AAV capsid design

Abstract

The present disclosure relates in some aspects to recombinant adeno-associated viruses with distinct tissue targeting capabilities. [Solution] In some aspects, the present disclosure relates to gene transfer methods using recombinant adeno-associated viruses, hi some aspects, the present disclosure relates to isolated AAV capsid proteins and isolated nucleic acids encoding the same.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of the filing dates of U.S. Provisional Application No. 62 / 486,642, entitled "AAV Capsid Design," filed April 18, 2017, No. 62 / 417,756, entitled "AAV Capsid Design," filed November 4, 2016, and No. 62 / 408,022, entitled "AAV Capsid Design," filed October 13, 2016. The entire contents of each of these applications are incorporated herein by reference.

[0002] Field of the Disclosure In some aspects, the present disclosure relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells. In some aspects, the present disclosure provides novel AAVs and methods of their use, as well as related kits. [Background technology]

[0003] background Recombinant AAV adeno-associated viruses (rAAV) can drive stable and sustained transgene expression in target tissues without significant toxicity and host immunogenicity. Thus, rAAV is a promising delivery vehicle for long-term therapeutic gene expression. However, the low transduction efficiency and limited tissue tropisms of currently available rAAV vectors may limit their application as viable and effective treatments. In addition, the reliable clinical translation of superior therapeutic AAV serotypes derived from non-human tissues remains a concern. Consequently, there remains a need for new AAV vectors for gene delivery. Summary of the Invention

[0004] overview In some aspects, the present disclosure relates to novel AAVs for gene therapy applications. In some embodiments, the AAVs described herein contain amino acid variations in one or more capsid proteins that confer new or enhanced tissue-tropic properties. According to some embodiments, variants of AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrids), and AAV8 that possess useful tissue-targeting properties have been identified and are disclosed herein. For example, AAV8 variants useful for transducing cells such as human hepatocytes (e.g., present in liver tissue), central nervous system cells (CNS cells), etc. are provided. In some embodiments, AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrids), and AAV8 variants useful for targeting cells of ophthalmic tissue (e.g., the eye), gastrointestinal tract, respiratory system, breast tissue, pancreatic tissue, urinary tract tissue, uterine tissue, tissues associated with certain cancers (e.g., breast cancer, prostate cancer, etc.), and other tissues are provided. In some embodiments, the variant AAVs described herein target tissues other than those targeted by their corresponding wild-type AAVs.

[0005] In some aspects, the present disclosure provides an isolated nucleic acid comprising a sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 1-409, 435-868, and 1726-1988, which encodes an AAV capsid protein. In some embodiments, a fragment of the isolated nucleic acid is provided. In some embodiments, the isolated nucleic acid fragment does not encode a peptide identical to the sequence of any one of SEQ ID NOs: 869, 870, or 871.

[0006] In some aspects, the present disclosure provides a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 410-434, 876-1718, and 1989-2251. In some embodiments, the nucleic acid encodes an AAV capsid protein or a variant thereof, and / or an AAV assembly-activating protein (AAP) or a variant thereof. In some embodiments, the AAP is in a different open reading frame of the nucleic acid than the AAV capsid protein. In some embodiments, the AAP is AAV2 AAP (AAP-2), or a variant thereof.

[0007] The disclosure provides, in some aspects, an isolated AAV capsid protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-409, 435-868, and 1726-1988. In some embodiments, the isolated AAV capsid protein comprises a sequence selected from SEQ ID NOs: 1-409, 837-852, or 1726-1814, wherein an amino acid in that sequence that is not identical to the corresponding amino acid in the sequence represented as SEQ ID NO: 869 is replaced with a conservative substitution.

[0008] In some aspects, the disclosure provides an AAV2 / 3 hybrid capsid protein. In some embodiments, the isolated AAV capsid protein comprises a sequence selected from SEQ ID NOs: 435-628 and 1815-1988, wherein an amino acid in that sequence that is not identical to the corresponding amino acid in the sequence set forth as SEQ ID NO: 869 or 870 is replaced with a conservative substitution.

[0009] In some embodiments, the isolated AAV capsid protein comprises a sequence selected from SEQ ID NOs: 629-836 or 853-868, wherein an amino acid in that sequence that is not identical to the corresponding amino acid in the sequence represented as SEQ ID NO: 871 is replaced with a conservative substitution.

[0010] In some aspects of the present disclosure, a composition is provided that comprises any of the above-mentioned isolated AAV capsid proteins. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a composition is provided that comprises one or more isolated AAV capsid proteins of the present disclosure and a physiologically compatible carrier.

[0011] In some aspects of the present disclosure, recombinant AAV (rAAV) is provided, which comprises any of the above-described isolated AAV capsid proteins. In some embodiments, a composition comprising the rAAV is provided. In some embodiments, the composition comprising the rAAV further comprises a pharmaceutically acceptable carrier. Recombinant AAVs comprising one or more of the isolated AAV capsid proteins of the present disclosure are also provided.

[0012] In some aspects of the disclosure, host cells are provided containing a nucleic acid comprising a coding sequence selected from the group consisting of SEQ ID NOs: 410-434, 876-1718, and 1989-2251 operably linked to a promoter. In some embodiments, compositions are provided comprising the host cells and a sterile cell culture medium. In some embodiments, compositions are provided comprising the host cells and a cryopreservative.

[0013] According to some aspects of the present disclosure, a method for delivering transgene to a subject is provided.In some embodiments, the method comprises administering the above-mentioned rAAV to a subject, wherein the rAAV comprises at least one transgene, and wherein the rAAV infects the target tissue cells of the subject.In some embodiments, the subject is selected from mice, rats, rabbits, dogs, cats, sheep, pigs, and non-human animals of the primate order.In one embodiment, the subject is a human.

[0014] In some embodiments, at least one transgene is a gene encoding a protein. In some embodiments, at least one transgene encodes a small interfering nucleic acid. In some embodiments, the small interfering nucleic acid is an miRNA. In some embodiments, the small interfering nucleic acid is an miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in a subject. In some embodiments, the miRNA is expressed in the cells of target tissue. In some embodiments, the target tissue is liver, central nervous system (CNS), eye, gastrointestinal, respiratory, breast, pancreatic, urinary tract, or uterine tissue.

[0015] In some embodiments, the transgene expresses a transcript that includes at least one binding site for an miRNA, which inhibits the activity of the transgene in tissues other than the target tissue by hybridizing to the binding site. In some embodiments, the rAAV is administered to a subject intravenously, transdermally, intraocularly, intrathecally, intracerebrally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.

[0016] According to some aspects of the present disclosure, there is provided a method for generating a somatic transgene animal model. In some embodiments, the method comprises administering any of the above-described rAAVs to a non-human animal, wherein the rAAV comprises at least one transgene, and wherein the rAAV infects cells of a target tissue of the non-human animal.

[0017] In some embodiments, the transgene is a gene encoding at least one protein. In some embodiments, the transgene encodes at least one small interfering nucleic acid. In some embodiments, the transgene encodes at least one reporter molecule. In some embodiments, the small interfering nucleic acid is an miRNA. In some embodiments, the small interfering nucleic acid is an miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in an animal. In some embodiments, the miRNA is expressed in the cells of the target tissue. In some embodiments, the target tissue is liver, central nervous system (CNS), eye, gastrointestinal, respiratory, breast, pancreatic, urinary tract, or uterine tissue.

[0018] In some embodiments, the transgene expresses a transcript that includes at least one binding site for an miRNA, which inhibits the activity of the transgene in tissues other than the target tissue by hybridizing to the binding site.

[0019] According to some aspects of the present disclosure, there is provided a method for generating a somatic gene transfer animal model comprising administering any of the rAAVs described above to a non-human animal, wherein the rAAV comprises at least one transgene, wherein the transgene expresses a transcript comprising at least one binding site for an miRNA, wherein the miRNA inhibits activity of the transgene in tissues other than the target tissue by hybridizing to the binding site in the transcript.

[0020] In some embodiments, the transgene comprises a tissue-specific promoter or an inducible promoter. In some embodiments, the tissue-specific promoter is a liver-specific thyroxine-binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a mucin-2 promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a retinoschisin promoter, a K12 promoter, a CC10 promoter, a surfactant protein C (SP-C) promoter, a PRCl promoter, an RRM2 promoter, a uroplakin 2 (UPII) promoter, or a lactoferrin promoter.

[0021] In some embodiments, the rAAV is administered to the animal intravenously, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation. According to some aspects of the present disclosure, there is provided a somatic gene transfer animal model produced by any of the above-described methods.

[0022] In another aspect of the present disclosure, a kit for producing rAAV is provided. In some embodiments, the kit includes a container containing an isolated nucleic acid having the sequence of any one of SEQ ID NOs: 410-434, 876-1718, and 1989-2251. In some embodiments, the kit includes a container containing an isolated nucleic acid encoding a polypeptide having the sequence of any one of SEQ ID NOs: 1-409, 435-868, or 1726-1988. In some embodiments, the kit further includes instructions for producing rAAV. In some embodiments, the kit further includes at least one container containing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene.

[0023] In another aspect of the present disclosure, a kit is provided that includes a container containing a recombinant AAV having any of the above-described isolated AAV capsid proteins. In some embodiments, the container of the kit is a syringe. In other aspects, the present disclosure relates to the use of AAV-based vectors as vehicles for gene delivery, for therapeutic, prophylactic, and research purposes, and for the development of somatic gene transfer animal models.

[0024] In some aspects, the present disclosure relates to AAV serotypes that have demonstrated distinct tissue / cell type tropism and can achieve stable somatic gene transfer in animal tissues at levels similar to adenoviral vectors (e.g., up to 100% in vivo tissue transformation, depending on the target tissue and vector dose) in the absence of vector-related toxicology. In other aspects, the present disclosure relates to AAV serotypes that have targeting capabilities for liver, central nervous system (CNS), ophthalmic, gastrointestinal, respiratory, breast, pancreatic, urinary tract, or uterine tissues. These tissues are associated with a wide range of human diseases, including neurological, metabolic, diabetic, ophthalmic, respiratory, gastrointestinal, urinary tract, and reproductive diseases, and certain cancers.

[0025] In some embodiments, the rAAV comprises at least one transgene. The transgene may cause a pathological condition. In some embodiments, the transgene encodes a protein that treats the pathological condition. In another aspect, the novel AAV of the present disclosure can be used in a method for delivering a transgene to a subject. The method is carried out by administering the rAAV of the present disclosure to a subject, wherein the rAAV comprises at least one transgene. In some embodiments, the rAAV targets a predetermined tissue of the subject.

[0026] In another aspect, the AAV of the present disclosure may be used in a method for generating a somatic gene transgenic animal model. The method is carried out by administering an rAAV of the present disclosure to an animal, wherein the rAAV comprises at least one transgene, wherein the transgene causes a pathological condition, and wherein the rAAV targets a predetermined tissue of the animal.

[0027] The transgene may express a number of genes, including cancer-related genes, pro-apoptotic genes, and apoptosis-related genes. In some embodiments, the transgene expresses a small interfering nucleic acid that can inhibit the expression of cancer-related genes. In other embodiments, the transgene expresses a small interfering nucleic acid that can inhibit the expression of apoptosis-related genes. In other embodiments, the small interfering nucleic acid is miRNA or shRNA. According to other embodiments, the transgene expresses a toxin, optionally wherein the toxin is DTA. In other embodiments, the transgene expresses a reporter gene, optionally a reporter enzyme such as beta-galactosidase, or a fluorescent protein such as GFP or luciferase.

[0028] The transgene may express an miRNA. In other embodiments, the transgene expresses an miRNA sponge, wherein the miRNA sponge inhibits the activity of one or more miRNAs in the animal. In some embodiments, the miRNA may be an endogenous miRNA, or it may be expressed in cells of liver, central nervous system (CNS), eye, gastrointestinal, respiratory, breast, pancreatic, urinary tract, or uterine tissues.

[0029] rAAV may transduce many different types of tissues, such as neurons, squamous epithelial cells, renal proximal or distal convoluted tubule cells, mucous gland cells, vascular endothelial cells, endometrial cells, retinal cells, or certain cancer cells (e.g., breast cancer cells, prostate cancer cells, etc.).

[0030] In some embodiments, the rAAV is administered in a dose of 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the rAAV is administered at a dose of 10 genome copies per kg. 10 , 10 11 , 1012 , 10 13 , or 10 14 The rAAV is administered in a dose of 1000 copies of the genome. The rAAV may be administered by any route. For example, in some embodiments, it may be administered intravenously (e.g., by portal vein injection).

[0031] In some embodiments, the transgene comprises a tissue-specific promoter such as the liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, mucin-2 promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, retinoschisin promoter, K12 promoter, CC10 promoter, surfactant protein C (SP-C) promoter, PRCl promoter, RRM2 promoter, uroplakin 2 (UPII) promoter, or lactoferrin promoter.

[0032] The somatic transgenic animal model may be a mammal, such as a mouse, rat, rabbit, dog, cat, sheep, pig, or non-human animal of the primate order. In some embodiments, a putative therapeutic agent may be administered to a somatic transgenic animal model to determine the effect of the putative therapeutic agent on a pathological condition in the animal. In another aspect, the present disclosure is a somatic transgenic animal produced by the methods described herein.

[0033] According to another aspect of the present disclosure, a kit for producing rAAV to generate somatically transgenic animals having a pathological condition in a predetermined tissue is provided, which includes at least one container containing a recombinant AAV vector, at least one container containing rAAV packaging components, and instructions for constructing and packaging the recombinant AAV.

[0034] The rAAV packaging components may include a host cell that expresses at least one rep gene and / or at least one cap gene. In some embodiments, the host cell is a 293 cell. In other embodiments, the host cell expresses at least one helper virus gene product that influences the production of the rAAV containing the recombinant AAV vector. The at least one cap gene may encode a capsid protein from an AAV serotype that targets a predetermined tissue.

[0035] In other embodiments, the rAAV packaging components include a helper virus, optionally wherein the helper virus is an adenovirus or a herpesvirus. The rAAV vector and its components may include any of the elements described herein. Illustratively, in some embodiments, the rAAV vector includes a transgene, such as any of the transgenes described herein. In some embodiments, the transgene expresses an miRNA inhibitor (e.g., an miRNA sponge or TuD RNA), where the miRNA inhibitor inhibits the activity of one or more miRNAs in the somatic transgenic animal.

