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Definitions

• the disclosure in some aspects relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells.
• the disclosure provides novel AAVs and methods of use thereof as well as related kits.
• rAAVs Recombinant AAV adeno-associated viruses
• rAAVs are capable of driving stable and sustained transgene expression in target tissues without notable toxicity and host immunogenicity.
• rAAVs are promising delivery vehicles for long-term therapeutic gene expression.
• low transduction efficiency and restricted tissue tropisms by currently available rAAV vectors can limit their application as feasible and efficacious therapies.
• faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues is a concern. Accordingly, a need remains for new AAV vectors for gene delivery.
• AAVs described herein comprise amino acid variations in one or more capsid proteins that confer new or enhanced tissue tropism properties.
• variants of AAV2, AAV2/3 e.g. , AAV2/3 hybrid
• AAV8 have been identified and are disclosed herein that possess useful tissue targeting properties.
• variants of AAV8 are provided that are useful for transducing cells, such as, human hepatocytes (e.g. , present in liver tissue), central nervous system cells (CNS cells), and others.
• variants of AAV2, AAV2/3 e.g.
• AAV2/3 hybrid AAV8 are provided that, in some embodiments, are useful for targeting cells of the ocular tissue (e.g. , the eye), gastrointestinal tract, respiratory system, breast tissue, pancreatic tissue, urinary tract tissue, uterine tissue, tissue associate with certain cancers (e.g. , breast cancer, prostate cancer, etc.), and other tissues.
• the variant AAVs described herein target tissue other than the tissue targeted by their corresponding wild-type AAVs.
• the disclosure in some aspects provides an isolated nucleic acid comprising a sequence encoding a polypeptide selected from the group consisting of: SEQ ID NO: 1-409, 435-868, and 1726-1988, which encodes an AAV capsid protein.
• a fragment of the isolated nucleic acid is provided.
• the fragment of the isolated nucleic acid does not encode a peptide that is identical to a sequence of any one of SEQ ID NOs: 869, 870, or 871.
• the disclosure provides a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 410-434, 876-1718, and 1989-2251.
• the nucleic acid encodes an AAV capsid protein, or a variant thereof and/or an AAV assembly-activating protein (AAP), or a variant thereof.
• AAP AAV assembly-activating protein
• AAP is in a different open reading frame of the nucleic acid than the AAV capsid protein.
• the AAP is AAV2 AAP (AAP-2), or variant thereof.
• the disclosure in some aspects provides 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.
• the isolated AAV capsid protein comprises a sequence selected from: SEQ ID NOs: 1-409, 837-852 or 1726-1814, wherein an amino acid of the sequence that is not identical to a corresponding amino acid of the sequence set forth as SEQ ID NO: 869 is replaced with a conservative substitution.
• the disclosure provides AAV2/3 hybrid capsid proteins.
• the isolated AAV capsid protein comprises a sequence selected from: SEQ ID NOs: 435-628 and 1815- 1988, wherein an amino acid of the sequence that is not identical to a corresponding amino acid of the sequence set forth as SEQ ID NO: 869 or 870 is replaced with a conservative substitution.
• the isolated AAV capsid protein comprises a sequence selected from: SEQ ID NOs: 629-836 or 853-868, wherein an amino acid of the sequence that is not identical to a corresponding amino acid of the sequence set forth as SEQ ID NO: 871 is replaced with a conservative substitution.
• a composition is provided that comprises any of the foregoing isolated AAV capsid proteins.
• the composition further comprises a pharmaceutically acceptable carrier.
• a composition of one or more of the isolated AAV capsid proteins of the disclosure and a physiologically compatible carrier is provided.
• a recombinant AAV (rAAV) is provided that comprises any of the foregoing isolated AAV capsid proteins.
• a composition comprising the rAAV is provided.
• the composition comprising the rAAV further comprises a pharmaceutically acceptable carrier.
• recombinant AAV is also provided, wherein the recombinant AAV includes one or more of the isolated AAV capsid proteins of the disclosure.
• a host cell contains a nucleic acid that comprises a coding sequence selected from the group consisting of: SEQ ID NO: 410- 434, 876-1718 and 1989-2251, that is operably linked to a promoter.
• a composition comprising the host cell and a sterile cell culture medium is provided.
• a composition comprising the host cell and a cryopreservative is provided.
• a method for delivering a transgene to a subject comprises administering any of the foregoing rAAVs to a subject, wherein the rAAV comprises at least one transgene, and wherein the rAAV infects cells of a target tissue of the subject.
• subject is selected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, and a non-human primate.
• the subject is a human.
• the at least one transgene is a protein coding gene. In certain embodiments, the at least one transgene encodes a small interfering nucleic acid. In certain embodiments, the small interfering nucleic acid is a miRNA. In certain embodiments, the small interfering nucleic acid is a miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in the subject. In certain embodiments, the miRNA is expressed in a cell of the target tissue In certain embodiments, the target tissue is liver, central nervous system (CNS), ocular, gastrointestinal, respiratory, breast, pancreas, urinary tract, or uterine tissue.
• CNS central nervous system
• the transgene expresses a transcript that comprises at least one binding site for a miRNA, wherein the miRNA inhibits activity of the transgene, in a tissue other than the target tissue, by hybridizing to the binding site.
• the rAAV is administered to the subject intravenously, transdermally, intraocularly, intrathecally, intracererbally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.
• a method for generating a somatic transgenic animal model comprises administering any of the foregoing 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.
• the transgene is at least one protein coding gene. In certain embodiments, the transgene encodes at least one small interfering nucleic acid. In some embodiments, the transgene encodes at least one reporter molecule. In certain embodiments, the small interfering nucleic acid is a miRNA. In certain embodiments, the small interfering nucleic acid is a miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in the animal. In certain embodiments, the miRNA is expressed in a cell of the target tissue In certain embodiments, the target tissue is liver, central nervous system (CNS), ocular, gastrointestinal, respiratory, breast, pancreas, urinary tract, or uterine tissue.
• CNS central nervous system
• the transgene expresses a transcript that comprises at least one binding site for a miRNA, wherein the miRNA inhibits activity of the transgene, in a tissue other than the target tissue, by hybridizing to the binding site.
• methods for generating a somatic transgenic animal model that comprise administering any of the foregoing rAAVs to a non-human animal, wherein the rAAV comprises at least one transgene, wherein the transgene expresses a transcript that comprises at least one binding site for a miRNA, wherein the miRNA inhibits activity of the transgene, in a tissue other than a target tissue, by hybridizing to the binding site of the transcript.
• the transgene comprises a tissue specific promoter or inducible promoter.
• the tissue specific promoter is a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, mucin-2 promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a retinoschisin promoter, a K12 promoter, a CCIO promoter, a surfactant protein C (SP-C) promoter, a PRC 1 promoter, a RRM2 promoter, uroplakin 2 (UPII) promoter, or a lactoferrin promoter.
• TSG liver- specific thyroxin binding globulin
• PPY pancreatic polypeptide
• Syn synapsin-1
• retinoschisin promoter a K12 promoter
• K12 promoter
• the rAAV is administered to the animal intravenously, transdermally, intraocularly, intrathecally, orally, intramuscularly, subcutaneously, intranasally, or by inhalation.
• a somatic transgenic animal model is provided that is produced by any of the foregoing methods.
• kits for producing a rAAV comprises a container housing an isolated nucleic acid having a sequence of any one of SEQ ID NO: 410-434, 876-1718, and 1989-2251.
• the kit comprises a container housing an isolated nucleic acid encoding a polypeptide having a sequence of any one of SEQ ID NO: 1-409, 435-868, or 1726-1988.
• the kit further comprises instructions for producing the rAAV.
• the kit further comprises at least one container housing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene.
• a kit comprising a container housing a recombinant AAV having any of the foregoing isolated AAV capsid proteins.
• the container of the kit is a syringe.
• the disclosure relates to the use of AAV based vectors as vehicles for, delivery of genes, therapeutic, prophylactic, and research purposes as well as the development of somatic transgenic animal models.
• the 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 those of adenoviral vectors (e.g., up to 100% in vivo tissue transduction depending upon target tissue and vector dose) in the absence of vector related toxicology.
• the disclosure relates to AAV serotypes having liver, central nervous system (CNS), ocular, gastrointestinal, respiratory, breast, pancreas, urinary tract, or uterine tissue- targeting capabilities. These tissues are associated with a broad spectrum of human diseases including neurological, metabolic, diabetic, ocular, respiratory, gastrointestinal, urinary tract, and reproductive diseases and certain cancers.
• the rAAV includes at least one transgene.
• the transgene may be one which causes a pathological state.
• the transgene encoding a protein that treats a pathological state.
• novel AAVs of the disclosure may be used in a method for delivering a transgene to a subject.
• the method is performed by administering a rAAV of the disclosure to a subject, wherein the rAAV comprises at least one transgene.
• the rAAV targets a predetermined tissue of the subject.
