Novel engineered capsid serotypes of recombinant adeno-associated viral vectors with reduced liver tropism

Abstract

Disclosed herein are engineered adeno-associated viral vectors and methods of their use.

Description

Attorney Docket No.103361-377WO1 NOVEL ENGINEERED CAPSID SEROTYPES OF RECOMBINANT ADENO- ASSOCIATED VIRAL VECTORS WITH REDUCED LIVER TROPISM CROSS-REFERENCE TO RELATED APPLICATION This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No.63 / 583,110, filed September 15, 2023, entitled “NOVEL ENGINEERED CAPSID SEROTYPES OF RECOMBINANT ADENO-ASSOCIATED VIRAL VECTOR WITH REDUCED LIVER TROPISM,” which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government Support under Grant No. CA166590 awarded by the National Institutes of Health. The Government has certain right in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on September 16, 2024, as an .XML file entitled “103361-377WO1_ST26.xml” created on September 16, 2024, and having a file size of 88,211 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD The present disclosure relates to engineered adeno-associated viral vectors and methods of their use. BACKGROUND

[0001] Recombinant adeno-associated virus (rAAV) vectors have found successful application in clinical trials to treat a range of diseases, because they are nonpathogenic with low immunogenicity and long-lasting transgene expression. In addition, rAAVs are widely applied in basic research for gene delivery to multiple tissues including liver, heart, skeleton muscle, brain and eyes. Yet applications of rAAVs for targeting some tissues with systemic delivery have been limited due to low transduction efficiency and high liver tropisms of naturally occurring AAV serotypes. Overcoming rAAVs liver tropism to reach target tissues has required large doses of rAAVs, which can lead to a narrow therapeutic window and has raised safety concerns.

[0002] Modifications of the capsid or the expression cassette are employed to improve efficiency of gene delivery and transgene restrictive expression with either local or systematicAttorney Docket No.103361-377WO1 delivery. For example, selecting more efficient naturally occurring serotypes plus the use of tissue specific promoter or micro-RNA targeting sequence enable specific tissue transduction, but require high dose to achieve therapeutic effects. Moreover, expression of recombinant therapies in off target tissues, particularly in liver can have undesirable consequences and deleterious effects. What are needed are new high transduction efficiency viral vectors that can avoid off target deleterious effects. SUMMARY

[0003] Disclosed are engineered adeno-associated viral vectors and uses thereof.

[0004] In one aspect, disclosed herein are engineered AAV capsid proteins, wherein the one or more amino acid substitutions occurs at residues that promote hepatic intracellular AAV degradation, ablate liver tropism, and / or disrupt AAV binding to hepatocytes.

[0005] In one aspect, disclosed herein are engineered adeno-associated virus (AAV) capsid proteins of any preceding aspect, wherein the one or more amino acid substitutions correspond to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof including, but not limited to one or more amino acid substitutions at residues 501, 713, and / or 714 of SEQ ID NO: 31. In one aspect, the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof. In one aspect, the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31, or any combination thereof. For example, in one aspect, the engineered AAV capsid protein wherein the substitution comprises F503Y, Y708D, and / or K709I. In some aspects, the vector does not transduce liver tissue. For example, in one aspect, the engineered AAV capsid protein comprises the sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or -SEQ ID NO: 44. In one aspect, the AAV capsid protein can comprise a recombinant 2 (Rec2), AAV8, or AAV9 capsid protein.

[0006] Also disclosed herein are engineered AAV of any preceding aspect, wherein the one or more amino acid substitutions is encoded by a nucleic acid substitution corresponding to position 1508, 2127, 2124, or any combination thereof within SEQ ID NO: 2 or SEQ ID NO: 5.Attorney Docket No.103361-377WO1

[0007] Also disclosed herein are vectors comprising the engineered AAV capsid protein of any preceding aspect. For example, disclosed herein are engineered AAV vectors of any preceding aspect, wherein the vector comprises a nucleic acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 18, or a variant thereof.

[0008] In one aspect, disclosed herein are vectors of any preceding aspect, wherein the vector further comprises a transgene (such as, for example, a leptin gene, an IL-15 gene, an IL- 15 / IL-15R complex gene, FGF21 gene, adiponectin gene, a therapeutic gene linking leptin and adiponectin by a 2A sequence, or a synthetic microRNA targeting the leptin). In some embodiments, the vector does not transduce into liver tissue.

[0009] In one aspect, disclosed herein are methods of reducing liver tropism of an adeno- associated virus (AAV) vector, wherein the AAV vector encodes an engineered AAV capsid protein; and wherein the method comprises substituting one or more amino acids corresponding to amino acid residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, including, but not limited to one or more amino acid substitutions at residues 501, 713, and / or 714 of SEQ ID NO: 31.

[0010] Also disclosed herein are methods of reducing liver tropism of an adeno-associated virus (AAV) vector of any preceding aspect, wherein the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof (such as for example, methods wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31). In some aspects, the methods further comprise introducing SEQ ID NO: 2 or SEQ ID NO: 5 into the AAV vector, wherein SEQ ID NO: 2 or SEQ ID NO: 5 comprise a nucleic acid substitution at codon position 1508, 2127, 2124, or any combination thereof, and wherein the nucleic acid substitution occurs at nucleotides that ablate liver tropism or disrupt AAV binding to hepatocytes. In some aspects, the capsid protein is a recombinant 2 (Rec2), AAV8, or AAV9 capsid protein.

[0011] In one aspect, disclosed herein are AAV capsid proteins made by the method of any preceding aspect.

[0012] Also disclosed herein are methods of delivering a gene to adipose tissue of a subject; methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a metabolic disorder or disease (such as, for example diabetes, obesity, metabolic syndrome, congenital leptin deficiency, or lipodystrophy) in a subject; and / or methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer in a subject, theAttorney Docket No.103361-377WO1 methods comprising administering to the engineered AAV vector (such as, for example, a Rec2, AAV8, or AAV9 vector including, but not limited to an AAV vector comprising a Rec2, AAV8, or AAV9 capsid protein) of any preceding aspect. For example, disclosed herein are methods of delivering a gene to adipose tissue of a subject of any preceding aspect; methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a metabolic disorder or disease (such as, for example diabetes, obesity, metabolic syndrome, congenital leptin deficiency, or lipodystrophy) of any preceding aspect; and / or methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer of any preceding aspect, wherein the amino acid substitution occurs at residues that ablate liver tropism or disrupt AAV binding to hepatocytes including, but not limited to F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof (such as for example, methods wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31). In one aspect, disclosed herein are methods of delivering a gene to adipose tissue of a subject of any preceding aspect; methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a metabolic disorder or disease (such as, for example diabetes, obesity, metabolic syndrome, congenital leptin deficiency, or lipodystrophy) of any preceding aspect; and / or methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer of any preceding aspect, wherein the AAV capsid protein comprise the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 44. In some aspects, wherein the AAV vector encodes a transgene or a therapeutic agent. BRIEF DESCRIPTION OF FIGURES

[0013] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0014] Figure 1A shows the schematic of the Rec2 variant V7 with three amino acid residue substitution on Rec2 capsid.

[0015] Figure 1B shows the amino acid sequence of Rec2 variant V7 (SEQ ID NO: 3).

[0016] Figures 2A, 2B, and 2C show the in vivo screening of Rec2 capsid variants. Figures 2A and 2B show the GFP fluorescence in eWAT (Figure 2A) and liver (Figure 2B) at 2-weeksAttorney Docket No.103361-377WO1 post i.p. injection of Rec-2-GFP or V1-7-GFP vectors at the dose of 2E10 vg / mouse. Figure 2C shows the GFP fluorescence ratio (adipose / liver). Data are means ± SEM. Sample size: Rec2- GFP n=5, V1-GFP n=3, V2-GFP n=3, V4-GFP n=3, V5-GFP n=7, V6-GFP n=5, V7-GFP n=7, V8-GFP n=5.

[0017] Figures 3A, 3B, 3C, 3D, and 3E show the intraperitoneal administration of V7 vector selectively transduces visceral adipose tissue and de-targets liver. Figure 3A shows the GFP fluorescence in eWAT and liver at 2-weeks post i.p. injection of Rec2-GFP or V7-GFP vectors. Figure 3B shows the western blotting in a separate experiment two weeks post AAV injection. Figure 3C shows the GFP fluorescence in dosing experiment at 2-weeks post i.p. injection. Figure 3D shows the western blotting in mice receiving high dose V7-GFP. Figure 3E shows the vector genome copy numbers in mice receiving high dose V7-GFP. Doses are indicated in each graph. Data are means±SEM. Sample size (biological replicates): Rec2-GFP 2E10 vg / mouse n=3-8, V7-GFP 2E10 vg / mouse n=8, V7-GFP 4E10 / mouse n=5, V7-GFP 8E10 vg / mouse n=4.

[0018] Figures 4A, 4B, and 4C show the intravenous injection of V7 vector preferentially transduces subcutaneous white adipose tissue and brown adipose tissue while de-targets liver. Figure 4A shows the western blotting at 2- weeks post tail vein injection of AAV8-GFP or V7- GFP. Figure 4B shows the western blotting of tissues from a mouse receiving V7-GFP (8E10 vg / mouse). Molecular weight markers are shown in kDa. Figure 4C shows the vector genome copy numbers in mice receiving high dose of AAV8 or V7-GFP. Doses are indicated in each graph. Data are means±SEM. Sample size (biological replicates): n=3 per group.

[0019] Figures 5A, 5B, 5C, 5D, 5E, 5F, and 5G show the intraperitoneal administration of V7-LEP / ADIPOQ vector rescues the excessive weight gain and impaired glycemic control in ob / ob mice. Figure 5A shows the schematic of the AAV vector containing human leptin and human adiponectin linking by 2A sequence. Figure 5B shows the body weight measurements. Figure 5C shows the area under the curve of body weight. Figure 5D shows the weight gain measurements. Figure 5E shows the area under the curve of weight gain. Figure 5F shows the glucose tolerance test at 8-weeks post AAV injection. Figure 5G shows the area under the curve of glucose tolerance test. Data are means±SEM. Sample size (biological replicates): ob / + buffer n=5, ob / ob GFP 8E10 n=5, ob / ob LEP / ADIPOQ 4E10 n=4, ob / ob LEP / ADIPOQ 8E10 n=4. * P<0.05, ** P<0.01 from One-way ANOVA followed by Tukey’s multiple comparison test.

[0020] Figures 6A and 6B show the intraperitoneal administration of V7-LEP / ADIPOQ vector normalizes body temperature and body composition in ob / ob mice. Figure 6A shows the rectal temperature at 8-weeks post AAV injection. Figure 6B shows the body composition byAttorney Docket No.103361-377WO1 EchoMRI at 9-weeks post AAV injection. Figure 6C shows the relative tissue mass calibrated to body weight at sacrifice 9-weeks post AAV injection. Data are means±SEM. Sample size (biological replicates): ob / + buffer n=5, ob / ob GFP 8E10 n=5, ob / ob LEP / ADIPOQ 4E10 n=4, ob / ob LEP / ADIPOQ 8E10 n=4. * P<0.05, ** P<0.01 from One-way ANOVA followed by Tukey’s multiple comparison test.

[0021] Figure 7 shows the biodistribution of transgene mRNA in a representative mouse of V7-LEP / ADIPOQ 8E10 group, (n=1).

[0022] Figures 8A, 8B, 8C, and 8D show the intraperitoneal administration of V7- LEP / ADIPOQ reverses hyperinsulinemia, hyperglycemia, and hepatic steatosis in ob / ob mice. Figure 8A shows the serum levels of human leptin and human adiponectin. No significant difference between the low dose and high dose. Figure 8B shows the serum level of insulin. Figure 8C shows the serum level of glucose. Figure 8D shows the liver triglyceride content. Data are means±SEM. Sample size (biological replicates): ob / + buffer n=5, ob / ob GFP 8E10 n=5, ob / ob LEP / ADIPOQ 4E10 n=4, ob / ob LEP / ADIPOQ 8E10 n=4. * P<0.05, ** P<0.01 from One-way ANOVA followed by Tukey’s multiple comparison test.