[0036] Each of the limitations of the present disclosure may cover various aspects of the present disclosure. Thus, it is anticipated that each of the limitations of the present disclosure involving any one element or combination of elements may be included in each aspect of the present disclosure. The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways. [Brief explanation of the drawings]

[0037] Brief description of the drawings [Figure 1A-1B]Figures 1A-1B show a workflow schematic for AAV variant identification. Figure 1A depicts high-throughput detection of novel AAV variants in selected human tissues. Proviral capsid sequences are amplified using high-cycle PCR followed by low-cycle PCR to barcode amplicon libraries for single-molecule real-time (SMRT) multiplexed sequencing. Figure 1B outlines the pipeline for bioinformatics analysis of sequencing data.

[0038] [Figures 2A-2D] Figures 2A-2D show data on in vivo detection of FFLuc transgene activity using various doses of selected AAV8 variants. Figure 2A shows that luciferase activity of various AAV8 variants was assessed 6 weeks after IV (intravenous), IM (intramuscular), or IN (intranasal) injection. Figures 2B-2D show data on the assessment of FFLuc activity for each variant, B2 (Figure 2B), B3 (Figure 2C), and B61 (Figure 2D), compared to AAV8 (mean ± SD, n = 3, t-test).

[0039] [Figure 3A-3B] Figures 3A-3B show data on the assessment of FFLuc transgene activity delivered by AAV8 variant B61 compared to AAV9 at day 21 after neonatal injection. Luciferase activity and genome copies were detected in the brain (Figure 3A) and spinal cord (Figure 3B) (mean ± SD, n = 5, t-test).

[0040] [Figure 4A-4B]Figures 4A-4B show data on in vivo detection of FFLuc transgene activity after right hindlimb intramuscular (IM) injection of AAV8 variant B44 compared with AAV8. Figure 4A shows whole-animal luciferase expression of variant B44 assessed at week 6 after IM injection. Figure 4B shows assessment of muscle (RTA, right tibialis anterior; LTA, left tibialis anterior), liver, and heart. Luciferase activity (left bar graph) and relative ratio (right bar graph) of B44 compared with AAV8 (mean ± SD, n = 3).

[0041] [Figure 5] Figure 5 shows a phylogenetic comparison of AAV8 variants (B2, B3, B61) with other AAV serotypes. [Figures 6A-6B] Figure 6A shows a schematic representation of the workflow for in vivo characterization of novel AAV variants by high-throughput directional screening. Figure 6B shows a schematic representation of the workflow for NHP characterization of novel AAV variants by high-throughput directional screening.

[0042] [Figure 7] Figure 7 shows a scatter plot displaying the distribution of distinct AAV2 capsid variants (409 total) and AAV2 / 3 variants (194 total) with one or more single amino acid variants. [Figure 8] Figure 8 shows a diagram of the vector construct used in the multiplexed screening of discovered capsid variants. A unique 6-bp barcode was cloned into the transgene and packaged into the candidate capsid variants.

[0043] [Figure 9]Figure 9 shows a schematic diagram of the indexed transgene and high-throughput sequencing library design for assessing capsid variant tropism profiling. An indexed and adapter cassette containing a 6-bp barcode (1° barcode) and a BstEII restriction site can be cloned into a vector construct using flanking BsrGI and SacI sites. Crude total DNA from tissue treated with rAAV containing both host and vector genomes was cleaved with BstEII enzyme. The resulting 5'-overhangs were used to specifically ligate to adapters containing a second barcode (allowing for further multiplexed sequencing and streamlining) and a 5'-biotin modification (which can be used to select adapter-containing fragments using magnetic bead enrichment). The enriched material can then be PCR amplified using primers specific for the adapter and transgene sequences to produce libraries for high-throughput sequencing. SEQ ID NOs: 1719-1725 are shown from top to bottom. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description Adeno-associated viruses (AAVs) are small (approximately 26 nm), replication-deficient, non-enveloped viruses that generally depend on the presence of a second virus, such as adenovirus or herpesvirus, for their growth in cells. AAVs are not known to cause disease and induce very weak immune responses. AAVs can infect both dividing and non-dividing cells and can integrate their genomes into the genome of host cells. These features make AAVs highly attractive candidates for creating viral vectors for gene therapy. Prototype AAV vectors based on serotype 2 provided evidence for non-toxic and stable gene transfer in murine and large animal models, but exhibited poor gene transfer efficiency in many key target tissues. The present disclosure, in some aspects, seeks to overcome this shortcoming by providing novel AAVs with distinct tissue targeting capabilities for gene therapy and research applications.

[0045] In some aspects of the present disclosure, new AAV capsid proteins with distinct tissue targeting capabilities are provided. In some embodiments, the AAV capsid proteins are isolated from tissues targeted by AAVs containing the capsid proteins. In some aspects, methods are provided for delivering transgenes to target tissues of a subject. The transgene delivery methods may be used for gene therapy (e.g., to treat disease) or research (e.g., to create somatic transgenic animal models).

[0046] Methods for AAV discovery Much of AAV biology is influenced by its capsid. As a result, methods for discovering novel AAVs have primarily focused on isolating the DNA sequence of the AAV capsid. A central feature of the adeno-associated virus (AAV) latent life cycle is its persistence in the form of integrated and / or episomal genomes in host cells. Methods used to isolate novel AAVs include PCR-based molecular rescue of latent AAV DNA genomes, infectious virus rescue of latent proviral genomes from tissue DNA in vitro in the presence of adenovirus helper functions, and rescue of circular proviral genomes from tissue DNA by rolling-circle linear amplification mediated by isothermal phage Phi-29 polymerase. All of these isolation methods focus on exploiting the latency of the AAV proviral DNA genome and rescuing the genomic DNA of the persistent virus.

[0047] In some aspects, the present disclosure relates to the discovery that novel AAV variants with desired tissue tropism can be identified from a subject's in vivo tissue. Without wishing to be bound by any particular theory, the use of in vivo tissue leverages the natural reservoir of genomic diversity observed among viral genome sequences isolated from both normal and tumor tissues of a subject. Thus, in some embodiments, in vivo tissue acts as a natural incubator for viral (e.g., viral capsid protein) diversity through selective pressure and / or immune evasion.

[0048] In some aspects, the present disclosure relates to the discovery that PCR products resulting from the amplification of AAV DNA (e.g., AAV DNA isolated or extracted from host cells or in vivo tissues of a subject) can be subjected to high-throughput single-molecule real-time (SMRT) sequencing to identify novel capsid protein variants. As used herein, "single-molecule real-time (SMRT) sequencing" refers to a parallelized single-minute sequencing method, as described, for example, by Roberts et al. (2013) Genome Biology 14:405, doi:10.1186 / gb-2013-14-7-405. While not wishing to be bound by any particular theory, the use of SMRT sequencing obviates the need to perform viral genome reconstruction and chimera prediction from aligned short-read fragments obtained from other conventional high-throughput genome sequencing methodologies.

[0049] Endogenous latent AAV genomes are transcriptionally active in mammalian cells (e.g., cells of non-human primate tissues such as the liver, spleen, and lymph nodes). Without wishing to be bound by theory, it is hypothesized that low levels of transcription from AAV genes may be required to maintain AAV persistence in the host, and that the resulting cap RNA may serve as a more suitable and abundant substrate for searching for functional cap sequences for vector development. Both rep and cap gene transcripts are detected in variable abundance by RNA detection methods (e.g., RT-PCR). The presence of cap gene transcripts and the ability of cap RNA to generate cDNA through reverse transcription (RT) significantly increases the abundance of templates for PCR-based rescue of novel cap sequences from tissues in vitro, enhancing the sensitivity of novel AAV discovery.

[0050] Novel cap sequences may also be identified by transfecting cells with total cellular DNA isolated from tissues that harbor proviral AAV genomes at extremely low abundance. The cells may further be transfected with a gene that provides helper virus function (e.g., adenovirus) to induce and / or increase AAV gene transcription in the transfected cells. In some embodiments, novel cap sequences of the present disclosure may be identified by isolating cap mRNA from transfected cells, creating cDNA from the mRNA (e.g., by RT-PCR), and sequencing the cDNA.

[0051] Isolated capsid proteins and nucleic acids encoding the same AAVs isolated from mammals, particularly non-human primates, are useful for creating gene transfer vectors for clinical development and human gene therapy applications. In some aspects, the present disclosure provides novel AAVs discovered in various in vivo tissues (e.g., liver, brain, gastric, respiratory, breast, pancreatic, rectal, prostate, urological, and cervical tissues) using the methods disclosed herein. In some embodiments, the tissue(s) in which the novel AAV variants were discovered are cancerous tissues (e.g., tumors or cancer cells). In some embodiments, nucleic acids encoding the capsid proteins of these novel AAVs were discovered in viral genomic DNA isolated from human tissues. Examples of tissues in which novel AAV capsid proteins were discovered are listed in Table 1. Nucleic acid and protein sequences and other information regarding AAVs are presented in Tables 3-5 and 8 and the Sequence Listing.

[0052] Isolated nucleic acids of the present disclosure that encode AAV capsid proteins include any nucleic acid having a sequence as set forth in any one of SEQ ID NOs: 410-435, 876-1718, or 1989-2251, as well as any nucleic acid having a sequence with substantial homology thereto. In some embodiments, isolated nucleic acids of the present disclosure include any nucleic acid having a sequence that encodes a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 1-409, 435-868, and 1726-1988. In some embodiments, the present disclosure provides isolated nucleic acids that have substantial homology to a nucleic acid having a sequence as set forth in any one of SEQ ID NOs: 410-435, 876-1718, and 1989-2251, but that do not encode a protein having an amino acid sequence as set forth in SEQ ID NO: 869, 870, or 871.

[0053] In some embodiments, isolated AAV capsid proteins of the present disclosure include any protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-409, 837-852, or 1726-1814, as well as any protein having substantial homology thereto. In some embodiments, the present disclosure provides an isolated capsid protein having substantial homology to a protein having a sequence as set forth in any one of SEQ ID NOs: 1-409, 837-852, or 1726-1814, but not having an amino acid sequence as set forth in SEQ ID NO: 869.

[0054] In some embodiments, an isolated AAV capsid protein of the present disclosure encompasses any protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 435-628 or 1815-1988, as well as any protein having substantial homology thereto. In some embodiments, the present disclosure provides an isolated capsid protein having substantial homology to a protein having a sequence as set forth in any one of SEQ ID NOs: 435-628 or 1815-1988, but not having an amino acid sequence as set forth in SEQ ID NO: 869 or 870.

[0055] In some embodiments, isolated AAV capsid proteins of the present disclosure include any protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 629-836 or 853-868, as well as any protein having substantial homology thereto. In some embodiments, the present disclosure provides an isolated capsid protein having substantial homology to a protein having a sequence as set forth in any one of SEQ ID NOs: 629-836 or 853-868, but not having an amino acid sequence as set forth in SEQ ID NO: 871.

[0056] "Homology" refers to the percent identity between two polynucleotides or two polypeptide moieties. The term "substantial homology," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is about 90-100% nucleotide sequence identity in the aligned sequences. When referring to a polypeptide or a fragment thereof, the term "substantial homology" indicates that when optimally aligned with another polypeptide, with appropriate gaps, insertions, or deletions, there is about 90-100% nucleotide sequence identity in the aligned sequences. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably greater than 97% identity. In some cases, highly conserved may refer to 100% identity. Identity is readily determined by those of skill in the art, for example, by using algorithms and computer programs known to those of skill in the art.

[0057] As described herein, alignment between nucleic acid or polypeptide sequences is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs, such as "Clustal W," accessible through web servers on the Internet. Alternatively, Vector NTI utilities may also be used. There are also numerous algorithms known in the art (including those contained in the above programs) that can be used to measure nucleotide sequence identity. As another example, polynucleotide sequences can be compared using BLASTN, which provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences. Similar programs (e.g., the "Clustal X" program, BLASTP) are also available for amino acid comparison.

[0058] Typically, any of these programs are used with default settings, although one skilled in the art may change these settings at any time. Alternatively, one skilled in the art may utilize another algorithm or computer program that provides at least the level of identity or alignment as provided by the referenced algorithm and program. Alignment may be used to identify corresponding amino acids between two proteins or peptides. A "corresponding amino acid" is an amino acid in a protein or peptide sequence that is aligned with an amino acid in another protein or peptide sequence. Corresponding amino acids may be identical or non-identical. Corresponding amino acids that are non-identical amino acids may be referred to as variant amino acids. Table 6 provides examples of variant amino acids.

[0059] Alternatively, for nucleic acids, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-strand-specific nuclease(s) and sizing of the digested fragments. Substantially homologous DNA sequences can be identified in Southern hybridization experiments under stringent conditions, as defined for the specific system. Defining appropriate hybridization conditions is within the skill of the art.

[0060] "Nucleic acid" sequence refers to a DNA sequence or an RNA sequence. In some embodiments, the term nucleic acid refers to, but is not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methyl The present invention captures sequences that include any of the known base analogs of DNA and RNA, such as 5'-aminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queuosine, 2-thiocytosine, and 2,6-diaminopurine.

[0061] In some embodiments, the proteins and nucleic acids of the present disclosure are isolated. As used herein, the term "isolated" means artificially obtained or produced. As used herein with respect to nucleic acids, the term "isolated" generally means: (i) amplified in vitro, e.g., by polymerase chain reaction (PCR); (ii) recombinantly produced, e.g., by cloning; (iii) purified, e.g., by cleavage and gel separation; or (iv) synthesized, e.g., by chemical synthesis. Isolated nucleic acids are those that can be readily manipulated by recombinant DNA techniques well known in the art.

[0062] Thus, a nucleotide sequence contained in a vector (whose 5' and 3' restriction sites are known or whose polymerase chain reaction (PCR) primer sequences are disclosed) is considered isolated, but a nucleic acid sequence existing in its natural state in its natural host is not. An isolated nucleic acid may, but need not, be substantially purified. For example, a nucleic acid isolated within a cloning or expression vector is not pure, in that it may comprise a small percentage of the material in the cell in which it is present. However, such a nucleic acid, as the term is used herein, is isolated because it is susceptible to ready manipulation by standard techniques known to those of skill in the art. When used herein with respect to a protein or peptide, the term "isolated" generally refers to a protein or peptide obtained or produced artificially (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0063] It should be understood that conservative amino acid substitutions can be made to provide functionally equivalent variants or homologs of the capsid protein. In some aspects, the present disclosure embraces sequence alterations that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art (such as those found in references summarizing such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus, conservative amino acid substitutions may be made in the amino acid sequences of the proteins and polypeptides disclosed herein.