• the AAVs of the disclosure may be used in a method for generating a somatic transgenic animal model.
• the method is performed by administering a rAAV of the disclosure to an animal, wherein the rAAV comprises at least one transgene, wherein the transgene causes a pathological state, and wherein the rAAV targets a predetermined tissue of the animal.
• the transgene may express a number of genes including cancer related genes, pro- apoptotic genes and apoptosis-related genes.
• the transgene expresses a small interfering nucleic acid capable of inhibiting expression of a cancer related gene.
• the transgene expresses a small interfering nucleic acid capable of inhibiting expression of an apoptosis-related gene.
• the small interfering nucleic acid in other embodiments is a miRNA or shRNA.
• the transgene expresses a toxin, optionally wherein the toxin is DTA.
• the transgene expresses a reporter gene that is optionally a reporter enzyme, such as Beta-Galactosidase or a Fluorescent protein, such as GFP or luciferase.
• the transgene may express a miRNA.
• the transgene expresses a miRNA sponge, wherein miRNA sponge inhibits the activity of one or more miRNAs in the animal.
• the miRNA may be an endogenous miRNA or it may be expressed in a cell of a liver, central nervous system (CNS), ocular, gastrointestinal, respiratory, breast, pancreas, urinary tract, or uterine tissue, in some embodiments.
• the rAAV may transduce many different types of tissue, such as neurons, squamous epithelial cells, renal proximal or distal convoluted tubular cells, mucosa gland cells, blood vessel endothelial cells, endometrial cells, retinal cells, or certain cancer cells (e.g. , breast cancer cells, prostate cancer cells, etc.).
• tissue such as neurons, squamous epithelial cells, renal proximal or distal convoluted tubular cells, mucosa gland cells, blood vessel endothelial cells, endometrial cells, retinal cells, or certain cancer cells (e.g. , breast cancer cells, prostate cancer cells, etc.).
• the rAAV is administered at a dose of 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some embodiments the rAAV is administered at a dose of 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 genome copies per kg.
• the rAAV may be administered by any route. For instance it may be administered intravenously (e.g. , by portal vein injection) in some embodiments.
• the transgene includes a tissue specific promoter such as a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, mucin-2 promoter, a pancreatic polypeptide (PPY) promoter, a synapsin- 1 (Syn) promoter, a retinoschisin promoter, a K12 promoter, a CC 10 promoter, a surfactant protein C (SP-C) promoter, a PRC 1 promoter, a RRM2 promoter, uroplakin 2 (UPII) promoter, or a lactoferrin promoter.
• TSG liver- specific thyroxin binding globulin
• PPY pancreatic polypeptide
• Syn synapsin- 1
• retinoschisin promoter a K12 promoter
• K12 promoter a CC 10 promoter
• SP-C
• the somatic transgenic animal model may be a mammal, such as a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, a non-human primate.
• a putative therapeutic agent may be administered to the somatic transgenic animal model to determine the effect of the putative therapeutic agent on the pathological state in the animal.
• the disclosure is a somatic transgenic animal produced by the methods described herein.
• a kit for producing a rAAV that generates a somatic transgenic animal having a pathological state in a predetermined tissue is provided according to another aspect of the disclosure.
• the kit includes at least one container housing a recombinant AAV vector, at least one container housing a rAAV packaging component, and instructions for constructing and packaging the recombinant AAV.
• the rAAV packaging component may include a host cell expressing at least one rep gene and/or at least one cap gene.
• the host cell is a 293 cell.
• the host cell expresses at least one helper virus gene product that affects the production of rAAV containing the recombinant AAV vector.
• the at least one cap gene may encode a capsid protein from an AAV serotype that targets the predetermined tissue.
• a rAAV packaging component includes a helper virus optionally wherein the helper virus is an adenovirus or a herpes virus.
• the rAAV vector and components therein may include any of the elements described herein.
• the rAAV vector comprises a transgene, such as any of the transgenes described herein.
• the transgene expresses a miRNA inhibitor (e.g. , a miRNA sponge or TuD RNA), wherein miRNA inhibitor inhibits the activity of one or more miRNAs in the somatic transgenic animal.
• a miRNA inhibitor e.g. , a miRNA sponge or TuD RNA
• FIGs. 1A-1B show workflow schematics for the identification of AAV variants.
• FIG. 1A depicts high-throughput detection of novel AAV variants in selected human tissues.
• FIG. IB shows a summary of the pipeline for bioinformatics analysis of sequencing data.
• FIGs. 2A-2D show data relating to in vivo detection of FFLuc transgene activity with different administrations of selected AAV8 variants.
• FIG. 2A shows luciferase activities of different AAV8 variants were evaluated at week 6 after IV (intravenous), IM (intramuscular), or IN (intranasal) injection.
• FIGs. 2B-2D data relating to evaluation of FFLuc activity for each variant, B2 (FIG. 2B), B3 (FIG. 2C), and B61 (FIG. 2D), compared to AAV8
• FIGs. 4A-4B show data relating to in vivo detection of FFLuc transgene activity after right hindlimb intramuscular (IM) injection of the AAV8 variant B44 compared to AAV8.
• FIG. 4A shows whole animal Luciferase expression of variant B44 was evaluated at week 6 after IM injection.
• FIG. 5 shows a phylogenic comparison of AAV8 variants (B2, B3, B61) to other AAV serotypes.
• FIG. 6A shows a schematic depiction of a workflow for the in vivo characterization of novel AAV variants by high-throughput tropism screening.
• FIG. 6B shows a schematic depiction of a workflow for the NHP characterization of novel AAV variants by high-throughput tropism screening.
• FIG. 7 shows a scatter plot displaying the distribution of distinct AAV2 capsid variants (409 total) and AAV2/3 variants (194 total) harboring one or more single-amino-acid variants.
• FIG. 8 shows diagrams of vector constructs used in the multiplexed screening of discovered capsid variants. Unique 6-bp barcodes were cloned into transgenes and packaged into candidate capsid variants.
• FIG. 9 shows a schematic of an indexed transgene and high-throughput sequencing library design to assess capsid variant tropism profiling.
• the indexed and adapter cassette containing a 6-bp barcode (1° barcode) and a BstEII restriction site can be cloned into vector constructs using flanking BsrGI and Sacl sites.
• Whole crude DNA from rAAV-treated tissues containing both host genome and vector genomes was cut with BstEII enzyme.
• the resulting 5'- overhang was used to specifically ligate to an adapter containing a second barcode, which allows for further multiplexed sequencing and streamlining; and a 5'-biotin modification, which can be used to select for adapter-containing fragments using magnetic bead
• Enriched material can then undergo PCR amplification using primers specific to adapter and transgene sequences to produce libraries for high-throughput sequencing.
• SEQ ID NOs.: 1719-1725 are shown from top to bottom.
• Adeno-associated virus is a small (-26 nm) replication-defective, non- enveloped virus that generally depends on the presence of a second virus, such as adenovirus or herpes virus, for its growth in cells.
• AAV is not known to cause disease and induces a very mild immune response.
• AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
• the disclosure in some aspects seeks to overcome this shortcoming by providing novel AAVs having distinct tissue targeting capabilities for gene therapy and research applications.
• new AAV capsid proteins are provided that have distinct tissue targeting capabilities.
• an AAV capsid protein is isolated from the tissue to which an AAV comprising the capsid protein targets.
• methods for delivering a transgene to a target tissue in a subject are provided. The transgene delivery methods may be used for gene therapy (e.g., to treat disease) or research (e.g., to create a somatic transgenic animal model) applications.
• a central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of integrated and/or episomal genomes in a host cell.
• Methods used for isolating novel AAV include PCR based molecular rescue of latent AAV DNA genomes, infectious virus rescue of latent pro viral genome from tissue DNAs in vitro in the presence of adenovirus helper function, and rescue of circular proviral genome from tissue DNAs by rolling-circle-linear amplification, mediated by an isothermal phage Phi-29 polymerase. All of these isolation methods take advantage of the latency of AAV proviral DNA genomes and focus on rescuing persistent viral genomic DNA.
• the disclosure relates to the discovery that novel AAV variants with desirable tissue tropisms can be identified from in vivo tissues of a subject.
• in vivo tissue exploits the natural reservoir of genomic diversity observed among viral genomic sequences isolated from both normal and tumor tissues of a subject.
• in vivo tissues act as natural incubators for viral (e.g., viral capsid protein) diversity through selective pressure and/or immune evasion.
• the disclosure relates to the discovery that PCR products resulting from amplification of AAV DNA (e.g., AAV DNA isolated or extracted from a host cell or in vivo tissue of a subject) can be subjected to high-throughput single-molecule, real-time (SMRT) sequencing to identify novel capsid protein variants.
• AAV DNA e.g., AAV DNA isolated or extracted from a host cell or in vivo tissue of a subject
• SMRT single-molecule, real-time sequencing
• Endogenous latent AAV genomes are transcriptionally active in mammalian cells (e.g., cells of nonhuman primate tissues such as liver, spleen and lymph nodes).
