (Hemophilia A A: FVIII, F8C, HEMA

[0716] (Classic), B (aka Christmas disease), and C) B: FVIX, HEME, FIX

[0717] C: F9, F11

[0718] Hepatic Adenoma TCF1, HNF1A, MODY3

[0719] Hepatic Failure, early onset, and Neurologic SCOD1, SCO1

[0720] Disorder

[0721]

[0722] ATTORNEY DOCKET NO.: 51772-007WO2

[0723] PATENT

[0724] Diseases Genes

[0725] Hepatic Lipase Deficiency LIPC

[0726] Hepatoblastoma, Cancer, and Carcinomas CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1,

[0727] AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPR1, MET, CASP8, MCH5

[0728] Hermansky-Pudlak Syndrome HPS1, HPS3, HPS4, HPS5, HPS6, HPS7, DTNBP1, BLOC1, BLOC1 S2, BLOC3 Human immunodeficiency viruses (HIV) IL10, CSIF, CMKBR2, CCR2, CMKBR5, susceptibility or infection CCCKR5 (CCR5), those in WG2015148670A1 Holoprosencephaly (HPE) (Alobar, Semilobar, ACVRL1, ENG, SMAD4

[0729] and Lobar)

[0730] Homocystinuria CBS, MTHFR, MTR, MTRR, MMADHC Human papillomavirus (HPV) HPV16, HPV18 E6 / E7

[0731] Herpes simplex virus (HSV)I, HSV2, and related HSV1 genes (immediate early and late HSV-1 Keratitis genes (UL1, 1.5, 5, 6, 8, 9, 12, 15, 16, 18, 19,

[0732] 22, 23, 26, 26.5, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 42, 48, 49.5, 50, 52, 54, S6, RL2, RS1, those described in WG2015153789, WG2015153791

[0733] Hunter's Syndrome (aka Mucopolysaccharidosis IDS

[0734] Type II)

[0735] Huntington's Disease (HD) and HD-like Disorders HD, HTT, IT15, PRNP, PRIP, JPH3, JP3, HDL2,

[0736] TBP, SCA17, PRKCE, BDNF, IGF1, EP300, RCOR1, PRKCZ, HDAC4, TGM2, those described in WG2013130824, WG2015089354 Hurler's Syndrome (aka Mucopolysaccharidosis IDUA, a-L-iduronidase

[0737] (MPS) Type IH, MPS IH)

[0738] Hurler-Scheie Syndrome (aka IDUA, a-L-iduronidase Mucopolysaccharidosis Type IH-S, MPS IH-S)

[0739] Hyaluronidase Deficiency (aka HYAL1

[0740] MPS IX)

[0741] Hypercholesterolaemia or Phenylketonuria (PKU) PAH, LDLR

[0742] Hyper IgM Syndrome CD40L

[0743] Hypertension caused Renal Damage Mineral corticoid receptor Immunodeficiencies CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5,

[0744] CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, All D, XPID, PIDX, TNFRSF14B, TACI

[0745] Inborn errors of metabolism: including urea cycle See also: Carbohydrate Metabolism Disorders disorders, organic acidemias, fatty acid oxidation (e.g., Galactosemia), Amino Acid Metabolism

[0746]

[0747] ATTORNEY DOCKET NO.: 51772-007WO2

[0748] PATENT

[0749] Diseases Genes

[0750] defects, amino acidopathies, carbohydrate Disorders (e.g., Phenylketonuria), Fatty Acid disorders, mitochondrial disorders Metabolism (e.g., MCAD deficiency), Urea Cycle Disorders (e.g., Citrullinemia), Organic Acidemias (e.g., Maple Syrup Urine Disease), Mitochondrial Disorders (e.g., MELAS), Peroxisomal Disorders (e.g., Zellweger syndrome)

[0751] Inflammation IL-10, IL-1 (IL-1 a, IL-1 b), IL-13, IL- 17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3c11 Inflammatory Bowel Diseases (e.g., Ulcerative NOD2, IRGM, LRRK2, ATG5, ATG16L1, IRGM, Colitis and Cronh's Disease) GATM, ECM1, CDH1, LAMB1, HNF4A, GNA12,

[0752] IL10, CARD9 / 15, CCR6, IL2RA, MST1, TNFSF15, REL, STAT3, IL23R, IL12B, FUT2 Interstitial Renal Fibrosis TGF-p type II receptor

[0753] Job's Syndrome (aka Hyper IgE Svndrome) STAT3, DOCK8

[0754] Juvenile Retinoschisis RS1, XLRS1

[0755] Kabuki Syndrome 1 MLL4, KMT2D

[0756] Kennedy Disease (aka SBMA / SMAX1 / AR

[0757] Spinobulbar Muscular Atrophy)

[0758] Klinefelter Syndrome Extra X chromosome in males

[0759] Lafora Disease EMP2A, EMP2B

[0760] Leber Congenital Amaurosis CRB1, RP12, CORD2, CRD, CRX, IMPDH1, OTX2, AIPL1, CABP4, CCT2, CEP290, CLUAP1, CRB1, CRX, DTHD1, GDF6, GUCY2D, IFT140, IQCB1, KCNJ13, LCA5, LRAT, NMNAT1, PRPH2, RD3, RDH12, RPE65, RP20, RPGRIP1, SPATA7, TULP1, LCA1, LCA4, GUC2D, CORD6, LCA3

[0761] Leber Hereditary Optic ND4

[0762] Lesch-Nyhan Syndrome HPRT1

[0763] Leukocyte deficiencies and disorders ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA,

[0764] EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4

[0765] Leukemia TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM,

[0766]

[0767] ATTORNEY DOCKET NO.: 51772-007WO2

[0768] PATENT

[0769] Diseases Genes

[0770] CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, MAY, CAIN, RUNX1, CBFA2, AML1, WHSC1 L1, NSD3, FLT3, AF1 Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, MAY, CAIN

[0771] Limb-Girdle Muscular Dystrophies LGMD

[0772] Lipoprotein lipase (LPL) deficiency LPL

[0773] Lowe Syndrome OCRL

[0774] Lupus Glomerulonephritis MAPK1

[0775] Lysosomal storage disorders GDNF

[0776] Machado-Joseph's Disease (also known as ATX3

[0777] Spinocerebellar Ataxia Type 3)

[0778] Macular Degeneration ABC4, CBC1, CHM1, APOE, C1QTNF5, C2, C3,

[0779] CCL2, CCR2, CD36, CFB, CFH, CFHR1, CFHR3, CNGB3, CP, CRP, CST3, CTSD, CX3CR1, ELOVL4, ERCC6, FBLN5, FBLN6, FSCN2, HMCN1, HTRA1, IL6, IL8, PLEKHA1, PROM1, PRPH2, RPGR, SERPING1, TCOF1, TIMP3, TLR3

[0780] Macular Dystrophy BEST1, C1QTNF5, CTNNA1, EFEMP1,

[0781] ELOVL4, FSCN2, GUCA1 B, HMCN1, IMPG1, OTX2, PRDM13, PROM1, PRPH2, RP1L1, TIMP3, ABCA4, CFH, DRAM2, IMG1, MFSD8, ADMD, STGD2, STGD3, RDS, RP7, PRPH, AVMD, AOFMD, VMD2

[0782] Malattia Leventinese EFEMP1, FBLN3

[0783] Malignant Melanoma CD86, B7-2, IL-12

[0784] Maple Syrup Urine Disease BCKDHA, BCKDHB, DBT

[0785] Marfan Syndrome FBN1

[0786] Maroteaux-Lamy Syndrome (aka MPS VI) ARSB

[0787] medium-chain acyl-CoA dehydrogenase (MCAD) MCAD

[0788] deficiency

[0789] McArdle's Disease (Glycogen Storage Disease PYGM

[0790]

[0791] ATTORNEY DOCKET NO.: 51772-007WO2

[0792] PATENT

[0793] Diseases Genes

[0794] Type V, myophosphorylase deficiency)

[0795] Medullary Cystic Kidney Disease UMOD, HNFJ, FJHN, MCKD2, ADMCKD2 Metachromatic Leukodystrophy ARSA

[0796] Methylmalonic Acidemia (MMA) MMAA, MMAB, MUT, MMACHC, MMADHC, LMBRD1

[0797] Mitochondrial Heteroplasmy, Myoclonic Epilepsy APALI

[0798] with ragged red fibers (MERRF), or Mitochondrial

[0799] Encephalomyopathy, Lactic Acidosis, and Stroke- Like Episodes (ME LAS)

[0800] Morquio Syndrome (aka MPS IVA and B) GALNS

[0801] Mucopolysaccharidosis Type VII (MPSVII) or Sly GUSB

[0802] Syndrome

[0803] Mucopolysaccharidosis diseases (Types I H / S, I See also Hurler / Scheie Syndrome, Hurler H, II, III A B and C, I S, IVA and B, IX, VII, and VI) Disease, Sanfilippo Syndrome, Scheie Syndrome, Morquio Syndrome, Hyaluronidase Deficiency, Sly Syndrome, and Maroteaux-Lamy Syndrome

[0804] Muscular Atrophy VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1

[0805] Muscular Dystrophy Sarcoglycan a, p, y, A, e, or L, (SGCA, SGCB, SGCG, SGCD, SGCE, or SGCZ), FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, MAYP3, DYSF, LGMD2B, LGMD2C, DMDA1, SCG3, ADL, DAG2, LGMD2D, DMDA2, LGMD2E, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, dystrophin, mini-dystrophin

[0806] Myotonic Dystrophy (Type 1 and Type 2) CNBP (Type 2), DMPK (Type 1)

[0807] Neoplasia PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, NOTCH1, NOTCH2, NOTCH3, NOTCH4, AKT, AKT2, AKT3, HIF, HIF1A, HIF3A, MET, HRG, BCL2, PPAR alpha, PPAR gamma, WT1 (Wilms Tumor), FGF Receptor

[0808]

[0809] ATTORNEY DOCKET NO.: 51772-007WO2

[0810] PATENT

[0811] Diseases Genes

[0812] Family members (5 members: 1, 2, 3, 4, 5), CDKN2A, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (Androgen Receptor), TSG101, IGF, IGF Receptor IGF1 (4 variants), IGF2 (3 variants), IGF1 Receptor, IGF 2 Receptor, BAX, BCL2, caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), KRAS, APC Neurofibromatosis (NF) (NF1, formerly NF1, NF2

[0813] Recklinghausen's NF, and NF2)

[0814] Niemann-Pick Lipidosis (Types A, B, and C) Types A and B: SMPD1, Type C: NPC1 or NPC2 Noonan Syndrome PTPN11, SOS1, RAF1, KRAS

[0815] Norrie Disease or X-linked Familial Exudative NDP

[0816] Vitreoretinopathv

[0817] North Carolina Macular Dystrophy MCDR1

[0818] Ornithine Carbamoyltransferase Deficiency OTC

[0819] Osteogenesis Imperfecta (Ol) (Types I, II, III, IV, COL1A1, COL1A2, CRTAP, P3H