[0064] Examples of isolated nucleic acids encoding polypeptides comprising AAV capsid proteins are nucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 410-434, 876-1718, and 1989-2251. Fragments of isolated nucleic acids encoding AAV capsid sequences may be useful for constructing nucleic acids encoding desired capsid sequences. Fragments may be of any suitable length. In some embodiments, fragments (portions) of isolated nucleic acids encoding AAV capsid sequences may be useful for constructing nucleic acids encoding desired capsid sequences. Fragments may be of any suitable length (e.g., at least 6, at least 9, at least 18, at least 36, at least 72, at least 144, at least 288, at least 576, at least 1152 nucleotides in length, or longer).

[0065] For example, a fragment of a nucleic acid sequence encoding a polypeptide of a first AAV capsid protein may be used to construct or incorporated into a nucleic acid sequence encoding a second AAV capsid sequence to alter the properties of the AAV capsid. In some embodiments, an AAV capsid protein comprising capsid sequence fragments from multiple AAV serotypes is referred to as a chimeric AAV capsid. The fragment may not encode a peptide identical to any one of SEQ ID NOs: 869, 870, or 871. For example, a fragment of a nucleic acid sequence encoding a variant amino acid (compared to a known AAV serotype) may be used to construct or incorporated into a nucleic acid sequence encoding an AAV capsid sequence to alter the properties of the AAV capsid.

[0066] In some embodiments, a nucleic acid sequence encoding an AAV variant may contain about 1 to about 100 amino acid variants compared to a known AAV serotype (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), or AAV8). In some embodiments, a nucleic acid sequence encoding an AAV variant may contain about 5 to about 50 amino acid variants compared to a known AAV serotype (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), or AAV8). In some embodiments, a nucleic acid sequence encoding an AAV variant may contain about 10 to about 30 amino acid variants compared to a known AAV serotype (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), or AAV8). In some embodiments, the nucleic acid sequence encoding the AAV variant may include 1, or 2, or 3, or 4, 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 amino acid variants compared to a known AAV serotype (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid) or AAV8).

[0067] For example, a nucleic acid sequence encoding an AAV variant (e.g., SEQ ID NO: 861) may contain three amino acid variants compared to a known AAV serotype (e.g., AAV8). Recombinant cap sequences having one or more of the three amino acid variants may be constructed by incorporating a fragment of a nucleic acid sequence containing the region encoding the variant amino acid into a nucleic acid sequence encoding a known AAV serotype. The fragment may be incorporated by any suitable method, including using site-directed mutagenesis. Thus, new AAV variants with new properties may be created.

[0068] In some aspects, the present disclosure provides isolated nucleic acids encoding AAV assembly activating proteins (AAPs) or variants thereof. As used herein, "assembly activating proteins" or "AAPs" are protein chaperones that target newly synthesized capsid proteins (e.g., AAV VP1, VP2, and VP3 VP proteins) to the nucleolus of a cell, thereby promoting encapsidation of the viral genome. Generally, AAPs are encoded in the cap gene of adeno-associated viruses. For example, AAP-2 is encoded in the cap gene of AAV2. Other examples of AAPs include, but are not limited to, AAP-1, AAP-3, AAP-4, AAP-5, AAP-8, AAP-9, AAP-11, and AAP-12, as described, for example, by Sonntag et al. J. Virol. 2011 Dec. 85(23): 12686-12697. In some embodiments, the AAP is translated from a different open reading frame (ORF) of the cap gene than the capsid proteins (e.g., VP1, VP2, VP3). For example, in some embodiments, the capsid proteins (e.g., AAV2 VP1, VP2, VP3) are translated from ORF1 of the cap gene, and the AAP (e.g., AAP-2) is translated from ORF2 of the cap gene. In some embodiments, the isolated nucleic acid encoding an AAP comprises or consists of a sequence selected from SEQ ID NOs: 410-434 and 876-1718.

[0069] Recombinant AAV In some aspects, the present disclosure provides isolated AAV. As used herein with respect to AAV, the term "isolated" refers to an AAV that has been artificially obtained or produced. Isolated AAV may be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV." Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities such that the transgene of the rAAV is specifically delivered to one or more predetermined tissue(s). The AAV capsid is an important factor in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the targeted tissue can be selected. In some embodiments, the rAAV comprises a capsid protein having an amino acid sequence as set forth in any one of SEQ ID NOS: 1-409, 435-852, 859-874, or 1726-1988, or a protein having substantial homology thereto.

[0070] Methods for obtaining recombinant AAVs with desired capsid proteins are well known in the art. (See, e.g., US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety.) Typically, the methods involve culturing host cells that contain a nucleic acid sequence encoding an AAV capsid protein (e.g., a nucleic acid encoding a polypeptide having a sequence as set forth in any one of SEQ ID NOS: 1-409, 435-868, or 1726-1988) or a fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to enable packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. In some embodiments, AAV comprises three capsid proteins, virion proteins 1 through 3 (designated VP1, VP2, and VP3), all of which may be expressed from a single cap gene.

[0071] Consequently, in some embodiments, the VP1, VP2, and VP3 proteins share a common core sequence. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, upon translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the protein shell is primarily composed of the VP3 capsid protein. In some embodiments, the function of the capsid protein is to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner. In some embodiments, the VP1 and / or VP2 capsid protein may contribute to the tissue tropism of the packaged AAV. In some embodiments, the tissue tropism of the packaged AAV is determined by the VP3 capsid protein. In some embodiments, the tissue tropism of AAV is enhanced or altered by mutations made in the capsid protein.

[0072] In some aspects, the present disclosure describes variants of wild-type AAV serotypes. In some embodiments, the variants have altered tissue tropism. In some embodiments, the AAV variants described herein contain amino acid variations (e.g., substitutions, deletions, insertions) within the cap gene. As discussed above, all three capsid proteins are transcribed from a single cap gene. Consequently, in some embodiments, an amino acid variation within the cap gene is present in all three capsid proteins encoded by the cap gene. Alternatively, in some embodiments, the amino acid variation may not be present in all three capsid proteins. In some embodiments, the amino acid variation occurs only in the VP1 capsid protein. In some embodiments, the amino acid variation occurs only in the VP2 capsid protein. In some embodiments, the amino acid variation occurs only in the VP3 capsid protein. In some embodiments, the AAV variants contain more than one variation in the cap gene. In some embodiments, more than one variation occurs within the same capsid protein (e.g., within VP3). In some embodiments, more than one variation occurs within different capsid proteins (eg, at least one variation in VP2 and at least one variation in VP3).

[0073] In some embodiments, the AAV variants described herein are variants of AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrids), or AAV8. AAV2 is known to efficiently transduce human central nervous system (CNS) tissue, kidney tissue, eye tissue (e.g., photoreceptor cells and retinal pigment epithelium (RPE)), and other tissues. Consequently, in some embodiments, the AAV3 variants described herein may be useful for delivering gene therapy to CNS tissue, kidney tissue, or eye tissue. AAV3 is also known to efficiently transduce cancerous human liver cells. Consequently, in some embodiments, the AAV3 variants described herein may be useful for delivering gene therapy to cancerous and normal human liver cells. AAV8 is known to target liver tissue, respiratory tissue, and eye tissue. Consequently, in some embodiments, the AAV8 variants described herein may be useful for delivering gene therapy to liver tissue, respiratory tissue, or eye.

[0074] It should be understood that AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), and AAV8 variants described herein may contain one or more variations in the cap gene compared to the corresponding wild-type AAV. Thus, in some embodiments, AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), and AAV8 variants described herein may have tissue tropism useful for delivering gene therapy to additional tissue types not targeted by wild-type AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrid) or AAV8. For example, in some embodiments, AAV8 variants described herein (e.g., B61; SEQ ID NO: 865) may be useful for delivering gene therapy to the central nervous system (CNS). In some embodiments, AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), or AAV8 variants described herein may be useful for targeting kidney cells or liver cells. In some embodiments, AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrids), or AAV8 variants described herein may be useful for targeting gene therapy to the liver, spleen, heart, or brain.

[0075] In some aspects, the AAV variants described herein may be useful for treating CNS-related disorders. As used herein, a "CNS-related disorder" is a disease or condition of the central nervous system. A CNS-related disorder may affect the spinal cord (e.g., myelopathy), the brain (e.g., encephalopathy), or tissues surrounding the brain and spinal cord. A CNS-related disorder may be of either inherited or acquired genetic cause through somatic mutation. A CNS-related disorder may be a psychological condition or disorder, such as attention deficit hyperactivity disorder, autism spectrum disorder, mood disorder, schizophrenia, depression, Rett syndrome, etc. A CNS-related disorder may also be an autoimmune disorder. A CNS-related disorder may also be a cancer of the CNS, such as brain cancer. A CNS-related disorder that is cancer may be a primary cancer of the CNS, such as astrocytoma, glioblastoma, etc., or a cancer that has metastasized to CNS tissue, such as lung cancer that has metastasized to the brain. Further non-limiting examples of CNS-related disorders include Parkinson's disease, lysosomal storage diseases, ischemia, neuropathic pain, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Canavan disease (CD).

[0076] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to cardiac cells (e.g., cardiac tissue). Consequently, in some embodiments, the AAV variants described herein may be useful for treating cardiovascular disorders. As used herein, a "cardiovascular disorder" refers to a disease or condition of the cardiovascular system. Cardiovascular diseases can affect the heart, circulatory system, arteries, veins, blood vessels, and / or capillaries. Cardiovascular disorders can be either inherited or acquired genetically through somatic mutations. Non-limiting examples of cardiovascular disorders include rheumatic heart disease, valvular heart disease, hypertensive heart disease, aneurysms, atherosclerosis, hypertension (e.g., high blood pressure), peripheral arterial disease (PAD), ischemic heart disease, angina, coronary heart disease, coronary artery disease, myocardial infarction, cerebrovascular disease, transient ischemic attack, inflammatory heart disease, cardiomyopathy, pericardial disease, congenital heart disease, heart failure, stroke, and myocarditis due to Chagas disease.

[0077] In some embodiments, the AAV variants described herein may target the lung and / or tissues of the pulmonary system (e.g., the respiratory system). Consequently, in some embodiments, the AAV variants described herein may be useful for treating pulmonary disease. As used herein, a "pulmonary disease" is a disease or illness of the pulmonary system. A pulmonary disease may affect the lungs or muscles involved in breathing. A pulmonary disease may have a genetic cause, either inherited or acquired through somatic mutation. A pulmonary disease may be a cancer of the lung, including, but not limited to, non-small cell lung cancer, small cell lung cancer, and pulmonary carcinoid tumors. Further non-limiting examples of pulmonary diseases include acute bronchitis, acute respiratory distress syndrome (ARDS), asbestosis, asthma, bronchiectasis, bronchiolitis, bronchiolitis obliterans organizing pneumonia (BOOP), bronchopulmonary dysplasia, byssinosis, chronic bronchitis, coccidioidomycosis (Cocci), chronic obstructive pulmonary disorder (COPD), cryptopathic organizing pneumonia (COP), cystic fibrosis, emphysema, hantavirus pulmonary syndrome, histoplasmosis, human metanephritis, and the like. These include: HIV, hypersensitivity pneumonitis, influenza, lymphangiocarcinoma, mesothelioma, Middle East respiratory syndrome, nontuberculous mycobacteria, whooping cough, pneumoconiosis (anthracosis), pneumonia, primary ciliary dyskinesia, primary pulmonary hypertension, pulmonary arterial hypertension, pulmonary fibrosis, pulmonary vascular disease, respiratory syncytial virus (RSV), sarcoidosis, severe acute respiratory syndrome (SARS), silicosis, sleep apnea, sudden infant death syndrome (SIDS), and tuberculosis.

[0078] In some embodiments, the AAV variants described herein may target liver tissue. Consequently, in some embodiments, the AAV variants described herein may be useful for treating liver disease. As used herein, "liver disease" refers to a disease or illness of the liver. Liver disease can be caused by either inherited or acquired genetic causes through somatic mutations. Liver disease can also be liver cancer, including, but not limited to, hepatocellular carcinoma (HCC), fibrolamellar carcinoma, cholangiocarcinoma, angiosarcoma, and hepatoblastoma. Further non-limiting examples of pulmonary diseases include Alagille syndrome, alpha 1 antitrypsin deficiency, autoimmune hepatitis, biliary atresia, cirrhosis, cystic disease of the liver, fatty liver disease, galactosemia, gallstones, Gilbert syndrome, hemochromatosis, liver disease during pregnancy, neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Reye's syndrome, sarcoidosis, toxic hepatitis, glycogen storage disease type 1, tyrosinemia, viral hepatitis A, B, C, Wilson's disease, and schistosomiasis.