[0023] Figures 9A, 9B, and 9C show the AAV8 capsid with F503Y / Y708D / K709I substitution loses liver-tropism. Figure 9A shows the schematic of AAV8 mutant trans plasmid. Figure 9B shows the GFP fluorescence in liver and eWAT at 2-weeks post i.p. injection of AAV8 or AAV8 mutant vector at 2E10 vg / mouse. Data are means±SEM. Sample size (biological replicates): AAV8 n=3, AAV8 mutant n=4. Figure 9C shows the western blotting of GFP in various tissues upon extended exposure. Molecular weight markers are shown in kDa. DETAILED DESCRIPTION

[0024] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.Attorney Docket No.103361-377WO1

[0025] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0027] The following definitions are provided for the full understanding of terms used in this specification.

[0028] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0029] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood thatAttorney Docket No.103361-377WO1 the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0030] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0031] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0032] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0033] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previouslyAttorney Docket No.103361-377WO1 observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0034] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0035] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0036] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0037] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0038] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.Attorney Docket No.103361-377WO1

[0039] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0040] "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0041] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of'' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0042] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0043] A “nucleic acid” is a chemical compound that serves as the primary information- carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.Attorney Docket No.103361-377WO1

[0044] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.215:403410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0045] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0046] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0047] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “aminoAttorney Docket No.103361-377WO1 acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, β- alanine, β-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3- Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N- Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2′- Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0048] Reference also is made herein to peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length≥100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0049] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.215:403410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0050] Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at leastAttorney Docket No.103361-377WO1 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0051] The term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position. In relation to substitutions, ‘immediately adjacent’ may be to the N-side (‘upstream’) or C-side (‘downstream’) of the amino acid occupying a position (‘the named amino acid’). Therefore, for an amino acid named / numbered ‘X,’ the insertion may be at position ‘X+1’ (‘downstream’) or at position ‘X−1’ (‘upstream’).

[0052] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or greater sequence identity over a certain defined length relative to a reference polypeptide such as, for example, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 44.

[0053] A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and / or binding DNA at a specific binding site.Attorney Docket No.103361-377WO1

[0054] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0055] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0056] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0057] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate,Attorney Docket No.103361-377WO1 metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0058] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of a neurological disease or disorder. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. Methods of Preventing liver tropism of a vector

[0060] Obesity and body fat distribution are important risk factors for type II diabetes, fatty liver, and other metabolic syndromes. Visceral adipose tissue (VAT) plays distinctive roles in metabolic homeostasis and disturbance. The type of obesity characterized by increased VAT is strongly associated with adverse metabolic outcomes whereas accumulation of subcutaneous fatAttorney Docket No.103361-377WO1 is thought to have neutral or even beneficial effects on metabolism. This relates to intrinsic functional differences of adipocytes in different depots, including insulin sensitivity, glucose uptake, rate of lipolysis, and adipokine and cytokine secretion.

[0061] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.

[0062] Adeno-associated viral (AAV) vectors are mainstream delivery platforms in gene therapy because of its safety profile and promising results in clinical trials. Today AAV vectors are utilized in at least 176 clinical trials (phase I, II, and III). AAV has been successfully used to deliver transgenes to liver, heart, skeletal muscle, brain, and eye. Yet the use of AAV-mediated gene transfer to adipose tissue has been limited due to the low transduction efficiency of naturally occurring AAV serotypes. The challenge of lacking efficient AAV platform for adipose gene delivery can be overcome by engineering the viral capsid through DNA shuffling and specific point mutation. Our previous study demonstrates that Rec2 capsid serotype, among a family of engineered hybrid capsid serotypes (Rec1~4) transduces adipose tissue with far superior efficiency than naturally occurring AAV serotypes. One caveat is that Rec2 capsid vector also transduces liver efficiently via intravenous or intraperitoneal administration. In the current study, in order to generate new serotypes with enhanced adipose tropism while eliminating liver tropism, we made several mutations in the capsid of Rec2 by analyzing amino acid sequence of capsid among AAV9, AAV8, Rec2, Rec3, and AAV2. Eight capsid variants (named LC.V1, 2, 3, 4, 5, 6, 7, 8) have been generated, packaged, and tested in vivo. Rec2 variant 7 showed the most tropism for adipose tissue without any tropism for liver tissue. By contrast Rec2 variants V2, V4, V5, V6 all had significant tropism for liver tissue in addition to the tropism for fat tissue. Variant 8 did not exhibited tropism for fat tissue or liver tissue. Compositions

[0063] Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of eachAttorney Docket No.103361-377WO1 various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular engineered adeno-associated viral capsid or vector is disclosed and discussed and a number of modifications that can be made to a number of molecules including the engineered adeno-associated viral capsid or vector are discussed, specifically contemplated is each and every combination and permutation of engineered adeno-associated viral capsid or vector and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0064] In one aspect, disclosed herein are engineered AAV capsid proteins, wherein the one or more amino acid substitutions occurs at residues that promote hepatic intracellular AAV degradation, ablate liver tropism, and / or disrupt AAV binding to hepatocytes.

[0065] In one aspect, disclosed herein are engineered adeno-associated virus (AAV) capsid proteins, wherein the one or more amino acid substitutions correspond to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof including, but not limited to one or more amino acid substitutions at residues 501, 713, and / or 714 of SEQ ID NO: 31. It is understood and herein contemplated that disclosed engineered AAV capsid proteins can comprise variants of SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 31, provided the vartiant also comprises at least one amino acid substitutions at a residue corresponding to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof including, but not limited to one or more amino acid substitutions at residues 501, 713, and / or 714 of SEQ ID NO: 31.

[0066] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences”Attorney Docket No.103361-377WO1 tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or greater sequence identity over a certain defined length relative to a reference polypeptide such as, for example, SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 31.

[0067] As used herein an amino acid residue is said to correspond to another residue or be an equivalent residue when it occupies the same position in a sequence alignment. For example, as shown in the sequence alignment below, residues 501, 713, and 714 of SEQ ID NO: 31 correspond to (i.e., are equivalent) to residues 503, 708, and 709 of SEQ ID NO: 1 and SEQ ID NO: 6. CLUSTAL O(1.2.4) multiple sequence alignment SEQIDNO:1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD 60 SEQIDNO:6 MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD 60 SEQIDNO:31 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60 *********************** ******:******:**:.************* **** SEQIDNO:1 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ 120 SEQIDNO:6 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ 120 SEQIDNO:31 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ 120 ***********************:*******:************:*************** SEQIDNO:1 AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKRLNFGQTGDS 180 SEQIDNO:6 AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDS 180 SEQIDNO:31 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQ-EPDSSAGIGKSGAQPAKKRLNFGQTGDT 179 ****:*********.************ *** .****:****.* ***:**********: SEQIDNO:1 ESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV 240 SEQIDNO:6 ESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV 240 SEQIDNO:31 ESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRV 239 ********:*****.***:* ***:*****:********************* ****** SEQIDNO:1 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ 300 SEQIDNO:6 ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ 300 SEQIDNO:31 ITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQ 299 ***********************.****::***:************************** SEQIDNO:1 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA 360 SEQIDNO:6 RLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA 360 SEQIDNO:31 RLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSA 359 **************.************:*:*.**********:******:**********Attorney Docket No.103361-377WO1 SEQIDNO:1 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFED 420 SEQIDNO:6 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFED 420 SEQIDNO:31 HEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFEN 419 *:**********************:***************************:*:* **: SEQIDNO:1 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW 480 SEQIDNO:6 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNW 480 SEQIDNO:31 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTID-GAATKNQTLKFSVAGPSNMAVQGRNY 478 ******************************* *.:: .* * ** .**..*: *.:*: SEQIDNO:1 LPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSN 540 SEQIDNO:6 LPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSN 540 SEQIDNO:31 IPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLS 538 :***.*********. *****:*** ..:.: ******* *** ***:**:.*:**** . SEQIDNO:1 GILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNT-------AP 593 SEQIDNO:6 GILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNT-------AP 593 SEQIDNO:31 GSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSDGTLAVPFKAQA 598 * ******.:.***.* ..**:*.*************.** ** * *.:.* SEQIDNO:1 QIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPLMGGFGLKHPPPQILIKN 653 SEQIDNO:6 QIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKN 653 SEQIDNO:31 QTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKN 658 * * *: ** *******:***********:******:***********:*********** SEQIDNO:1 TPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSV 713 SEQIDNO:6 TPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSV 713 SEQIDNO:31 TPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV 718 *********:**:.*******************************************..* SEQIDNO:1 DFAVNTEGVYSEPRPIGTRYLTRNL 738 SEQIDNO:6 DFAVNTEGVYSEPRPIGTRYLTRNL 738 SEQIDNO:31 EFAVNTEGVYSEPRPIGTRYLTRNL 743 :***********************

[0068] In one aspect, the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof. In one aspect, the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31, or any combination thereof. For example, in one aspect, the engineered AAV capsid protein wherein the substitution comprises F503Y, Y708D, and / or K709I. In some aspects, the vector does not transduce liver tissue. For example, in one aspect, the engineered AAV capsid protein comprises the sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or -SEQ ID NO: 44. In one aspect, the AAV capsid protein can comprise a recombinant 2 (Rec2), AAV8, or AAV9 capsid protein.Attorney Docket No.103361-377WO1

[0069] Also disclosed herein are engineered AAV capsid proteins, wherein the one or more amino acid substitutions is encoded by a nucleic acid substitution corresponding to position 1508, 2127, 2124, or any combination thereof within SEQ ID NO: 2 or SEQ ID NO: 5.

[0070] In one aspect disclosed herein are vectors comprising any of the engineered AAV capsid proteins disclosed herein. In particular, disclosed herein are adeno-associated virus (AAV) recombinant 2 (Rec2), AAV8, or AAV9 vectors comprising capsid proteins comprising one or more amino acid substitutions that reduces liver tropism (such as, for example a nucleic acid substitution at position 1508, 2124 and / or 2127 of SEQ ID NO: 2 or SEQ ID NO: 5 resulting in a substitution at residue 503, 708, and / or 709 (or the equivalent residues 501, 713, and / or 714 for AAV9) including, but not limited to F503Y, F503T, F503X, Y708D, Y708E, K709I, K709L, and / or K709G substitution for Rec2 and AAV8 and F501Y, Y713D, and / or K714I for AAV9). For example, in one aspect, the capsid protein of the engineered AAV vector comprises a substitution F503Y, Y708D, and / or K709I. In some aspects, the vector does not transduce liver tissue. For example, in one aspect, the engineered AAV capsid protein comprises the sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 44.

[0071] In one aspect, disclosed herein are AAV capsid proteins or vectors comprising said capsid proteins, wherein the one or more amino acid substitutions occurs at residues that promote hepatic intracellular AAV degradation or disrupt AAV binding to hepatocytes.

[0072] Also disclosed herein are engineered AAV vectors, further comprising a transgene (such as, for example, a leptin gene, an IL-15 gene, an IL-15 / IL-15R complex gene, FGF21 gene, adiponectin gene, a therapeutic gene linking leptin and adiponectin by a 2A sequence, or a synthetic microRNA targeting the leptin). In some embodiments, the vector does not transduce into liver tissue.

[0073] In some aspects, the transgene can be operatively linked to a promoter or a tissue specific promoter operatively linked to a RNA silencing element.

[0074] As used herein, plasmid or viral vectors are agents that transport a nucleic acid of interest, such as leptin or IL-15 into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus,Attorney Docket No.103361-377WO1 neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non- dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.