[0820] V, VI, and VII)

[0821] Osteopetrosis LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1

[0822] Patau's Syndrome (Trisomv 13) Additional copy of chromosome 13 Parkinson's Disease (PD) SNCA (PARK1), UCHL1 (PARK 5), and LRRK2

[0823] (PARK8), PARK3, PARK2, PARK4, PARK7, PINKI (PARK6), x-Synuclein, DJ-1, Parkin, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, NCAP, PRKN, PDJ, DBH, NDUFV2, BDNF, GDNF, GBA, AADC, GAD1, GAD2, NRTN, NPY

[0824] Pattern Dystrophy of the retinal pigment RDS / peripherin

[0825] epithelium (RPE)

[0826] Phenylketonuria (PKU) PAH, PKU1, QDPR, DHPR, PTS

[0827] Polycystic Kidney and Hepatic Disease FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4,

[0828] PKDTS, PRKCSH, G19P1, PCLD, SEC63 Pompe Disease GAA

[0829] Porphyria (actually refers to a group of different ALAD, ALAS2, CPOX, FECH,

[0830] diseases all having a specific heme production HMBS, PPOX, UROD, UROS

[0831] process abnormality)

[0832] Posterior Polymorphous Corneal Dystrophy TCF4, COL8A2

[0833] Prader-Willi Syndrome Deletion of region of short arm of

[0834]

[0835] ATTORNEY DOCKET NO.: 51772-007WO2

[0836] PATENT

[0837] Diseases Genes

[0838] chromosome 15, including UBE3A

[0839] Primary Hyperoxaluria (e.g., Type 1) LDHA (lactate dehydrogenase A) and hydroxyacid oxidase 1 (HAO1)

[0840] Primary Open Angle Glaucoma (POAG) MYOC

[0841] Primary Sclerosing Cholangitis TCF4, COL8A2

[0842] Progeria (also called Hutchinson-Gilford Progeria LMNA

[0843] Syndrome)

[0844] Progressive External Ophthalmoplegia ANT-I

[0845] Propionic Acidaemias PCCA

[0846] Prostate Cancer HOXB13, MSMB, GPRC6A, TP53, GM-CSF Pyruvate Dehydrogenase Deficiency PDHA1

[0847] Kidney / Renal Carcinoma RLIP76, VEGF

[0848] Rett Syndrome MECP2, RTT, PPMX, MRX16, MRX79, CDKL5,

[0849] STK9, MECP2, RTT, PPMX, MRX16, MRX79, x- Synuclein, DJ-1

[0850] Retinitis Pigmentosa (RP) ADIPOR1, ABCA4, AGBL5, ARHGEF18,

[0851] ARL2BP, ARL3, ARL6, BEST1, BBS1, BBS2, C2ORF71, C8ORF37, CA4, CERKL, CLRN1, CNGA1, CMGB1, CRB1, CRX, CYP4V2, DHDDS, DHX38, EMC1, EYS, FAM161A, FSCN2, GPR125, GUCA1B, HK1, HPRPF3, HGSNAT, IDH3B, IMPDH1, IMPG2, IFT140, IFTI72, KLHL7, KIAA1549, KIZ, LRAT, MAK, MERTK, MVK, NEK2, NUROD1, NR2E3, NRL, OFD1, PDE6A, PDE6B, PDE6G, POMGNT1, PRCD, PROM1, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, PRPH2, RPB3, RDH12, REEP6, RP39, RGR, RHO, RLBP1, ROM1, RP1, RP1L1, RPY, RP2, RP9, RPE65, RPGR, SAMD11, SAG, SEMA4A, SLC7A14, SNRNP200, SPP2, SPATA7, TRNT1, TOPORS, TTC8, TULP1, USH2A, ZNF408, ZNF513, see also 20120204282

[0852] Sanfilippo Syndrome or Mucopolysaccharidosis NAGLU

[0853] Type III B (MPS IIIB)

[0854] short-chain acyl-CoA dehydrogenase (SCAD) SCAD

[0855] deficiency

[0856] Scheie Syndrome (also known as IDUA, a-L-iduronidase Mucopolysaccharidosis Type I S (MPS l-S))

[0857]

[0858] ATTORNEY DOCKET NO.: 51772-007WO2

[0859] PATENT

[0860] Diseases Genes

[0861] Schizophrenia Neuregulinl (NRG1 ), ERB4 (receptor for Neuregulin), Complexinl (CPLX1), TPH1 Tryptophan hydroxylase, TPH2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBP1, Dao (Dao1), TCF4, COL8A2

[0862] Secretase Related Disorders APH-1 (alpha and beta), PSEN1,

[0863] NCSTN, PEN-2, Nos1, Parpl, Natl, Nat2, CTSB, APP, APH1 B, PSEN2, PSENEN, BACE1, ITM2B, CTSD, NOTCH1, TNF, INS, DYT10, ADAM17, APOE, ACE, STN, TP53, IL6, NGFR, IL1 B, ACHE, CTNNB1, IGF1, IFNG, NRG1, CASP3, MAPK1, CDH1, APBB1, HMGCR, CREB1, PTGS2, HES1, CAT, TGFB1, ENO2, ERBB4, TRAPPC10, MAOB, NGF, MMP12, JAG1, CD40LG, PPARG, FGF2, LRP1, NOTCH4, MAPK8, PREP, NOTCH3, PRNP, CTSG, EGF, REN, CD44, SELP, GHR, ADCYAP1, INSR, GFAP, MMP3, MAPK10, SP1, MYC, CTSE, PPARA, JUN, TIMP1, IL5, IL1 A, MMP9, HTR4, HSPG2, KRAS, CYCS, SMG1, IL1 R1, PROK1, MAPK3, NTRK1, IL13, MME, TKT, CXCR2, CHRM1, ATXN1, PAWR, NOTCJ2, M6PR, CYP46A1, CSNK1 D, MAPK14, PRG2, PRKCA, L1 CAM, CD40, NR1I2, JAG2, CTNND1, CMA1, SORT1, DLK1, THEM4, JUP, CD46, CCL11, CAV3, RNASE3, HSPA8, CASP9, CYP3A4, CCR3, TFAP2A, SCP2, CDK4, JOF1A, TCF7L2, B3GALTL, MDM2, RELA, CASP7, IDE, FANP4, CASK, ADCYAP1 R1, ATF4, PDGFA, C21 ORF33, SCG5, RMF123, NKFB1, ERBB2, CAV1, MMP7, TGFA, RXRA, STX1A, PSMC4, P2RY2, TNFRSF21, DLG1, NUMBL, SPN, PLSCR1, UBQLN2, UBQLN1, PCSK7, SPON1, SILV, QPCT, HESS, GCC1

[0864] Selective IgA Deficiency Type 1: MSH5, Type 2: TNFRSF13B Severe Combined Immunodeficiency (SCID), JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, SCID-XI, and Adenosine deaminase (ADA)-SCID RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R,

[0865]

[0866] ATTORNEY DOCKET NO.: 51772-007WO2

[0867] PATENT

[0868] Diseases Genes

[0869] CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4, those identified in US Pat. App. Pub.

[0870] 20110225664, 20110091441, 20100229252, 20090271881 and 20090222937

[0871] Sickle Cell Disease HBB, BCL11A, BCL11Ae, cis- regulatory elements of the B-globin locus, HBG 1 / 2 promoter, HBG distal CCAAT box region between -92 and -130 of the HBG Transcription Start Site, those described in WG2015148863, WO 2013 / 126794,

[0872] US Pat. Pub. 20110182867 Spinocerebellar Ataxias (SCA Types 1, 2, 3, 6, 7, ATXN1, ATXN2, ATX3

[0873] 8, 12, and 17)

[0874] Sorsby Fundus Dystrophy TIMP3

[0875] Stargardt Disease ABCR, ELOVL4, ABCA4, PROM1 Thalassemia (Alpha, Beta, Delta) HBA1, HBA2 (Alpha), HBB (Beta), HBB and HBD (delta), LCRB, BCL11 A, BCL11 Ae, cis- regulatory elements of the B-globin locus, HBG 1 / 2 promoter, those described in

[0876] WG2015148860, US Pat. Pub.

[0877] 20110182867, 2015 / 148860

[0878] Thymic Aplasia (DiGeorge Syndrome; 22q 11.2 Deletion of 30 to 40 genes in the middle of Deletion Syndrome) chromosome 22 at a location known as 22q11.2, including TBX1, DGCR8 Transthyretin Amyloidosis (ATTR) TTR (transthyretin)

[0879] Trimethylaminuria FMO3

[0880] Trinucleotide Repeat Disorders (generally) HTT, SBMA / SMAX1 / AR, FXN / X25 ATX3, ATXN1, ATXN2, DMPK, Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP - global instability), VLDLR, Atxn7, Atxnl 0, FEN1, TNRC6A, PABPN1, JPH3, MED15, ATXN3, TBP, CACNA1A, ATXN80S, PPP2R2B, ATXN7, TNRC6B, TNRC6C, CELF3, MAB21 L1, MSH2, TMEM185A, SIX5, CNPY3, RAXE, GNB2, RPL14, ATXN8, ISR, TTR, EP400, GIGYF2, OGG1, STC1, CNDP1, C1 OORF2, MAML3, DKC1, PAXIP1, CASK, MAPT, SP1, POLG, AFF2, THBS1, TP53, ESR1, CGGBP1, ABT1, KLK3, PRNP, JUN, KCNN3, BAX, FRAXA,

[0881]

[0882] ATTORNEY DOCKET NO.: 51772-007WO2

[0883] PATENT

[0884] Diseases Genes

[0885] KBTBD10, MBNL1, RAD51, NCOA3, ERDA1, TSC1, COMP, GGLC, RRAD, MSH3, DRD2, CD44, CTCF, CCND1, CLSPN, MEF2A, PTPRU, GAPDH, TRIM22, WT1, AHR, GPX1, TPMT, NDP, ARX, TYR, EGR1, UNG, NUMBL, FABP2, EN2, CRYGC, SRP14, CRYGB, PDCD1, HOXA1, ATXN2L, PMS2, GLA, CBL, FTH1, IL12RB2, OTX2, HOXA5, POLG2, DLX2, AHRR, MANF, RMEM158, see also 20110016540

[0886] Triple X (XXX) Syndrome X chromosome trisomy

[0887] Turner's Syndrome (XO) Monosomy X

[0888] Tuberous Sclerosis TSC1, TSC2

[0889] Usher Syndrome (Types I, II, and III) ABHD12, CDH23, CIB2, CLRN1, DFNB31, GPR98, HARS, MYO7A, PCDH15, USH1C, USH1G, USH2A, USHI11A, those described in WO2015134812A1

[0890] Velocardiofacial Syndrome (aka 22q11.2 Deletion Many genes are deleted, COM, TBX1, and other Syndrome, DiGeorge Syndrome, Conotruncal are associated with symptoms

[0891] Anomaly Face Syndrome (CTAF), Autosomal

[0892] Dominant Opitz G / BB Syndrome or Cayler CardioFacial Syndrome)

[0893] very long-chain acyl-CoA dehydrogenase VLCAD

[0894] (VLCAD) deficiency

[0895] Von Gierke's Disease (Glycogen Storage Disease G6PC, SLC37A4

[0896] Type I)

[0897] Von Hippel-Lindau Syndrome VHL

[0898] Von Willebrand Disease (Types I, II, and III) VWF

[0899] Wilson Disease ATP7B

[0900] Wiskott-Aldrich Syndrome WAS

[0901] Xeroderma Pigmentosum POLH

[0902] X-linked myotubular myopathy MTM1

[0903]

[0904] In some embodiments, a subject treated by administering an AAV particle or pharmaceutical composition described herein is a mammal such as a mouse, a rat, or a primate (e.g., monkey, chimpanzee, or human). In some embodiments, the subject is a primate. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human.