[0079] In some embodiments, the AAV variants described herein may target kidney tissue. Consequently, in some embodiments, the AAV variants described herein may be useful for treating kidney disease. As used herein, "kidney disease" refers to a disease or condition of the liver. Kidney disease can be of either inherited or acquired genetic cause through somatic mutation. Kidney disease can also be cancer of the kidney, including, but not limited to, renal cell carcinoma, clear cell carcinoma, papillary carcinoma type 1, papillary carcinoma type 2, chromophobe carcinoma, oncocell carcinoma, collecting duct carcinoma, transitional cell carcinoma of the renal pelvis, and Wilms' tumor. Further non-limiting examples of kidney diseases include Abderhalden-Kaufmann-Lignac Syndrome (Nephropathic Cystinosis), Acute Renal Failure / Acute Kidney Injury, Acute Lobar Nephronia, Acute Phosphate Nephropathy, Acute Tubular Necrosis, Adenine Phosphoribosyltransferase Deficiency, Adenovirus Nephritis, Alport Syndrome, Amyloidosis, Angiomyolipoma, Analgesic Nephropathy, Angiotensin Antibodies and Focal Segmental Glomerulosclerosis, Antiphospholipid Syndrome, Anti-TNF-α Therapy Associated Glomerulonephritis, APOL1 Mutations, Apparent Mineralocorticoid Excess Syndrome, Aristolochic Acid Nephropathy,

[0080] Balkan Endemic Nephropathy, Bartter Syndrome, Beet Urinary Tract, Beta-Thalassemia Kidney Disease, Bile Cast Nephropathy, BK Polyoma, C1q Nephropathy, Cardiorenal Syndrome, CFHR5 Nephropathy, Cholesterol Embolism, Churg-Strauss Syndrome, Chyluria, Collapsing Glomerulopathy, CMV-Associated Glomerular Collapse, Congenital Nephrotic Syndrome, Conorenal Syndrome (Mainzer-Sardino Syndrome or Sardino-Mainzer Disease), Contrast-Induced Nephropathy, Copper Sulfate Intoxication, Cortical Necrosis Necrosis, Cryoglobulinemia, Crystal-Induced Acute Kidney Injury, Cystic Kidney Disease, Acquired Cystinuria, Dense Deposit Disease (MPGN Type 2), Dent Disease (X-Linked Recessive Nephrolithiasis), Dialysis Disequilibrium Syndrome, Diabetic Kidney Disease, Diabetes Insipidus, EAST Syndrome, Ectopic Ureter, Edema, Erdheim-Chester Disease, Fabry Disease, Familial Hypocalciuric Hypercalcemia, Fanconi Syndrome, Fraser Syndrome, Fibronectin Glomerulopathy, Fibrillary and Immunotactoid Glomerulopathy, Fraley Syndrome, Glomerulosclerosis, Focal Sclerosis, Focal-Spectral Glomerulopathy,

[0081] Galloway-Mowat syndrome, Gitelman syndrome, glomerular disease, tubular reflux Reflux, Glycosuria, Goodpasture's syndrome, Hemolytic uremic syndrome (HUS), Atypical hemolytic uremic syndrome (aHUS), Hemophagocytic syndrome, Hemorrhagic cystitis, Paroxysmal nocturnal hemoglobinuria and hemosiderinosis associated with hemolytic anemia, Hepatic veno-occlusive disease, Sinusoidal obstruction syndrome, Hepatitis C-related kidney disease, Hepatorenal syndrome, HIV-associated nephropathy (HIVAN), Horseshoe kidney (renal fusion), Hunners ulcer, Hyperaldosteronism, Hypercalcemia, Hyperkalemia, Hypermagnesemia, Hypernatremia, Hyperoxaluria, Hyperphosphatemia, Hypocalcemia, Hypokalemia, Hypokalemia-induced renal dysfunction, Hypomagnesemia, Hyponatremia, Hypophosphatemia, IgA nephropathy, IgG4 nephropathy, Interstitial cystitis, Bladder pain syndrome, Interstitial nephritis, Ivemark's syndrome, Kidney stones Stones, Nephrolithiasis, Leptospirosis kidney disease, Light Chain Deposition Disease, Monoclonal Immunoglobulin Deposition Disease, Liddle syndrome, Lightwood-Albright syndrome, Lipoprotein glomerulopathy, Lithium nephrotoxicity, Hereditary FSGS caused by LMX1B mutations, Lower back pain and hematuria, Lupus, Systemic lupus erythematosus, Lupus kidney disease, Lupus nephritis, Lyme disease-associated glomerulonephritis,

[0082] Malarial nephropathy, malignant hypertension, malakoplakia, meatal stenosis, medullary cystic kidney disease, medullary sponge kidney, megaureter, melamine toxicity and kidney, membranoproliferative glomerulonephritis, membranous nephropathy, Mesoamerican nephropathy, metabolic acidosis, metabolic alkalosis, microscopic polyangiitis, milk-alkali syndrome, minimal change disease, polycystic dysplastic kidney, multiple myeloma, myeloproliferative neoplasms and glomerulopathies, nail-patella syndrome, nephrocalcinosis, nephrogenic systemic fibrosis, nephroptosis (migrating kidney, nephroptosis) Ptosis), Nephrotic syndrome, Neurogenic bladder, Nodular glomerulosclerosis, Nongonococcal, Nutcracker syndrome, Orofacial-digital syndrome, Orthostatic hypotension, Orthostatic proteinuria, Osmotic diuresis, Page kidney, Papillary necrosis, Papillary renal syndrome (renal coloboma syndrome, isolated renal hypoplasia), Peritoneal-Renal syndrome, Posterior urethral valve, Postinfectious glomerulonephritis, Poststreptococcal glomerulonephritis, Polyarteritis nodosa, Polycystic kidney disease, Posterior urethral valve, Preeclampsia, Proliferative glomerulonephritis with monoclonal IgG deposits (Nasr disease), Proteinuria (protein in the urine), Pseudohyperaldosteronism, Pseudohypoparathyroidism, Pulmonary-renal syndrome, Pyelonephritis (kidney infection), Pyonephrosis

[0083] Radiation nephropathy, refeeding syndrome, reflux nephropathy, rapidly progressive glomerulonephritis, renal abscess, perinephric abscess, renal agenesis, renal artery aneurysm, renal artery stenosis, renal cell carcinoma, renal cyst, renal hypouricemia with exercise-induced acute renal failure, renal infarction, renal osteodystrophy, renal tubular acidosis, Reset Osmostat, retrocaval ureter, retroperitoneal fibrosis, rhabdomyolysis, rhabdomyolysis related to bariatric surgery, rheumatoid arthritis-related renal disease, sarcoidosis renal disease, salt-losing, renal and cerebral, Schimke's immune dyskinesia, scleroderma renal crisis, serpentine Fibula-polycystic kidney syndrome, Exner syndrome, sickle cell nephropathy, silica exposure and chronic kidney disease, kidney disease following hematopoietic cell transplantation, kidney disease associated with stem cell transplantation, thin basement membrane disease, benign familial hematuria, trigonitis, tuberous sclerosis, renal tubular dysgenesis, tumor lysis syndrome, uremia, uremic optic neuropathy, ureterocele, urethral caruncle, urethral stricture, urinary incontinence, urinary tract infection, urinary tract obstruction, vesicoenteric fistula, vesicoureteral reflux, von Hippel-Lindau disease, warfarin-associated nephropathy, Wegener's granulomatosis, granulomatosis with polyangiitis, and Wunderlich syndrome.

[0084] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to ocular tissue (e.g., ocular tissue or cells). Consequently, in some embodiments, the AAV variants described herein may be useful for treating ocular disorders. As used herein, an "ocular disorder" is an ocular disease or condition. Ocular diseases can affect the eye, sclera, cornea, anterior chamber, posterior chamber, iris, pupil, lens, vitreous humor, retina, or optic nerve. Ocular disorders can be genetic in origin, either inherited or acquired through somatic mutation. Non-limiting examples of ocular diseases and disorders include, but are not limited to, the following: age-related macular degeneration, retinopathy, diabetic retinopathy, macular edema, glaucoma, retinitis pigmentosa, and ocular cancer.

[0085] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to gastrointestinal tissue (e.g., tissues of the gastrointestinal tract). Consequently, in some embodiments, the AAV variants described herein may be useful for treating gastrointestinal disorders. As used herein, a "gastrointestinal disorder" is a disease or illness of the gastrointestinal tract. Gastrointestinal disorders can affect the mucosa (e.g., epithelium, lamina propria, muscularis mucosae, etc.), submucosa (e.g., submucosal plexus, myenteric plexus, etc.), muscularis, serosa and / or adventitia of the gastrointestinal tract, oral cavity, esophagus, pylorus, stomach, duodenum, small intestine, cecum, appendix, colon, anal canal, or rectum. Gastrointestinal disorders can be of genetic origin, either inherited or acquired through somatic mutation. Non-limiting examples of gastrointestinal diseases and disorders include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome, celiac disease, gastroesophageal reflux disease (GERD), achalasia, diverticulitis, diarrhea, and certain cancers (e.g., intestinal cancer, stomach cancer, colon cancer, rectal cancer, etc.).

[0086] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to breast tissue (e.g., breast tissue). Consequently, in some embodiments, the AAV variants described herein may be useful for treating breast disorders. As used herein, a "breast disorder" is a disease or condition of the breast. Breast diseases may affect the fibrous tissue, fatty tissue, lobules, or ducts of the breast. Breast disorders may be of genetic origin, either inherited or acquired through somatic mutation. Non-limiting examples of breast diseases and disorders include, but are not limited to, the following: mastitis, breast calcinosis, fat necrosis, fibroadenoma, fibrosis and simple cysts, galactorrhea, hyperplasia, and breast cancer.

[0087] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to pancreatic tissue (e.g., pancreatic tissue). Consequently, in some embodiments, the AAV variants described herein may be useful for treating pancreatic disorders. As used herein, a "pancreatic disorder" refers to a disease or condition of the pancreas. Pancreatic diseases may affect the head, neck, body, tail, islets (e.g., islets of Langerhans), acini, or columnar epithelium. Pancreatic disorders may be of genetic origin, either inherited or acquired through somatic mutation. Non-limiting examples of pancreatic diseases and disorders include, but are not limited to, diabetes (e.g., diabetes mellitus type 1 and diabetes mellitus type 2), pancreatitis (e.g., acute pancreatitis, chronic pancreatitis), and pancreatic cancer.

[0088] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to urinary tract tissue (e.g., tissue of the urinary tract, such as bladder tissue). Consequently, in some embodiments, the AAV variants described herein may be useful for treating urinary tract disorders. As used herein, a "urinary tract disorder" is a disease or illness of the urinary tract. Urinary tract diseases can affect the bladder, ureters, urethra, or prostate. Urinary tract disorders can be of genetic origin, either inherited or acquired through somatic mutation. Non-limiting examples of urinary tract diseases and disorders include, but are not limited to, the following: urinary tract infection, kidney stones, bladder control problems (e.g., urinary retention, urinary incontinence, etc.), cystitis, and bladder cancer.

[0089] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to uterine tissue (e.g., tissue of the uterus). Consequently, in some embodiments, the AAV variants described herein may be useful for treating uterine disorders. As used herein, a "uterine disorder" is a disease or illness of the uterus. Uterine disorders can affect the cervix, cervical canal, uterine body (fundus), endometrium, myometrium, or perimetrium. Uterine disorders can be of genetic cause, either inherited or acquired through somatic mutation. Non-limiting examples of uterine diseases and disorders include, but are not limited to, the following: adenomyosis, endometriosis, endometrial hyperplasia, Asherman's syndrome, and endometrial cancer.

[0090] The components to be cultured in the host cell for packaging the rAAV vector into an AAV capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell engineered using methods known to those skilled in the art to contain one or more of the required components. Most preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may also be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, the selected stable host cell may contain the selected component(s) under the control of a constitutive promoter and the selected other component(s) under the control of one or more inducible promoters. For example, stable host cells may be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter), but which contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated by one skilled in the art.

[0091] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to produce the rAAV of the present disclosure may be delivered to a packaging host cell using any suitable genetic elements (vectors). In some embodiments, a single nucleic acid encoding all three capsid proteins (e.g., VP1, VP2, and VP3) is delivered in a single vector. In some embodiments, the nucleic acids encoding the capsid proteins are delivered to a packaging host cell by two vectors: a first vector containing a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2) and a second vector containing a second nucleic acid encoding a single capsid protein (e.g., VP3).

[0092] In some embodiments, three vectors, each containing nucleic acid encoding a different capsid protein, are delivered to the host cell for packaging. The selected genetic elements may be delivered by any suitable method, including those described herein. The methods used to construct any of the disclosed embodiments are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and selecting a suitable method is not limited by this disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0093] In some embodiments, recombinant AAV may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (e.g., rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes).

[0094] Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19 (described in U.S. Pat. No. 6,001,650) and the pRep6cap6 vector (described in U.S. Pat. No. 6,156,303), both of which are incorporated herein by reference in their entireties. Accessory function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (e.g., "accessory functions") on which AAV depends for replication. Accessory functions include those functions required for AAV replication, but also include, without limitation, those moieties involved in transcriptional activation of AAV genes, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0095] In some aspects, the present disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell; a cell is "transfected" when exogenous DNA is introduced into the cell (e.g., across the cell membrane). Numerous transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids (such as nucleotide integration vectors and other nucleic acid molecules) into a suitable host cell.

[0096] "Host cell" refers to any cell that contains or is capable of containing a substance of interest. Often, host cells are mammalian cells. Host cells may be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNA involved in the production of recombinant AAV. The term encompasses the progeny of the original transfected cell. Thus, as used herein, "host cell" may refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent cell due to natural, accidental, or deliberate mutation.

[0097] As used herein, the term "cell line" refers to a population of cells capable of sustained or long-term growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is also known in the art that spontaneous or induced changes may occur in the karyotype during the storage or transfer of such clonal populations. Thus, cells derived from the cell line may not be exactly identical to the ancestral cell or culture, and the cell line referred to encompasses such variants.

[0098] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that leads to the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid (such as RNA).

[0099] The cells may also be transfected with a vector (e.g., a helper vector) that provides helper functions to the AAV. The vector that provides the helper functions may provide adenoviral functions, including, for example, E1a, E1b, E2a, and E4 ORF6. The adenoviral gene sequences that provide these functions may be obtained from any known adenoviral serotype, such as serotypes 2, 3, 4, 7, 12, and 40 (further including any of the currently identified human types known in the art). Thus, in some embodiments, the methods involve transfecting cells with a vector that expresses one or more genes required for AAV replication, AAV gene transcription, and / or AAV packaging.

[0100] As used herein, the term "vector" encompasses any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with appropriate control elements and that can transfer genetic sequences between cells. Thus, the term encompasses cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated as those vectors in which a nucleic acid segment (e.g., a nucleic acid sequence) to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery and required to initiate the specific transcription of a gene.

[0101] The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct location and orientation relative to the nucleic acid that controls the initiation of RNA polymerase and expression of the gene. The term "expression vector or construct" refers to any type of genetic construct containing a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of a nucleic acid, for example, to produce a biologically active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from the transcribed gene.

[0102] In some cases, the isolated capsid gene can be used to construct and package a recombinant AAV using methods well known in the art and to determine functional characteristics associated with the capsid protein encoded by the gene. For example, the isolated capsid gene can be used to construct and package a recombinant AAV (rAAV) containing a reporter gene (e.g., B-galactosidase, GFP, luciferase, etc.). The rAAV can then be delivered to an animal (e.g., a mouse), and the tissue targeting properties of the isolated novel capsid gene can be determined by examining the expression of the reporter gene in various tissues of the animal (e.g., heart, liver, kidney). Other methods for characterizing the isolated novel capsid gene are disclosed herein, and further methods are well known in the art.