[0075] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

[0076] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0077] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.Attorney Docket No.103361-377WO1

[0078] Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site- specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No.6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0079] Herein is disclosed a novel engineered hybrid serotype Rec2 vector which leads to high transduction of adipose tissue superior to naturally occurring serotypes (AAV1 and AAV8) and other engineered serotypes (Rec1, Rec3, Rec4) when the vectors are injected directly to the fat pads at a dose 1 to 2 orders lower than previously reported studies. Interestingly, the administration route substantially influences the tropism and efficacy of Rec2 vector. Intravenous administration of Rec2 vector primarily transduces liver at the doses ranging from 2x109to 2x1010vg per mouse. In contrast, oral administration of Rec2 vector leads to preferential transduction of brown fat with absence of transduction in the gastrointestinal track at the doses lower than 2x1010vg per mouse. To improve gene delivery to VAT, it was firstly determined whether Rec2 vector, through IP administration, a simple and less invasive procedure, could efficiently transduce VAT. Accordingly, in one aspect, disclosed herein are methods of targeting a nucleic acid based treatment to visceral adipose tissue in a subject comprising administering to the subject any of the engineered adeno-associated virus (AAV) vectors disclosed herein (for example, the disclosed Rec2 vector). For example, disclosed herein are methods of targeting a nucleic acid based treatment to visceral adipose tissue in a subject comprising administering to the subject an engineered adeno-associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a leptin transgene or IL-15 transgene operatively linked to a promoter; and wherein the AAV vector is administered intraperitoneally.

[0080] To prevent transgene expression in a tissue, a new AAV expression plasmid was generated that contained two expression cassettes: one comprising the transgene (for example, leptin, adiponectin, or IL-15), and the other cassette used the tissue-specific promoter to drive a specific RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) targeting a regulatory element (such as, for example, WPRE) sequence which only exists in the transgene expression cassette.

[0081] The inserted genes in viral and retroviral usually contain promoters, and / or enhancers to help control the expression of the desired gene product (for example, a chicken β-actin (CBA) promoter). A promoter is generally a sequence or sequences of DNA that function when in aAttorney Docket No.103361-377WO1 relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0082] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. a ^-actin promoter, such as, for example, the chicken ^-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein. Thus, in one aspect, disclosed herein are engineered adeno-associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a transgene operatively linked to a promoter (such as, for example, the chicken ^-actin promoter); and wherein the second cassette comprises a tissue-specific and microRNA (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette.

[0083] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio.3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio.4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.Attorney Docket No.103361-377WO1

[0084] The promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0085] In certain embodiments the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

[0086] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin. By using, in the second cassette of the engineered AAV vectors disclosed herein, tissue specific promoters operatively linked to the RNA silencing element, expression of an RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) which targets the regulatory element in the first cassette can be used to limit off-target transduction of the transgene (for example leptin, adiponectin, and / or IL-15). For example, disclosed herein are engineered AAV vectors, further comprising a first expression cassette comprising a regulatory element (such as, for example, a woodchuck posttranscriptional regulatory element (WPRE) sequence) and a transgene (such as, for example, a leptin gene, an IL-15 gene, an IL-15 / IL-15R complex gene, FGF21 gene, adiponectin gene, a therapeutic gene linking leptin and adiponectin by a 2A sequence, or a synthetic microRNA targeting the leptin) operatively linked to a promoter and a second expression cassette comprising a tissue specific promoter; wherein the first cassette comprises a regulatory element and a transgene (such as, for example, leptin, adiponectin, and / or IL-15) operatively linked to a promoter (such as, for example CBA); and wherein the second cassette comprises a tissue specific promoter and a RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette. It is understood and herein contemplated that the tissue-specific promoter in the second cassette is operatively linked to the RNA silencing element such that transcription of the RNA silencingAttorney Docket No.103361-377WO1 element only occurs when in the appropriate tissue for the tissue specific promoter. Thus, in one aspect, disclosed herein are methods of expressing a transgene in the VAT while restricting off- target transduction of the transgene comprising administering to subject an engineered adeno- associated virus (AAV) vectors comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a transgene (such as, for example, leptin, adiponectin, and / or IL-15) operatively linked to a promoter (such as, for example CBA); and wherein the second cassette comprises a tissue specific promoter and a RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette; and wherein the RNA silencing element is operatively linked to the tissue specific promoter.

[0087] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

[0088] The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes ß-galactosidase, and green fluorescent protein.

[0089] In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cellAttorney Docket No.103361-377WO1 line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

[0090] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet.1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol.5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0091] In one aspect, the disclosed AAV vectors can also comprise a regulatory element (such as, for example the woodchuck posttranscriptional regulatory element (WPRE)) which would not be found in the host endogenously and only present on the first cassette. The regulatory element can be operatively linked to the promoter and / or transgene in the first cassette such that disruption of the regulatory element (such as, through the use of a microRNA, including, but not limited to smRNA, shRNA, RNAi, siRNA, and / or piRNA) turns off expression of the transgene. In one aspect, disruption of the regulator element can be via an RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) which is specific for the regulator element. By linking said RNA silencing element to a tissue specific promoter, expression of the transgene can be prevented from occurring in tissues where expression of the transgene would be deleterious. Thus, in one aspect, disclosed herein are engineered adeno-associated virus (AAV) vectors comprising two expression cassettes; wherein the first cassette comprises a regulatory element (such as, for example WPRE) and a transgene (such as, for example, leptin, adiponectin, and / or IL-15) operatively linked to a promoter (such as, for example, the chicken β-actinAttorney Docket No.103361-377WO1 promoter); and wherein the second cassette comprises a tissue specific promoter and a RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette (see Figure 2A); wherein the RNA silencing element is operatively linked to the tissue specific promoter. Homology / identity

[0092] It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 percent sequence identity to the stated sequence. Those of skill in the art readily understand how to determine the homology / sequence identity of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0093] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol.48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0094] The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol.183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. Delivery of the compositions to cells

[0095] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viralAttorney Docket No.103361-377WO1 vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier. Nucleic acid based delivery systems

[0096] Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.53:83-88, (1993)).

[0097] As used herein, viral vectors are agents that transport a transgene into a cell (such as, for example a brain cell) without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the engineered viral vectors disclosed herein are derived from adeno-associated vectors (AAV). Adeno-associated viral vectors

[0098] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb of DNA and wild type AAV is known to stably insert into chromosome 19 (such as, for example at AAV integration site 1 (AAVS1)). In one aspect, the disclosed AAV vector can comprise a selectable marker such as, for example, the gene encoding the green fluorescent protein, GFP or enhanced GFP (EGFP).

[0099] In one aspect, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0100] Typically, the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs (typically from AAV2), or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-Attorney Docket No.103361-377WO1 specific expression. United states Patent No.6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0101] The disclosed vectors thus are designed to persist in the nucleus as extrachromosomal and not integrate into the human genome. This ability to persist without integration adds to the safety of these vectors.

[0102] The inserted genes in viral and retroviral usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. In vivo / ex vivo

[0103] As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject’s cells in vivo and / or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

[0104] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. Expression systems

[0105] The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.Attorney Docket No.103361-377WO1 Viral Promoters and Enhancers

[0106] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

[0107] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio.3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio.4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers f unction to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0108] The promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0109] In certain embodiments the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferredAttorney Docket No.103361-377WO1 promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

[0110] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0111] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. Markers

[0112] The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes ß-galactosidase, and green fluorescent protein.

[0113] In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genesAttorney Docket No.103361-377WO1 necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

[0114] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet.1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol.5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin. Peptides Protein variants

[0115] As discussed herein there are numerous variants of the AAV capsid proteins (VP1, VP2, and VP3) that are known and herein contemplated. In addition, to the known functional strain variants there are derivatives of the AAV capsid proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and, in some instances, can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, therebyAttorney Docket No.103361-377WO1 producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.

[0116] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and / or glycosylation.

[0117] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.Attorney Docket No.103361-377WO1

[0118] Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

[0119] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86

[1983] ), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.

[0120] It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology / identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0121] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol.48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0122] The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol.183:281-306, 1989.Attorney Docket No.103361-377WO1

[0123] It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

[0124] As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein from which that protein arises are also known and herein disclosed and described.

[0125] It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 1 and Table 2. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site-specific way.

[0126] Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH--, --CH2S--, --CH2--CH2 --, --CH=CH-- (cis and trans), --COCH2 --, -- CH(OH)CH2--, and --CHH2SO—(These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A. F., Vega Data (March 1983), Vol.1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp.463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (--CH2NH--, CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H2--S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (--COCH2--);Attorney Docket No.103361-377WO1 Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (--COCH2--); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (--CH(OH)CH2--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH2--); and Hruby Life Sci 31:189-199 (1982) (--CH2--S--); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is --CH2NH--. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0127] Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

[0128] D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L- lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. Pharmaceutical carriers / Delivery of pharmaceutical products

[0129] As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0130] The compositions (i.e., the engineered AAV vectors disclosed herein) may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, by cerebrospinal fluid injection, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth viaAttorney Docket No.103361-377WO1 delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0131] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein.

[0132] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins,Attorney Docket No.103361-377WO1 dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). Pharmaceutically Acceptable Carriers

[0133] The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0134] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0135] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0136] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0137] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, cerebrospinal fluid injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. TheAttorney Docket No.103361-377WO1 disclosed engineered viral vectors can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0138] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0139] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0140] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0141] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Therapeutic Uses

[0142] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in theAttorney Docket No.103361-377WO1 regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389. A typical daily dosage of the antibody used alone might range from about 1 µg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above. Methods of Treating Diseases and / or Disorders Associated with Metabolism or Cancer

[0143] The disclosed AAV vectors can be used for targeted gene disruption, restoration, and modification in any animal that can undergo these events. Gene modification and gene disruption refer to the methods, techniques, and compositions that surround the selective removal, integration, or alteration of a gene or stretch of chromosome in an animal. In general, a cell is transformed with a vector which is designed for homologous recombination with a region of a particular chromosome contained within the cell, as for example, described herein. This homologous recombination event can produce a chromosome which has exogenous DNA introduced, for example in frame, with the surrounding DNA. This type of protocol allows for very specific mutations, to be introduced into the genome contained within the cell.

[0144] In one aspect, disclosed herein are methods of reducing liver tropism of an adeno- associated virus (AAV) vector, wherein the AAV vector encodes an engineered AAV capsid protein; and wherein the method comprises substituting one or more amino acids corresponding to amino acid residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, including, but not limited to one or more amino acid substitutions at residues 501, 713, and / or 714 of SEQ ID NO: 31.

[0145] Also disclosed herein are methods of reducing liver tropism of an adeno-associated virus (AAV) vector, wherein the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof (such as for example, methods wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31). In some aspects, the methods further comprise introducing SEQ ID NO: 2 or SEQ ID NO: 5 into the AAV vector,Attorney Docket No.103361-377WO1 wherein SEQ ID NO: 2 or SEQ ID NO: 5 comprise a nucleic acid substitution at codon position 1508, 2127, 2124, or any combination thereof, and wherein the nucleic acid substitution occurs at nucleotides that ablate liver tropism or disrupt AAV binding to hepatocytes. In some aspects, the capsid protein is a recombinant 2 (Rec2), AAV8, or AAV9 capsid protein. In one aspect, disclosed herein are AAV capsid proteins made by any method disclosed herein.