[0905] so ATTORNEY DOCKET NO.: 51772-007WO2

[0906] PATENT

[0907] Pharmaceutical Compositions

[0908] Pharmaceutical compositions of the present disclosure may include a vehicle for administration into a patient, such as a human patient suffering from a disorder or disease, as described herein. The pharmaceutical composition described herein may be prepared using methods known in the art. For example, pharmaceutical compositions may be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

[0909] Pharmaceutical compositions may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0910] For example, a pharmaceutical composition of the present disclosure may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for administration by a route selected from intramuscular, intrahepatic, intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intraocular (e.g., intravitreal), intraventricular, intraiumbar, intrahippocampal, intrastriatal (putamen and / or caudate), intracerebral, intracerebrospinal, intracranial, intracortical, intradermal, transdermal, parenteral, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intraputaminal, intramidbrain, intra cisterna magna, intra substantia nigra, intra ventral tegmental area, intrathalamic, intravascular, inhalation, perfusion, lavage, and / or oral administration. In this connection, sterile aqueous media that may be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for ATTORNEY DOCKET NO.: 51772-007WO2

[0911] PATENT

[0912] human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.

[0913] A pharmaceutical composition containing, for example, an AAV viral particle described herein, typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and a nucleic acid. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and a nucleic acid. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and a nucleic acid. The sterile PBS is typically a pharmaceutical grade PBS.

[0914] In certain embodiments, pharmaceutical compositions include one or more AAV viral particles described herein and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In certain embodiments, AAV viral particles described herein may be mixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0915] In certain embodiments, pharmaceutical compositions including an AAV viral particle described herein encompass any pharmaceutically acceptable salts or esters. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of inhibitors. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0916] In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used. Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the AAV capsid proteins of the invention may be introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

[0917] In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

[0918] In certain embodiments, pharmaceutical compositions include a co-solvent system. Certain of such co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent ATTORNEY DOCKET NO.: 51772-007WO2

[0919] PATENT

[0920] system, which is a solution of absolute ethanol including 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant Polysorbate 80™ and 65% w / v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0921] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intramuscular, intraocular (e.g., intravitreal), intravenous, subcutaneous, intrathalamic, intrathecal, intracerebroventricular, etc.). In certain of such embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0922] In some embodiments, the AAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high AAV concentrations are present. Methods for reducing aggregation of AAVs are well known in the art and include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.). Formulation of pharmaceutically acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active ingredient or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active ingredient in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0923] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ATTORNEY DOCKET NO.: 51772-007WO2

[0924] PATENT

[0925] ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by using a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by using surfactants. For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, a sterile aqueous medium that may be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0926] Sterile injectable solutions are prepared by incorporating the active AAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fi Itered solution thereof. The agents, compositions, and / or systems described herein may, in some embodiments, be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the components of the invention and instructions for use. Kits for research purposes may contain the components in appropriate concentrations or quantities for running various experiments.

[0927] Routes of Administration

[0928] The AAV viral particle or pharmaceutical composition of the present disclosure, containing a transgene described herein may be administered to a patient (e.g., a human patient) by a variety of routes of administration. Administration of an effective dose of the AAV particle or pharmaceutical composition may be by exemplary routes of administration standard in the art, including, but not limited to, systemic (e.g., by intravenous), local (e.g., intracortical), and direct injection (e.g., delivery to a particular muscle or other tissue). The route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., intramuscular, intrahepatic, intravenous, intrathecal, intracerebroventricular, intracisternal, intrastriatal (putamen and / or caudate), intracerebral, intracerebrospinal, intracranial, intracortical, intraparenchymal, intradermal, transdermal, parenteral, intranasal, subcutaneous, percutaneous, intratracheal, intraocular (e.g., intravitreal), intraventricular, intralumbar, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and / or oral ATTORNEY DOCKET NO.: 51772-007WO2

[0929] PATENT

[0930] administration. Intravascular administration may include delivery into the vasculature of a patient. In some embodiments, the administration is into a vessel considered to be a vein (intravenous), and in some administration, the administration is into a vessel considered to be an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein.

[0931] Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and / or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary. Administration may be performed by intrathecal injection with or without Trendelenberg tilting. In some cases, the AAV viral particle or pharmaceutical composition may be administered, e.g., in a single administration.

[0932] Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient. Treatment may include administration of viral particles or other agents described herein as useful for the introduction of a transgene into a target cell in various unit doses. Each unit dose will ordinarily contain a predetermined quantity of the therapeutic composition.

[0933] Kits

[0934] The compositions described herein, such as an AAV viral particle or pharmaceutical composition of the present disclosure may be provided in a kit. In some embodiments, the kit may include an AAV viral particle as described herein. In some embodiments, the kit may include a pharmaceutical composition as described herein. The kit may include a package insert that instructs a user of the kit, such as a physician of skill in the art, to perform any one of the methods of treatment described herein. The kit may optionally include a syringe or other device for administering the AAV viral particle or pharmaceutical composition of the present disclosure. In some embodiments, the kit may include one or more additional therapeutic agents.

[0935] EXAMPLES

[0936] Example 1. Discovery of the peptide family of the invention

[0937] Objective

[0938] This Example describes the results of a series of experiments undertaken to discover and characterize a family of 7-mer peptide inserts that may be incorporated into an AAV capsid protein (such as AAV9, among others) in order to enhance transduction, particularly of muscle cells.

[0939] Materials and Methods

[0940] Peptide insert AAV library

[0941] A large, randomized library of 7-mer peptide inserts was constructed for incorporation into the AAV9 VP1 capsid protein. Briefly, a library oligo was generated by solid phase synthesis. The method avoided the introduction of Cys and stop codons. The redundancy at the nucleic acid level (due to synonymous codons) that is often introduced using degenerate base approaches was also avoided with ATTORNEY DOCKET NO.: 51772-007WO2

[0942] PATENT

[0943] our approach. Standard techniques were used to preserve the diversity of the library through oversampling at all stages of library construction, propagation, and vector production.

[0944] The library oligo between the codons for amino acids 588 and 589 (corresponding to AAV9 VP1 numbering) was cloned in the AAV9 Cap gene, which was situated between inverted terminal repeats (ITRs) in the library plasmid. The library of Cap variants was driven by a CMV promoter and contained a fragment of Rep upstream of the Cap ORF to ensure proper Cap VP ratios.

[0945] After sequence-verifying the correct assembly of the Cap9 variant library, the library plasmid was transfected along with an adenovirus helper plasmid and a plasmid containing a full-length copy of Rep2. The plasmid library was then converted to a vector library in which each AAV9 peptide insertion capsid variant packaged its own genome. This linkage between genotype and phenotype was essential for capsid directed evolution since it allowed for the tracking and propagation of the most successful variants through rounds of selection.

[0946] Primary capsid library screen

[0947] The unbiased AAV9 peptide insert capsid library was subjected to several rounds of selection to identify the ability to infect muscle cells. Next generation sequencing (NGS)-based scoring was performed on the screen output, and clustering of the resulting peptide sequences revealed distinct peptide insert families. Next, enrichment criteria was chosen to define hit peptides from the screen. Clustering of the hit peptide sequences revealed distinct peptide families.

[0948] Results

[0949] Subjecting the top 506 hit peptides from the primary screen to clustering analysis led to the discovery of the peptide family of the invention (Figure 1). This family of peptides generally is at least seven residues in length has two cationic residues (i.e., Lys or Arg), which are distributed between positions 2 and 5 of the polypeptide sequence. The cationic residues may be adjacent to one another or separated by one or more amino acids. Peptide inserts of the invention generally terminate in a glycine residue at the C-terminus. Of the 506 unique peptides, those with sequences of the peptide family of the invention are shown in Table 5 along with their NGS-based enrichment score from the primary screen.

[0950] Table 5. Round 2 enrichment score

[0951] Insert sequence NGS score from primary screen of unbiased library SEQ ID NO.

[0952] TKSRPAG 27.72371339 17 SIRKPAG 25.99814541 18 SIRQKVG 25.72273699 19 SRNKPIG 23.71085807 20 TFKKPAG 19.01075256 21 LRNKPAG 18.13644886 22 TLRKPAG 15.43068536 23 SKTRPAG 13.19305625 24 TRIKPVG 12.67126338 25

[0953]

[0954] ATTORNEY DOCKET NO.: 51772-007WO2

[0955] PATENT

[0956] Insert sequence NGS score from primary screen of unbiased library SEQ ID NO.

[0957] MIKKPVG 12.47119729 26 NRIKPAG 12.28933774 27 TIKRPVG 10.08252844 28 TITKKPG 10.00945584 29 SIRRPAG 9.744206432 30

[0958]

[0959] Subsequent to the initial clustering, the criteria for hit peptides were relaxed, expanding the pool to >4000 sequences. Searching this expanded group of sequences for matches to the peptide family of the invention revealed additional examples, shown in Table 6.

[0960] Table 6. Round 2 enrichment score

[0961] Insert sequence NGS score from primary screen of unbiased library SEQ ID NO.

[0962] TKNKPVG 6.6334277 31 MKTKPVG 2.153520665 32 TKAKPAG 3.652397624 33 TRAKPVG 4.716596396 34 MMKKPVG 5.523070079 35 TKIKPSG 5.710957606 36 GKTKPAG 7.27319404 37 SKMRPVG 6.173166121 38 TKIRPMG 2.692756096 39 SRIKPVP 2.49523074 40 TKIRPQG 2.231789777 41 TRTKVAG 4.506257934 42 SKIRPGG 5.133324955 43 SKTKPQG 4.273996741 44 TLRKIAG 4.652664925 45 SKSKPIG 4.719632733 46 TRTKVVG 5.364403679 47 TIIKKIG 1.85838114 48 TIKFKVG 3.669837012 49

[0963]

[0964] A closer examination of the expanded list of sequences revealed sub-groups within the family according to the location of the cationic (i.e., Lys or Arg) residues at the N-terminal portion of the peptide. Sequence logos for these subfamily groupings are depicted in Figure 2.