[0103] The above-described methods for packaging a recombinant vector into a desired AAV capsid to produce an rAAV of the present disclosure are not intended to be limiting, and other suitable methods will be apparent to those of skill in the art.

[0104] Recombinant AAV vectors A "recombinant AAV (rAAV) vector" of the present disclosure typically consists, at a minimum, of a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector that is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and / or expression of the transgene in the cells of the target tissue.

[0105] The AAV sequences of the vector typically include cis-acting 5' and 3' inverted terminal sequences (see, for example, BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are tolerated. The ability to modify these ITR sequences is within the knowledge of the art. (See, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such a molecule employed in the present disclosure is a "cis-acting" plasmid in which a selected transgene sequence and associated regulatory elements contain the transgene flanked by 5' and 3' AAVITR sequences, which may be obtained from any known AAV, including currently identified mammalian AAV types.

[0106] In some embodiments, the present disclosure provides a self-complementary AAV vector. As used herein, the term "self-complementary AAV vector" (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the AAV ITRs. The absence of a TR prevents replication initiation at the vector end where the TR is absent. Generally, scAAV vectors generate a single-stranded inverted sequence genome with a wild-type (wt) AAV TR at each end and a mutant TR (mTR) in the middle.

[0107] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. Pseudotyping is the process of producing a virus or viral vector by combining it with an envelope protein from a foreign virus. The result is a pseudotyped viral particle. With this method, the envelope protein from the foreign virus can also be used to alter the host tropism or increase / decrease the stability of the viral particle. In some aspects, the pseudotyped rAAV contains nucleic acids from two or more different AAVs, where the nucleic acid from one AAV encodes a capsid protein and the nucleic acid from at least one other AAV encodes a protein and / or genome of another virus. In some embodiments, pseudotyped rAAV refers to an AAV that contains the inverted terminal repeats (ITRs) of one AAV serotype and the capsid protein of another AAV serotype. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with proteins of Y would be designated AAV X / Y (e.g., AAV2 / 1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs can be useful to combine the tissue-specific targeting ability of capsid proteins from one AAV serotype with viral DNA from another AAV serotype, thereby enabling targeted delivery of a transgene to a target tissue.

[0108] In addition to the key elements identified above for recombinant AAV vectors, the vectors also include conventional control elements that are necessary to be operably linked to the transgene elements in a manner that permits its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences encompass both expression control sequences proximal to a gene of interest and expression control sequences acting in trans or at a distance to regulate the gene of interest.

[0109] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., Kozak consensus sequences); sequences that improve protein stability; and, when desired, sequences that improve secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be used.

[0110] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably" linked when they are covalently linked such that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, and (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein.

[0111] Thus, a promoter region is said to be operably linked to a DNA sequence (so that the resulting transcript is translated into a desired protein or polypeptide) if the promoter region is capable of directing transcription of that DNA sequence. Similarly, two or more coding regions are operably linked when their transcription from a common promoter results in the expression of two or more proteins translated in-frame. In some embodiments, operably linked coding sequences produce a fusion protein. In some embodiments, operably linked coding sequences produce a functional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

[0112] In nucleic acids encoding proteins, a polyadenylation sequence is generally inserted after the transgene sequence and before the 3'AAVITR sequence. rAAV constructs useful in the present disclosure may also contain an intron, preferably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences may be used to produce proteins containing more than one polypeptide chain. The selection of these and other common vector elements is conventional, and many such sequences are available (see, e.g., Sambrook et al., and references cited therein, e.g., pages 3.18, 3.26, and 16.17, 16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989).

[0113] In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994;4:928-933; Mattion, NM et al., J Virology, November 1996;p. 8124-8127; Furler, S et al., Gene Therapy, 2001;8:864-873; and Halpin, C et al., The Plant Journal, 1999;4:453-459). The cleavage activity of 2A sequences has previously been demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994;4:928-933; Mattion, NM et al., J Virology, November 1996;p. 8124-8127; Furler, S et al., Gene Therapy, 2001;8:864-873; and Halpin, C et al., The Plant Journal, 1999;4:453-459; de Felipe, P et al., Gene Therapy, 1999;6:198-208; de Felipe, P et al., Human Gene Therapy, 2000;11:1921-1931; and Klump, H et al., Gene Therapy, 2001;8:811-817).

[0114] The precise nature of regulatory sequences required for gene expression in host cells may vary between species, tissues, or cell types but will generally include 5' non-transcribed and 5' non-translated sequences involved with initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer element, etc., as necessary. In particular, such 5' non-transcribed regulatory sequences will include a promoter region containing a promoter sequence for transcriptional control of an operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activating sequences, as desired. Vectors of the present disclosure may optionally include 5' leader or signal sequences. The selection and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

[0115] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen].

[0116] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological states (e.g., acute phase, specific differentiation state of cells), or only during cell replication. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Numerous other systems have also been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), and the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)). (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only during replicating cells.

[0117] In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that the expression of the transgene mimics the native expression. The native promoter may be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic the native expression.

[0118] In some embodiments, regulatory sequences confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: the liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, alpha-myosin heavy chain (a-MHC) promoter, gastrointestinal-specific mucin-2 promoter, eye-specific retinoschisin promoter, eye-specific K12 promoter, respiratory tissue-specific CC10 promoter, respiratory tissue-specific surfactant protein C (SP-C) promoter, breast tissue-specific PRCl promoter, breast tissue-specific RRM2 promoter, urinary tract tissue-specific uroplakin 2 (UPII) promoter, uterine tissue-specific lactoferrin promoter, or cardiac troponin T (cTnT) promoter.

[0119] Other exemplary promoters include the beta-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)); the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); the immunoglobulin heavy chain promoter; the T cell receptor α-chain promoter, and neuronal promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), as would be apparent to one skilled in the art.

[0120] In some embodiments, one or more binding sites for one or more miRNAs are incorporated into a transgene in an rAAV vector to inhibit transgene expression in one or more tissues of a transgene-carrying subject. Those skilled in the art will appreciate that binding sites may be selected to control transgene expression in a tissue-specific manner. For example, a binding site for liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. Target sites in an mRNA can be in the 5' UTR, 3' UTR, or coding region. Typically, target sites are in the 3' UTR of an mRNA. Furthermore, transgenes may be designed so that multiple miRNAs regulate mRNA by recognizing the same site or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly efficient inhibition of expression. The target site sequence may contain a total of 5 to 100, 10 to 60, or more nucleotides. The target site sequence may include at least 5 nucleotides of the target gene binding site sequence.

[0121] Recombinant AAV vectors: transgene coding sequences The composition of the transgene sequence of an rAAV vector will depend on the application for which the resulting vector will be used. For example, one type of transgene sequence includes a reporter sequence that produces a detectable signal upon expression. In another example, the transgene encodes a therapeutic protein or a therapeutic functional RNA. In another example, the transgene encodes a protein or functional RNA intended to be used for research purposes, e.g., to create a somatic transgenic animal model carrying the transgene, e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA intended to be used to create an animal model of a disease. Suitable transgene coding sequences will be apparent to those skilled in the art.

[0122] Reporter sequences that may be provided in a transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When linked to regulatory elements that drive their expression, reporter sequences provide signals that can be detected by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays (including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry). For example, if the marker sequence is a LacZ gene, the presence of the signal-transmitting vector is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-transmitting vector can be visually measured by color or light production in a luminometer. Such reporters can be useful, for example, to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of rAAV.

[0123] In some aspects, the present disclosure provides rAAV vectors for use in methods for preventing or treating one or more genetic deficiencies or dysfunctions in a mammal, such as, for example, a polypeptide deficiency or polypeptide excess in a mammal, and specifically for treating or reducing the severity or degree of a deficiency in a human exhibiting one or more disorders that result in a deficiency of such polypeptide in cells and tissues. The methods involve administering to a subject an rAAV vector encoding one or more therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc., in a pharmaceutically acceptable carrier in an amount and for a duration sufficient to treat such disorder in the subject suffering from the deficiency or disorder.

[0124] Thus, the present disclosure embraces the delivery of rAAV vectors encoding one or more peptides, polypeptides, or proteins useful for treating or preventing disease states in mammalian subjects. Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptotic factors, cytokines, anti-diabetic factors, anti-apoptotic agents, clotting factors, and anti-tumor factors. Other non-limiting examples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropins, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

[0125] The rAAV vector may contain a gene to be transferred to a subject to treat a disease associated with reduced expression, lack of expression, or dysfunction of the gene. Exemplary genes and associated conditions include, but are not limited to, glucose-6-phosphatase, associated with glycogen storage deficiency type 1A; phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency; galactose-1-phosphate uridyltransferase, associated with galactosemia; phenylalanine hydroxylase, associated with phenylketonuria; branched-chain alpha-ketoacid dehydrogenase, associated with maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium-chain acyl-CoA dehydrogenase, associated with medium-chain acetyl-CoA deficiency; and ornithine transcarbamylase, associated with ornithine transcarbamylase deficiency. argininosuccinate synthetase, associated with citrullinemia; low-density lipoprotein receptor protein, associated with familial hypercholesterolemia; UDP-glucuronosyltransferase, associated with Crigler-Najjar disease; adenosine deaminase, associated with severe combined immunodeficiency; hypoxanthine guanine phosphoribosyltransferase, associated with gout and Lesch-Nyhan syndrome; biotinidase, associated with biotinidase deficiency; beta-glucocerebrosidase, associated with Gaucher disease; beta-glucuronidase, associated with Sly syndrome; peroxisomal membrane protein 70 kDa, associated with Zellweger syndrome;

[0126] erythropoietin for the treatment of anemia due to thalassemia or renal failure; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic disease; thrombomodulin and tissue factor pathway inhibitors for the treatment of blocked blood vessels, e.g., as seen in atherosclerosis, thrombosis, or embolism; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; beta-adrenergic receptors, antisense to phospholamban, or mutant forms of phospholamban, and the sarco(endo)plasmic reticulum (the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and cardiac adenylyl cyclase; tumor suppressor genes such as p53 for the treatment of various cancers; cytokines such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancer; dystrophin or mini-dystrophin and utrophin or mini-utrophin for the treatment of muscular dystrophies; and insulin for the treatment of diabetes.

[0127] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, conditions, or disorders related to the central nervous system (CNS). The following is a non-limiting list of genes associated with CNS diseases: DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3Rtau / 4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, which are associated with Alzheimer's disease; UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β, which are associated with Parkinson's disease; IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin1, FTL, TITF-1, which are associated with Huntington's disease; FXN, which is associated with Friedreich's ataxia; ASPA, which is associated with Canavan disease; DMD, which is associated with muscular dystrophy; and SMN1, UBE1, DYNC1H1, which are associated with spinal muscular atrophy. In some embodiments, the present disclosure relates to a recombinant AAV comprising a nucleic acid that expresses one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to a recombinant AAV comprising a nucleic acid that expresses one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0128] In some embodiments, the present disclosure relates to nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or disorders related to the cardiovascular system. The following is a non-limiting list of genes associated with cardiovascular disease: VEGF, FGF, SDF-1, connexin 40, connexin 43, SCN4a, HIF1α, SERCa2a, ADCY1, and ADCY6. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0129] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or disorders associated with the pulmonary system. The following is a non-exhaustive list of genes associated with pulmonary diseases: TNFα, TGFβ1, SFTPA1, SFTPA2, SFTPB, SFTPC, HPS1, HPS3, HPS4, ADTB3A, IL1A, IL1B, LTA, IL6, CXCR1, and CXCR2. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0130] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating liver-related diseases, disorders, or disorders. The following is a non-exhaustive list of genes associated with liver disease: α1-AT, HFE, ATP7B, fumarylacetoacetate hydrolase (FAH), glucose-6-phosphatase, NCAN, GCKR, LYPLAL1, and PNPLA3. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0131] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating kidney-related diseases, disorders, or disorders. The following is a non-exhaustive list of genes associated with kidney disease: PKD1, PKD2, PKHD1, NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, SCARB3, FN1, COL4A5, COL4A6, COL4A3, COL4A4, FOX1C, RET, UPK3A, BMP4, SIX2, CDC5L, USF2, ROBO2, SLIT2, EYA1, MYOG, SIX1, SIX5, FRAS1, FREM2, GATA3, KAL1, PAX2, TCF2, and SALL1. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to a recombinant AAV comprising a nucleic acid that expresses one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0132] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating eye-related diseases, disorders, or disorders. The following is a non-exhaustive list of genes associated with eye diseases: CFH, C3, MT-ND2, ARMS2, TIMP3, CAMK4, FMN1, RHO, USH2A, RPGR, RP2, TMCO, SIX1, SIX6, LRP12, ZFPM2, TBK1, GALC, myocilin, CYP1B1, CAV1, CAV2, optineurin, and CDKN2B. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0133] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating breast-related diseases, disorders, or disorders. The following is a non-exhaustive list of genes associated with breast diseases: BRCA1, BRCA2, Tp53, PTEN, HER2, BRAF, and PARP1. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0134] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or disorders associated with the gastrointestinal tract. The following is a non-exhaustive list of genes associated with gastrointestinal diseases: CYP2C19, CCL26, APC, IL12, IL10, and IL-18. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0135] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or disorders related to the pancreas. The following is a non-exhaustive list of genes associated with pancreatic diseases: PRSS1, SPINK1, STK11, MLH1, KRAS2, p16, p53, and BRAF. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0136] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or disorders related to the urinary tract. The following is a non-exhaustive list of genes associated with urinary tract diseases: HSPA1B, CXCR1 & 2, TLR2, TLR4, TGF-1, FGFR3, RB1, HRAS, TP53, and TSC1. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids expressing one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0137] In some embodiments, the present disclosure relates to AAVs comprising nucleic acids encoding proteins or functional RNAs useful for treating diseases, disorders, or conditions associated with the uterus. The following is a non-exhaustive list of genes associated with ocular diseases: DN-ER, MLH1, MSH2, MSH6, PMS1, and PMS2. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the above-mentioned genes or fragments thereof. In some embodiments, the present disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit the expression of one or more of the above-mentioned genes.