[0146] In one aspect, it is understood, that the disclosed vectors can be used to rescue deficiencies of gene / protein expression that lead to a disease. In one aspect, the AAV vectors disclosed herein can be used to treat obesity, diabetes, congenital leptin deficiency, lipodystrophy (such as, for example, congenital generalized lipodystrophy (also known as Beradinelli-Seip syndrome), familial partial lipodystrophy, acquired partial lipodystrophy (also known as Barraquer-Simons syndrome), acquired generalized lipodystrophy, centrifugal abdominal lipodystrophy, lipoatrophia annularis, localized lipodystrophy, and HIV-associated lipodystrophy), or metabolic syndromes. Thus, in one aspect, disclosed herein are methods of delivering a gene to adipose tissue of a subject comprising administering to the subject any of the engineered AAV vectors disclosed herein. Also disclosed herein methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing diabetes, obesity, metabolic syndrome, or lipodystrophy in a subject comprising administering to the subject any of the engineered AAV vectors disclosed herein. For example, disclosed herein methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing diabetes, obesity, metabolic syndrome, or lipodystrophy in a subject comprising administering to the subject an AAV vector comprising an engineered AAV capsid protein (such as, for example, a Rec2, AAV8, or AAV9 vector including, but not limited to an AAV vector comprising a Rec2, AAV8, or AAV9 capsid protein) comprising an amino acid substitution occurs at residues that ablate liver tropism or disrupt AAV binding to hepatocytes including, but not limited to F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof (such as for example, methods wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31). In some aspects, the AAV capsid protein comprises the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 44. In some aspects, the engineered vector encodes a therapeutic agent.Attorney Docket No.103361-377WO1

[0147] It is further understood and herein contemplated that activation of NK cell precursors and NK cells resident in adipose tissue can facilitate treatment of a cancer as the NK cells will attack the cancer. The activation of NK precursors and NK cells in adipose tissue can be accomplished by providing IL-15 to the NK cells. The engineered AAV vectors disclosed herein being tissue specific can provide this IL-15.

[0148] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and / or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non- small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma / glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers.

[0149] Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing cancer and / or metastasis (such as, for example, lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, and / or rectal cancer) in a subject comprising administering to the subject any of the engineered AAV vector of disclosed herein. For example, disclosed herein methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer in a subject comprising administering to the subject an AAV vector comprising an engineered AAV capsid protein (suchAttorney Docket No.103361-377WO1 as, for example, a Rec2, AAV8, or AAV9 vector including, but not limited to an AAV vector comprising a Rec2, AAV8, or AAV9 capsid protein) comprising an amino acid substitution occurs at residues that ablate liver tropism or disrupt AAV binding to hepatocytes including, but not limited to F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G relative to SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof (such as for example, methods wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I relative to SEQ ID NO: 31). In some aspects, the AAV capsid protein comprises the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 44. In some aspects, the engineered vector encodes a therapeutic agent.

[0150] Thus, in one aspect, the AAV vectors disclosed herein can be used to treat a cancer in a subject by administering an IL-15 encoding AAV vector described herein. Accordingly, in one aspect, disclosed herein are methods of treating obesity, diabetes, cancer, lipodystrophy, and / or metabolic syndromes in a subject comprising administering to the subject an engineered adeno-associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element (such as, for example WPRE) and a leptin transgene and / or IL-15 transgene operatively linked to a promoter (such as the CBA promoter); and wherein the second cassette comprises a tissue specific promoter and a RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette; wherein the RNA silencing element is operatively linked to the tissue specific promoter.

[0151] Also disclosed herein are methods of targeting a nucleic acid based treatment to visceral adipose tissue in a subject comprising administering to the subject an engineered adeno- associated virus (AAV) vector comprising two expression cassettes; wherein the first cassette comprises a regulatory element and a leptin transgene operatively linked to a promoter; and wherein the second cassette comprises a tissue specific promoter (such as, for example, albumin) and a RNA silencing element (such as, for example, microRNA, including, but not limited to, smRNA, shRNA, RNAi, siRNA, and / or piRNA) that targets the regulatory element in the first expression cassette; wherein the RNA silencing element is operatively linked to the tissue specific promoter; and wherein the AAV vector is administered intraperitoneally.Attorney Docket No.103361-377WO1

[0152] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0153] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0154] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES

[0155] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0156] Example 1: Development of an adipose-tropic AAV capsid ablating liver tropism

[0157] AAV vectors are mainstream delivery platforms in gene therapy, yet AAV-mediated gene transfer to adipose tissue is underdeveloped due to low efficiency of natural AAVs. The present disclosure demonstrates that an engineered capsid Rec2 displayed improved adipo- tropism but with the caveat of liver transduction. To generate highly adipo-tropic capsid, a Rec2 capsid was modified by site-specific mutagenesis and found the variant V7 with F503Y, Y708D and K709I substitution to harbor highly selective adipo-tropism while diminishing liver transduction. Intraperitoneal injection favored transduction to visceral fat while intravenous administration favored subcutaneous fat. Intraperitoneal administration of V7 vector harboringAttorney Docket No.103361-377WO1 human leptin and adiponectin as single transcript normalized the metabolic dysfunction of ob / ob mice at a low dose. Moreover, introducing the same mutagenesis to AAV8 capsid diminished liver transduction suggesting F503, Y708 and K709 critical for liver transduction. The Rec2.V7 vector provides a powerful tool for basic research and potent vehicle for adipose-targeting gene therapy.

[0158] Adeno-associated virus (AAV) is a single-stranded DNA virus with non-enveloped capsid. Recombinant AAV vectors are replication defective AAV particles serving as an invaluable tool for genetic manipulation of specific tissues and have been used safely in clinical trials. AAV has become the most popular delivery system for gene therapy due to their non- pathogenic and broad tropism being used in more than 200 ongoing clinical trials. AAV vectors have been successful in delivering transgenes to liver, heart, skeletal muscle, brain, and eye. In contrast, adipose tissue, one of largest organs in the body, playing a pivotal role in physiology and pathologies of multiple diseases, is under investigated in the field of AAV gene therapy largely due to the low transduction efficiency in adipose tissue of natural AAV serotypes. A comprehensive literature review finds AAV8 the most efficient capsid to transduce adipose tissue among natural AAV serotypes that have been evaluated. However, AAV8 transduces liver far more efficiently than adipose tissue, necessitating methods to restrict off-target transgene expression in the liver.

[0159] It is known that modulating AAV capsids affects the viral tissue tropism and the functional role of a provided transgene. To specifically manipulate the adipose tissue, a recombinant AAV (rAAV) vector was generated to achieve highly efficient transduction of adipose tissue. The engineered hybrid capsid Rec2, generated by capsid shuffling of AAV8 and rhesus 20 (rh20), can transduce white adipose tissue (WAT) and brown adipose tissue (BAT) far more efficiently than the natural (AAV1, 8, and 9) and engineered capsids (Rec1, Rec3, Rec4) being compared via direct fat pad injection. One limitation is the transduction of liver when the Rec2 vector is injected systemically. To prevent transgene expression in the liver, the AAV vector genome was engineered to contain two expression cassettes: one uses ubiquitous hybrid cytomegalovirus enhancer / chicken β-actin (CBA) promoter to drive the transgene, and the other uses the liver-specific albumin promoter to drive a specific microRNA targeting woodchuck posttranscriptional regulatory element (WPRE) sequence which only exists in the transgene expression cassette. The efficacy of this adipose-specific (AS) dual-cassettes design (named AS / Rec2) was demonstrated.Attorney Docket No.103361-377WO1

[0160] Although a significant advancement in AAV technology targeting the adipose tissue, the AS / Rec2 system has drawbacks: 1) Rec2 vectors are present in the liver and therefore bearing the risk of viral vector-associated toxicity at extremely high dose; 2) the dual-cassette design is unable to restrict transgene expression in the liver if the transgenes are miRNA and shRNA; 3) the introduction of the second cassette further exacerbate the size limitation of a transgene (<1750 bp in the current dual-cassette design) and thereby excluding its use for many genes and CRISPR / Cas9 genome editing; 4) restriction of liver transgene expression is less efficient in conditions where the albumin promoter is weakened. For example, it was found that AS / Rec2-Fgf21 led to 4.5-fold increase of transgene Fgf21 expression in the liver in 24-months- old mice. In contrast, the same AS / Rec2-Fgf21 vector did not alter Fgf21 expression in the liver of young mice. As such, a capsid with improved adipose tropism and truly diminishing liver tropism is highly desirable.

[0161] To overcome the limitations of the AS / Rec2 system, Rec2 was used herein as a template for capsid engineering to generate truly adipose-tropic AAVs while ablating liver tropism. AAV capsid mediates cellular entry and post entry processing (intracellular trafficking), that can dictate the tropism of the vector and level of transgene expression. Capsid modification in many serotypes have been reported to achieve tropism alteration. Herein, the Rec2 capsid was modified by site-specific mutagenesis and generated 8 variants of Rec2 capsid. In vivo screening found the Rec2 capsid variant with F503Y, Y708D and K709I substitution (referred to Rec2.V7, V7 in short) led to substantial transduction of adipose tissue while no transgene expression was detected in the liver. The V7 capsid was further characterized via different routes of administration and doses and conducted a proof-of-concept study in the congenital leptin deficiency model ob / ob mice. RESULTS Rec2 capsid modification results in adipo-tropic V7 vector with ablating liver tropism

[0162] To search for adipo-tropic vector while ablating liver tropism, 8 variants were generated by mutagenesis in Rec2 capsid sequence (Table 3), named V1~8. V3 failed to package and was discarded. For in vivo screening, pseudo-AAV vectors: cis plasmid with AAV2 ITRs and a single cassette to express enhanced green fluorescent protein (referred to GFP), were packaged to native Rec2, and Rec2 capsid variants using the same methods of triple transfection and purification. To screen the variants, tissue lysates were used for direct GFP fluorescence count read-out at a 485-nm excitation wavelength with cut-off 528-nm emission wavelength.Attorney Docket No.103361-377WO1 Adipose tissue is distributed in various location, including visceral or intraperitoneal and subcutaneous fat depots. Commonly referred visceral fat depots in rodent include epididymal (male) or gonadal (female) white adipose tissue (eWAT or gWAT), mesentery white adipose tissue (mWAT), and retroperitoneal white adipose tissue (rWAT). Subcutaneous fat depots include inguinal white adipose tissue (iWAT) and intrascapular brown adipose tissue (BAT). Intraperitoneal (i.p.) delivery

[0163] Upon initial in vivo screening at the dose of 2E10 viral genome (vg) / mouse via i.p. injection, V7 capsid vector (Figure 1) led to substantial GFP expression in the eWAT (Figure 2A) while no GFP fluorescence was detected in the liver (Figure 2B). Other variants or vectors at the same dose either failed to alter liver tropism or lost adipose transduction (Figure 2A-C). For example, the variant 1, namely Rec2.V1 capsid lost transduction efficiency to adipose tissue by 76% via intra-adipose injection and by 83% via intraperitoneal injection compared to native Rec2 capsid. Moreover, V1 capsid showed 50% lower transduction efficiency to liver compared to native Rec2 capsid. These variants were deemed failure of the original intention to generate adipose-specific AAV serotypes, and later were screened for transduction to other tissues. Surprisingly, robust transduction of the rat brain was observed in many brain structures including striatum (targeting structure), cortex, septum, hippocampus, thalamus, subthalamic nucleus, substantia nigra, etc. As such, V7 capsid was selected for further characterization. Mutations in V7 are indicated in Figure 1A and highlighted in its capsid amino acid sequence in Figure 1B.