[0965] Conclusion

[0966] The above results describe a library-based approach and further analysis to discover the peptide family of the invention. ATTORNEY DOCKET NO.: 51772-007WO2

[0967] PATENT

[0968] Example 2. In vitro validation of capsid variants of the invention in HEK293 cells

[0969] Objective

[0970] This Example describes the results of experiments undertaken to validate the activity of capsid variants of the invention in vitro.

[0971] Materials and Methods

[0972] Exemplary peptides of the invention were sourced from the primary screen results, and additional peptides were inferred from consensus alignments of the primary hits. Individual lab scale vector preps of capsids presenting peptides of the invention and a luciferase reporter were produced. The titer of the vector preps was quantified to assess the intrinsic manufacturability of the capsids. Capsid yield is frequently compromised by peptide insertion engineering, impacting cost-of-goods for gene therapy products. Assessment of this property of various engineered variants can thus be relevant to variant selection. In addition to titer, the relative transduction activity of each variant was evaluated compared to AAV9 using transduction of HEK293 cells.

[0973] Results

[0974] Table 7 presents a summary of HEK293 cell transduction activity and production yields, both normalized to the AAV9 comparator.

[0975] Table 7. Summary of HEK293 cell transduction activity and production yields

[0976] HEK 293 in vitro Vector

[0977] Insert peptide

[0978] transduction activity manufacturing titer SEQ ID NO. sequence

[0979] (fold change over AAV9) (fraction of AAV9)

[0980] TKSRPAG 81 0.46 17 SIRKPAG 115 0.34 18 SRNKPIG 118 0.14 20 TFKKPAG 112 0.32 21 TKIKPAG 8.1 0.2 50 TKIKPVG 5.2 0.2 51 TKTKPAG 2.8 0.2 52 TKTKPVG 1.3 0.2 53 SKI KPAG 2.5 0.1 54 SKIKPVG 1.7 0.2 55 SKTKPAG 10.1 0.2 56

[0981]

[0982] ATTORNEY DOCKET NO.: 51772-007WO2

[0983] PATENT

[0984] Conclusion

[0985] The above results demonstrated that many capsid variants with a peptide insert of the invention successfully enhanced the transduction of human-kidney-origin HEK293 cells relative to AAV9 (up to 118-fold) while retaining acceptable production yields.

[0986] Example 3. In vitro confirmation of cross-species potential of capsid variants of the invention in human and mouse muscle cells

[0987] Objective

[0988] This Example describes the results of experiments undertaken to confirm the cross-species potential of peptides of the invention in vitro.

[0989] Materials and Methods

[0990] To confirm the potential of these capsids for cross-species intramuscular AAV activity, several motif-containing capsid variants were generated. The transduction activity of each of variant and AAV9 vectors was determined using a luciferase reporter in primary muscle cell cultures derived from human and mouse.

[0991] Results

[0992] All tested capsid variants exhibited broad enhancement over AAV9 in transduction of primary muscle cell cultures from human and mouse origin (Table 8).

[0993] Table 8. In vitro transduction activity in muscle cells

[0994] In vitro transduction activity on In vitro transduction activity on

[0995] Insert peptide SEQ ID primary human muscle cells primary mouse muscle cells sequence NO.

[0996] (fold change over AAV9) (fold change over AAV9) SIRKPAG 21.74049944 80.03338 18 TKSRPAG 39.15875905 76.34824 17 SRNKPIG 369.8220932 531.05 20 TFKKPAG 13.99445546 55.14309 21

[0997]

[0998] Conclusion

[0999] The above results demonstrated the potential of capsid variants with a peptide insert of the invention for cross-species intramuscularly administered AAV activity.

[1000] Example 4. In vivo validation of capsid variants of the invention in mice

[1001] Objective

[1002] This Example describes the results of experiments undertaken to validate the activity of capsid variants of the invention in vivo. ATTORNEY DOCKET NO.: 51772-007WO2

[1003] PATENT

[1004] Materials and Methods

[1005] To test the in vivo potential of peptide inserts of the invention, a capsid variant containing a peptide insert of the invention (SIRKPAG, SEQ ID NO: 18) was compared to AAV9 vectors for intramuscular AAV activity in mice. SIRKPAG and AAV9 (each encapsulating identical CMV-GLP-1-Fc reporter constructs) were dosed into Rag mice at several doses intramuscularly. Fourteen days later, serum was collected from a terminal bleed for GLP-1-Fc reporter protein determination (Figure 3A). Muscle (Figure 3B) and liver (Figure 3C) were collected for reporter mRNA quantification.

[1006] Results

[1007] SIRKPAG reporter protein determination and mRNA quantification are shown in Figure 3A, Figure 3B, and Figure 3C. The SIRKPAG capsid enhanced serum production of the reporter protein across a wide range of doses (Figure 3A). Local enhancement at the muscle injection site was pronounced, particularly at higher doses, for SIRKPAG (Figure 3B). The lower serum protein enhancement relative to muscle mRNA enhancement may be partially explained by partially reduced liver targeting by SIRKPAG (Figure 3C), another clinically beneficial attribute. Remarkably, the apparent liver transduction by SIRKPAG changed very little over a 10-fold dose escalation, whereas AAV9 spillover from muscle to liver increased more than 10-fold over the same dose range. High tissue-specific targeting of SIRKPAG from intramuscular delivery is desirable clinically, as liver spillover from the injection site presents a potential safety risk. Moreover, liver spillover complicates the ability of clinicians to predict serum expression levels across patient populations, as typical serum neutralizing antibodies against AAVs -which differ in prevalence across individuals - would be predicted to impact liver spillover transduction but not muscle directed transduction.

[1008] Conclusion

[1009] This results in this Example established the intramuscular AAV potential for capsid variants containing peptide inserts of the invention.

[1010] Example 5. In vivo screen of variant libraries in mice

[1011] Objective

[1012] This Example describes the results of experiments undertaken to discover variant peptides of the invention with the potential for high intramuscular AAV activity in vivo.

[1013] Materials and Methods

[1014] An initial library of peptide variants (“MS”) was constructed from 510 sequences sourced directly from the primary screen output and novel sequences generated using the observed peptide family of the invention. A mini-library of these variants (and a few control sequences) was then cloned in the same capsid library format as the original primary screen (Cap9 variant genes encoded between AAV2 ITRs with a CMV promoter). An AAV library was generated in which each of these capsid insert variants packaged its own genome.

[1015] The purified AAV library was then delivered intramuscularly to 5 Rag mice. Fourteen days postinjection, the injected leg muscles were collected at necropsy, and mRNA was extracted. Deep ATTORNEY DOCKET NO.: 51772-007WO2

[1016] PATENT

[1017] sequencing was conducted on the variable region of the capsid genes from the purified AAV library and cDNA libraries from muscle of mice receiving the AAV library.

[1018] NGS sequencing data was used to derive a muscle score for each variant by taking the log of the ratio of each variant’s abundance in muscle cDNA libraries normalized to that variant’s abundance in the injected AAV library.

[1019] Results

[1020] The results are tabulated in Table 9. Variants not detected in muscle mRNA could not be scored and are as (ND).

[1021] In terms of library muscle enrichment scores, several sequences outperformed SIRKPAG (SEQ ID NO: 18), a sequence with excellent known intramuscular AAV activity in mice.

[1022] Table 9. In vivo muscle transduction scores for the MS library

[1023] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1024] TIRKPAG 3.7 57 TIRKPQG 3.5 58 TRSKPQG 3.2 59 SIRKPAG 2.9 18 SIRRPAG 2.8 30 TRNKPAG 2.5 60 SRKKPVG 2.5 61 SRSRPAG 2.3 62 TIRRPIG 2.3 63 TKRKKVG 2.3 64 SIRRPVG 2.2 65 TRTKPQG 1.9 66 TIRRPAG 1.8 67 TRSKPVG 1.8 68 SRSKPVG 1.7 69 TRNKPVG 1.5 70 SRSKPAG 1.3 71 TIIKPAG 1.3 72 TRTRPIG 1.2 73 TIRKIVG 1.2 74 SRTKPQG 1.1 75 MRTRPAG 1.1 76 TRIRPIG 1.1 77 TLRKIAG 1.1 45 GRRKPAG 1.1 78 SRTKPAG 1.0 79

[1025]

[1026] ATTORNEY DOCKET NO.: 51772-007WO2

[1027] PATENT

[1028] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1029] TRSKPIG 1.0 80 MISKPAG 0.9 81 TRTRIVG 0.9 82 SRNKPIG 0.9 20 TRAKPQG 0.9 83 TRAKPGG 0.9 84 SRIKPQG 0.9 85 SRTRPAG 0.9 86 TIRRPQG 0.8 87 TRNRPAG 0.8 88 TRAKPAG 0.8 89 TRTKVAG 0.8 42 TRIRPVG 0.8 90 NFIKPAG 0.8 91 TFTKIAG 0.8 92 NRTRPIG 0.7 93 TIRKKVG 0.7 94 NRSKPAG 0.7 95 SITKKQG 0.7 96 TRNRPQG 0.7 97 TLIRPAG 0.6 98 SRIRPAG 0.6 99 TMTRPVG 0.6 100 SLIRPAG 0.6 101 TRAKPVG 0.6 34 MLTKPVG 0.6 102 MLIRPAG 0.6 103 SRIRIQG 0.5 104 TIIRPAG 0.5 105 TIIRPVG 0.5 106 TRTKPIG 0.5 107 SRIKPAG 0.4 108 TLIKPAG 0.4 109 TIRKVVG 0.4 110 TIRRPSP 0.4 111 TIMRPAG 0.4 112 TRIKPAG 0.4 113 MFAKPAG 0.4 114 SIKRPAG 0.3 115

[1030]

[1031] ATTORNEY DOCKET NO.: 51772-007WO2

[1032] PATENT

[1033] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1034] TRTKPVG 0.3 116 TRTMVVG 0.3 117 SLIRPVG 0.3 118 SRIKPVP 0.3 40 SIRKPIG 0.3 119 TITKPAG 0.3 120 TFTKPAG 0.3 121 MITRPAG 0.3 122 MITRPVG 0.3 123 SIIRVAG 0.3 124 TLAKKIG 0.3 125 SLIKPVG 0.2 126 SIMKPAG 0.2 127 TIRMPAP 0.2 128 TKSRPAG 0.2 17 SIRQKVG 0.2 19 SFTKPVG 0.2 129 TKTKKAG 0.2 130 QMPRTPG 0.2 131 MRIRPAG 0.2 132 TRMKPAG 0.1 133 TKSRPSG 0.1 134 TITRPIG 0.1 135 NFARPIG 0.1 136 TLTKPAG 0.1 137 SIIRPVG 0.1 138 TLIKPVG 0.1 139 TLTKPVG 0.1 140 NIIRPIG 0.1 141 MLNRPAG 0.1 142 TKIRPQG 0.0 41 SRIRVAG 0.0 143 SIIKPVG 0.0 144 TFKKPAG 0.0 21 TLAKPMG 0.0 145 TITRPAG 0.0 146 NLRRPVG -0.1 147 MRNMPAG -0.1 148 NRTKPAP -0.1 149