• mammalian cells e.g., cells of nonhuman primate tissues such as liver, spleen and lymph nodes.
• both rep and cap gene transcripts are detected with variable abundances by RNA detection methods (e.g., RT-PCR).
• RT-PCR RNA detection methods
• the presence of cap gene transcripts and ability to generate cDNA of cap RNA through reverse transcription (RT) in vitro significantly increases abundance of templates for PCR-based rescue of novel cap sequences from tissues and enhances the sensitivity of novel AAV discovery.
• Novel cap sequences may also be identified by transfecting cells with total cellular DNAs isolated from the tissues that harbor proviral AAV genomes at very low abundance, The cells may be further transfected with genes that provide helper virus function (e.g., adenovirus) to trigger and/or boost AAV gene transcription in the transfected cells.
• helper virus function e.g., adenovirus
• novel cap sequences of the disclosure may be identified by isolating cap mRNA from the transfected cells, creating cDNA from the mRNA (e.g., by RT-PCR) and sequencing the cDNA.
• AAVs isolated from mammals, particularly non-human primates are useful for creating gene transfer vectors for clinical development and human gene therapy applications.
• the disclosure provides in some aspects novel AAVs that have been discovered in various in vivo tissues (e.g., liver, brain, gastric, respiratory, breast, pancreatic, rectal, prostate, urologic, and cervical tissues) using the methods disclosed herein.
• the tissue(s) in which a novel AAV variant is discovered is a cancerous tissue (e.g., a tumor or a cancer cell).
• nucleic acids encoding capsid proteins of these novel AAVs have been discovered in viral genomic DNA isolated from the human tissues. Examples of tissues in which novel AAV capsid proteins have been discovered are described in Table 1. Nucleic acid and protein sequences as well as other information regarding the AAVs are set forth in Tables 3-5 and 8, and in the sequence listing.
• Isolated nucleic acids of the 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.
• isolated nucleic acids of the disclosure include any nucleic acid having a sequence encoding a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 1-409, 435-868, and 1726-1988.
• the disclosure provides an isolated nucleic acid that has substantial homology with 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 does not encode a protein having an amino acid sequence as set forth in SEQ ID NOs: 869, 870, or 871.
• isolated AAV capsid proteins of the 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.
• the disclosure provides an isolated capsid protein that has substantial homology with a protein having a sequence as set forth in any one of SEQ ID NOs 1-409, 837-852, or 1726-1814, but that does not have an amino acid sequence as set forth in SEQ ID NO: 869.
• isolated AAV capsid proteins of the disclosure include 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.
• the disclosure provides an isolated capsid protein that has substantial homology with a protein having a sequence as set forth in any one of SEQ ID NOs 435-628 or 1815-1988, but that does not have an amino acid sequence as set forth in SEQ ID NO: 869 or 870.
• isolated AAV capsid proteins of the 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.
• the disclosure provides an isolated capsid protein that has substantial homology with a protein having a sequence as set forth in any one of SEQ ID NOs 629-836 or 853-868, but that does not have an amino acid sequence as set forth in SEQ ID NO: 871.
• “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties.
• nucleic acid indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in about 90 to 100% of the aligned sequences.
• polypeptide indicates that, when optimally aligned with appropriate gaps, insertions or deletions with another polypeptide, there is nucleotide sequence identity in about 90 to 100% of the aligned sequences.
• highly conserved means at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. In some cases, highly conserved may refer to 100% identity. Identity is readily determined by one of skill in the art by, for example, the use of algorithms and computer programs known by those of skill in the art.
• sequences of nucleic acids or polypeptides are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as "Clustal W", accessible through Web Servers on the internet.
• Multiple Sequence Alignment Programs such as "Clustal W”, accessible through Web Servers on the internet.
• Vector NTI utilities may also be used.
• algorithms known in the art can be used to measure nucleotide sequence identity, including those contained in the programs described above.
• polynucleotide sequences can be compared using BLASTN, which provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Similar programs are available for the comparison of amino acid sequences, e.g. , the "Clustal X" program, BLASTP.
• any of these programs are used at default settings, although one of skill in the art can alter these settings as needed.
• one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. Alignments may be used to identify corresponding amino acids between two proteins or peptides.
• corresponding amino acid is an amino acid of a protein or peptide sequence that has been aligned with an amino acid of another protein or peptide sequence. Corresponding amino acids may be identical or non-identical. A corresponding amino acid that is a non-identical amino acid may be referred to as a variant amino acid. Table 6 provides examples of variant amino acids.
• homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
• nucleic acid sequence refers to a DNA or RNA sequence.
• nucleic acid captures sequences that include any of the known base analogues of DNA and RNA such as, but 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- methylpseudo-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2- methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylgu
• proteins and nucleic acids of the disclosure are isolated.
• isolated means artificially obtained or produced.
• isolated generally means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
• PCR polymerase chain reaction
• recombinantly produced by cloning recombinantly produced by cloning
• purified as by cleavage and gel separation
• synthesized by, for example, chemical synthesis.
• An isolated nucleic acid is one that is readily manipulable by recombinant DNA techniques well known in the art.
• nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not.
• An isolated nucleic acid may be substantially purified, but need not be.
• a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
• isolated generally refers to a protein or peptide that has been artificially obtained or produced (e.g. , by chemical synthesis, by recombinant DNA technology, etc.).
• conservative amino acid substitutions may be made to provide functionally equivalent variants, or homologs of the capsid proteins.
• the disclosure embraces sequence alterations that result in conservative amino acid substitutions.
• 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 altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile 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.
• Conservative substitutions of amino acids include substitutions made among 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. Therefore, one can make conservative amino acid substitutions to the amino acid sequence of the proteins and polypeptides disclosed herein.
• An example of an isolated nucleic acid that encodes a polypeptide comprising an AAV capsid protein is a nucleic acid having a sequence selected from the group consisting of: SEQ ID NO: 410-434, 876- 1718, and 1989-2251.
• a fragment of an isolated nucleic acid encoding an AAV capsid sequence may be useful for constructing a nucleic acid encoding a desired capsid sequence. Fragments may be of any appropriate length.
• a fragment (portion) of an isolated nucleic acid encoding an AAV capsid sequence may be useful for constructing a nucleic acid encoding a desired capsid sequence. Fragments may be of any appropriate 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 or more nucleotides in length). For example, a fragment of nucleic acid sequence encoding a polypeptide of a first AAV capsid protein may be used to construct, or may be incorporated within, a nucleic acid sequence encoding a second AAV capsid sequence to alter the properties of the AAV capsid.
• AAV capsid proteins that comprise capsid sequence fragments from multiple AAV serotypes are referred to as chimeric AAV capsids.
• the fragment may be a fragment that does not encode a peptide that is identical to a sequence of any one of SEQ ID NOs: 869, 870, or 871.
• a fragment of nucleic acid sequence encoding a variant amino acid may be used to construct, or may be incorporated within, a nucleic acid sequence encoding an AAV capsid sequence to alter the properties of the AAV capsid.
• a nucleic acid sequence encoding an AAV variant may comprise about 1 to about 100 amino acid variants compared with 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 comprise about 5 to about 50 amino acid variants compared with a known AAV serotype (e.g., AAV serotype 2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8).
• AAV serotype e.g., AAV serotype 2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8.
• a nucleic acid sequence encoding an AAV variant may comprise about 10 to about 30 amino acid variants compared with a known AAV serotype (e.g., AAV serotype 2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8).
• a nucleic acid sequence encoding an AAV variant may comprise 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 with a known AAV serotype (e.g., AAV serotype 2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8).
• a nucleic sequence encoding an AAV variant may comprise 3 amino acid variants compared with a known AAV serotype (e.g. , AAV8).
• a recombinant cap sequence may be constructed having one or more of the 3 amino acid variants by incorporating fragments of a nucleic acid sequence comprising a region encoding a variant amino acid into the sequence of a nucleic acid encoding the known AAV serotype.
• the fragments may be incorporated by any appropriate method, including using site directed mutagenesis.
• new AAV variants may be created having new properties.
• the disclosure provides isolated nucleic acids encoding AAV assembly-activating proteins (AAPs), or variants thereof.
• AAP AAV assembly-activating proteins
• an “assembly activating protein” or “AAP” is a protein chaperone that functions to target newly synthesized capsid proteins (e.g. , VP proteins, such as AAV VP1, VP2, and VP3) to the nucleolus of a cell thereby promoting encapsidation of viral genomes.
• capsid proteins e.g. , VP proteins, such as AAV VP1, VP2, and VP3
• an AAP is encoded in the cap gene of an adeno-associated virus.
• AAP-2 is encoded in the cap gene of AAV2.
• an AAP is translated from a different open reading frame (ORF) of the cap gene than a capsid protein ⁇ e.g., VP1, VP2, VP3).