[0164] Next, i.p. experiments were repeated at the dose of 2E10 vg / mouse and verified that GFP fluorescence was detected in eWAT but not in liver in mice receiving V7 vector (Figure 3A). In contrast, GFP fluorescence was detected in both eWAT and liver from mice receiving parent Rec2 vector at same dose. Consistent with GFP fluorescence reading, Western blotting also confirmed V7 vector-mediated transgene expression in adipose tissue, but not liver, while Rec2 resulted in transgene expression in both WAT and liver (Figure 3B). Because V7 vector resulted in lower GFP expression in WAT compared to that of native Rec2 at the dose of 2E10 vg / mouse (Figures 3A and 3B), a dosing experiment was conducted to validate the adipo- tropism of V7 and to estimate the adipose transduction efficiency. Increasing the dose of V7 vector to 4E10 vg / mouse improved transgene expression in eWAT, reaching approximately 40% of native Rec2 at 2E10 vg / mouse (Figure 3C). Of note, i.p. injection of V7 vector led to similar GFP expression in the mesenteric WAT (mWAT) compared to native Rec2. Importantly, V7Attorney Docket No.103361-377WO1 vector via i.p. injection displayed specific WAT transgene expression with no GFP fluorescence detected in liver, pancreas, heart, skeletal muscle, spleen, and kidney even at higher dose of 8E10 vg / mouse (Figure 3C). In contrast, Rec2 vector led to strong GFP expression in both WAT and liver, and noticeable amount of GFP in pancreas (Figure 3C). The adipo-tropism of V7 vector was confirmed by a different investigator who packaged the V7 vector and repeated the high dose experiment (Figure 3D). Next, the viral vector genome copy numbers were quantified in eWAT, liver, and other peripheral tissues from mice receiving i.p. injection of V7 vector at high dose (8E10 vg / mouse). V7 vector genome was mostly detected in eWAT (56791 ± 1302 copy number / 50 ng genomic DNA) and barely found in liver (629 ± 8 copy number / 50 ng genomic DNA) (Figure 3E). Intravenous (i.v.) administration

[0165] Next, the biodistribution of V7 vector was examined by i.v. administration. AAV8 is reported to be the most efficient natural serotype for liver-directed gene transfer. AAV8 was used as a positive control in this experiment. Two doses (low dose of 4E10, high dose of 8E10 vg / mouse) were administered via tail vein injection. Western blotting showed V7 vector transduced inguinal WAT (iWAT) and BAT at both low dose and high dose. AAV8 at high dose resulted in GFP protein level in iWAT and BAT comparable to the levels of V7 at low dose (Figure 4A). As expected, AAV8 yielded robust GFP expression in liver with high dose (Figure 4A). In contrast, no liver transgene expression was detected in mice receiving V7 vector even at high dose (Figure 4A). Interestingly, both V7 and AAV8 vector did not yield GFP expression in eWAT. Next, V7 biodistribution was further examined in additional tissues from a mouse receiving high dose V7 vector by Western blotting. GFP expression was detected in fat depots (iWAT, BAT and rWAT) but not in liver, spleen, pancreas, lung, intestine, heart, and kidney (Figure 4B). To further confirm diminished liver tropism of V7 vector, total RNA was isolated from iWAT and liver, and quantified transgene GFP mRNA levels by qRT-PCR. Saline injection served as negative control. Cycle threshold (Ct) values of GFP from mice receiving V7 and AAV8 vectors were subtracted from saline control. β-actin was house-keeping gene for qRT-PCR. As shown in Table 4, GFP mRNA expression was detected in iWAT but not liver in mice receiving V7. In contrast, GFP mRNA was detected in both liver and iWAT for AAV8. In addition, the vector genome copy numbers were measured in iWAT and liver of mice receiving i.v. injection of either V7 or AAV8 vector at high dose (8E10 vg / mouse). V7 vector genome was mostly detected in iWAT (127216 ± 14778 genome copy number / 50 ng genomic DNA) and wasAttorney Docket No.103361-377WO1 found barely in liver (1034 ± 16 genome copy number / 50 ng genomic DNA) (Figure 4C). Consistent with protein and mRNA data, the vast majority AAV8 vector was accumulated in liver (199099 ± 3877 genome copy number / 50 ng genomic DNA) vs in iWAT (25111 ± 3332 genome copy number / 50 ng genomic DNA) (Figure 4C). V7-based dual adipokines gene therapy rescues metabolic syndromes in congenital leptin deficiency

[0166] To investigate the therapeutic potential of V7 vector, a proof-of-concept study was conducted in ob / ob mice, a model for congenital leptin deficiency in humans. Previously, it was reported that a single injection of AS / Rec2-leptin (4E10 vg / mouse, i.p.) completely rescued the metabolic abnormalities including obesity, hyperphagia, low energy expenditure, fatty liver, glucose intolerance, and hyperinsulinemia. To facilitate clinical translation, a new gene therapy was developed based on the V7 capsid. Because V7 vector ablates liver tropism, the liver- restricting cassette in the AS / Rec2 system becomes unnecessary and therefore avoiding concerns of introducing exogenous regulatory elements that interfere with endogenous microRNA machinery especially at high expression levels. Moreover, absence of transduction to the liver by V7 vector could minimize the severe adverse events related to liver toxicity that has been a major challenge to the field of systemic gene therapies. In addition, AAV gene therapy allows delivery of more than one therapeutic gene. Adiponectin is a major adipokine produced exclusively by adipocytes and plays a crucial role in glucose and lipid metabolism, inflammation, and oxidative stress. Adiponectin level is decreased in obesity and lipodystrophy. It was contemplated that a gene therapy delivering both leptin and adiponectin will alleviate the metabolic abnormalities in lipodystrophy more efficiently than delivery of leptin alone. The LEP / ADIPOQ transgene was constructed using the 2A sequence to link human leptin cDNA and human adiponectin cDNA. The 2A sequence allows expression of the two adipokines from one transcript. Such design has been successfully applied to the IL15 / Il15Rα gene therapy in a previous study (Xiao, R., Mansour, A.G., Huang, W., Chrislip, L.A., Wilkins, R.K., Queen, N.J., Youssef, Y., Mao, H.C., Caligiuri, M.A., and Cao, L. (2019). Adipocytes: A Novel Target for IL-15 / IL-15Ralpha Cancer Gene Therapy. Mol Ther 27, 922-932. 10.1016 / j.ymthe.2019.02.011). The LEP / ADIPOQ gene was cloned to the ubiquitous CBA expression plasmid and packaged to V7 capsid (Figure 5A).

[0167] Male ob / ob mice, 7 weeks of age, were randomized to receive an i.p. administration of V7-LEP / ADIPOQ (4E10 vg or 8E10 vg / mouse) or V7-GFP (8E10 vg / mouse). Age-matchedAttorney Docket No.103361-377WO1 male ob heterozygote (ob / +) from the colony was used as a control for normal metabolic phenotypes. The body weight of ob / ob mice receiving V7-GFP were observed to increase rapidly over 9 weeks period of the study (Figure 5B and 5C). In contrast, the weight gain of ob / ob mice was leveled off two weeks after injection of V7-LEP / ADIPOQ at both doses (4E10 and 8E10 vg / mouse) (Figures 5B, 5C, 5D, and 5E). High dose (8E10 vg / mouse) of V7- LEP / ADIPOQ almost brought the body weight back to the starting point (Figure 5B and 5C). When the weight gain per week was compared among four groups, the LEP / ADIPOQ gene delivery showed a dose dependent effect on restricting body weight gain of ob / ob mice (Figure 5E). High dose (8E10 vg / mouse) ob / ob group was comparable to heterozygote group over the duration of the study (Figures 5D and 5E), indicating complete rescue of excessive weight gain in ob / ob mice. A glucose tolerance test was performed at 7 weeks post AAV injection. V7- LEP / ADIPOQ treatment, both low dose and high dose, completely normalized the fast-state glucose disposal (Figures 5F and 5G). Abnormal low body temperature is a known feature of ob / ob mice and can be restored by leptin replacement. It is reported that ob / ob mice do not exhibit reduced BAT thermogenic capacity, but thermoregulatory thresholds are shifted downward. Leptin replacement can normalize ob / ob body temperature to wildtype mice level by reducing heat loss. V7-LEP / ADIPOQ treatment corrected the abnormal low body temperature of ob / ob mice independent of doses (Figure 6A). Body composition was determined by echoMRI at 9 weeks post AAV injection. V7-LEP / ADIPOQ treatment significantly reduced adiposity and increased lean mass compared to ob / ob mice treated with V7-GFP, with high dose trending to larger changes of body composition (Figure 6B). Consistent with echoMRI data, at sacrifice 9 weeks after V7 vectors administration, the relative weight of adipose depots, such as eWAT, iWAT, rWAT and BAT in V7-LEP / ADIPOQ mice (both doses) were all reduced significantly compared to ob / ob mice treated with V7-GFP ranging from 40% to 70% (Figure 6C). Relative liver mass was reduced by 50% with both doses of V7-LEP / ADIPOQ (Figure 6C).

[0168] The biodistribution of the V7-LEP / ADIPOQ vector was examined in major peripheral organs / tissues plus brain of a representative mouse from the ob / ob V7-LEP / ADIPOQ 8E10 vg / mouse group by qRT-PCR. The primers were designed to detect human ADIPOQ transgene without cross reaction to mouse Adipoq. Human ADIPOQ transgene expression was detected in adipose tissues located exclusively in intraperitoneal cavity, including eWAT, mWAT and rWAT (Figure 7). No transgene mRNA was detected in brain, liver, or other peripheral organs / tissues (Figure 7). Serum levels of human adipokines were measured with ELISAs without cross reaction to mouse counterparts. The serum levels of human leptin andAttorney Docket No.103361-377WO1 adiponectin showed a dose dependent effect although not reaching statistical significance (Figure 8A). Human leptin level reached 0.86 ± 0.15 ng / ml and 1.65 ± 0.6 ng / ml with low and high dose, respectively (Figure 8A), a comparable level to that of wildtype mice serum level or Rec2-dual cassette leptin vector, and exceeding the serum level of mouse leptin in the ob / + control mice (0.70 ± 0.05 ng / ml). Human adiponectin serum level was 2.5 ± 0.8 ng / ml and 4.4 ± 2.0 ng / ml with low and high dose, respectively (Figure 8A). ob / ob mice exhibited hyperinsulinemia, whose serum insulin levels were robustly reduced by V7-LEP / ADIPOQ treatment ranging from 3 to 9 folds dependent of dose (Figure 8B). Of note, high dose V7- LEP / ADIPOQ brought down serum insulin of ob / ob mice to the level comparable to ob heterozygote mice. Serum glucose level was also decreased significantly with both doses of V7- LEP / ADIPOQ in comparison to ob / ob V7-GFP mice. High dose, in particular, completely normalized serum glucose to the level of ob heterozygote mice (Figure 8C). Hepatic triglyceride content was significantly decreased in V7-LEP / ADIPOQ mice, both doses, when compared to ob / ob V7-GFP control, indicating liver steatosis was significantly diminished. High dose reduced the hepatic lipid content to a comparable level of ob heterozygote mice (Figure 8D). AAV8 capsid with F503Y, Y708D and K709I substitution ablates liver tropism

[0169] To investigate whether the mutations in generating V7 capsid are critical for liver- tropism in other AAV capsid, the F503Y, Y708D and K709I substitutions were made in AAV8 capsid (Figure 9A). GFP reporter vector was packaged to the resulting AAV8 mutant capsid as in the V7 experiment and i.p. injected to wildtype mice at the dose of 2E10 vg / mouse. Robust GFP fluorescence was detected in the livers of mice receiving native AAV8 vector while negligible GFP fluorescence was found in the livers of AAV8 mutant vector (Figure 9B). Biodistribution was further assessed by Western blotting. Strong GFP expression in the liver was observed in mice receiving native AAV8, which was completely diminished in mice receiving AAV8 mutant vector even upon extended exposure (Figure 9C). In contrast, GFP was detected in the eWAT of mice receiving AAV8 mutant but at reduced level compared to native AAV8 (Figure 9C). DISCUSSION

[0170] The Rec2 platform has become the leading AAV vector system for adipose gene delivery and has been used by multiple labs worldwide (with or without liver-restricting dual- cassette design). However, while being aware of its drawbacks, the present disclosure putsAttorney Docket No.103361-377WO1 considerable efforts on generating new capsids with improved adipo-tropism while de-targeting liver and other tissues. Herein, the V7 capsid with three mutations (F503Y, Y708D and K709I) on Rec2 capsid achieved selective transduction of adipose tissue while eliminating transgene expression in the liver and other tissues when non-restricted expression cassette was used. The adipo-tropism of the V7 vector were validated by assessments of vector genome, transgene mRNA, and transgene protein. Based on current knowledge, there is no AAV capsid that exclusively transduces adipose tissue while ablating liver tropism has been reported. As such, the V7 capsid may represent the first of this new class of AAV capsid.