[1035]

[1036] ATTORNEY DOCKET NO.: 51772-007WO2

[1037] PATENT

[1038] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1039] SKIRPAG -0.1 150 SRIKPVG -0.1 151 TRIKPIG -0.1 152 TRIKPVG -0.1 25 TKSKPSG -0.1 153 GKIKPIG -0.1 154 SLSKPAG -0.1 155 MLIKPAG -0.1 156 SIIKPAG -0.1 157 SIARPSG -0.1 158 TRTKVVG -0.2 47 TKNRPAG -0.2 159 TKIRPMG -0.2 39 TKSKPAG -0.2 160 TLIKKAG -0.2 161 LIRKPIG -0.2 162 SITKPSG -0.2 163 TKNKPAG -0.2 164 TLKRPAG -0.2 165 LIRKPAG -0.2 166 TMIRPMG -0.3 167 TITKPVG -0.3 168 TRIRVAG -0.3 169 TIAKPAG -0.3 170 TRMKVAG -0.3 171 TRMKPVG -0.3 172 SKIRPVG -0.3 173 TIIKPVG -0.3 174 TITKPSG -0.3 175 TITKPIG -0.3 176 SLKKPAG -0.3 177 TLKKPAG -0.3 178 NLRRPIG -0.3 179 TIKRPAG -0.3 180 SIKMPAG -0.3 181 TISRPAG -0.3 182 MRAKPVG -0.3 183 GRNKPAG -0.3 184 LIRKPVG -0.4 185

[1040]

[1041] ATTORNEY DOCKET NO.: 51772-007WO2

[1042] PATENT

[1043] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1044] SIIKVAG -0.4 186 MITKPSG -0.4 187 TKTRIAG -0.4 188 SKSKPAG -0.4 189 SIIQKVG -0.4 190 SINKPVG -0.5 191 SMIKPVG -0.5 192 TLIKPIG -0.5 193 TLNKPVG -0.5 194 MITKPVG -0.5 195 TISKPVG -0.5 196 TKIKPSG -0.5 36 SMKKPSG -0.5 197 TIIKPIG -0.5 198 SITKPVG -0.5 199 TIIKKIG -0.6 48 TKIKPAP -0.6 200 SIKKPAG -0.6 201 SKIRPIG -0.6 202 LRNRPVP -0.6 203 SINRPVG -0.6 204 SITKPAG -0.6 205 SKAKVVG -0.6 206 TLNKPAG -0.6 207 SRMKPAG -0.6 208 TKTKPMG -0.6 209 TIKKPMG -0.6 210 TRAQPAG -0.6 211 TRRKPAG -0.6 212 TRRRPVG -0.6 213 TKTRPVG -0.6 214 NIKKPAG -0.6 215 SKTRVAG -0.7 216 MLKKPQG -0.7 217 SLNRPAG -0.7 218 SRTKPVG -0.7 219 SLTRPIG -0.7 220 SIAKPAG -0.7 221 TIKKPAG -0.7 222

[1045]

[1046] ATTORNEY DOCKET NO.: 51772-007WO2

[1047] PATENT

[1048] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1049] NIIKPIG -0.7 223 TMMKPVG -0.7 224 TLRKPAG -0.7 23 TMRKPVG -0.7 225 TIKKPQG -0.7 226 LRNKPAG -0.7 22 TIIRVVP -0.7 227 SKIRVVG -0.7 228 SKTKPSG -0.7 229 TKTRPIG -0.7 230 TKNRPSG -0.7 231 TIIKKVG -0.8 232 NKIKPVG -0.8 233 TFKKPVG -0.8 234 LITKPAG -0.8 235 SKMRPVG -0.8 38 SIMKPVG -0.8 236 MIKKPVG -0.8 26 MKTKPGG -0.8 237 SKMRVQG -0.8 238 TKIRPAG -0.8 239 SIIKKVG -0.9 240 NRIKPAG -0.9 27 SIKKPSG -0.9 241 MKIFPAG -0.9 242 TKTRPQG -0.9 243 SKIRPGG -0.9 43 SINKPAG -0.9 244 NLRMKAG -0.9 245 TMRKPIG -0.9 246 GRIKPAG -0.9 247 TIKRPIG -0.9 248 TKARPAG -0.9 249 TKMRVIG -0.9 250 TIN KPAG -0.9 251 MKTRPIP -0.9 252 TLAVPFK -0.9 253 MIAKPAG -0.9 254 TKIRPVG -1.0 255

[1050]

[1051] ATTORNEY DOCKET NO.: 51772-007WO2

[1052] PATENT

[1053] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1054] TIMRKVG -1.0 256 SKTRPVG -1.0 257 GMTKPAG -1.0 258 TRTKKVG -1.0 259 TINKPVG -1.0 260 TKTKPQG -1.0 261 TKTKPAG -1.1 52 MMKKPVG -1.1 35 TIKKPVG -1.1 262 GLTKPIG -1.1 263 TLKQPAG -1.1 264 SKTRPAG -1.1 24 TKNKIVG -1.1 265 MIKRPAP -1.1 266 SIAKVVG -1.1 267 SKNKPAG -1.1 268 TLMFPGG -1.1 269 TIKKPIG -1.1 270 GIRKPAG -1.1 271 SKTKPVG -1.1 272 TLNKIAG -1.1 273 SKTKPQG -1.1 44 MINKPVG -1.1 274 SIKKPVG -1.1 275 NLTKPVG -1.1 276 MKTKPVP -1.1 277 TISKIAG -1.1 278 SKIKPVG -1.2 55 MKIRPVG -1.2 279 TKIKPAG -1.2 50 TIKRPVG -1.2 28 SKIKPAG -1.2 54 SKTKPAG -1.2 56 TKAKPAG -1.2 33 TKMKPAG -1.2 280 TIMKPVG -1.2 281 LITKVVG -1.2 282 SKTRKAG -1.2 283 TKIKPQG -1.2 284

[1055]

[1056] ATTORNEY DOCKET NO.: 51772-007WO2

[1057] PATENT

[1058] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1059] TKIKVVG -1.2 285 GLKKPMG -1.3 286 TRTMKQG -1.3 287 MKIKPAG -1.3 288 TIIKKAG -1.3 289 TIIQPVG -1.3 290 TKNKPIG -1.3 291 NIIRKVG -1.3 292 TIKRPQP -1.3 293 TKSKPVG -1.3 294 TKTFIAG -1.4 295 SITQPAG -1.4 296 TKTKPSG -1.4 297 RMPVQKG -1.4 298 TKTKPVG -1.4 53 SMSRPIG -1.4 299 SKTKPIG -1.4 300 TRRKVAG -1.4 301 MKIKPSG -1.4 302 NKIKPGG -1.4 303 SRTQPAG -1.5 304 SKARPVG -1.5 305 LFIKPVG -1.5 306 LKIKPVG -1.5 307 TKIKPIG -1.5 308 MIIKPVG -1.5 309 SLKKVAG -1.5 310 SITQPVG -1.5 311 MKNKPVG -1.5 312 LKTRPVG -1.5 313 SINKIVG -1.5 314 TKARPGG -1.5 315 TLRKPVG -1.5 316 TIIFPAG -1.6 317 MKTKPQG -1.6 318 TMRKPQG -1.6 319 TIRRVPG -1.6 320 LINKPVG -1.6 321 MKTKPVG -1.6 32

[1060]

[1061] ATTORNEY DOCKET NO.: 51772-007WO2

[1062] PATENT

[1063] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1064] TLRQPVG -1.6 322 MKIKPVG -1.6 323 TKNKPVG -1.6 31 SIRRPPG -1.7 324 TLRKPIG -1.7 325 SISKPVG -1.7 326 SKIKPVP -1.7 327 TKSKPIG -1.7 328 TKIMKSG -1.7 329 TFKKPIG -1.7 330 TKTKPIG -1.8 331 TKIKPVG -1.8 51 NIKFPAG -1.8 332 SKKKVAG -1.8 333 MKSMVAG -1.8 334 TKAKIMG -1.8 335 TLKQPQG -1.8 336 SRMRPVG -1.8 337 SISKKVG -1.8 338 TITKKVG -1.8 339 SRTKKAG -1.8 340 SKSKPIG -1.9 46 MKIKPVP -1.9 341 SKAKPVG -1.9 342 TKRQPAG -1.9 343 GKARPAG -1.9 344 NIAKPVG -1.9 345 MIIRPVG -1.9 346 MKTKPAG -2.0 347 SLAKPIG -2.0 348 TLKKPIG -2.0 349 TKTKVAP -2.0 350 SRTRPVG -2.0 351 NNSTRGG -2.0 352 TMSKIVG -2.0 353 TRRKPVG -2.0 354 SRAKPAG -2.0 355 SIKKPAP -2.1 356 SKKFPMG -2.1 357

[1065]

[1066] ATTORNEY DOCKET NO.: 51772-007WO2

[1067] PATENT

[1068] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1069] TFTKKVG -2.1 358 NRTKPVG -2.1 359 TIKKVAG -2.1 360 NKTKPVG -2.1 361 TITKKPG -2.1 29 GKIKPAG -2.1 362 SIAKPIG -2.1 363 TIKFKVG -2.2 49 GIAKPVG -2.2 364 GKTKPAG -2.2 37 TIKKPSP -2.3 365 SITFPIG -2.3 366 MKSRPVG -2.3 367 TKIMPVG -2.4 368 G KN KPAG -2.4 369 LNTTKPI -2.4 370 SLTRKQG -2.4 371 SLTKKAG -2.4 372 SITMPAG -2.4 373 TKRKPIG -2.5 374 TKKQPIG -2.5 375 TKTKKVG -2.5 376 MKSKVVG -2.5 377 LIKKPAG -2.5 378 SIIQPAG -2.5 379 SKRKPAG -2.6 380 LKIKPIG -2.6 381 KNPAKPP -2.6 382 TKIKPPG -2.6 383 SNYVKQT -2.7 384 TKIMPIG -2.7 385 KNPTKPA -2.7 386 LINKPIG -2.7 387 TKRRPAG -2.8 388 TLMQPIG -2.8 389 GLRKPVG -2.9 390 SIIMPVG -2.9 391 GIKQKPG -3.0 392 SRRKPQG -3.0 393

[1070]