[0138] The rAAV of the present disclosure can be used to restore expression of a gene whose expression is reduced, silenced, or otherwise dysfunctional in a subject (e.g., a tumor suppressor silenced in a subject with cancer). The rAAV of the present disclosure can also be used to knock down the expression of aberrantly expressed genes in a subject (e.g., an oncogene expressed in a subject with cancer). In some embodiments, an rAAV vector containing a nucleic acid encoding a cancer-associated gene product (e.g., a tumor suppressor) may be used to treat cancer by administering the rAAV vector containing the nucleic acid to a subject with cancer. In some embodiments, an rAAV vector containing a nucleic acid encoding a small interfering nucleic acid (e.g., shRNA, miRNA) that inhibits the expression of a cancer-associated gene product (e.g., an oncogene) may be used to treat cancer by administering the rAAV vector containing the nucleic acid to a subject with cancer. In some embodiments, rAAV vectors containing nucleic acids encoding gene products associated with cancer (or functional RNA that inhibits expression of genes associated with cancer) may be used for research purposes, for example, to study cancer or to identify therapeutics that treat cancer.

[0139] The following is a non-exhaustive list of exemplary genes (e.g., oncogenes and tumor suppressors) known to be associated with cancer development: AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF , BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, C DC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT 1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61 , DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6 , F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, G DF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB, HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1,<h2 style=";text-align:left;direction:ltr">ICAM1、ID1、ID2、IDUA、IER3、IFITM1、IGF1R、IGF2R、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、IRF3、ITGA3、ITGA6、ITGB4、JAK1、JARID1A、JUN、JUNB、J UND、K-アルファ-1、KIT、KITLG、KLK10、KPNA2、KRAS2、KRT18、KRT2A、KRT9、LAMB 1、LAMP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MA P3K8、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MAS1、MCC、MCM2、MCM4、MDM 2、MDM4、MET、MGST1、MICB、MLLT3、MME、MMP1、MMP14、MMP17、MMP2、MNDA、MSH2 、MSH6、MT3、MYB、MYBL1、MYBL2、MYC、MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、 NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINJ1、NMBR、NME1、NME2、NME3、NOTCH1、 NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、 PA2G4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PE A15、PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT 1、PLK2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKC BP1、PRNP、PRSS15、PSMA1、PTCH、PTEN、PTGS1、PTMA、PTN、PTPRN、RAB5A、RAC 1、RAD50、RAF1、RALBP1、RAP1A、RARA、RARB、RASGRF1、RB1、RBBP4、RBL2、REA、 REL、RELA、RELB、RET、RFC2、RGS19、RHOA、RHOB、RHOC、RHOD、RIPK1、RPN2、RP S6KB1、RRM1、SARS、SELENBP1、SEMA3C、SEMA4D、SEPP1、SERPINH1、SFN、SFPQ、<h2 style=";text-align:left;direction:ltr">SFRS7、SHB、SHH、SIAH2、SIVA、SIVA TP53、SKI、SKIL、SLC16A1、SLC1A4、SLC20A1、SMO、SMPD1、SNAI2、SND1、SNRPB2 、SOCS1、SOCS3、SOD1、SORT1、SPINT2、SPRY2、SRC、SRPX、STAT1、STAT2、STAT3、 STAT5B、STC1、TAF1、TBL3、TBRG4、TCF1、TCF7L2、TFAP2C、TFDP1、TFDP2、TGFA、TGFB1、TGFBI、TGFBR2、TGFBR3、THBS1、TIE、TIMP1、TIMP3、TJP1、TK1、TLE1、TNF 、TNFRSF10A、TNFRSF10B、TNFRSF1A、TNFRSF1B、TNFRSF6、TNFSF7、TNK1、TOB1、 TP53、TP53BP2、TP53I3、TP73、TPBG、TPT1、TRADD、TRAM1、TRRAP、TSG101、TUFM、 TXNRD1、TYRO3、UBC、UBE2L6、UCHL1、USP7、VDAC1、VEGF、VHL、VIL2、WEE1、WNT1 、WNT2、WNT2B、WNT3、WNT5A、WT1、XRCC1、YES1、YWHAB、YWHAZ、ZAP70、およびZNF9。、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> The rAAV vector may include as a transgene a nucleic acid encoding a protein or functional RNA that modulates apoptosis. The following is a non-exhaustive list of nucleic acids encoding genes associated with apoptosis and their gene products and their homologs, as well as small interfering nucleic acids (e.g., shRNAs, miRNAs) that inhibit the expression of these genes and their homologs, useful as transgenes in certain embodiments of the present disclosure: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1. , BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARD8, CARD9, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, G ADD45A, GDNF, HRK, IGF1R, LTA, LTBR, ​​MCL1, NOL3, PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFR SF11B, TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF1 0, TNFSF14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 DRD2, GRIA1, GRIA2,GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3Rtau / 4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β, IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin1, FTL, TITF-1, FXN, ASPA, DMD, and SMN1, UBE1, DYNC1H1. ,

[0141] Those skilled in the art will also recognize that in the case of a transgene encoding a protein or polypeptide, mutations resulting in conservative amino acid substitutions can be made in the transgene to provide functionally equivalent variants or homologs of the protein or polypeptide. In some aspects, the present disclosure embraces sequence alterations that result in conservative amino acid substitutions in the transgene. In some embodiments, the transgene includes a gene with a dominant-negative mutation. For example, the transgene may express a mutant protein that interacts with the same elements as the wild-type protein, thereby blocking the same aspects of the wild-type protein's function.

[0142] Useful transgene products also include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression through cleavage / degradation of target RNA transcripts or translational repression of target messenger RNAs (mRNAs). miRNAs are typically naturally expressed as 19-25 untranslated RNA products that ultimately exert their activity through sequence-specific interactions with the 3' untranslated region (UTR) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into miRNA duplexes and then into "mature" single-stranded miRNA molecules. This mature miRNA guides the multiprotein complex miRISC, which identifies target sites (e.g., in the 3' UTR region) of target mRNAs based on their complementarity to the mature miRNA.

[0143] The following non-limiting list of miRNA genes and their homologs are useful as transgenes or as targets for the small interfering nucleic acids (e.g., miRNA sponges, antisense oligonucleotides, TuD RNAs) encoded by the transgene in certain embodiments of the present methods: hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-10 1, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b,

[0144] hsa-miR-106b*, hsa-miR-107, hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178, hsa-miR-1179, hsa-miR-1180, hsa-miR- 1181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201, hsa-miR-1202, hsa-miR-1203, hs a-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-1224-3p, h sa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR-12 29, hsa-miR-1231, hsa-miR-1233, hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124, hsa-miR-124*, hsa-miR-1243, hsa-m iR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR-1253 、hsa-miR-1254、hsa-miR-1255a、hsa-miR-1255b、hsa-miR-1256、hsa-miR -1257、hsa-miR-1258、hsa-miR-1259、hsa-miR-125a-3p、hsa-miR-125a-5 p、hsa-miR-125b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-miR-126、hsa -miR-126*、hsa-miR-1260、hsa-miR-1261、hsa-miR-1262、hsa-miR-1263、hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267, hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-mi R-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278, hsa-mi R-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282, hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287 hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299, hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1 303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a, hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, h sa-miR-132, hsa-miR-132*, hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-1 35a、hsa-miR-135a*、hsa-miR-135b、hsa-miR-135b*、hsa-miR-136、hsa-miR-136*、hsa-miR-137、hsa-miR-138、hsa-miR-138-1*、hsa-miR-138-2*、hsa-miR-139-3p, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*, hsa-miR-146b-3p, hsa-miR- 146b-5p、hsa-miR-147、hsa-miR-147b、hsa-miR-148a、hsa-miR-148a*、hsa-miR-148b、hsa-miR-148b*、hsa-miR-149、hsa-miR-149*、hsa-miR-150、hsa-miR-150*、hsa-miR-151-3p、hsa-miR-151-5p、hsa-miR-152、hsa-miR-153、hsa-miR-154、hsa-miR-154*、hsa-miR-155、hsa-miR-155*、

[0145] hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*, hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*, hsa-miR-181a hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*, hsa-miR-1825, hs a-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*, hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*, hsa-miR-187, h sa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a, hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b, hsa-miR- 191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194 *, hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p, h sa-miR-199b-5p、hsa-miR-19a、hsa-miR-19a*、hsa-miR-19b、hsa-miR-19b-1*、hsa-miR-19b-2*、hsa-miR-200a、hsa-miR-200a*、hsa-miR-200b、hs a-miR-200b*、hsa-miR-200c、hsa-miR-200c*、hsa-miR-202、hsa-miR-202*、hsa-miR-203、hsa-miR-204、hsa-miR-205、hsa-miR-206、hsa-miR-208a、hsa-miR-208b、hsa-miR-20a、hsa-miR-20a*、hsa-miR-20b、hsa-miR-20b*、hsa-miR-21、hsa-miR-21*、hsa-miR-210、hsa-miR-211、hsa-miR-212、h sa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221, hsa-miR- 221*、hsa-miR-222、hsa-miR-222*、hsa-miR-223、hsa-miR-223*、hsa-miR-224、hsa-miR-23a、hsa-miR-23a*、hsa-miR-23b、hsa-miR-23b*、hsa-miR -24、hsa-miR-24-1*、hsa-miR-24-2*、hsa-miR-25、hsa-miR-25*、hsa-miR-26a、hsa-miR-26a-1*、hsa-miR-26a-2*、hsa-miR-26b、hsa-miR-26b*、h sa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-m iR-298, hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b, hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR- 29c*、hsa-miR-300、hsa-miR-301a、hsa-miR-301b、hsa-miR-302a、hsa-mi R-302a*、hsa-miR-302b、hsa-miR-302b*、hsa-miR-302c、hsa-miR-302c*、hsa-miR-302d, hsa-miR-302d*, hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b, hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d, hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hs a-miR-31*, hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p, hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329, hsa-miR-330-3p

[0146] hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p, hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p, hsa-miR-33 8-5p、hsa-miR-339-3p、hsa-miR-339-5p、hsa-miR-33a、hsa-miR-33a*、hsa-miR-33b、hsa-miR-33b*、hsa-miR-340、hsa-miR-340*、hsa-miR-342-3p、 hsa-miR-342-5p、hsa-miR-345、hsa-miR-346、hsa-miR-34a、hsa-miR-34a*、hsa-miR-34b、hsa-miR-34b*、hsa-miR-34c-3p、hsa-miR-34c-5p、hsa-mi R-361-3p, hsa-miR-361-5p, hsa-miR-362-3p, hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-3 69-3p、hsa-miR-369-5p、hsa-miR-370、hsa-miR-371-3p、hsa-miR-371-5p、hsa-miR-372、hsa-miR-373、hsa-miR-373*、hsa-miR-374a、hsa-miR-374a *, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375, hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377, hsa-miR-377*, hsa-mi R-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsa-miR- 409-3p、hsa-miR-409-5p、hsa-miR-410、hsa-miR-411、hsa-miR-411*、hsa-miR-412、hsa-miR-421、hsa-miR-422a、hsa-miR-423-3p、hsa-miR-423-5p、hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*, hsa-miR-433 hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-m iR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p, hsa-m iR-485-5p、hsa-miR-486-3p、hsa-miR-486-5p、hsa-miR-487a、hsa-miR-487b、hsa-miR-488、hsa-miR-488*、hsa-miR-489、hsa-miR-490-3p、hsa-miR -490-5p、hsa-miR-491-3p、hsa-miR-491-5p、hsa-miR-492、hsa-miR-493、hsa-miR-493*、hsa-miR-494、hsa-miR-495、hsa-miR-496、hsa-miR-497、h sa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p, hsa-miR-502-3p, h sa-miR-502-5p、hsa-miR-503、hsa-miR-504、hsa-miR-505、hsa-miR-505*、hsa-miR-506、hsa-miR-507、hsa-miR-508-3p、hsa-miR-508-5p、hsa-miR- 509-3-5p、hsa-miR-509-3p、hsa-miR-509-5p、hsa-miR-510、hsa-miR-511、hsa-miR-512-3p、hsa-miR-512-5p、hsa-miR-513a-3p、hsa-miR-513a-5p、hsa-miR-513b、hsa-miR-513c、hsa-miR-514、hsa-miR-515-3p、hsa-miR-515-5p、hsa-miR-516a-3p、hsa-miR-516a-5p、hsa-miR-516b、hsa-miR-517*、hsa-miR-517a、hsa-miR-517b、hsa-miR-517c、hsa-miR-518a-3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、hsa-miR-518d-3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa-miR-518f、hsa-miR-518f*、hsa-miR-519a、hsa-miR-519b-3p、hsa-miR-519c-3p、hsa-miR-519d、hsa-miR-519e、hsa-miR-519e*、hsa-miR-520a-3p、hsa-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、hsa-miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR-520h、hsa-miR-521、hsa-miR-522、

[0147] hsa-miR-523, hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-53 2-5p、hsa-miR-539、hsa-miR-541、hsa-miR-541*、hsa-miR-542-3p、hsa-miR-542-5p、hsa-miR-543、hsa-miR-544、hsa-miR-545、hsa-miR-545*、hsa- miR-548a-3p、hsa-miR-548a-5p、hsa-miR-548b-3p、hsa-miR-548b-5p、hsa-miR-548c-3p、hsa-miR-548c-5p、hsa-miR-548d-3p、hsa-miR-548d-5p、 hsa-miR-548e、hsa-miR-548f、hsa-miR-548g、hsa-miR-548h、hsa-miR-548i、hsa-miR-548j、hsa-miR-548k、hsa-miR-548l、hsa-miR-548m、hsa-miR- 548n、hsa-miR-548o、hsa-miR-548p、hsa-miR-549、hsa-miR-550、hsa-miR-550*、hsa-miR-551a、hsa-miR-551b、hsa-miR-551b*、hsa-miR-552、hsa- miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa -miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR -574-3p、hsa-miR-574-5p、hsa-miR-575、hsa-miR-576-3p、hsa-miR-576-5p、hsa-miR-577、hsa-miR-578、hsa-miR-579、hsa-miR-580、hsa-miR-581、hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR-588, hsa-miR-589, hsa-miR-5 89*, hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595, hsa-miR-596, hsa-miR-597, hsa-m iR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606, hsa-miR-607, hsa-miR-6 08, hsa-miR-609, hsa-miR-610, hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p, hsa-miR-615-5p, hsa-miR-616, hsa-miR -616*、hsa-miR-617、hsa-miR-618、hsa-miR-619、hsa-miR-620、hsa-miR-621、hsa-miR-622、hsa-miR-623、hsa-miR-624、hsa-miR-624*、hsa-miR- 625, hsa-miR-625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa- miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR- 642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668, hsa-miR-671-3p, hsa -miR-671-5p、hsa-miR-675、hsa-miR-7、hsa-miR-708、hsa-miR-708*、hsa-miR-7-1*、hsa-miR-7-2*、hsa-miR-720、hsa-miR-744、hsa-miR-744*、hsa-miR-758、hsa-miR-760、hsa-miR-765、hsa-miR-766、hsa-miR-767-3p、hsa-miR-767-5p、hsa-miR-768-3p、hsa-miR-768-5p、