[0171] Rec2 vector has shown differential tropism based on delivery route. I.V. administration leads to primarily liver transduction. I.P. injection transduces both liver and visceral WAT. Surprisingly, oral administration of Rec2 transduces the intrascapular BAT but not gastric intestinal track or liver. V7 vector clearly displayed a distinct biodistribution compared to the parent Rec2 capsid. Systemic administration of V7 vector led to no liver transduction while the transduction of fat depots depended on the administration routes: i.p. delivery favoring visceral fat depots while i.v. administration favoring subcutaneous WAT and BAT. The different pattern of fat transduction by i.v. versus i.p. administration is likely attributed to mouse anatomy of adipose tissue and their vasculature. Previous study reported that i.v. administration of AAV8- or AAV9-GFP yielded varying transduction efficiency to fat depots with highest GFP expression in mWAT, BAT and iWAT, and least GFP expression in eWAT. Of note, the doses used for AAV8 or AAV9 vectors were 5E12 vg / mouse, much higher than the dose of 8E10 vg / mouse used in the V7 study. It is possible that increasing dose of V7 vector can improve the transduction of visceral fat by i.v. injection. Alternatively, combining i.v. and i.p. administration may achieve whole body adipose transduction, worthy of further investigation.

[0172] One of the main objectives of this disclosure was to eliminate the liver tropism of Rec2 capsid. Rec2 is a chimeric capsid generated via shuffling between capsid of AAV8 and rh20. AAV8 and rh20 have been shown to efficiently transduce liver and brain tissue, respectively. The amino acid sequences of capsid among AAV2, AAV8, AAV9, Rec2, Rec3 were analyzed, and chose the locations to make point mutations or insertional mutation in Rec2 capsid based on the functional readout data in the publications for AAV2 capsid residues. Capsid (cap) gene encodes structural viral proteins VP1-3 via an alternative splicing and initiation. All three VP proteins share identical carboxyl-terminal amino acids. Tissue tropism of AAV depends on the specific interactions between capsid-specific structures and cellularAttorney Docket No.103361-377WO1 glycans and receptors. AAV2 makes a cell entry by using membrane-associated heparan sulfate proteoglycan (HSPG) as its primary receptor. Other candidates have also been reported to participate in AAV attachment. The atomic structure of AAV2 capsid has been determined at a resolution of 3.0 micron. Based on the computer modeling, some genetic capsid modifications can be tolerated for AAV2 capsid. For example, previous work showed heparin binding loci that include a cluster between position 561 and 591, which was re-confirmed by recent comprehensive AAV capsid fitness landscape study. Further studies identify residues R585 and R588 as two primary residues responsible for HSPG binding. Additional studies report genetic mutation on position 587 and surrounding residues for vector re-targeting, and map some of amino acid residues at extreme C-terminus, which is essential for liver transduction by AAV2 and AAV9. Thus, mutations on Rec2 capsid were made in the region equivalent to the region surrounding R585 and R588 of AAV2 capsid and to screen new capsids with adipose tropism and de-targeting liver. Additional modifications were expanded beyond the R585~R588 region (Table 3). Regarding liver transduction, the F503Y, Y708D and K709I substitution resulted in ablating liver tropism in both Rec2 and AAV8 capsids suggesting F503, Y708 and K709 together play a critical role in hepatic transduction of Rec2 and AAV8 vectors. There is further testing whether these substitutions can be applied to additional commonly used AAV capsids such as AAV9. AAV9 capsid shares 85% amino acid homology with Rec2 or AAV8. Importantly, F503, Y 708, and K709 are conserved among Rec2, AAV8 and AAV9 capsids. Notably, although being able to maintain adipo-tropism, the efficiency of V7 capsid was lower than Rec2 capsid but still higher than AAV8. It has been contemplated that the domains in Rec2 capsid critical for the vector entry into adipose tissue and liver likely reside at C-terminal of VP of Rec2 capsid and are possibly overlapped partially. As such, when the three-residue substitution (F503Y / Y708D / K709I) disrupts liver binding, it likely affects adipose binding domain as well.

[0173] In addition to serving as a tool for basic research on adipose biology, adipo-tropic AAV vectors can provide an avenue to developing gene therapies for genetic disorders and disease conditions involving adipose tissue. The pathology of adipose tissue is most often associated with obesity or diabetes in humans. Six forms of human monogenic obesity are primarily due to inactivating mutations in leptin-melanocortin signaling. Congenital leptin deficiency is a monogenic defect found in adipose tissue, causing severe obesity, hyperphagia, and hyperinsulinemia. Moreover, leptin deficiency shares many symptoms with lipodystrophy. Leptin replacement therapy is necessary in patients with homozygous leptin mutations orAttorney Docket No.103361-377WO1 acquired leptin deficiency derived from congenital or acquired lipodystrophy. Recombinant leptin protein analog (Metreleptin) is an approved drug but requires frequent injection for life and cost an average of $565,000 per patient per year. Gene therapy based on an adipo-tropic AAV vector may have significant advantages compared to current standard of care. This adipo- tropic AAV platform has enabled lower overall systemic doses to patients, resulting in fewer toxicities and much lower cost of goods for systemically delivered gene therapies.

[0174] Prior preclinical study has demonstrated using the Rec2 / dual cassette vector in ob / ob mice (Huang, W., Liu, X., Queen, N.J., and Cao, L. (2017). Targeting Visceral Fat by Intraperitoneal Delivery of Novel AAV Serotype Vector Restricting Off-Target Transduction in Liver. Mol Ther Methods Clin Dev 6, 68-78.10.1016 / j.omtm.2017.06.002). Here, a proof-of- concept was conducted for a V7-based gene therapy in the ob / ob mice mirroring the published study. A single i.p. injection of V7 vector carrying human leptin and human adiponectin normalized excessive weight gain, glucose intolerance, abnormal low core temperature, hyperinsulinemia, and hepatic steatosis in the ob / ob mice. Noteworthy, V7 vector at the low dose of 4E10 vg / mouse resulted in the circulating level of human leptin comparable to the normal level of mouse leptin in WT mice. Importantly, the dose of V7 vector was 50-100 folds lower than standard gene therapies but achieved higher level of leptin expression. Because restoring the circulating leptin level to 10% of normal levels is sufficient to mitigate the metabolic syndromes associated with congenital leptin deficiency, the dose of V7-LEP / ADIPOQ could be further reduced. Moreover, it was contemplated that a gene therapy delivering both leptin and adiponectin will alleviate the metabolic abnormalities more efficiently than leptin alone in lipodystrophy models because adiponectin promotes adipogenesis.

[0175] Studies are underway to determine the percentage of adipocytes being transduced by V7 vector and whether V7 vector can transduce other cell populations residing in the adipose tissue (e.g. immune cells, progenitor cells), which are critical for some applications such as knock-down endogenous adipose genes and investigations on interaction between mature adipocytes and stromal vascular fraction (SVF). In a preliminary study, mature adipocytes were isolated and SVF from eWAT of mice receiving IP injection of V7-GFP (8E10 vg / mouse) and performed qRT-PCR to quantify GFP mRNA. The majority of GFP mRNA was detected in the mature adipocytes while the GFP expression in SVF was 4.4% of that in the mature adipocytes, showing V7 vector mainly transduces mature adipocytes. Moreover, ablating liver tropism of V7 capsid presumably reduces viral vector-associated toxicity. Because severe adverse events were rarely observed in clinical and nonclinical studies and mostly occurred after administration ofAttorney Docket No.103361-377WO1 very high dose (>1 to 3E14 vg / kg i.v.), the current disclosure uses low doses (<3E12 vg / kg) was unable to assess toxicity. Admittedly, biodistribution of transgene mRNA was obtained from one representative mouse of V7-LEP / ADIPOQ 8E10 group (Figure 7).

[0176] In summary, the data demonstrate that the engineered Rec2 capsid variant V7 with F503Y, Y708D and K709I substitution achieved selective transduction to adipose tissue and ablating liver tropism upon systemic administration in mice. These substitutions can be applied to AAV8 capsid for liver de-targeting indicating F503, Y708 and K709 critical for liver transduction. A single administration of V7-LEP / ADIPOQ led to reversal of metabolic abnormalities in a murine model of congenital leptin deficiency at a dose substantially lower than those commonly used for AAV systemic gene therapy. Further characterization and refinement of V7 vector platform provides a powerful tool kit enabling genetic manipulation of adipose tissue in basic research and gene therapy for adipose-originated diseases and beyond. EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Mice

[0177] Wildtype C57 / BL6 mice, 12-18 weeks of age, were purchased from Charles River Laboratories.7-week-old ob / ob mice on a C57 / BL6 background were purchased from Jackson Laboratories. All mice were housed in a temperature-controlled room (20-23 ºC) with a 12-hr. light / 12 hr. dark cycle. Mice were maintained on a standard chow diet (7912 rodent chow, Teklad), with free access to food and water ad libitum. Male mice were used in all studies. All use of animal was approved and in accordance with the Ohio State University Animal Care and Use Committee (IACUC), protocol approval 2012A00000060-R3. METHOD DETAILS Capsid modifications by site-directed mutagenesis Rec2 capsid V7 mutagenesis

[0178] The Rec2 capsid was mutagenized at sites indicated in Figure1A and full capsid sequence is listed in Figure 1B. We name Rec2 variants as V1 to V8 in numerical order. GeneArt site-directed mutagenesis Plus kit (A14604, Invitrogen) was used to carry out the designated mutagenesis. Rec2 capsid sequence is based on published data (Charbel Issa, P., De Silva, S.R., Lipinski, D.M., Singh, M.S., Mouravlev, A., You, Q., Barnard, A.R., Hankins, M.W., During, M.J., and Maclaren, R.E. (2013). Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina. PLoSAttorney Docket No.103361-377WO1 One 8, e60361.10.1371 / journal.pone.0060361). Primers for mutagenesis was synthesized by Sigma-Aldrich: F503Y (ACAACAATAGCAACTATGCCTGGACTGCTGG (SEQ ID NO: 32), CCAGCAGTCCAGGCATAGTTGCTATTGTTGT (SEQ ID NO: 33)); Y708D and K709I (TACACCTCCAACTACGACATATCTACAAGTGTGGA (SEQ ID NO: 34), TCCACACTTGTAGATATGTCGTAGTTGGAGGTGTA (SEQ ID NO:35)).

[0179] Mutagenesis was set up and carried out according to the manufacturer’s instruction, and the nucleotide replacement at each site was confirmed by sequencing at the Core Facility of The Ohio State University, Comprehensive Cancer Center. The whole capsid was also sequenced and confirmed without unintended mutation. AAV8 capsid mutation

[0180] Rec2 capsid was generated from capsid domain shuffling between rh20 and AAV8 whose C-terminal including those three amino acids mutagenized for V7 is identical to AAV8 capsid C-terminal. As such, AAV8 capsid mutagenesis of F503Y, Y708D and K709I was conducted as described for V7 mutagenesis. AAV vectors packaging

[0181] The rAAV vector backbone contains CBA (hybrid cytomegalovirus–chicken β-actin) promoter, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal flanked by AAV2-inverted terminal repeats. Reporter gene—enhanced green fluorescent protein (referred as GFP) or therapeutic gene—human leptin cDNA and human adiponectin cDNA linked with a 2A sequence (referred as LEP / ADIPOQ) was cloned into polylinker sites of cis-plasmid. LEP / ADIPOQ sequence was synthesized by gBlock of IDT. Plasmids used for viral packaging were prepared by using EndoFree plasmid Maxi and Mega Kit (Qiagen). Human embryonic kidney 293 cells were co-transfected with three plasmids—rAAV plasmid or cis-plasmid, AAV trans-plasmid encoding rep and cap genes including Rec2, Rec2.V1 to V8, AAV8, and AAV8 mutant, and adenoviral helper pF Δ6—using standard CaPO4transfection. AAV was purified from the cell lysate by ultracentrifugation through an iodixanol density gradient (OptiPrep Density Gradient Medium, D1556, Sigma). AAV was tittered using quantitative PCR using Step OnePlus Real-Time PCR System (Applied Biosystems) with the Power SYBR Green PCR Master Mix (Applied Biosystems#A25742). Administration of AAV vectorsAttorney Docket No.103361-377WO1