[1071] ATTORNEY DOCKET NO.: 51772-007WO2

[1072] PATENT

[1073] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1074] TKTMPQG -3.0 394 SISKKQG -3.0 395 GIKKPAG -3.0 396 TKKFPVG -3.0 397 TKRKPVG -3.1 398 SRTKPIG -3.1 399 TIKKPPG -3.3 400 NKKRPVG -3.3 401 SKRRPVP -3.3 402 TRRRPAG -3.5 403 SRIMKAG -3.6 404 SFRQPVG -3.6 405 TRKKPAG -3.6 406 MITMPVG -3.6 407 MKKKPIG -3.7 408 MLRKPSG -3.7 409 TMRRPQG -3.7 410 MINKPPG -4.0 411 TFIKPVG -4.0 412 IKVARPP -4.0 413 TIKKKVG -4.0 414 TKKRPAG -4.1 415 NRRKPVG -4.1 416 GRKKPIG -4.1 417 SLARPVP -4.3 418 SLRKPIG -4.3 419 SKRKPIG -4.5 420 SKKKPVG -4.6 421 SMAKPIG -4.9 422 SIRKKIG -5.2 423 TKKKPVG -5.3 424 TKKKPMG -5.3 425 SKRKPVG -5.6 426 NKRKPVG -5.6 427 NKKKPVG -6.2 428 TRKKVGG -6.6 429 TRKKPVG -6.7 430 NKNMKGG -6.8 431 TKKKPQG -6.8 432

[1075]

[1076] ATTORNEY DOCKET NO.: 51772-007WO2

[1077] PATENT

[1078] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1079] TKKKPSG -6.9 433 TLSQPVG -7.2 434 SKKKPAG -7.4 435 MKIKPPG -9.2 436 SRKKPQG -9.5 437 TKKKPAG ND 438 SKKRPSG ND 439 LMTMPVG ND 440 TKRKPAG ND 441 NKIKKVG ND 442 SKIRPPG ND 443 TKIKKVG ND 444 SKIKKVG ND 445 SKRKPQG ND 446 TFTRKIG ND 447 SRMKPVG ND 448 SFSKPPG ND 449 SKRRPVG ND 450 TKRRPVG ND 451 SKKKVVG ND 452 MIIRKAG ND 453 SRKKPIG ND 454 SKIKKAG ND 455 TKKKPPG ND 456 SISKPPG ND 457 TKIKKAG ND 458 NRKRPVG ND 459 MKRKPVG ND 460 TKAKKVG ND 461 SKTKKAG ND 462 NKRMKVG ND 463 SRKKVVG ND 464 SLRRKVP ND 465 TIIRPPG ND 466 GKKKPQG ND 467 MKKKPGG ND 468 LLRKIPG ND 469 SIRKKVG ND 470 MKTKKAG ND 471

[1080]

[1081] ATTORNEY DOCKET NO.: 51772-007WO2

[1082] PATENT

[1083] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1084] SKSKKAG ND 472 TKNKKVG ND 473 MRARPVG ND 474 TKSRKPG ND 475 TLKKKPG ND 476 SKARKVG ND 477 TKTRKAG ND 478 TRIKPPG ND 479 TLRKKAG ND 480 SRRKPVG ND 481 GKRKPIG ND 482 SKTQPAG ND 483 GRKRPVG ND 484 LKIMPPG ND 485 TRIKKQG ND 486 MKRRPVG ND 487 LRKKPVG ND 488 LRKRPAG ND 489 TRNKKAG ND 490 NIRKKAG ND 491 TIRKKAG ND 492 TRIKKAG ND 493 SKNKKAG ND 494 MKRKPAG ND 495 TKRKVAG ND 496 TRRMKVG ND 497 LIRKKVG ND 498 MRIKPPG ND 499 TRRRPVP ND 500 TKRKKAG ND 501 TRARKIG ND 502 SRIKKAG ND 503 SRAKKAG ND 504 TKRKIAG ND 505 TRRKVVG ND 506 MRRMVQG ND 507 MKRKPGG ND 508 TRKRKVG ND 509 LKRRPAG ND 510

[1085]

[1086] ATTORNEY DOCKET NO.: 51772-007WO2

[1087] PATENT

[1088] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1089] SKKRKAG ND 511 NIKRKGG ND 512 TRRKKQG ND 513 TRKKKAG ND 514 TKIRKAG ND 515 SKKKKVG ND 516 GKSRKIG ND 517 MKRRPPG ND 518 TKRRKVG ND 519 MRRKPVG ND 520 TKKKKAG ND 521 TRRKKVG ND 522 TKRRKAG ND 523 TMRKKAG ND 524 LKRKKAG ND 525

[1090]

[1091] Conclusion

[1092] An in vivo screen can be used to discover many additional capsid variants of the invention that exhibit muscle enrichment.

[1093] Example 6. In vivo screen of saturation mutagenesis libraries of a peptide insert of the invention in mice

[1094] Objective

[1095] This Example describes the results of experiments undertaken to discover variant peptides of the invention with the potential for high intramuscular AAV activity in vivo.

[1096] Materials and Methods

[1097] A saturation mutagenesis library was constructed based on the active insert sequence SIRKPAG (SEQ ID NO: 18). DNA oligos encoding every possible single amino acid variant of SIRKPAG (SEQ ID NO: 18) were designed (20 amino acids plus a stop codon at 7 positions or 21 x 7 = 147 peptide insert variants). A mini-library of these variants was cloned in the same capsid library format as the original primary screen (Cap9 variant genes encoded between AAV2 ITRs with a CMV promoter). An AAV library was generated in which each of these capsid insert variants packaged its own genome.

[1098] The purified AAV library was then delivered intramuscularly to 3 Rag mice. Fourteen days postinjection, the injected leg muscles were collected at necropsy, and mRNAwas extracted. Deep sequencing was conducted on the variable region of the capsid genes from the purified AAV library, and cDNA libraries from muscle of mice receiving the AAV library.

[1099] NGS sequencing data were used to derive a muscle score for every variant by taking the log of the ratio of each variant’s abundance in muscle cDNA libraries normalized to that variant’s abundance in the injected AAV library. ATTORNEY DOCKET NO.: 51772-007WO2

[1100] PATENT

[1101] Results

[1102] The results are tabulated in Table 10. Variants not detected in muscle mRNA could not be scored and are noted here as (ND). In terms of library muscle enrichment scores, several sequences outperformed SIRKPAG, a sequence with excellent known intramuscular AAV activity in mice.

[1103] Table 10. In vivo muscle transduction scores for the saturation mutagenesis library Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1104] SKRKPAG 4.7 380 SQRKPAG 2.7 526 SIRRPAG 2.6 30 SFRKPAG 2.6 527 SIRKTAG 2.5 528 WIRKPAG 2.5 529 STRKPAG 2.2 530 TIRKPAG 2.2 57 SVRKPAG 1.3 531 SIRKPNG 1.3 532 SIRKPPG 1.2 533 SIYKPAG 1.1 534 SIRKPAG 1.1 18 SARKPAG 1.0 535 SSRKPAG 0.9 536 SNRKPAG 0.9 537 SHRKPAG 0.8 538 SIRKPSG 0.7 539 HIRKPAG 0.6 540 SIRYPAG 0.5 541 SIRHPAG 0.5 542 SIRKPQG 0.4 543 SIRKAAG 0.3 544 SIRKPGG 0.0 545 SIFKPAG -0.2 546 SIGKPAG -0.3 547 SIRKPMG -0.3 548 SIRKPTG -0.4 549 SGRKPAG -0.5 550 EIRKPAG -0.5 551 LIRKPAG -0.7 166 SIRLPAG -0.9 552

[1105]

[1106] ATTORNEY DOCKET NO.: 51772-007WO2

[1107] PATENT

[1108] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1109] SIRKPVG -0.9 553 SIRKPIG -1.2 119 DIRKPAG -1.2 554 SIIKPAG -1.2 157 SI RS PAG -1.2 555 SICKPAG -1.4 556 SILKPAG -1.4 557 SITKPAG -1.4 205 SIPKPAG -1.5 558 NIRKPAG -1.5 559 SIRKPFG -1.6 560 SIRKPAS -1.6 561 PIRKPAG -1.9 562 SIRQPAG -1.9 563 SIRNPAG -1.9 564 SIAKPAG -2.0 221 SIVKPAG -2.1 565 SIRKPEG -2.1 566 SIRKPXG -2.2 567 SIRVPAG -2.2 568 SLRKPAG -2.3 569 SIQKPAG -2.3 570 FIRKPAG -2.4 571 SISKPAG -2.5 572 SIRTPAG -2.6 573 SXRKPAG -2.7 574 SIRKPLG -2.7 575 SIRKPAV -2.8 576 SIMKPAG -2.8 127 SIRKPHG -2.9 577 SIRMPAG -3.0 578 SIHKPAG -3.0 579 S IRC PAG -3.0 580 SIRKRAG -3.1 581 SIRAPAG -3.1 582 SIRFPAG -3.1 583 SIRIPAG -3.3 584 SIRKPDG -3.4 585 SIRKEAG -3.5 586

[1110]

[1111] ATTORNEY DOCKET NO.: 51772-007WO2

[1112] PATENT

[1113] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1114] SIRKLAG -3.6 587 SRRKPAG -3.7 588 SIRKPYG -3.8 589 SIRKPAW -3.8 590 SIRDPAG -3.8 591 SIRPPAG -3.9 592 GIRKPAG -3.9 271 SIRKPRG -4.0 593 SIRKDAG -4.0 594 SMRKPAG -4.1 595 SINKPAG -4.2 244 SIKKPAG -4.4 201 SIRGPAG -4.5 596 RIRKPAG -4.7 597 KIRKPAG -5.4 598 SIRKPAA -6.1 599 SERKPAG -6.4 600 YIRKPAG -6.6 601 SIRKSAG -6.7 602 AIRKPAG -7.2 603 MIRKPAG -7.4 604 SPRKPAG -7.5 605 SIRKQAG -7.6 606 SIWKPAG -8.0 607 SIRKPAD -8.0 608 IIRKPAG -8.3 609 SIRKMAG -8.6 610 SIRKNAG -9.9 611 SIRWPAG -10.8 612 SI EKPAG -10.9 613 SIREPAG -12.0 614 SIDKPAG -14.6 615 SDRKPAG -14.9 616 QIRKPAG ND 617 XIRKPAG ND 618 VIRKPAG ND 619 CIRKPAG ND 620 SWRKPAG ND 621 SYRKPAG ND 622

[1115]

[1116] ATTORNEY DOCKET NO.: 51772-007WO2

[1117] PATENT

[1118] Amino Acid Sequence muscle enrichment score (Iog2(fold-change)) SEQ ID NO.