• ORF open reading frame
• a capsid protein ⁇ e.g., AAV2 VP1, VP2, VP3 is translated from ORF 1 of a cap gene and an AAP ⁇ e.g., AAP-2) is translated from ORF 2 of the cap gene.
• an isolated nucleic acid encoding an AAP comprises or consists of a sequence selected from SEQ ID NO: 410-434 and 876-1718. Recombinant AAV 's
• the disclosure provides isolated AAVs.
• isolated refers to an AAV that has been artificially obtained or produced. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as "recombinant AAVs".
• Recombinant AAVs preferably have tissue-specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s).
• the AAV capsid is an important element in determining these tissue- specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.
• 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.
• the methods involve culturing a host cell which contains 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 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 permit packaging of the recombinant AAV vector into the AAV capsid proteins.
• capsid proteins are structural proteins encoded by a cap gene of an AAV.
• AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which may be expressed from a single cap gene. Accordingly, 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 respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the protein shell is primarily comprised of a VP3 capsid protein.
• the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
• capsid proteins deliver the viral genome to a host in a tissue specific manner.
• VP1 and/or VP2 capsid proteins may contribute to the tissue tropism of the packaged AAV.
• the tissue tropism of the packaged AAV is determined by the VP3 capsid protein.
• the tissue tropism of an AAV is enhanced or changed by mutations occurring in the capsid proteins.
• the instant disclosure describes variants of wild-type AAV serotypes
• the variants have altered tissue tropism.
• the AAV variants described herein comprise amino acid variations (e.g. , substitution, deletion, insertion) within the cap gene.
• amino acid variations e.g. , substitution, deletion, insertion
• all three capsid proteins are transcribed from a single cap gene.
• an amino acid variation within a cap gene is present in all three capsid proteins encoded by said cap gene.
• an amino acid variation may not be present in all three capsid proteins.
• an amino acid variation occurs only in the VP1 capsid protein.
• an amino acid variation occurs only in the VP2 capsid protein.
• an amino acid variation occurs only within the VP3 capsid protein.
• an AAV variant comprises more than one variation in a cap gene.
• the more than one variation occur within the same capsid protein (e.g., within VP3).
• the more than one variation occur within different capsid proteins (e.g., at least one variation in VP2 and at least one variation in VP3).
• the AAV variants described herein are variants of AAV2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8.
• AAV2 is known to efficiently transduce human central nervous system (CNS) tissue, kidney tissue, ocular tissue (e.g. , photoreceptor cells and retinal pigment epithelium (RPE)), and other tissues. Accordingly, in some
• the AAV3 variants described herein may be useful for delivering gene therapy to CNS tissue, kidney tissue, or ocular tissue. It is also known that AAV3 efficiently transduces cancerous human hepatocytes. Accordingly, in some embodiments, the AAV3 variants described herein may be useful for delivering gene therapy to cancerous and normal human hepatocytes.
• AAV8 is known to target tissue of the liver tissue, respiratory tissue, and the eye. Accordingly, in some embodiments, the AAV8 variants described herein may be useful for delivering gene therapy to the liver tissue, respiratory tissue or the eye.
• the AAV2, AAV2/3 (e.g. , AAV2/3 hybrid) and AAV8 variants described herein may comprise one or more variations within the cap gene compared with a corresponding wild-type AAV. Therefore, in some embodiments, the AAV2, AAV2/3 (e.g. , AAV2/3 hybrid) and AAV8 variants described herein may have a tissue tropism useful for delivering gene therapy to additional tissue types that are not targeted by wild-type AAV2, AAV2/3 (e.g. , AAV2/3 hybrid) or AAV8.
• AAV8 variants described herein may be useful for delivering gene therapy to the central nervous system (CNS).
• CNS central nervous system
• AV2, AAV2/3 e.g. , AAV2/3 hybrid
• AAV8 variants described herein may be useful for targeting cells of the kidney or cells of the liver.
• AAV2, AAV2/3 e.g. , AAV2/3 hybrid
• AAV8 variants described herein may be useful for targeting gene therapy to the liver, spleen, heart or brain.
• AAV variants described herein may be useful for the treatment of CNS-related disorders.
• 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. , a myelopathy), brain (e.g. , a encephalopathy) or tissues surrounding the brain and spinal cord.
• a CNS-related disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• a CNS-related disorder may be a psychological condition or disorder, e.g. , Attention Deficient Hyperactivity Disorder, Autism Spectrum Disorder, Mood Disorder, Schizophrenia, Depression, Rett Syndrome, etc.
• a CNS-related disorder may be an autoimmune disorder.
• a CNS-related disorder may also be a cancer of the CNS, e.g. , brain cancer.
• a CNS-related disorder that is a cancer may be a primary cancer of the CNS, e.g. , an astrocytoma, glioblastomas, etc., or may be a cancer that has metastasized to CNS tissue, e.g. , a lung cancer that has metastasized to the brain.
• Further non-limiting examples of CNS- related disorders include Parkinson' s Disease, Lysosomal Storage Disease, Ischemia, Neuropathic Pain, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), and
• AAV variants described herein may be useful for delivering gene therapy to cardiac cells (e.g., heart tissue). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of cardiovascular disorders.
• a "cardiovascular disorder” is a disease or condition of the cardiovascular system.
• a cardiovascular disease may affect the heart, circulatory system, arteries, veins, blood vessels and/or capillaries.
• a cardiovascular disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• Non-limiting examples of cardiovascular disorders include rheumatic heart disease, valvular heart disease, hypertensive heart disease, aneurysm, atherosclerosis, hypertension (e.g., high blood pressure), peripheral arterial disease (PAD), ischemic heart disease, angina, coronary heart disease, coronary artery disease, myocardial infarction, cerebral vascular disease, transient ischemic attack, inflammatory heart disease, cardiomyopathy, pericardial disease, congenital heart disease, heart failure, stroke, and myocarditis due to Chagas disease.
• AAV variants described herein may target the lung and/or tissue of the pulmonary system (e.g. , respiratory system). Accordingly, in some embodiments, in some
• AAV variants described herein may be useful for treatment of pulmonary disease.
• a "pulmonary disease” is a disease or condition of the pulmonary system.
• a pulmonary disease may affect the lungs or muscles involved in breathing.
• a pulmonary disease may be of a genetic origin, either inherited or acquired through a 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 lung carcinoid tumor.
• 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), cryptogenic organizing pneumonia (COP), cystic fibrosis, emphysema, Hantavirus Pulmonary Syndrome, histoplasmosis, Human Metapneumovirus, hypersensitivity pneumonitis, influenza, lymphangiomatosis, mesothelioma, Middle Eastern Respiratory Syndrome, non-tuberculosis Mycobacterium, Pertussis, Pneumoconiosis (Black Lung Disease), pneumonia, primary ciliary dyskinesia, primary pulmonary hypertension, pulmonary arterial hypertension, pulmonary fibrosis,
• AAV variants described herein may target liver tissue.
• AAV variants described herein may be useful for treatment of hepatic disease.
• a "hepatic disease” is a disease or condition of the liver.
• a hepatic disease may be of a genetic origin, either inherited or acquired through a somatic mutation.
• a hepatic disease may be a cancer of the liver, including but not limited to hepatocellular carcinoma (HCC), fibrolamellar carcinoma, cholangiocarcinoma,
• pulmonary diseases include Alagille Syndrome, Alpha 1 Anti-Trypsin Deficiency, autoimmune hepatitis, biliary atresia, cirrhosis, cystic disease of the liver, fatty liver disease, galactosemia, gallstones, Gilbert's Syndrome, hemochromatosis, liver disease in pregnancy, neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Reye's Syndrome, sarcoidosis, toxic hepatitis, Type 1 Glycogen Storage Disease, tyrosinemia, viral hepatitis A, B, C, Wilson Disease, and schistosomiasis.
• AAV variants described herein may target kidney tissue. Accordingly, in some embodiments, AAV variants described herein may be useful for treatment of kidney disease.
• a "kidney disease" is a disease or condition of the liver.
• a kidney disease may be of a genetic origin, either inherited or acquired through a somatic mutation.
• a kidney disease may be a cancer of the kidney, including but not limited to renal cell cancer, clear cell cancer, papillary cancer type 1, papillary cancer type 2, chromophobe cancer, oncocytic cell cancer, collecting duct cancer, transitional cell cancer of the renal pelvis and Wilm' s tumor.