[0182] AAV vectors were administered into mice either through i.p. injection in 150 µL AAV dilution buffer or via tail vein injection in 50 µL AAV dilution buffer. Doses ranging from 2E10 to 8E10 vg / mouse were used and specified in each experiment in figure legend. GFP content measurement

[0183] GFP content was measured. In brief, tissues were homogenized in ice-cold RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with proteinase inhibitors cocktail, followed by briefly sonication. The mixture was then spun at 13,000 rpm for 15 min at 4 °C. The supernatant was collected. GFP fluorescence in 150 µL supernatant was measured with microreader (Synergy H1, Bio Tek Instrument) using a 485-nm excitation wavelength and cut-off 528-nm emission wavelength. GFP content was represented as readings after subtracting auto-fluorescence from GFP-null corresponding tissue and calibrated by either protein content in the samples or wet tissues weight. ob / ob mice experiment

[0184] Male homozygous ob / ob mice (JAX 000632 B6.Cg-Lepob / J), seven weeks old, were randomly assigned to receive V7-GFP or V7-LEP / ADIPOQ. Sex and age-matched heterozygous ob / + mice received AAV buffer as another control. Body weight and food intake were monitored weekly over the period of 9 weeks. One mouse from V7-LEP / ADIPOQ 4E10 group and one mouse from V7-LEP / ADIPOQ 8E10 group were excluded due to technical errors of administration. Glucose tolerance test (GTT)

[0185] Eight weeks after i.p. administration of AAV vectors, mice were i.p. injected with glucose solution (1mg glucose per gram body weight) after overnight fast with water ad libitum. Blood was collected from the tail at various time points and the blood glucose concentrations were measured with a portable glucose meter (Bayer Contour Next). Rectal temperature measurement

[0186] Temperature probe (Physitemp, model BAT-12) topped with lubricant (white petroleum jelly) was used to measure the rectal temperature of ob / ob mice at 8-weeks after i.p. administration of AAV vectors.Attorney Docket No.103361-377WO1 Body composition by EchoMRI

[0187] EchoMRI was used to measure body composition of fat and lean masses in live mice without anesthesia at 9-weeks post AAV injection. EchoMRI imaging was performed with a 3- in-1 Analyzer (EchoMRI LLC, Houston, TX) according to manufacturer instructions at Small Animal Imaging Core of The Dorothy M. Davis Heart & Lung Research Institute, Ohio State University. Mice were subjected to a 5-Gauss magnetic field and whole-body masses of fat, lean, free water, and total water were determined during separate cycles by manufacturer software comparison to a canola oil standard. Serum harvest and analysis

[0188] Trunk blood was collected at euthanasia, clotted on ice, and centrifuged at 12,000 rpm for 15 min at 4°C. The serum was collected and stored at -20°C until further analysis. R&D Systems DuoSet ELISA kits were used to assay serum human leptin (R&D Systems, #DY398) and human adiponectin / Acrp30 (R&D Systems, # DY1065). Mouse serum insulin was measured with ELISA kit from ALPCO (ALPCO #80-INSMSU-E01). A Caymen Chemical colorimetric assay was used to determine serum glucose levels (#10009582). Liver lipid extraction and triglycerides assay

[0189] Hepatic lipid was extracted from 50 mg of liver tissue from each animal by chloroform / methanol (2:1 v / v), followed by rinse in 50 mM NaCl and CaCl2(0.36M) / Methanol (1:1 v / v). An aliquot of the extract was mixed with Triton X100 and cold acetone, then was dried up and redissolved in PBS. Hepatic triglyceride quantification was performed with a Caymen Chemical triglyceride assay kit (Caymen Chemical, #10010303). Immunoblotting

[0190] Tissues were homogenized in ice-cold Pierce RIPA buffer containing 1x Roche Phosstop and Calbiochem protease inhibitor cocktail III, then was subjected to brief sonication on ice. Tissues lysates were spun at 13,000 rpm for 15 min at 4 °C. The supernatant was collected, and the protein concentration was assayed with BCA protein assay kit (Piece). 20 µg of protein from each sample was loaded and separated by gradient gel (4-20%, Mini-PROTEAN TGX, Bio-Rad), then transferred to a nitrocellulose membrane (BIO-RAD). Blots were incubated overnight at 4 °C with the following primary antibodies, β-actin (Cell SignalingAttorney Docket No.103361-377WO1 #4970, 1:1000), GFP (Santa Cruz Biotechnology, sc-9986 HRP, 1:1000). Chemiluminescence signal was detected and visualized by LI-COR Odyssey Fc imaging system (LI-COR Biotechnology, Lincoln, NE). Quantification analysis was carried out with image studio software version 5.2 (LI-COR Biotechnology). Quantitative RT-PCR

[0191] Total RNA was isolated using the RNeasy Mini kit (QIAGEN) with RNase-free DNase treatment. cDNA was reverse transcribed using Taqman Reverse Transcription Reagents (Applied Biosystems). qPCR was carried out on StepOnePlus Real-Time PCR System using Power SYBR Green PCR Master Mix (Applied Biosystems). Primers were designed to detect GFP (GACTTCTTCAAGTCCGCCAT (SEQ ID NO: 36) , TGTGGCTGTTGTAGTTGTACTC (SEQ ID NO: 37)), ADIPOQ (AGGCAGGAAAGGAGAACCT (SEQ ID NO: 38), GTGGAGCCATCATAGTGGT (SEQ ID NO: 39)), Gapdh (TCCCACTCTTCCACCTTCGA (SEQ ID NO: 40), TGCTGTAGCCGTATTCATTGTCA (SEQ ID NO: 41)), and Actinb (ACCCGCGAGCACAGCTT (SEQ ID NO: 42), ATATCGTCATCCATGGCGAACT (SEQ ID NO: 43)). Data were calibrated to Actinb and quantified using the 2-∆∆CTmethod. Viral vector copy number measurement

[0192] To assess vector biodistribution, viral vector copy number was measured as previously described. Briefly, total DNA from various tissues was isolated using DNesay Blood and Tissue kit (Qiagen). GFP fragment was amplified and quantified by qPCR to determine the copy number of viral particle.50 ng of DNA from each sample was used for real-time PCR with either duplicate or triplicate. Mouse nucleic genomic DNA fragment of GAPDH gene was used as control for mouse genetic DNA. Genomic DNA of untreated animals was also used as negative control for the comparison. Data are expressed as copies of V7-GFP / 50 ng of genomic DNA. Quantification and Statistical analysis

[0193] Data are expressed as means ± SEM. GraphPad Prism 9 was used to analyze data. One-way ANOVAs with Tukey’s post hoc test was utilized for comparisons between three or four groups. Time course data (BWs, GTT) were analyzed using a mixed ANOVA and area under the curve calculations were performed where applicable. Normality was tested using the Shapiro-Wilk method. * Indicates p value < 0.05; ** indicates p value < 0.01 in figures.Attorney Docket No.103361-377WO1