[0148] hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*, hsa-miR-885-3p, hsa-miR -885-5p、hsa-miR-886-3p、hsa-miR-886-5p、hsa-miR-887、hsa-miR-888、hsa-miR-888*、hsa-miR-889、hsa-miR-890、hsa-miR-891a、hsa-miR-891b、hsa-miR-892a、hsa-miR-892b、hsa-miR-9、hsa-miR-9*、hsa-miR-920、hsa- miR-921, hsa-miR-922, hsa-miR-923, hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934, hsa-miR-935, hsa-miR-936, hsa-mi R-937、hsa-miR-938、hsa-miR-939、hsa-miR-940、hsa-miR-941、hsa-miR-942、hsa-miR-943、hsa-miR-944、hsa- miR-95、hsa-miR-96、hsa-miR-96*、hsa-miR-98、hsa-miR-99a、hsa-miR-99a*、hsa-miR-99b、およびhsa-miR-99b*。

[0149] miRNA inhibits the function of the mRNA it targets, and as a result, inhibits the expression of the polypeptide coded by mRNA.Therefore, blocking (partially or completely) the activity of miRNA (for example, silencing miRNA) can effectively induce or restore the expression of the polypeptide whose expression is inhibited (de-repressing the polypeptide).In one embodiment, de-repression of the polypeptide coded by the mRNA target of miRNA is achieved by inhibiting miRNA activity in cells through any one of various methods.

[0150] For example, blocking the activity of miRNA can be achieved by hybridization of a small interfering nucleic acid (for example, antisense oligonucleotide, miRNA sponge, TuD RNA) that is complementary or substantially complementary to miRNA, thereby blocking the interaction between miRNA and its target mRNA.As used herein, a small interfering nucleic acid that is substantially complementary to miRNA is one that can hybridize with miRNA and block the activity of miRNA.In some embodiments, a small interfering nucleic acid that is substantially complementary to miRNA is a small interfering nucleic acid that is complementary to miRNA at all bases except 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases.In some embodiments, a small interfering nucleic acid that is substantially complementary to miRNA is a small interfering nucleic acid sequence that is complementary to miRNA at at least one base.

[0151] "miRNA inhibitors" are agents that block miRNA function, expression, and / or processing. Illustratively, these molecules include, but are not limited to, microRNA-specific antisense molecules, microRNA sponges, tough decoy RNAs (TuD RNAs), and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit miRNA interaction with the Drosha complex. As discussed above, microRNA inhibitors can be expressed in cells from transgenes in rAAV vectors. MicroRNA sponges specifically inhibit miRNAs through complementary heptameric seed sequences (Ebert, MS Nature Methods, Epub August 12, 2007). In some embodiments, an entire family of miRNAs can be silenced using a single sponge sequence. TuD RNA achieves efficient and long-term repression of specific miRNAs in mammalian cells (see, for example, Takeshi Haraguchi, et al., Nucleic Acids Research, 2009, Vol. 37, No. 6 e43, the contents of which regarding TuD RNA are incorporated herein by reference). Other methods for silencing miRNA function (derepressing miRNA targets) in cells will also be apparent to those skilled in the art.

[0152] In some embodiments, the cloning capacity of a recombinant RNA vector may limit the desired coding sequence and require the complete replacement of the virus's 4.8 kilobase genome. Thus, large genes may in some cases not be suitable for use in standard recombinant AAV vectors. Those skilled in the art will appreciate that options are available in the art for overcoming limited coding capacity. For example, the AAV ITRs of two genomes can anneal to form head-to-tail concatemers, nearly doubling the capacity of the vector. Insertion of splice sites allows for removal of the ITRs from the transcript. Other options for overcoming limited cloning capacity will also be apparent to those skilled in the art.

[0153] Somatic transgenic animal models produced using rAAV-based gene transfer The present disclosure also relates to the production of somatically transgenic animal models of disease using recombinant adeno-associated virus (rAAV)-based methods. The methods are based, at least in part, on the observation that AAV serotypes and their variants mediate efficient and stable gene transfer in a tissue-specific manner in adult animals. rAAV elements (capsid, promoter, transgene product) are combined to obtain somatically transgenic animal models that express stable transgenes in a time- and tissue-specific manner. Somatically transgenic animals produced by the methods of the present disclosure can serve as useful models of human diseases, pathological conditions, and / or characterize the effects of genes whose function (e.g., tissue-specific, disease role) is unknown or not fully understood.

[0154] For example, an animal (e.g., a mouse) can be infected at a distinct developmental stage (e.g., age) with an rAAV containing a capsid with specific tissue targeting capability (e.g., liver, heart, pancreas) and a transgene with a tissue-specific promoter driving expression of a gene involved in disease. Upon infection, the rAAV infects distinct cells of the target tissue and produces the transgene product.

[0155] In some embodiments, the sequence of the coding region of the transgene is modified. The modification may alter the function of the product encoded by the transgene. The effect of the modification can then be studied in vivo by generating a somatic transgenic animal model using the methods disclosed herein. In some embodiments, the modification of the coding region sequence is a nonsense mutation resulting in a fragment (e.g., a truncated version). In other cases, the modification is a missense mutation resulting in an amino acid substitution. Other modifications are possible and will be apparent to those skilled in the art.

[0156] In some embodiments, the transgene causes a pathological condition. A pathological transgene is a gene whose product is involved in a disease or disorder (e.g., causes a disease or disorder, predisposes an animal to a disease or disorder) and / or can induce a disease or disorder in an animal. The animal can then be observed to assess several aspects of the disease (e.g., progression, response to treatment, etc.). These examples are not intended to be limiting, and other aspects and examples are disclosed herein and described in more detail below.

[0157] In some aspects, the present disclosure provides methods for producing somatic transgenic animal models through the targeted destruction of specific cell types. For example, a model of type 1 diabetes can be produced by the targeted destruction of pancreatic beta-islets. In another example, the targeted destruction of specific cell types can be used to evaluate the role of specific cell types in human disease. In this regard, transgenes encoding cellular toxins (e.g., diphtheria toxin A (DTA)) or pro-apoptotic genes (NTR, Box, etc.) can be useful as transgenes for the functional ablation of specific cell types. Other exemplary transgenes whose products kill cells are encompassed by the methods disclosed herein and will be apparent to those skilled in the art.

[0158] In some aspects, the present disclosure provides methods for producing somatically transgenic animal models for studying the long-term effects of gene overexpression or knockdown. Long-term gene overexpression or knockdown (e.g., by shRNA, miRNA, miRNA inhibitors, etc.) in specific target tissues can disrupt normal metabolic balance and establish pathological conditions, thereby producing animal models of diseases such as cancer. In some aspects, the present disclosure provides methods for producing somatically transgenic animal models for studying the long-term effects of gene overexpression or knockdown, potentially of oncogenes and other genes for studying tumorigenesis and gene function in target tissues. Useful transgene products include proteins known to be associated with cancer and small interfering nucleic acids that inhibit the expression of such proteins.

[0159] Other suitable transgenes can be readily selected by one of skill in the art, provided they are useful for creating animal models of tissue-specific pathological conditions and / or diseases.

[0160] Recombinant AAV administration method The rAAV in the composition can be delivered to a subject by any suitable method known in the art. The rAAV is preferably suspended in a physiologically compatible carrier (for example, in a composition) and can be administered to a subject, for example, a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human animal of the primate order (for example, macaque). In some embodiments, the host animal does not include a human.

[0161] Delivery of rAAV to a mammalian subject can be, for example, by intramuscular injection or by administration into the mammalian subject's bloodstream. Administration into the bloodstream can be by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream via isolated limb perfusion, a technique well known in the surgical arts (a method that essentially allows the artisan to isolate a limb from the systemic circulation prior to administration of rAAV virions). A variant of the isolated limb perfusion technique described in U.S. Pat. No. 6,177,403 can also be employed by those skilled in the art to administer virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissues.

[0162] Moreover, in some instances, it may be desirable to deliver virions to the CNS of a subject. "CNS" refers to all cells and tissues of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neurons (neuronal cells), glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAV may be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as by injection into the ventricular region and into the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule (such as by stereotactic injection), with a needle, catheter, or related device (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

[0163] Compositions of the present disclosure may include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs (each carrying one or more different transgenes).

[0164] Suitable carriers can be easily selected by those skilled in the art, taking into account the indications for which rAAV is intended. For example, one suitable carrier includes saline, which may be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of a carrier is not a limitation of the present disclosure.

[0165] Optionally, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0166] rAAV is administered in an amount sufficient to transfect the cells of desired tissue and provide sufficient level of gene transfer and expression without undue adverse effects.Conventional and pharmaceutically acceptable administration routes include but are not limited to direct delivery to selected organ (for example, intraportal delivery to the liver), oral, inhalation (including nasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral and other parenteral administration routes.Administration routes can be combined as desired.

[0167] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., a dose unit in genome copies per kilogram body weight (GC / kg), will vary based on several factors, including, but not limited to: the route of administration of the rAAV virions, the gene or RNA expression level required to achieve the therapeutic effect, the particular disease or disorder being treated, and the gene or RNA product stability. One of skill in the art can readily determine the rAAV virion dose range for treating a patient with a particular disease or disorder based on the aforementioned factors as well as other factors well known in the art.

[0168] The effective amount of rAAV is sufficient to target infection of animals and target desired tissues.In some embodiments, the effective amount of rAAV is sufficient to produce a stable somatic gene transfer animal model.The effective amount mainly depends on factors such as the subject's species, age, weight, health status, and tissue to be targeted, and therefore may vary between animals and tissues.For example, the effective amount of rAAV is generally about 10 9 ~10 16 In some embodiments, the rAAV is administered in a volume ranging from about 1 ml to about 100 ml of solution containing genome copies. 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the rAAV is administered at a dose of 10 genome copies per kg. 10 , 10 11 , 10 12 , 10 13 , or 10 14 In some cases, the dose is about 10 genome copies. 11 ~10 12 Dosage between 10 and 20 copies of the rAAV genome is appropriate. 12 rAAV genome copies are effective in targeting heart, liver, and pancreatic tissues, and in some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0169] In some embodiments, the rAAV composition may be specifically formulated to contain rAAV at a high concentration (e.g., at a concentration of 10 13 The rAAV is formulated to reduce aggregation of AAV particles in compositions present at concentrations of 0.01 GC / ml or greater. Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of detergents, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the entire contents of which are incorporated herein by reference.)

[0170] The formulation of pharmaceutically acceptable excipient and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for use with the specific compositions described herein in various treatment regimens.

[0171] Typically, these preparations may contain at least about 0.1% or more of active compound, but the percentage of active ingredient(s) may of course vary, and may conveniently be between about 1 or 2% and about 70% or 80% or more by weight or volume of the total preparation. Of course, the amount of active compound in each therapeutically useful composition may be adjusted so that a suitable dosage is obtained in any unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, administration route, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical preparations. In light of this, various dosages and treatment regimens may be desired.

[0172] In some situations, it may be desirable to deliver a rAAV-based therapeutic construct in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities described in U.S. Patent Nos. 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver the rAAV. In some embodiments, the preferred mode of administration is via portal vein injection.

[0173] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0174] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0175] For administration of injectable aqueous solutions, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermic injection fluid or injected at the proposed injection site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0176] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV into the appropriate solvent, along with various other ingredients listed herein (if required), followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient, plus any desired additional ingredients, from a previously sterile-filtered solution thereof.

[0177] The rAAV compositions disclosed herein may also be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the protein's free amino groups) and acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are conveniently administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like.

[0178] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a host.

[0179] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. may be used for the introduction of compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by rAAV vectors may be formulated for delivery encapsulated in either lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, or the like.

[0180] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-times have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for the preparation of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0181] Liposomes have been successfully used with numerous cell types that are normally resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints typical of viral-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the efficacy of liposome-mediated drug delivery have been completed.

[0182] Liposomes are formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 angstroms, which contain aqueous solution in their cores.

[0183] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally encapsulate substances stably and reproducibly. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0184] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and is described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and efficiency of drug penetration into and through the circulatory system. Other contemplated drug delivery alternatives are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0185] Kits and Related Compositions In some embodiments, the agents described herein may be assembled into pharmaceutical, diagnostic, or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kits may include one or more containers housing the components of the present disclosure and instructions for use. That is, such kits may include one or more agents described herein along with instructions describing the intended application and suitable use of these agents. In some embodiments, the agents in the kits may be in pharmaceutical formulations and dosages suitable for specific applications and methods of administering the agents. Kits for research purposes may contain components in concentrations or amounts appropriate for carrying out various experiments.

[0186] Kits may be designed to facilitate researchers' use of the methods described herein and may take a number of forms. Each of the components of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), where applicable. In some cases, some of the compositions may be constitutable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" may define instructional and / or promotional components, typically accompanied by written instructions on or associated with the packaging of the present disclosure. Instructions may also encompass any oral or electronic instructions, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., provided in any manner that would clearly identify the instructions to the user as being related to the kit. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, and these instructions may also reflect approval by the agency of the manufacture, use, or sale for animal administration.

[0187] The kit may contain any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or for isolating, mixing, and applying a sample to a subject. The kit may include a container containing an agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be aseptically prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may contain another agent that is aseptically prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may also include one or more or all of the components required to administer the agent to an animal, such as a syringe, topical application device, or IV needle tubing and bag, particularly in the case of a kit for producing a particular somatic cell animal model.