[1119] SCRKPAG ND 623 SIXKPAG ND 624 SIRXPAG ND 625 SIRKKAG ND 626 SIRKXAG ND 627 SIRKGAG ND 628 SIRKWAG ND 629 SIRKVAG ND 630 SIRKHAG ND 631 SIRKYAG ND 632 SIRKCAG ND 633 SIRKIAG ND 634 SIRKFAG ND 635 SIRKPKG ND 636 SIRKPWG ND 637 SIRKPCG ND 638 SIRKPAK ND 639 SIRKPAQ ND 640 SIRKPAE ND 641 SIRKPAX ND 642 SIRKPAT ND 643 SIRKPAP ND 644 SIRKPAR ND 645 SIRKPAM ND 646 SIRKPAL ND 647 SIRKPAN ND 648 SIRKPAH ND 649 SIRKPAY ND 650 SIRKPAC ND 651 SIRKPAI ND 652 SIRKPAF ND 653

[1120]

[1121] ATTORNEY DOCKET NO.: 51772-007WO2

[1122] PATENT

[1123] Conclusion

[1124] An in vivo screen using saturation mutagenesis of a peptide of the invention can be used to discover many additional capsid variants of the invention that exhibit muscle enrichment.

[1125] Example 7. In vivo validation of enhanced activity of capsid variants of the invention in mice Objective

[1126] This Example describes the results of experiments undertaken to validate the activity of a peptide insert of the invention for intramuscular AAV activity in vivo.

[1127] Materials and Methods

[1128] Using library screens in vivo, several family variants were discovered with apparently enhanced in vivo intramuscular AAV transduction activity as compared to SIRKPAG (SEQ ID NO: 18). One such variant, TIRKPAG (SEQ ID NO: 57), was enhanced in multiple library screens. To test the in vivo potential of the TIRKPAG (SEQ ID NO: 57) variant, this variant was compared to AAV9 for intramuscular AAV activity in mice. TIRKPAG (SEQ ID NO: 57) and AAV9 (each encapsulating identical CMV-GLP-1-Fc reporter constructs) were dosed into Rag mice intramuscularly at 1 E9 GC / mouse. Twenty-one days later, serum was collected from a terminal bleed for GLP-1-Fc reporter protein determination (Figure 4A). Muscle (Figure 4B) and liver (Figure 4C) were collected for reporter mRNA quantification.

[1129] Results

[1130] TIRKPAG (SEQ ID NO: 57) reporter protein determination and mRNA quantification are shown in Figure 4A, Figure 4B, and Figure 4C. The TIRKPAG capsid enhanced serum production of the reporter protein by 24-fold (Figure 4A). Local enhancement at the muscle injection site was pronounced for TIRKPAG (Figure 4B), reaching 71 -fold over the AAV9 muscle expression level. As with other peptide family members, TIRKPAG (SEQ ID NO: 57) was highly liver de-targeted, with only 2% of the liver transduction observed for AAV9 (Figure 4C), another clinically beneficial attribute. High tissue-specific targeting of TIRKPAG (SEQ ID NO: 57) from intramuscular delivery is desirable clinically, as liver spillover from the injection site presents a potential safety risk. Moreover, liver spillover complicates the ability of clinicians to predict serum expression levels across patient populations, since typical serum neutralizing antibodies against AAVs - which differ in prevalence across individuals - would be predicted to impact liver spillover transduction but not muscle directed transduction.

[1131] Conclusion

[1132] This results in this Example established the intramuscular AAV potential for the TIRKPAG (SEQ ID NO: 57) variant.

[1133] OTHER EMBODIMENTS

[1134] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. ATTORNEY DOCKET NO.: 51772-007WO2

[1135] PATENT

[1136] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations described herein following, in general, the principles described herein and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Claims

ATTORNEY DOCKET NO.: 51772-007WO2PATENTWhat is claimed is:CLAIMS1. A recombinant adeno-associated virus (AAV) capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (I):(X1)(X2)(X3)(X4)(X5)(X6)GFormula (I)wherein each of (X2) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X3), (X5), and (X6), independently, represents any naturally occurring amino acid.

2. The recombinant AAV capsid protein of claim 1, wherein (X1 ) represents Thr, Ser, Asn, Gly, Leu, or Met.

3. The recombinant AAV capsid protein of claim 2, wherein (X1) represents Thr or Ser.

4. The recombinant AAV capsid protein of any one of claims 1-3, wherein (X3) represents Thr, lie, Ser, Asn, Ala, Arg, Met or Lys.

5. The recombinant AAV capsid protein of claim 4, wherein (X3) represents Thr. lie, Ser, Asn, or Ala.

6. The recombinant AAV capsid protein of any one of claims 1-5, wherein (X5) represents Pro, Lys, Vai, or lie.

7. The recombinant AAV capsid protein of claim 6, wherein (X5) represents Pro, Lys, or Vai.

8. The recombinant AAV capsid protein of claim 7, wherein (X5) represents Pro.

9. The recombinant AAV capsid protein of any one of claims 1 -8, wherein (X6) represents Ala, Gly, lie, Met, Gin, Ser, or Vai.

10. The recombinant AAV capsid protein of claim 9, wherein (X6) represents Ala, lie, Gin, or Vai.

11. The recombinant AAV capsid protein of any one of claims 1 -10, wherein (X2) represents Lys.

12. The recombinant AAV capsid protein of any one of claims 1-10, wherein (X2) represents Arg.

13. The recombinant AAV capsid protein of any one of claims 1-12, wherein (X4) represents Lys.

14. The recombinant AAV capsid protein of any one of claims 1-12, wherein (X4) represents Arg.ATTORNEY DOCKET NO.: 51772-007WO2PATENT15. A recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (II):(X1)(X2)(X3)(X4)(X5)(X6)GFormula (II)wherein each of (X3) and (X4), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X5), and (X6), independently, represents any naturally occurring amino acid.

16. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Thr, Ser, Met, His, Gly, or Trp.

17. The recombinant AAV capsid protein of claim 16, wherein (X1 ) represents Thr or Ser.

18. The recombinant AAV capsid protein of any one of claims 15-17, wherein (X2) represents lie, Phe, Leu, Lys, Arg, Ala, His, Met, Asn, Gin, Ser, Thr, or Vai.

19. The recombinant AAV capsid protein of claim 18, wherein (X2) represents lie, Phe or Leu.

20. The recombinant AAV capsid protein of claim 19, wherein (X2) represents lie.

21. The recombinant AAV capsid protein of any one of claims 15-20, wherein (X5) represents Pro, lie, Vai, Ala, or Thr.

22. The recombinant AAV capsid protein of claim 21, wherein (X5) represents Pro or lie.

23. The recombinant AAV capsid protein of claim 22, wherein (X5) represents Pro.

24. The recombinant AAV capsid protein of any one of claims 15-23, wherein (X6) represents Ala, Vai, Gin, lie, Asn, Pro, or Ser.

25. The recombinant AAV capsid protein of claim 24, wherein (X6) represents Ala or Vai.

26. The recombinant AAV capsid protein of any one of claims 15-25, wherein (X3) represents Lys.

27. The recombinant AAV capsid protein of any one of claims 15-25, wherein (X3) represents Arg.

28. The recombinant AAV capsid protein of any one of claims 15-27, wherein (X4) represents Lys.

29. The recombinant AAV capsid protein of any one of claims 15-27, wherein (X4) represents Arg.ATTORNEY DOCKET NO.: 51772-007WO2PATENT30. A recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (III):(X1)(X2)(X3)(X4)(X5)(X6)GFormula (III)wherein each of (X4) and (X5), independently, represents an amino acid selected from the group consisting of Lys and Arg, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid.

31. The recombinant AAV capsid protein of claim 30, wherein (X1 ) represents Thr or Ser.

32. The recombinant AAV capsid protein of claim 31, wherein (X1 ) represents Thr.

33. The recombinant AAV capsid protein of any one of claims 30-32, wherein (X2) represents lie, Lys, or Leu.

34. The recombinant AAV capsid protein of claim 33, wherein (X2) represents lie.

35. The recombinant AAV capsid protein of any one of claims 30-34, wherein (X3) represents Thr, Arg, Ala, or lie.

36. The recombinant AAV capsid protein of claim 35, wherein (X3) represents Thr or Arg.

37. The recombinant AAV capsid protein of any one of claims 30-36, wherein (X6) represents Vai, lie, Ala, Pro, or Gin.

38. The recombinant AAV capsid protein of claim 37, wherein (X6) represents Vai or lie.

39. The recombinant AAV capsid protein of any one of claims 30-38, wherein (X4) represents Lys.

40. The recombinant AAV capsid protein of any one of claims 30-38, wherein (X4) represents Arg.

41. The recombinant AAV capsid protein of any one of claims 30-40, wherein (X5) represents Lys.

42. The recombinant AAV capsid protein of any one of claims 30-40, wherein (X5) represents Arg.

43. A recombinant AAV capsid protein comprising an artificial peptide insert, wherein the peptide insert has an amino acid sequence represented by Formula (IV):(X1)(X2)(X3)(X4)P(X6)(X7)Formula (IV)ATTORNEY DOCKET NO.: 51772-007WO2PATENTwherein (X4) represents an amino acid selected from the group consisting of Lys and Arg, (X7) represents an amino acid selected from the group consisting of Gly and Pro, and each of (X1), (X2), (X3), and (X6), independently, represents any naturally occurring amino acid.

44. The recombinant AAV capsid protein of claim 43, wherein (X1) represents Thr, Ser, Met, or Asn.

45. The recombinant AAV capsid protein of claim 44, wherein (X1) represents Thr, Ser, or Met.

46. The recombinant AAV capsid protein of any one of claims 43-45, wherein (X2) represents lie, Leu, Phe, Arg, or Met.

47. The recombinant AAV capsid protein of claim 46, wherein (X2) represents lie, Leu, or Phe.

48. The recombinant AAV capsid protein of any one of claims 43-47, wherein (X3) represents lie, Thr, Ala, Met, Asn, Arg, Ser, or Tyr.

49. The recombinant AAV capsid protein of claim 48, wherein (X3) represents lie or Thr.

50. The recombinant AAV capsid protein of any one of claims 43-49, wherein (X6) represents Ala, Vai, lie, or Ser.

51. The recombinant AAV capsid protein of claim 50, wherein (X6) represents Ala or Vai.

52. The recombinant AAV capsid protein of any one of claims 43-51, wherein (X4) represents Lys.

53. The recombinant AAV capsid protein of any one of claims 43-51, wherein (X4) represents Arg.

54. The recombinant AAV capsid protein of any one of claims 43-53, wherein (X7) represents Gly.

55. The recombinant AAV capsid protein of any one of claims 43-53, wherein (X7) represents Pro.

56. The recombinant AAV capsid protein of claim 1, wherein (X1 ) represents Thr.

57. The recombinant AAV capsid protein of claim 1, wherein (X1) represents Ser.

58. The recombinant AAV capsid protein of claim 1, wherein (X1) represents Asn.

59. The recombinant AAV capsid protein of claim 1, wherein (X1) represents Gly.

60. The recombinant AAV capsid protein of claim 1, wherein (X1) represents Leu.ATTORNEY DOCKET NO.: 51772-007WO2PATENT61. The recombinant AAV capsid protein of claim 1, wherein (X1) represents Met.

62. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Thr.

63. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents lie.

64. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Ser.

65. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Asn.

66. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Ala.

67. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Arg.

68. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Met.

69. The recombinant AAV capsid protein of any one of claims 1 and 56-61, wherein (X3) represents Lys.

70. The recombinant AAV capsid protein of any one of claims 1 and 56-69, wherein (X5) represents Pro.

71. The recombinant AAV capsid protein of any one of claims 1 and 56-69, wherein (X5) represents Lys.

72. The recombinant AAV capsid protein of any one of claims 1 and 56-69, wherein (X5) represents Vai.

73. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Ala.

74. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Gly.ATTORNEY DOCKET NO.: 51772-007WO2PATENT75. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents lie.

76. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Met.

77. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Gin.

78. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Ser.

79. The recombinant AAV capsid protein of any one of claims 1 and 56-72, wherein (X6) represents Vai.

80. The recombinant AAV capsid protein of any one of claims 1 and 56-79, wherein (X2) represents Lys.

81. The recombinant AAV capsid protein of any one of claims 1 and 56-79, wherein (X2) represents Arg.

82. The recombinant AAV capsid protein of any one of claims 1 and 56-81, wherein (X4) represents Lys.

83. The recombinant AAV capsid protein of any one of claims 1 and 56-81, wherein (X4) represents Arg.

84. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Thr.

85. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Ser.

86. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Met.

87. The recombinant AAV capsid protein of claim 15, wherein (X1) represents His.

88. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Gly.

89. The recombinant AAV capsid protein of claim 15, wherein (X1) represents Trp.

90. The recombinant AAV capsid protein of any one of claims 15 and 84-89, wherein (X2) represents lie.ATTORNEY DOCKET NO.: 51772-007WO2PATENT91. The recombinant AAV capsid protein of any one of claims 15 and 84-89, wherein (X2) represents Phe.

92. The recombinant AAV capsid protein of any one of claims 15 and 84-89, wherein (X2) represents Leu.

93. The recombinant AAV capsid protein of any one of claims 15 and 84-92, wherein (X5) represents Pro.

94. The recombinant AAV capsid protein of any one of claims 15 and 84-92, wherein (X5) represents lie.

95. The recombinant AAV capsid protein of any one of claims 15 and 84-92, wherein (X5) represents Vai.

96. The recombinant AAV capsid protein of any one of claims 15 and 84-92, wherein (X5) represents Ala.

97. The recombinant AAV capsid protein of any one of claims 15 and 84-92, wherein (X5) represents Thr.

98. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Ala.

99. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Vai.

100. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Gin.

101. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents lie.

102. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Asn.

103. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Pro.ATTORNEY DOCKET NO.: 51772-007WO2PATENT104. The recombinant AAV capsid protein of any one of claims 15 and 84-97, wherein (X6) represents Ser.

105. The recombinant AAV capsid protein of any one of claims 15 and 84-104, wherein (X3) represents Lys.

106. The recombinant AAV capsid protein of any one of claims 15 and 84-104, wherein (X3) represents Arg.

107. The recombinant AAV capsid protein of any one of claims 15 and 84-106, wherein (X4) represents Lys.

108. The recombinant AAV capsid protein of any one of claims 15 and 84-106, wherein (X4) represents Arg.

109. The recombinant AAV capsid protein of claim 30, wherein (X1) represents Thr.

110. The recombinant AAV capsid protein of claim 30, wherein (X1 ) represents Ser.

111. The recombinant AAV capsid protein of any one of claims 30, 109, and 110, wherein (X2) represents lie.

112. The recombinant AAV capsid protein of any one of claims 30, 109, and 110, wherein (X2) represents Lys.

113. The recombinant AAV capsid protein of any one of claims 30, 109, and 110, wherein (X2) represents Leu.

114. The recombinant AAV capsid protein of any one of claims 30 and 109-113, wherein (X3) represents Thr.

115. The recombinant AAV capsid protein of any one of claims 30 and 109-113, wherein (X3) represents Arg.

116. The recombinant AAV capsid protein of any one of claims 30 and 109-113, wherein (X3) represents Ala.

117. The recombinant AAV capsid protein of any one of claims 30 and 109-113, wherein (X3) represents lie.ATTORNEY DOCKET NO.: 51772-007WO2PATENT118. The recombinant AAV capsid protein of any one of claims 30 and 109-117, wherein (X6) represents Vai.

119. The recombinant AAV capsid protein of any one of claims 30 and 109-117, wherein (X6) represents lie.

120. The recombinant AAV capsid protein of any one of claims 30 and 109-117, wherein (X6) represents Ala.

121. The recombinant AAV capsid protein of any one of claims 30 and 109-117, wherein (X6) represents Pro.

122. The recombinant AAV capsid protein of any one of claims 30 and 109-117, wherein (X6) represents Gin.

123. The recombinant AAV capsid protein of any one of claims 30 and 109-122, wherein (X4) represents Lys.

124. The recombinant AAV capsid protein of any one of claims 30 and 109-122, wherein (X4) represents Arg.

125. The recombinant AAV capsid protein of any one of claims 30 and 109-124, wherein (X5) represents Lys.

126. The recombinant AAV capsid protein of any one of claims 30 and 109-124, wherein (X5) represents Arg.

127. The recombinant AAV capsid protein of claim 43, wherein (X1) represents Thr.

128. The recombinant AAV capsid protein of claim 43, wherein (X1) represents Ser.

129. The recombinant AAV capsid protein of claim 43, wherein (X1) represents Met.

130. The recombinant AAV capsid protein of claim 43, wherein (X1 ) represents Asn.

131. The recombinant AAV capsid protein of any one of claims 43 and 127-130, wherein (X2) represents lie.

132. The recombinant AAV capsid protein of any one of claims 43 and 127-130, wherein (X2) represents Leu.ATTORNEY DOCKET NO.: 51772-007WO2PATENT133. The recombinant AAV capsid protein of any one of claims 43 and 127-130, wherein (X2) represents Phe.

134. The recombinant AAV capsid protein of any one of claims 43 and 127-130, wherein (X2) represents Arg.

135. The recombinant AAV capsid protein of any one of claims 43 and 127-130, wherein (X2) represents Met.

136. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents lie.

137. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Thr.

138. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Ala.

139. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Met.

140. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Asn.

141. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Arg.

142. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Ser.

143. The recombinant AAV capsid protein of any one of claims 43 and 127-135, wherein (X3) represents Tyr.

144. The recombinant AAV capsid protein of any one of claims 43 and 127-143, wherein (X6) represents Ala.

145. The recombinant AAV capsid protein of any one of claims 43 and 127-143, wherein (X6) represents Vai.

146. The recombinant AAV capsid protein of any one of claims 43 and 127-143, wherein (X6) represents lie.ATTORNEY DOCKET NO.: 51772-007WO2PATENT147. The recombinant AAV capsid protein of any one of claims 43 and 127-143, wherein (X6) represents Ser.

148. The recombinant AAV capsid protein of any one of claims 43 and 127-147, wherein (X4) represents Lys.

149. The recombinant AAV capsid protein of any one of claims 43 and 127-147, wherein (X4) represents Arg.

150. The recombinant AAV capsid protein of any one of claims 43 and 127-149, wherein (X7) represents Gly.

151. The recombinant AAV capsid protein of any one of claims 43 and 127-149, wherein (X7) represents Pro.

152. A recombinant AAV capsid protein comprising a peptide insert having the amino acid sequence of any one of ID NOs: 17-653.

153. The recombinant AAV capsid protein of any one of claims 1-152, wherein the peptide insert is incorporated into a wild-type AAV capsid protein.

154. The recombinant AAV capsid protein of claim 153, wherein the wild-type AAV capsid protein is a capsid protein that occurs naturally in an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhl 0, or AAVrh74.

155. The recombinant AAV capsid protein of claim 154, wherein the wild-type AAV capsid protein is a capsid protein that occurs naturally in AAV9.

156. The recombinant AAV capsid protein of any one of claims 153-155, wherein the wild-type AAV capsid protein is a VP1, VP2, or VP3 capsid protein.

157. The recombinant AAV capsid protein of claim 156, wherein the wild-type AAV capsid protein is a VP1 capsid protein.

158. The recombinant AAV capsid protein of any one of claims 153-157, wherein the peptide insert is incorporated into the wild-type AAV capsid protein between a pair of consecutive amino acid residues that are located within a variable region (VR) selected from the group consisting of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX.ATTORNEY DOCKET NO.: 51772-007WO2PATENT159. The recombinant AAV capsid protein of claim 158, wherein the peptide insert is incorporated into the wild-type AAV capsid protein between a pair of consecutive amino acid residues that are located within VR-VIII.

160. The recombinant AAV capsid protein of claim 159, wherein the pair of consecutive amino acid residues is selected from the group consisting of (a) amino acids 581 and 582, (b) amino acids 582 and 583, (c) amino acids 583 and 584, (d) amino acids 584 and 585, (e) amino acids 585 and 586, (f) amino acids 586 and 587, (g) amino acids 587 and 588, (h) amino acids 588 and 589, (i) amino acids 589 and 590, (j) amino acids 590 and 591, (k) amino acids 591 and 592, or (I) amino acids 592 and 593, wherein the amino acids are numbered relative to the amino acid sequence of wild-type AAV9 VP1 (SEQ ID NO: 492).

161. An AAV particle comprising the AAV capsid protein of any one of claims 1 -160.

162. The AAV particle of claim 161, wherein the AAV capsid protein encapsulates an AAV genome comprising, in the 5’-to-3’ direction:a) a first inverted terminal repeat (ITR);b) a transgene of interest; andc) a second ITR.

163. The AAV particle of claim 162, wherein the AAV genome further comprises, between the first ITR and the transgene, one or more transcription regulatory elements that modulate expression of the transgene.

164. A method of expressing a transgene in a subject, the method comprising administering to the subject the AAV particle of any one of claims 161-163.

165. The method of claim 164, wherein the AAV particle is administered to the subject by way of intramuscular, intrahepatic, intravenous, intrathecal, intracerebroventricular, intracisternal, intrastriatal, intracerebral, intracerebrospinal, intracranial, intracortical, intradermal, transdermal, parenteral, intranasal, subcutaneous, percutaneous, intratracheal, intraocular, or intravascular administration.

166. The method of claim 165, wherein the AAV particle is administered to the subject by way of intramuscular administration.

167. The method of any one of claims 164-166, wherein the subject is a mammal.

168. The method of claim 167, wherein the subject is a human.ATTORNEY DOCKET NO.: 51772-007WO2PATENT169. A kit comprising the AAV particle of any one of claims 161 -163 and a package insert, wherein the package insert instructs a user of the kit to administer the AAV particle to a subject in accordance with the method of any one of claims 164-168.

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