• kidney disease examples include Abderhalden-Kaufmann-Lignac syndrome (Nephropathic Cystinosis), Acute Kidney 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-a Therapy-related Glomerulonephritis, APOL1 Mutations, Apparent Mneralocorticoid Excess Syndrome, Aristolochic Acid Nephropathy, Balkan Endemic Nephropathy, Bartter Syndrome, Beeturia, ⁇ -Thalassemia Renal Disease, Bile Cast Nephropathy, BK Polyoma, Clq Nephropathy, Cardio
• Hemoglobinuria and Hemolytic Anemia Hepatic Veno-Occlusive Disease, Sinusoidal
• Obstruction Syndrome Hepatitis C-Associated Renal Disease, Hepatorenal Syndrome, HIV- Associated Nephropathy (HIV AN), Horseshoe Kidney (Renal Fusion), Hunner's Ulcer, Hyperaldosteronism, Hypercalcemia, Hyperkalemia, Hypermagnesemia, Hypernatremia, Hyperoxaluria, Hyperphosphatemia, Hypocalcemia, Hypokalemia, Hypokalemia-induced renal dysfunction, Hypomagnesemia, Hyponatremia, Hypophosphatemia, IgA Nephropathy, IgG4 Nephropathy, Interstitial Cystitis, Painful Bladder Syndrome, Interstitial Nephritis, Ivemark's syndrome, Kidney Stones, Nephrolithiasis, Leptospirosis Renal Disease, Light Chain Deposition Disease, Monoclonal Immunoglobulin Deposition Disease, Liddle Syndrome, Lightwood- Albright Syndrome, Lipoprotein Glomerulopathy, Lithium
• Glomerulonephritis Membranous Nephropathy, MesoAmerican Nephropathy, Metabolic Acidosis, Metabolic Alkalosis, Microscopic Polyangiitis, Milk-alkalai syndrome, Minimal Change Disease, Multicystic dysplastic kidney, Multiple Myeloma, Myeloproliferative Neoplasms and Glomerulopathy, Nail-patella Syndrome, Nephrocalcinosis, Nephrogenic Systemic Fibrosis, Nephroptosis (Floating Kidney, Renal Ptosis), Nephrotic Syndrome, Neurogenic Bladder, Nodular Glomerulosclerosis, Non-Gonococcal, Nutcracker syndrome, Orofaciodigital Syndrome, Orthostatic Hypotension, Orthostatic Proteinuria, Osmotic Diuresis, Page Kidney, Papillary Necrosis, Papillorenal Syndrome (Renal-Coloboma
• Granulomatosis Granulomatosis with Polyangiitis, and Wunderlich syndrome.
• AAV variants described herein may be useful for delivering gene therapy to ocular tissue (e.g. , tissue or cells of the eye). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of ocular disorders.
• an "ocular disorder" is a disease or condition of the eye. An ocular disease may affect the eye, sclera, cornea, anterior chamber, posterior chamber, iris, pupil, lens, vitreous humor, retina, or optic nerve. An ocular disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• Non-limiting examples of ocular diseases and disorders include but are not limited to: age-related macular degeneration, retinopathy, diabetic retinopathy, macular edema, glaucoma, retinitis pigmentosa and eye cancer.
• AAV variants described herein may be useful for delivering gene therapy to gastrointestinal tissue (e.g. , tissue of the gastrointestinal tract). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of gastrointestinal tract disorders.
• a "gastrointestinal tract disorder" is a disease or condition of the gastrointestinal tract. A gastrointestinal disease may affect the mucosa (e.g. , epithelium, lamina basement, muscularis mucosae, etc. ), submucosa (e.g.
• a gastrointestinal tract disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• Non-limiting examples of gastrointestinal tract diseases and disorders include but are not limited to:
• IBD inflammatory bowel disease
• Crohn' s disease Crohn' s disease
• ulcerative colitis irritable bowel syndrome
• Celiac disease Celiac disease
• gastroesophageal reflux disease GDD
• achakasua diverticulitus
• diarrhea and certain cancers (e.g. , bowel cancer, stomach cancer, colon cancer, rectal cancer, etc.).
• certain cancers e.g. , bowel cancer, stomach cancer, colon cancer, rectal cancer, etc.
• AAV variants described herein may be useful for delivering gene therapy to breast tissue (e.g. , tissue of the breast). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of breast disorders.
• a breast disorder is a disease or condition of the breast.
• a breast disease may affect the fibrous tissue, fatty tissue, lobules, or ducts of the breast.
• a breast disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• Non-limiting examples of breast diseases and disorders include but are not limited to: mastitis, breast calcification, fat necrosis, fibroadenoma, fibrosis and simple cysts, galactorrhea, hyperplasia and breast cancer.
• AAV variants described herein may be useful for delivering gene therapy to pancreatic tissue (e.g. , tissue of the pancreas). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of pancreatic disorders.
• a "pancreatic disorder" is a disease or condition of the pancreas.
• a pancreatic disease may affect the head of the pancreas, neck of the pancreas, body of the pancreas, tail of the pancreas, pancreatic islets (e.g. , islets of Langerhans), acini, or columnar epithelium.
• a pancreatic disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• 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.
• diabetes e.g. , diabetes mellitus type 1 and diabetes mellitus type 2
• pancreatitis e.g. , acute pancreatitis, chronic pancreatitis
• pancreatic cancer e.g., diabetes mellitus type 2
• pancreatitis e.g. , acute pancreatitis, chronic pancreatitis
• 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). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of urinary tract disorders.
• a "urinary tract disorder" is a disease or condition of the urinary tract.
• a urinary tract disease may affect the bladder, ureters, urethera, or prostate.
• a urinary tract disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• urinary tract diseases and disorders include but are not limited to: urinary tract infections, kidney stones, bladder control problems (e.g. , urinary retention, urinary incontinence, etc.), cystitis, and bladder cancer.
• AAV variants described herein may be useful for delivering gene therapy to uterine tissue (e.g. , tissue of the uterus). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of uterine disorders.
• a "uterine disorder" is a disease or condition of the uterus.
• a uterine disease may affect the cervix, cervical canal, body of the uterus (fundus), endometrium, myometrium, or perimetrium.
• a uterine disorder may be of a genetic origin, either inherited or acquired through a somatic mutation.
• Non-limiting examples of uterine diseases and disorders include but are not limited to: adenomyosis, endometriosis, endometrial hyperplasia, Asherman' s syndrome, and endometrial cancer.
• the components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans.
• any one or more of the required components e.g. , recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
• a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
• a stable host cell will contain the required component(s) under the control of an inducible promoter.
• the required component(s) may be under the control of a constitutive promoter.
• a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
• a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
• the recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).
• a single nucleic acid encoding all three capsid proteins e.g., VP1, VP2 and VP3 is delivered into the packaging host cell in a single vector.
• nucleic acids encoding the capsid proteins are delivered into the packaging host cell by two vectors; a first vector comprising a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2) and a second vector comprising a second nucleic acid encoding a single capsid protein (e.g., VP3).
• three vectors, each comprising a nucleic acid encoding a different capsid protein are delivered to the packaging host cell.
• the selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.
• recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650).
• the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector.
• An AAV helper function vector encodes the "AAV helper function" sequences (e.g. , rep and cap), which function in trans for productive AAV replication and encapsidation.
• the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g.
• AAV virions containing functional rep and cap genes include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6, 156,303, the entirety of both incorporated by reference herein.
• the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g. , "accessory functions").
• the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, 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.
• the disclosure provides transfected host cells.
• transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been
• transfected when exogenous DNA has been introduced inside the cell (e.g. , across the cell membrane).
• a number of transfection techniques are generally known in the art. See, e.g. , 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 a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
• a “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell that has been transfected. Thus, a "host cell” as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
• cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
• the terms "recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
• Cells may also be transfected with a vector (e.g. , helper vector) that provides helper functions to the AAV.
• the vector providing helper functions may provide adenovirus functions, including, e.g. , Ela, Elb, E2a, and E40RF6.
• the sequences of adenovirus gene providing these functions may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types known in the art.
• the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging.
• vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. , that is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
• the term includes cloning and expression vehicles, as well as viral vectors.
• useful vectors are contemplated to be those vectors in which the nucleic acid segment (e.g. , 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 the cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene.
• the phrases “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
• expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
• expression includes transcription of the nucleic acid, for example, to generate a biologically- active polypeptide product or inhibitory RNA (e.g. , shRNA, miRNA, miRNA inhibitor) from a transcribed gene.
• an isolated capsid gene can be used to construct and package recombinant AAVs, using methods well known in the art, to determine functional
• isolated capsid genes can be used to construct and package a recombinant AAV (rAAV) comprising a reporter gene (e.g. , B-Galactosidase, GFP, Luciferase, etc. ).
• rAAV recombinant AAV
• the rAAV can then be delivered to an animal (e.g. , mouse) and the tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g. , heart, liver, kidneys) of the animal.
• tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g. , heart, liver, kidneys) of the animal.
• Other methods for characterizing the novel isolated capsid genes are disclosed herein and still others are well known in the art.
• Recombinant AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell.
• the transgene is a nucleic acid sequence, heterologous to the vector sequences, 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 operatively linked to regulatory components in a manner that permits transgene transcription, translation, and/or expression in a cell of a target tissue.
• the AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g. , B. J. Carter, in “Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)).
• the ITR sequences are about 145 bp in length.
• substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g. , texts such as Sambrook et al, "Molecular Cloning.
• a Laboratory Manual 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)).
• An example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences.
• the AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.