[0188] The kit may have various forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum-sealable pouch, a sealable thermoformed tray, or similar pouch or tray form, with the accessories loosely packed within a pouch, one or more tubes, containers, boxes, or bags. The kit may be sterilized after the accessories are added, allowing the individual accessories in the container to be opened in an otherwise convenient manner. The kit may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths (such as gauze) for applying or removing a disinfecting agent, disposable gloves, a support for the agent prior to administration, etc., depending on the particular application.

[0189] The instructions included in the kit may accompany methods for detecting latent AAV from cells. Additionally, kits of the present disclosure may include instructions, negative and / or positive controls, containers, diluents and buffers for the samples, sample preparation tubes, and a printed or electronic table of reference AAV sequences for sequence comparison.

[0190] example Example 1: Isolation from human tissue of transcriptionally active novel AAV capsid sequences with desired tissue tropism and properties. This example describes novel AAV capsid sequences isolated through the following steps: 1) PCR amplification of wtAAV genomes present in normal and diseased human tissues; 2) high-throughput single-molecule real-time (SMRT) sequencing of PCR amplicon libraries; 3) variant identification / profiling through bioinformatic analyses; and 4) selection of high-confidence ORFs that can be translated into full-length capsid proteins. A schematic depiction of the workflow used in this example is shown in Figure 1A-1B.

[0191] This approach leverages the natural pool of genomic diversity observed among viral genomes isolated from both normal and tumor tissues. Conceptually, in vivo tissues act as natural incubators for viral genomic diversity through selective pressure and / or immune evasion. Thus, discoveries of inter- and intra-tissue variability, as well as inter-patient diversity, benefit from methods that can profile the full spectrum of AAV variants found among tissues and organs of human origin.

[0192] PCR amplification of AAV genomes from human tissues To isolate diverse AAV variants, potentially identifying new serotypes with unique tropisms, 844 human surgical specimens from 455 patients were collected from West China Hospital (Sichuan University, Chengdu, China). These tissues encompassed a wide range of tissue / organ types and various tumor types (Table 1). Specifically, AAV variants were identified from nine normal liver tissues, seven liver tumors, four benign prostatic hyperplasia tissues, two normal lung tissues, one pancreatic tumor tissue, one breast cancer tissue, one normal breast tissue, one gastric cancer tissue, one normal gastric tissue, one brain tissue, and one glioma sample.

[0193] Total genomic DNA was extracted from human tissues and subjected to PCR amplification of AAV capsid sequences. The PCR primers used in this example are listed in Table 2. Briefly, either universal AAV primers (e.g., RepF318, AV2cas) for amplification of the 4.1-kb AAVrep-cap sequence or universal AAV primers (e.g., CapF, CapR) for amplification of the 2.3-kb AAVcap sequence were used for PCR.

[0194] Table 1: Clinical specimens for wtAAV genome amplification [Table 1]

[0195] Table 2: PCR primer sequences [Table 2]

[0196] High-throughput sequencing and bioinformatics analysis of AAV PCR products AAV PCR products were subjected to high-throughput single-molecule real-time (SMRT) sequencing. This approach obviates the need to perform viral genome reconstruction and chimera prediction from aligned short read fragments obtained from other conventional high-throughput genome sequencing methodologies.

[0197] Using a variant analysis pipeline developed from open-source bioinformatics tools, we identified over 600 previously undescribed high-confidence AAV2, AAV2 / 3 hybrid, and AAV8 capsid sequence variants. Specifically, we identified 224 AAV8 variants (with 1–10 single amino acid variants); 425 AAV2 variants (with 1–20 single amino acid variants); and 194 AAV2 / 3 hybrid variants (with 10–50 single amino acid variants). Tables 3, 4, and 5 summarize the unique capsid protein variants. For comparison, the wild-type AAV2, AAV3, and AAV8 capsid amino acid sequences are listed in SEQ ID NOs: 869, 870, and 871, respectively. Figure 7 is a scatter plot displaying the distribution of distinct AAV2 capsid variants and AAV2 / 3 variants with one or more single amino acid variants.

[0198] Table 3: Unique AAV2 and AAV2 / 3 hybrid variants identified by SMRT sequencing (amino acid sequences) and bioinformatics analysis. [Table 3]

[0199] Table 4: Unique AAV8 variants (amino acid sequences) identified by SMRT sequencing and bioinformatics analysis. [Table 4]

[0200] Table 5: Additional AAV8 variant capsid proteins [Table 5]

[0201] Example 2: Identification of AAV8 variants with improved in vivo tropism. A subset of candidate AAV8 variants (e.g., B2, B3, B44, and B61) were cloned into AAV packaging vectors using standard molecular cloning methods and packaged with a luciferase reporter gene driven by the CB6 promoter. The resulting vectors were injected into mice, and the in vivo level of luciferase transgene expression was analyzed by whole-animal imaging and relative luminescence quantification. It was observed that the B2 (SEQ ID NO: 854) and B3 (SEQ ID NO: 855) variants had higher expression in the liver after intramuscular injection (Figures 2A-2D), whereas the B61 (SEQ ID NO: 865) variant had higher transduction efficiency in the brain and spinal cord compared to AAV9 after IV injection of neonatal mice (Figures 3A-3B). This is noteworthy, given that wild-type AAV8 was observed to cross the blood-brain barrier less than AAV9. One AAV8 variant, B44 (SEQ ID NO: 861), has better liver transduction ability compared to AAV8 after IM injection (Figures 4A-4B).

[0202] Phylogenetic analysis was performed to compare AAV8 capsid variants B2, B3, B44, and B61 with other AAV serotypes. Briefly, the amino acid sequences of AAV8 variants were aligned with other publicly available AAV sequences using ClustalW, and phylogenetic trees were inferred using the Minimum Evolution method in MEGA 6.06. Bioinformatics analysis results indicate that the B2, B3, B44, and B61 sequences are related to the Clade E [AAV8] capsid protein (Figure 5). Representative amino acid substitutions of AAV8 variants are listed in Table 6.

[0203] Table 6: Representative amino acid substitutions in AAV8 variants compared to wild-type AAV8 [Table 6]

[0204] Example 3: In vitro assessment of rAAV genome packaging efficiency and initial characterization of candidate capsid variants. Molecular cloning of packaging plasmid constructs containing selected AAV capsid variants AAV2 and AAV2 / 3 hybrid capsid variants identified by SMRT sequencing were cloned into packaging plasmids by replacing the conventional viral capsid genes using standard molecular cloning strategies (e.g., site-directed mutagenesis of parental AAV2 or AAV2 / 3 capsid expression plasmids, PCR-based cloning and Gibson assembly, or outsourced synthesis). Figure 8 shows the vector constructs to be used in multiplexed screening of the discovered capsid variants. An overview of proposed transgene cassettes to be used for various diagnostic strategies is shown in Table 7.

[0205] Table 7: Transgene cassettes for various diagnostic strategies [Table 7]

[0206] High-throughput small-scale vector production and multiplexed assessment of packaging efficiency by titration for vector genomes Quantification of rAAV vector genomes in crude lysates was used to directly test the packaging efficiency of rAAV variants with both first-generation (single-stranded AAV) and second-generation (self-complementary AAV) vectors immediately after triple transfection of HEK293 packaging cells. This provides a streamlined alternative to performing the entire workflow of small-scale vector production, followed by silver staining and traditional PCR titration of vector genomes to assess viral quality for all discovered variants. This method, which can be adapted for implementation in a 96-well format, was used to rapidly identify variants that produce high-titer vectors.

[0207] Serological evaluation of novel AAV variants Candidate variants with high packaging efficiency were screened against the ongoing AAV for antibody cross-reactivity by standard means, such as capsid immunological assays to test the novel rAAV against serum from AAV-immunized rabbits. In addition, pooled human IgG (IVIG) neutralization assays were performed for each candidate variant to determine the likelihood of pre-existing humoral immunity in the human population.

[0208] Example 4: In vivo analysis of rAAV2 and rAAV2 / 3 variants to study vector transformation biology, prevalence of pathotoxicity, tissue / organ tropism, and biodistribution profiles. Mouse studies Candidate capsid variants are classified based on tissue distribution and prioritized by organ of interest. The group of candidate variants is subjected to clustered indexing (Figure 6A), whereby multiple packaging plasmids expressing candidate capsid variants are mixed and expressed to package unique DNA-barcoded transgenes by triple transfection (e.g., F9 coagulation factor IX (F.IX) to assess liver targeting and expression efficiency of secreted factors, EGFP to assess biodistribution and extent of tissue-specific transformation via organ / tissue sections and comparative immunofluorescence microscopy, or luciferase (Luc) to assess the quality of CNS and liver transformation via live animal imaging).

[0209] For studies quantifying the capacity of rAAV variants for liver-targeted transgene expression and secretion, we designed rAAV constructs containing the thyroxine-binding globulin (TGB) liver-specific promoter. For studies profiling whole-animal vector transduction, we designed constructs containing the CMV-enhancer and chicken β-actin promoter (CB6) regulatory cassettes.

[0210] Vectors encapsulating indexed transgenes are injected into adult and newborn mice by different administration routes and screened for secreted F.IX expression, EGFP expression, or Luc expression in a one-month longitudinal study to profile AAV variant-mediated transgene expression. Administration routes for the CNS / brain include intraperipheral (IV, to test transduction across the blood-brain barrier), intracerebroventricular (ICV), intraparenchymal, and intrathecal. Administration for the retina is performed via subretinal injection. In some embodiments, IV injection also targets the liver.

[0211] Animals exhibiting unique transgene expression (e.g., transgenes delivered by AAV2, AAV2 / 3, or AAV8) compared to control animals are sacrificed and organs harvested. Individual organs are assayed for the presence and abundance of the barcoded transgene by conventional PCR amplification of bulk DNA extracts or cDNA libraries containing the transgene message, followed by Illumina sequencing to identify barcoded transgenes enriched in each tissue. Figure 9 outlines the general strategy for transgene indexing design. The detectable barcoded transgene abundance and tissue / organ distribution reflect the tropism and transduction efficiency of each group of candidate rAAV variants. A group of high-efficiency candidates with desired vector properties is selected. Individual candidate variants from the selected group are used to package barcoded transgenes for a second round of screening to identify individual high-efficiency variants. Clustering-indexing can be performed iteratively in multiple rounds of hierarchical selection to reduce the workload.

[0212] Non-human primate (NHP) research Candidate rAAV variants will be screened for biodistribution in non-human primates using a clustering-indexing methodology similar to that outlined in the mouse study (Figure 6B). Organ-targeting transduction efficiency via different administration routes will be assessed again in NHPs to validate the rAAV variant profile observed in the previous mouse study.

[0213] Immunogenicity, prevalence of neutralizing antibodies in the human population, capacity for genotoxicity, and general aspects of pathogenicity will be quantified in parallel with primary assessments to determine the transformation profile in non-human primate (NHP) animals (e.g., histopathology of multiple tissues and organs to examine T cell or neutrophil infiltrates, monitoring hepatotoxicity by ALT / AST activity, and analyzing inflammation by examination of tissue sections).

[0214] Example 5: Isolation of novel AAV capsid sequences. Additional AAV capsid sequences were isolated. Using a variant analysis pipeline developed from bioinformatics tools, an additional 263 high-confidence AAV2 and AAV2 / 3 hybrid capsid sequence variants that have not previously been described were identified. For comparison, the wild-type AAV2 and AAV3 capsid amino acid sequences are listed in SEQ ID NOs: 869 and 870, respectively.

[0215] Table 8: Additional unique AAV2 and AAV2 / 3 hybrid variants identified by SMRT sequencing and bioinformatics analysis (amino acid sequences). [Table 8]

[0216] The corresponding DNA sequences are provided for all libraries. The nucleic acid sequences of the AAV2 capsid variants correspond to SEQ ID NOs: 1989-2077. The nucleic acid sequences of the AAV2 / 3 capsid variants correspond to SEQ ID NOs: 2078-2251.

[0217] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. Also, the phraseology and terminology used herein is for purposes of description and should not be regarded as limiting. The use of "comprising," "including," or "having," "containing," "involving," and variations thereof is intended to cover the items listed thereafter in this specification and equivalents thereof and additional items.

[0218] Having thus described several aspects of at least one embodiment of this disclosure, it should be understood that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of this disclosure. Consequently, the foregoing description and drawings are by way of example only.

Claims

1. A recombinant expression vector comprising a nucleic acid encoding a polypeptide having a sequence consisting of SEQ ID NO:

66.

2. An isolated AAV capsid protein comprising the amino acid sequence of SEQ ID NO:

66.

3. A recombinant expression vector comprising a nucleic acid sequence encoding the isolated AAV capsid protein of claim 2.

4. A recombinant AAV (rAAV) comprising the isolated AAV capsid protein of claim 2.

5. A host cell containing a nucleic acid comprising a coding sequence for a polypeptide consisting of SEQ ID NO: 66, wherein said coding sequence is operably linked to a promoter.

6. The rAAV of claim 4 for use in a treatment method comprising administering the rAAV to a subject, wherein the rAAV contains a transgene, and wherein the rAAV infects cells of a target tissue of the subject and delivers the transgene.

7. 7. The rAAV for use according to claim 6, wherein the subject is selected from humans, mice, rats, rabbits, dogs, cats, sheep, pigs, and non-human animals of the primate order.

8. A method for generating a somatic gene transfer animal model, comprising administering the rAAV described in claim 4 to a non-human animal, wherein the rAAV contains a transgene, and wherein the rAAV infects cells of a target tissue of the non-human animal.

9. 8. The rAAV for use according to claim 6 or 7, wherein the transgene expresses a transcript comprising at least one binding site for a miRNA, and the miRNA inhibits the activity of the transgene in tissues other than the target tissue by hybridizing to the binding site.

10. 8. The rAAV for use according to claim 6 or 7, wherein the transgene is a protein-coding gene or encodes a small interfering nucleic acid.

11. 11. The rAAV for use according to claim 10, wherein the small interfering nucleic acid is a miRNA, a miRNA sponge, or a TuD RNA that inhibits the activity of a miRNA in a subject or animal.

12. 12. The rAAV for use according to any one of claims 6, 7 or 9-11, wherein the rAAV is administered intravenously, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.

13. 10. A kit comprising a container containing a recombinant AAV (rAAV) having the isolated AAV capsid protein of claim 2, Optionally, the container is a syringe.

Images