[0194] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. TABLESAttorney Docket No.103361-377WO1TABLE 3. Genetic modification of Rec2 capsid.A yield above 1E11 per 150 mm plate is considered as good packaging whereas yield below 1E10 per 150 mm plate is considered as poor packaging. TABLE 4. qRT-PCR data of i.v. administration.Attorney Docket No.103361-377WO1Neg: GFP Ct ≥ the Ct value of no reverse transcriptase controlAttorney Docket No.103361-377WO1 SEQUENCES BOLD nucleotides and amino acids indicate where substitution mutations are located. 1. SEQ ID NO: 1 – Amino acid sequence of Rec2 capsid protein MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 2. SEQ ID NO: 2 - Nucleic acid sequence of Rec2 capsid protein ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATT CGCGAGTGGTGGGACCTGAAACCTGGAGCCCCCAAGCCCAAGGCCAACCAGCAGA AGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTC AACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGC ACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTAT AACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGG CAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTC TGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATC ACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGACAGGCCAGCAGCCCG CGAAAAAGAGACTCAACTTTGGGCAGACTGGCGACTCAGAGTCAGTGCCCGACCCT CAACCAATCGGAGAACCCCCCGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGC TGCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT AGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCAC CACCAGCACCCGAACCTGGGCCCTCCCCACCTACAACAACCACCTCTACAAGCAAA TCTCCAACGGGACATCGGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCAttorney Docket No.103361-377WO1 ACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGAC TGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAA GCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCG CCAATAACCTTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGT ACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCA TGATTCCTCAGTACGGGTACCTGACTCTGAACAACGGCAGTCAGGCCGTGGGCCGT TCCTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAAC TTTGAGTTCAGCTACCAGTTTGAGGACGTGCCTTTTCACAGCAGCTACGCGCACAGC CAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCT CGGACTCAGTCCACGGGAGGTACCGCAGGAACTCAGCAGTTGCTATTTTCTCAGGC CGGGCCTAATAACATGTCGGCTCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACC GGCAGCAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTG GACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCA TCGCTATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACGGGATC CTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCAT GCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATAC GGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGT CAACAGGCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGC AGGGGCCCATCTGGGCCCAGATTCCTCACACGGACGGGGACTTCCACCCGTCTCCG CTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACAC GCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCAT CACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAG GAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATC TACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCAT TGGCACCCGTTACCTCACCCGTAATCTGTAA 3. SEQ ID NO: 3 – Amino acid sequence of Rec2.V7 capsid ^ Rec2 capsid protein with F503Y, Y708D, and K709I substitution mutations. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFAttorney Docket No.103361-377WO1 NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLM GGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYDISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 4. SEQ ID NO: 4 – Nucleic acid sequence of Rec2.V7 capsid ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATT CGCGAGTGGTGGGACCTGAAACCTGGAGCCCCCAAGCCCAAGGCCAACCAGCAGA AGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTC AACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGC ACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTAT AACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGG CAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTC TGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATC ACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGACAGGCCAGCAGCCCG CGAAAAAGAGACTCAACTTTGGGCAGACTGGCGACTCAGAGTCAGTGCCCGACCCT CAACCAATCGGAGAACCCCCCGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGC TGCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT AGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCAC CACCAGCACCCGAACCTGGGCCCTCCCCACCTACAACAACCACCTCTACAAGCAAA TCTCCAACGGGACATCGGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGC ACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGAC TGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAA GCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCG CCAATAACCTTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGT ACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCA TGATTCCTCAGTACGGGTACCTGACTCTGAACAACGGCAGTCAGGCCGTGGGCCGT TCCTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAAC TTTGAGTTCAGCTACCAGTTTGAGGACGTGCCTTTTCACAGCAGCTACGCGCACAGCAttorney Docket No.103361-377WO1 CAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCT CGGACTCAGTCCACGGGAGGTACCGCAGGAACTCAGCAGTTGCTATTTTCTCAGGC CGGGCCTAATAACATGTCGGCTCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACC GGCAGCAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTATGCCTG GACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCA TCGCTATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACGGGATC CTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCAT GCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATAC GGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGT CAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGC AGGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGC TGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACG CCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATC ACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGG AAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACGACATATCT ACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATT GGCACCCGTTACCTCACCCGTAATCTGTAA 5. SEQ ID NO: 5 – Nucleic acid sequence of AAV8 capsid ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATT CGCGAGTGGTGGGCGCTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAA AGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTC AACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGC ACGACAAGGCCTACGACCAGCAGCTGCAGGCGGGTGACAATCCGTACCTGCGGTAT AACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGG CAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTC TGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATC ACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAACAGCCCG CCAGAAAAAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTTCCAGACCCT CAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGC TGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT AGTTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCAC CACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAAAttorney Docket No.103361-377WO1 TCTCCAACGGGACATCGGGAGGAGCCACCAACGACAACACCTACTTCGGCTACAGC ACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGAC TGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAA GCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCG CCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCG TACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTC ATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACG CTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAA CTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAG CCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTC TCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAG GTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTAC CGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTG GACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCA TCGCTATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACGGGATC CTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCAT GCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATAC GGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGT CAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGC AGGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGC TGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACG CCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATC ACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGG AAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCT ACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATT GGCACCCGTTACCTCACCCGTAATCTGTAA 6. SEQ ID NO: 6 - Amino acid sequence of AAV8 capsid MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFAttorney Docket No.103361-377WO1 DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 7. SEQ ID NO: 7 – Amino acid sequence of Rec2 capsid protein with F503Y substitution MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 8. SEQ ID NO: 8 - Amino acid sequence of Rec2 capsid protein with Y708D substitution MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLAttorney Docket No.103361-377WO1 FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 9. SEQ ID NO: 9 – Amino acid sequence of Rec2 capsid protein with K709I substitution MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 10. SEQ ID NO: 10 – Amino acid sequence of Rec2 capsid protein with F503Y and Y708D substitutions MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYAttorney Docket No.103361-377WO1 GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 11. SEQ ID NO: 11 – Amino acid sequence of Rec2 capsid protein with F503Y and K709I substitutions. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 12. SEQ ID NO: 12 – Amino acid sequence of Rec2 capsid protein with Y708D and K709I substitutions. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPLAttorney Docket No.103361-377WO1 MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 13. SEQ ID NO: 13 – Amino acid sequence of Rec2 capsid protein with a F503T substitution. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNTAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 14. SEQ ID NO: 14 – Amino acid sequence of Rec2 capsid protein with a F503K substitution. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNKAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNLAttorney Docket No.103361-377WO1 15. SEQ ID NO: 15 – Amino acid sequence of Rec2 capsid protein with a Y708E substitution. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYEKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 16. SEQ ID NO: 16 – Amino acid sequence of Rec2 capsid protein with a K709L substitution. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYLSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 17. SEQ ID NO: 17 – Amino acid sequence of Rec2 capsid protein with a K709G substitution. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGAttorney Docket No.103361-377WO1 NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKTGQQPAKKR LNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVF TDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLL FSQAGPNNMSAQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLA NPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNRQGALPGMVWQNRDVYLQGPIWAQIPHTDGDFHPSPL MGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYGSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 18. SEQ ID NO: 18 – Nucleic acid sequence of AAV8 capsid protein with F503Y, Y708D, and K709I substitutions. ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATT CGCGAGTGGTGGGCGCTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAA AGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTC AACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGC ACGACAAGGCCTACGACCAGCAGCTGCAGGCGGGTGACAATCCGTACCTGCGGTAT AACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGG CAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTC TGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATC ACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAACAGCCCG CCAGAAAAAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTTCCAGACCCT CAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGC TGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT AGTTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCAC CACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAA TCTCCAACGGGACATCGGGAGGAGCCACCAACGACAACACCTACTTCGGCTACAGC ACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGAC TGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAA GCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCG CCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGAttorney Docket No.103361-377WO1 TACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTC ATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACG CTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAA CTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAG CCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTC TCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAG GTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTAC CGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTATGCCT GGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGC ATCGCTATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACGGGAT CCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCA TGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATAC GGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGT CAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGC AGGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGC TGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACG CCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATC ACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGG AAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACGACATATCT ACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATT GGCACCCGTTACCTCACCCGTAATCTGTAA The bold nucleotides indicate where amino acid codes are changed by mutagenesis. 1) 1508 T->A (TTT->TAT) or Phe(F)->Tyr(Y) (F503Y) 2) 2124 T->G (TAC->GAC) or Tyr(Y)->Asp(D) (Y708D) 3) 2127 A->T (AAA->ATA) or Lys(K)->Iso-lle(I) (K709I) 19. SEQ ID NO: 19 – Amino acid sequence of AAV8 capsid protein with F503Y, Y708D, and K709I substitutions. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNAttorney Docket No.103361-377WO1 WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 20. SEQ ID NO: 20 – Amino acid sequence of AAV8 capsid with F503Y substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 21. SEQ ID NO: 21 – Amino acid sequence of AAV8 capsid with Y708D substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSAttorney Docket No.103361-377WO1 QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 22. SEQ ID NO: 22 – Amino acid sequence of AAV8 capsid with K709I substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 23. SEQ ID NO: 23 – Amino acid sequence of AAV8 capsid with F503Y and Y708D substitutions. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNSAttorney Docket No.103361-377WO1 LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 24. SEQ ID NO: 24 – Amino acid sequence of AAV8 capsid with F508Y and K709I substitutions. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNYAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 25. SEQ ID NO: 25 – Amino acid sequence of AAV8 capsid with Y708D and K709I substitutions. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPAttorney Docket No.103361-377WO1 LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYDISTSVDFAVNTEGVYSEPRPIGTRYLTRNL 26. SEQ ID NO: 26 – Amino acid sequence of AAV8 capsid with F503T substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNTAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 27. SEQ ID NO: 27 – Amino acid sequence of AAV8 capsid with F503K substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNKAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNLAttorney Docket No.103361-377WO1 28. SEQ ID NO: 28 – Amino acid sequence of AAV8 capsid with Y708E substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYEKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 29. SEQ ID NO: 29 – Amino acid sequence of AAV8 capsid with K709L substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNTAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYLSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 30. SEQ ID NO: 30 – Amino acid sequence of AAV8 capsid with K709G substitution. MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGAttorney Docket No.103361-377WO1 NLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKR LNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQ VFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQT LGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNTAWTAGTKYHLNGRNS LANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATE EYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYGSTSVDFAVNTEGVYSEPRPIGTRYLTRNL 31. SEQ ID NO: 31 - Amino acid sequence of AAV9 capsid MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGY KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEF QERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGS LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYF PSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT IDGAATKNQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSDGTLAVPFKAQAQT GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHP PPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL 32. SEQ ID NO: 44 - Amino acid sequence of AAV9 capsid with F501Y, Y713D, and K714I substitutions. MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGAttorney Docket No.103361-377WO1 NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKR LNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNW HCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQV FTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTIDGAATKNQTLK FSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEYAWPGASSWALNGRNSLMN PGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYG QVATNHQSDGTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDG NFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYDISNNVEFAVNTEGVYSEPRPIGTRYLTRNL

Claims

Attorney Docket No.103361-377WO1 CLAIMS What is claimed is:

1. An engineered adeno-associated virus (AAV) capsid protein comprising an amino acid substitution at one or more amino acid residues that ablate liver tropism or disrupt AAV binding to hepatocytes.

2. The engineered AAV capsid protein of claim 1, wherein the one or more amino acid substitutions correspond to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6, or any combination thereof.

3. The engineered AAV capsid protein of claim 1 or 2, wherein the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G, or any combination thereof.

4. The engineered AAV capsid protein of claim 3, wherein the amino acid substitution comprises a F503Y, Y708D or Y708E, and K709I.

5. The engineered AAV capsid protein of any one of claims 1-4, wherein the one or more amino acid substitutions is encoded by a nucleic acid substitution corresponding to position 1508, 2127, 2124, or any combination thereof within SEQ ID NO: 2 or SEQ ID NO:

5.

6. The engineered AAV capsid protein of any one of claims 1-4, wherein the capsid protein is a recombinant 2 (Rec2) or AAV8 capsid protein.

7. The engineered capsid protein of any of claims 1-4, wherein the capsid protein comprises an AAV9 capsid protein and wherein the amino acid substitutions corresponding to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6 comprise residues 501, 713, and / or 714 of SEQ ID NO:

31.

8. The engineered AAV9 capsid protein of claim 7, wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I, or any combination thereof 9. The engineered AAV capsid protein of any of claims 1-8, wherein the capsid protein comprise the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,Attorney Docket No.103361-377WO1 SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 44, or at variant thereof.

10. An adeno-associated virus (AAV) vector encoding the capsid protein of any one of claims 1-9.

11. The AAV vector of claim 9, wherein the vector comprises a nucleic acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 18, or a variant thereof.

12. The AAV vector of claim 10 or 11, wherein the AAV vector does not transduce into liver tissue.

13. The AAV vector of any one of claims 10-12, wherein the AAV vector comprises a transgene.

14. The AAV vector of claim 13, wherein the transgene is a leptin gene.

15. The AAV vector of claim 13, wherein the transgene is an IL-15 gene or an IL-15 / IL15R complex gene.

16. The AAV vector of claim 13, wherein the transgene is a FGF21 gene.

17. The AAV vector of claim 13, wherein the transgene is a therapeutic gene linking leptin and adiponectin by a 2A sequence.

18. The AAV vector of claim 13, wherein the transgene is a synthetic microRNA targeting the leptin.

19. A method of reducing liver tropism of an adeno-associated virus (AAV) vector, wherein the AAV vector encodes an engineered AAV capsid protein; and wherein the method comprises substituting one or more amino acids corresponding to amino acid residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO:

6.

20. The method of claim 19, wherein the amino acid substitution comprises F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G, or any combination thereof.Attorney Docket No.103361-377WO1 21. The method of claim 20, wherein the amino acid substitution comprises a F503Y, Y708D or Y708E, and K709I.

22. The method of any of claims 19-21, the method further comprising introducing SEQ ID NO: 2 or SEQ ID NO: 5 into the AAV vector, wherein SEQ ID NO: 2 or SEQ ID NO: 5 comprise a nucleic acid substitution at codon position 1508, 2127, 2124, or any combination thereof, and wherein the nucleic acid substitution occurs at nucleotides that ablate liver tropism or disrupt AAV binding to hepatocytes.

23. The method of claim 22, wherein the capsid protein is a recombinant 2 (Rec2) or AAV8 capsid protein.

24. The method of claim 19, wherein AAV vector encodes an engineered adeno-associated virus (AAV) 9 (AAV9) capsid protein; and wherein the amino acid substitutions corresponding to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6 comprise residues 501, 713, and / or 714 of SEQ ID NO:

31.

25. The method of claim 24, wherein the amino acid substitution comprises F501Y, Y713D, or K714I, or any combination thereof.

26. An AAV capsid protein made by the method of any one of claims 19-25.

27. A method of delivering a gene to adipose tissue of a subject, the method comprising administering to the subject the engineered AAV vector of any of claims 10-18.

28. A method of treating a metabolic disorder or disease in a subject, the method comprising administering to the subject the engineered AAV vector of any of claims 10-18.

29. A method of treating a cancer in a subject comprising administering to the engineered AAV vector of any of claims 10-18.

30. The method of any one of claims 27-29, wherein the engineered AAV vector encodes an engineered adeno-associated virus (AAV) recombinant 2 (Rec2), AAV8, or AAV9 capsid protein.Attorney Docket No.103361-377WO1 31. The method of any one of claims 27-30, wherein the engineered adeno-associated virus (AAV) capsid protein comprise an amino acid substitution to one or more amino acids corresponding to amino acid residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO:

6.

32. The method of claim 31, wherein the amino acid substitution comprises a F503Y, F503T, F503K, Y708D, Y708E, K709I, K709L, and / or K709G, or any combination thereof.

33. The method of claim 32, wherein the amino acid substitution comprises a F503Y, Y708D or Y708E, and K709I.

34. The method of claim 33, wherein the capsid protein is a recombinant 2 (Rec2) or AAV8 capsid protein.

35. The method of claim 31, wherein the capsid protein comprises an AAV9 capsid protein and wherein the amino acid substitutions corresponding to residues 503, 708, and / or 709 of SEQ ID NO: 1 or SEQ ID NO: 6 comprise residues 501, 713, and / or 714 of SEQ ID NO:

31.

36. The method of claim 35, wherein the amino acid substitution comprises F501Y, Y713D, and / or K714I, or any combination thereof 37. The method of any one of claims 27-36, wherein the amino acid substitution occurs at residues that ablate liver tropism or disrupt AAV binding to hepatocytes.

38. The method of any one of claims 27-37, wherein the AAV capsid protein comprise the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

44.

39. The method of any one of claims 27-38, wherein the AAV vector encodes a transgene or a therapeutic agent.

40. The method of claim 39, wherein the metabolic disorder or disease comprises diabetes, obesity, metabolic syndrome, congenital leptin deficiency, or lipodystrophy.

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