• the disclosure provides a self-complementary AAV vector.
• scAAV 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 ITRs of the AAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present.
• TR terminal resolution site
• scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle.
• the rAAVs of the present disclosure are pseudotyped rAAVs.
• Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle.
• the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles.
• a pseudotyped rAAV comprises nucleic acids from two or more different AAVs, wherein the nucleic acid from one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and/or the viral genome.
• a pseudotyped rAAV refers to an AAV comprising an inverted terminal repeat (ITR) of one AAV serotype and a capsid protein of a different AAV serotype.
• ITR inverted terminal repeat
• a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX/Y (e.g., AAV2/1 has the ITRs of AAV2 and the capsid of AAV1).
• pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.
• the vector also includes conventional control elements necessary which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure.
• "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
• 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 enhance translation efficiency (e.g. , Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
• polyA polyadenylation
• a great number of expression control sequences, including promoters that are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
• nucleic acid sequence e.g. , coding sequence
• regulatory sequences are said to be "operably” linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences.
• nucleic acid sequences be translated into a functional protein
• two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
• a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
• operably linked coding sequences yield a fusion protein.
• operably linked coding sequences yield a functional RNA (e.g. , shRNA, miRNA, miRNA inhibitor).
• a polyadenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence.
• a rAAV construct useful in the present disclosure may also contain an intron, desirably 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).
• An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
• An IRES sequence would be used to produce a protein that contains more than one polypeptide chains.
• a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M 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 the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M 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
• regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like.
• 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for
• Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
• the vectors of the disclosure may optionally include 5' leader or signal sequences.
• constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the
• CMV cytomegalovirus
• PGK phosphoglycerol kinase
• Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g. , acute phase, a particular differentiation state of the cell, or in replicating cells only.
• Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
• inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (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.
• MT zinc-inducible sheep metallothionine
• Dex dexamethasone
• MMTV mouse mammary tumor virus
• T7 polymerase promoter system WO 98/10088
• ecdysone insect promoter No et al, Proc. Natl. Acad. Sci. USA, 93:3346- 3351 (1996)
• inducible promoters that may be useful in this context are those that are regulated by a specific physiological state, e.g. , temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
• the native promoter for the transgene will be used.
• the native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression.
• the native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli.
• other native expression control elements such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
• the regulatory sequences impart tissue-specific gene expression capabilities.
• the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
• tissue-specific regulatory sequences e.g. , promoters, enhancers, etc.
• tissue-specific regulatory sequences are well known in the art.
• tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, a gastrointestinal-specific mucin-2 promoter, an eye-specific retinoschisin promoter, an eye- specific K12 promoter, a respiratory tissue-specific CC10 promoter, a respiratory tissue-specific surfactant protein C (SP-C) promoter, a breast tissue-specific PRC1 promoter, a breast tissue-specific RRM2 promoter, a urinary tract tissue- specific uroplakin 2 (
• Beta-actin promoter hepatitis B virus core promoter, Sandig et al., Gene Ther., 3: 1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7: 1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24: 185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.
• AFP alpha-fetoprotein
• Immunol., 161: 1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), 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)), among others which will be apparent to the skilled artisan.
• NSE neuron-specific enolase
• one or more bindings sites for one or more of miRNAs are incorporated in a transgene of a rAAV vector, to inhibit the expression of the transgene in one or more tissues of an subject harboring the transgene.
• binding sites may be selected to control the expression of a transgene in a tissue specific manner.
• binding sites for the liver- specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver.
• the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region. Typically, the target site is in the 3' UTR of the mRNA.
• the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same 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 comprise a total of 5-100, 10-60, or more nucleotides.
• the target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.
• the composition of the transgene sequence of the rAAV vector will depend upon the use to which the resulting vector will be put.
• one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal.
• the transgene encodes a therapeutic protein or therapeutic functional RNA.
• the transgene encodes a protein or functional RNA that is intended to be used for research purposes, e.g. , to create a somatic transgenic animal model harboring the transgene, e.g. , to study the function of the transgene product.
• the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of disease. Appropriate transgene coding sequences will be apparent to the skilled artisan.
• Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
• the reporter sequences When associated with regulatory elements which drive their expression, the reporter sequences, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for ⁇ -galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
• Such reporters can, for example, be useful in verifying the tissue-specific targeting capabilities and tissue specific promoter regulatory activity of an rAAV.
• the disclosure provides rAAV vectors for use in methods of 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 particularly for treating or reducing the severity or extent of deficiency in a human manifesting one or more of the disorders linked to a deficiency in such polypeptides in cells and tissues.
• the method involves administration of an rAAV vector that encodes one or more therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc. in a pharmaceutically-acceptable carrier to the subject in an amount and for a period of time sufficient to treat the deficiency or disorder in the subject suffering from such a disorder.
• the disclosure embraces the delivery of rAAV vectors encoding one or more peptides, polypeptides, or proteins, which are useful for the treatment or prevention of disease states in a mammalian subject.
• exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors.
• therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAPl, 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.
• the rAAV vectors may comprise a gene to be transferred to a subject to treat a disease associated with reduced expression, lack of expression or dysfunction of the gene.
• genes and associated disease states 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 uridyl transferase, 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 Co A dehydrogenase, associated with medium chain acetyl CoA deficiency; ornith
• hypoxanthine guanine phosphoribosyl transferase associated with Gout and Lesch-Nyan syndrome
• biotinidase associated with biotinidase deficiency
• beta-glucocerebrosidase associated with Gaucher disease
• beta-glucuronidase associated with Sly syndrome
• peroxisome membrane protein 70 kDa associated with Zellweger syndrome
• porphobilinogen deaminase associated with acute intermittent porphyria
• alpha- 1 antitrypsin for treatment of alpha- 1 antitrypsin deficiency (emphysema)
• erythropoietin for treatment of anemia due to thalassemia or to renal failure
• vascular endothelial growth factor vascular endothelial growth factor
• angiopoietin-1 angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases
• thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; the beta adrenergic receptor, anti-sense to, or a mutant form of, phospholamban, the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for the treatment of congestive heart failure; a tumor suppessor gene such as p53 for the treatment of various cancers; a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; dystrophin or
• minidystrophin and utrophin or miniutrophin for the treatment of muscular dystrophies; and, insulin for the treatment of diabetes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the central nervous system (CNS).
• CNS disease DRD2, GRIA1, GRIA2,GRIN1, SLC1A1, SYP, SYTl, CHRNA7, 3Rtau/4rTUS, APP, BAX, BCL-2, GRIKl, GFAP, IL-1, AGER, associated with Alzheimer's Disease; UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstml, S 106p, associated with Parkinson's Disease; IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin 1, FTL, TITF-1, associated with Huntington's Disease; FXN, associated with Freidrich's ataxia; ASPA, associated with Canavan's
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof. In some embodiments, the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with 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, HIFla, SERCa2a, ADCY1, and ADCY6.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the pulmonary system.
• the following is a non-limiting list of genes associated with pulmonary disease: TNFa, TGFpl, SFTPA1, SFTPA2, SFTPB, SFTPC, HPS 1, HPS 3, HPS 4, ADTB3A, ILIA, IL1B, LTA, IL6, CXCR1, and CXCR2.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the liver.
• the following is a non-limiting list of genes associated with liver disease: al-AT, HFE, ATP7B, fumarylacetoacetate hydrolase (FAH), glucose-6- phosphatase, NCAN, GCKR, LYPLAL1, and PNPLA3.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the kidney.
• AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the kidney.
• the following is a non-limiting list of genes associated with kidney disease: PKD1, PKD2, PKHD1, NPHS 1, NPHS2, PLCE1, CD2AP, LAMB2, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, SCARB3, FN1, COL4A5, COL4A6, COL4A3, COL4A4, FOX1C, RET, UPK3A, BMP4, SIX2, CDC5L, USF2, ROB02, SLIT2, EYA1, MYOG, SIX1, SIX5, FRAS 1, FREM2, GATA3, KALI, PAX2, TCF2, and SALL1.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof. In some embodiments, the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the eye.
• AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the eye.
• genes associated with ocular disease CFH, C3, MT-ND2, ARMS2, TIMP3, CAMK4, FMN1, RHO, USH2A, RPGR, RP2, TMCO, SIX1, SIX6, LRP12, ZFPM2, TBK1, GALC, myocilin, CYP1B 1, CAV1, CAV2, optineurin and CDKN2B.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof. In some embodiments, the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with breast.
• the following is a non-limiting list of genes associated with breast disease: BRCA1, BRCA2, Tp53, PTEN, HER2, BRAF, and PARP1.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the gastrointestinal tract.
• the following is a non-limiting list of genes associated with gastrointestinal disease: CYP2C19, CCL26, APC, IL12, IL10, and IL- 18.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the pancreas.
• the following is a non-limiting list of genes associated with pancreatic disease: PRSS 1, SPINK1, STK11, MLH1, KRAS2, pl6, p53, and BRAF.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the urinary tract.
• AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the urinary tract.
• the following is a non-limiting list of genes associated with urinary tract disease: HSPA1B, CXCR1 & 2, TLR2, TLR4, TGF- 1,
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof. In some embodiments, the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the disclosure relates to an AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the uterus.
• AAV comprising a nucleic acid encoding a protein or functional RNA useful for the treatment of a condition, disease or disorder associated with the uterus.
• genes associated with ocular disease DN-ER, MLH1, MSH2, MSH6, PMS 1, and PMS2.
• the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more of the foregoing genes or fragments thereof. In some embodiments, the disclosure relates to recombinant AAVs comprising nucleic acids that express one or more functional RNAs that inhibit expression of one or more of the foregoing genes.
• the rAAVs of the disclosure can be used to restore the expression of genes that are reduced in expression, silenced, or otherwise dysfunctional in a subject (e.g., a tumor suppressor that has been silenced in a subject having cancer).
• the rAAVs of the disclosure can also be used to knockdown the expression of genes that are aberrantly expressed in a subject (e.g., an oncogene that is expressed in a subject having cancer).
• an rAAV vector comprising a nucleic acid encoding a gene product associated with cancer (e.g., tumor suppressors) may be used to treat the cancer, by administering a rAAV harboring the rAAV vector to a subject having the cancer.
• an rAAV vector comprising a nucleic acid encoding a small interfering nucleic acid (e.g., shRNAs, miRNAs) that inhibits the expression of a gene product associated with cancer (e.g. , oncogenes) may be used to treat the cancer, by administering a rAAV harboring the rAAV vector to a subject having the cancer.
• a small interfering nucleic acid e.g., shRNAs, miRNAs
• an rAAV vector comprising a nucleic acid encoding a gene product associated with cancer (or a functional RNA that inhibits the expression of a gene associated with cancer) may be used for research purposes, e.g. , to study the cancer or to identify therapeutics that treat the cancer.
• exemplary genes known to be associated with the development of cancer e.g.
• AARS AARS, ABCB 1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP50, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS 1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDK
• TNFRSF10B TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1, TOB 1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101, TUFM,
• a rAAV vector may comprise as a transgene, a nucleic acid encoding a protein or functional RNA that modulates apoptosis.
• the following is a non-limiting list of genes associated with apoptosis and nucleic acids encoding the products of these genes and their homologues and encoding small interfering nucleic acids (e.g.
• shRNAs, miRNAs that inhibit the expression of these genes and their homologues are useful as transgenes in certain embodiments of the 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,
• transgenes encoding proteins or polypeptides
• that mutations that results in conservative amino acid substitutions may be made in a transgene to provide functionally equivalent variants, or homologs of a protein or polypeptide.
• the disclosure embraces sequence alterations that result in conservative amino acid substitution of a transgene.
• the transgene comprises a gene having a dominant negative mutation.
• a transgene may express a mutant protein that interacts with the same elements as a wild-type protein, and thereby blocks some aspect of the function of the wild-type protein.
• Useful transgene products also include miRNAs.
• miRNAs and other small interfering nucleic acids regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA).
• miRNAs are natively expressed, typically as final 19-25 non-translated RNA products. miRNAs exhibit their activity through sequence- specific interactions with the 3' untranslated regions (UTR) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into a miRNA duplex, and further into a "mature" single stranded miRNA molecule.
• This mature miRNA guides a multiprotein complex, miRISC, which identifies target site, e.g. , in the 3' UTR regions, of target mRNAs based upon their complementarity to the mature miRNA.
• miRISC multiprotein complex
• miRNA genes are useful as transgenes or as targets for small interfering nucleic acids encoded by transgenes (e.g. , miRNA sponges, antisense oligonucleotides, TuD RNAs) in certain embodiments of the 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-l*, hsa-let-7f-2*, hsa-let-7g, hsa-let- 7g*, hsa-let-7i, hsa-let-7i*, hsa-miR
• a miRNA inhibits the function of the mRNAs it targets and, as a result, inhibits expression of the polypeptides encoded by the mRNAs.
• blocking partially or totally
• the activity of the miRNA e.g. , silencing the miRNA
• derepression of polypeptides encoded by mRNA targets of a miRNA is accomplished by inhibiting the miRNA activity in cells through any one of a variety of methods.
• blocking the activity of a miRNA can be accomplished by
• an small interfering nucleic acid that is substantially complementary to a miRNA is one that is capable of hybridizing with a miRNA, and blocking the miRNA' s activity.
• a small interfering nucleic acid that is substantially complementary to a miRNA is a small interfering nucleic acid that is complementary to the miRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases.
• a "miRNA Inhibitor” is an agent that blocks miRNA function, expression and/or processing.
• these molecules include but are not limited to microRNA specific antisense, microRNA sponges, tough decoy RNAs (TuD RNAs) and microRNA
• oligonucleotides double-stranded, hairpin, short oligonucleotides
• MicroRNA inhibitors can be expressed in cells from a transgenes of a rAAV vector, as discussed above.
• MicroRNA sponges specifically inhibit miRNAs through a complementary heptameric seed sequence (Ebert, M.S. Nature Methods, Epub August, 12, 2007).
• an entire family of miRNAs can be silenced using a single sponge sequence.
• TuD RNAs achieve efficient and long-term-suppression of specific miRNAs in mammalian cells (See, e.g., Takeshi Haraguchi, et al., Nucleic Acids Research, 2009, Vol. 37, No. 6 e43, the contents of which relating to TuD RNAs are incorporated herein by reference).
• Other methods for silencing miRNA function See, e.g., Takeshi Haraguchi, et al., Nucleic Acids Research, 2009, Vol. 37, No
• the cloning capacity of the recombinant RNA vector may limit a desired coding sequence and may require the complete replacement of the virus's 4.8 kilobase genome. Large genes may, therefore, not be suitable for use in a standard recombinant AAV vector, in some cases.
• the skilled artisan will appreciate that options are available in the art for overcoming a limited coding capacity. For example, the AAV ITRs of two genomes can anneal to form head to tail concatamers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. Other options for overcoming a limited cloning capacity will be apparent to the skilled artisan.
• the disclosure also relates to the production of somatic 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 variants thereof mediate efficient and stable gene transfer in a tissue specific manner in adult animals.
• the rAAV elements capsid, promoter, transgene products
• the somatic transgenic animal produced by the methods of the disclosure can serve as useful models of human disease, pathological state, and/or to characterize the effects of gene for which the function (e.g., tissue specific, disease role) is unknown or not fully understood.
• an animal e.g., mouse
• a rAAV comprising a capsid having a specific tissue targeting capability (e.g., liver, heart, pancreas) and a transgene having a tissue specific promoter driving expression of a gene involved in disease.
• the rAAV infects distinct cells of the target tissue and produces the product of the transgene.
• the sequence of the coding region of a 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.
• modification of the sequence of coding region is a nonsense mutation that results in a fragment (e.g. , a truncated version).
• the modification is a mis sense mutation that results in an amino acid substitution.
• Other modifications are possible and will be apparent to the skilled artisan.
• the transgene causes a pathological state.
• a transgene that causes a pathological state is a gene whose product has a role in a disease or disorder (e.g. , causes the disease or disorder, makes the animal susceptible to the disease or disorder) and/or may induce the disease or disorder in the animal. The animal can then be observed to evaluate any number of aspects of the disease (e.g. , progression, response to treatment, etc.). These examples are not meant to be limiting, other aspects and examples are disclosed herein and described in more detail below.
• models of type 1 diabetes can be produced by the targeted destruction of pancreatic Beta-islets.
• the targeted destruction of specific cell types can be used to evaluate the role of specific cell types on human disease.
• transgenes that encode cellular toxins e.g. , diphtheria toxin A (DTA)
• DTA diphtheria toxin A
• NTR pro-apoptotic genes
• Other exemplary transgenes, whose products kill cells are embraced by the methods disclosed herein and will be apparent to one of ordinary skill in the art.
• the disclosure in some aspects, provides methods for producing somatic transgenic animal models to study the long-term effects of over-expression or knockdown of genes.
• the long term over expression or knockdown e.g. , by shRNA, miRNA, miRNA inhibitor, etc.
• the disclosure in some aspects, provides methods for producing somatic transgenic animal models to study the long-term effects of over-expression or knockdown of gene of potential oncogenes and other genes to study tumorigenesis and gene function in the targeted tissues.
• Useful transgene products include proteins that are known to be associated with cancer and small interfering nucleic acids inhibiting the expression of such proteins.
• transgenes may be readily selected by one of skill in the art provided that they are useful for creating animal models of tissue-specific pathological state and/or disease.
• the rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art.
• the rAAV preferably suspended in a physiologically compatible carrier (e.g., in a composition) may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque).
• a host animal does not include a human.
• Delivery of the rAAVs to a mammalian subject may be by, for example,
• the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions.
• isolated limb perfusion technique described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue.
• CNS all cells and tissue of the brain and spinal cord of a vertebrate.
• the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cereobro spinal fluid (CSF), interstitial spaces, bone, cartilage and the like.
• Recombinant AAVs may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection (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.
• compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g. , a second rAAV encoding having one or more different transgenes).
• a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.
• Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed.
• one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g. , phosphate buffered saline).
• Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
• compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
• suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
• Suitable chemical stabilizers include gelatin and albumin.
• the rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.
• Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g. , intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.
• the dose of rAAV virions required to achieve a particular "therapeutic effect,” e.g. , the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.
• a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.
• an effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary between animals or tissues. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10 9 to 10 16 genome copies. In some embodiments the rAAV is administered at a dose of 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some embodiments the rAAV is administered at a dose of 10 10 , 10 11 , 10 12 , 10 13 , or 10 14
• 10 rAAV genome copies is appropriate.
• 10 rAAV genome copies is effective to target heart, liver, and pancreas tissues.
• stable transgenic animals are produced by multiple doses of an rAAV.
• rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present
• rAAVs e.g. , -10 GC/ml or more.
• Methods for reducing aggregation of rAAVs include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g. , Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
• Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
• these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
• the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
• Factors such as solubility,
• 5,641,515 and 5,399,363 may be used to deliver rAAVs.
• a preferred mode of administration is by portal vein injection.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. , glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
• polyol e.g. , glycerol, propylene glycol, and liquid polyethylene glycol, and the like
• suitable mixtures thereof e.g. , glycerol, propylene glycol, and liquid polyethylene glycol, and the like
• vegetable oils e.g. , glycerol, propylene glycol, and liquid polyethylene glycol, and the like
• Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, paraben
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• a sterile aqueous medium that can be employed will be known to those of skill in the art.
• one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570- 1580).
• Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
• Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the rAAV compositions disclosed herein may also be formulated in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• solutions 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 easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
• 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.
• solvents dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
• Supplementary active ingredients can also be incorporated into the compositions.
• “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
• Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
• the rAAV vector delivered trangenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
• Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
• the formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
• Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively 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 effectiveness of liposome-mediated drug delivery have been completed.
• Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
• MLVs generally have diameters of from 25 nm to 4 ⁇ . Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500.ANG., containing an aqueous solution in the core.
• SUVs small unilamellar vesicles
• Nanocapsule formulations of the rAAV may be used.
• Nanocapsules can generally entrap substances in a stable and reproducible way.
• ultrafine particles sized around 0.1 ⁇
• Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
• Sonophoresis i.e., ultrasound
• U.S. Pat. No. 5,656,016 has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system.
• Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
• kits may include one or more containers housing the components of the disclosure and instructions for use.
• kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents.
• agents in a kit may be in a
• Kits for research purposes may contain the components in appropriate concentrations or quantities for running various experiments.
• the kit may be designed to facilitate use of the methods described herein by researchers and can take many forms.
• Each of the compositions of the kit may be provided in liquid form (e.g. , in solution), or in solid form, (e.g. , a dry powder).
• some of the compositions may be constitutable or otherwise processable (e.g. , to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
• a suitable solvent or other species for example, water or a cell culture medium
• Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g. , videotape, DVD, etc.), Internet, and/or web-based
• the written instructions may be in a form prescribed by a
• the kit may contain any one or more of the components described herein in one or more containers.
• the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
• the kit may include a container housing agents described herein.
• the agents may be in the form of a liquid, gel or solid (powder).
• the agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely.
• the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
• the kit may have one or more or all of the components required to administer the agents to an animal, such as a syringe, topical application devices, or iv needle tubing and bag, particularly in the case of the kits for producing specific somatic animal models.
• the kit may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag.
• the kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
• the kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.
• the kit may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
• other components for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
• kits of the disclosure may involve methods for detecting a latent AAV in a cell.
• kits of the disclosure may include, instructions, a negative and/or positive control, containers, diluents and buffers for the sample, sample preparation tubes and a printed or electronic table of reference AAV sequence for sequence comparisons.
• Example 1 Isolation Of Transcriptionally Active, Novel AAV Capsid Sequences With Desired Tissue Tropisms and Properties From Human Tissues.
• This example describes novel AAV capsid sequences isolated by 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 by bioinformatic analyses; and 4) the selection of high- confidence ORFs that can be translated into full length capsid proteins.
• SMRT single-molecule, real-time
• AAV variants were identified from nine normal liver tissues, 7 liver tumors, four enlarged prostate 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.
• PCR primers used in this example are described in Table 2. Briefly, either panAAV primers for the amplification of 4.1-kb AAV rep-cap sequence ⁇ e.g., RepF318, AV2cas), or panAAV primers for amplification of 2.3-kb AAV cap sequence ⁇ e.g., CapF, CapR) were used for PCR.
• Table 1 Clinical specimens for wtAA V genome amplification
• AAV PCR products were subjected to high-throughput single-molecule, real-time (SMRT) sequencing. This approach removes the need to perform viral genome
• variant analysis pipelines developed from open source bioinformatic tools more than 600 previously undescribed, high-confidence AAV2, AAV2/3 hybrid, and AAV8 capsid sequence variants were identified. Specifically, 224 AAV8 variants (harboring 1 to 10 single amino acid variants); 425 AAV2 variants (harboring 1 to 20 single amino acid variants); and 194 AAV2/3 hybrid variants (harboring 10 to 50 single amino acid variants) were identified. Tables 3, 4 and 5 summarize the unique capsid protein variants. For purposes of comparison, wild-type AAV2, AAV3, and AAV8 capsid amino acid sequences are described in SEQ ID NOs: 869, 870, and 871, respectively. FIG.
• AAV7 is a scatter plot displaying the distribution of distinct AAV2 capsid variants and AAV2/3 variants harboring one or more single-amino-acid variants.
• Table 3 Unique AAV2 and AAV2/3 hybrid variants (amino acid sequences) identified by
• DNA sequences are provided for 4.1 kb libraries.
• Table 4 Unique AAV8 variants (amino acid sequences) identified by SMRT sequencing and bioinformatics analyses.
• the B61 (SEQ ID NO: 865) variant has higher transduction efficiencies in brain and spinal cord compared to AAV9 (FIGs 3A-3B). This is notable since wild-type AAV8 has been observed to cross the blood brain barrier less than AAV9.
• B44 SEQ ID NO: 861 has better ability transduced to liver after IM injection compared to AAV8 (FIGs. 4A-4B).
• Example 3 In vitro assessment of rAAV genome packaging efficiency and initial characterization of candidate capsid variants.
• FIG. 8 shows vector constructs to be used in multiplexed screening of discovered capsid variants.
• Table 7 A summary of the proposed transgene cassettes to be used for various diagnostic strategies is shown in Table 7.
• Table 7 Transgene cassettes for various diagnostic strate
• Quantification of vector genomes of rAAV in crude lysate is used to directly test rAAV variant packaging efficiency of both first-generation (single- strand AAV) and second- generation (self-complementary AAV) vectors directly following triple-transfection of HEK 293 packaging cells.
• This provides a streamlined alternative to performing the full workflow for small-scale vector production followed by silver staining and conventional PCR titration of vector genomes to assess virus quality for all discovered variants. Since this method can be scaled for performance in 96-well formats, it us used to quickly identify variants that produce high titer vectors.
• Candidate variants with high packaging efficiency are screened for antibody cross- reactivity to current AAVs by standard means, such as capsid immunology assays to test novel rAAVs against serum from AAV- immunized rabbits.
• capsid immunology assays to test novel rAAVs against serum from AAV- immunized rabbits.
• pooled human IgG (IVIG) neutralizing assays are performed for each candidate variant to determine the potential for pre-existing humoral immunity in the human population.
• Example 4 In vivo analyses of rAAV2 and rAAV2/3 variants to study vector
• Candidate capsid variants are grouped based on tissue distribution, and prioritized by organs of interest. Groups of candidate variants are subjected to clustered-indexing (FIG. 6A), whereby multiple packaging plasmids expressing candidate capsid variants are mixed and expressed to package uniquely DNA barcoded transgenes by triple-transfection ⁇ e.g., F9 coagulation factor IX (F.IX), to assess liver targeting and expression efficacy of secreted factors; EGFP, to assess bio-distribution and the extent of tissue-specific transduction via organ/tissue sectioning and comparative immunofluorescence microscopy; or Luciferase (Luc), to assess the quality of CNS and liver transduction via live animal imaging
• F.IX F9 coagulation factor IX
• EGFP to assess bio-distribution and the extent of tissue-specific transduction via organ/tissue sectioning and comparative immunofluorescence microscopy
• Luciferase Luciferase (Luc)
• rAAV constructs comprising the thyroxine-binding globulin (TGB), a liver specific promoter, are designed.
• TGB thyroxine-binding globulin
• CB6 chicken ⁇ -actin promoter
• Vectors encapsidating indexed transgenes are injected into adult and newborn mice by different routes of administration, and screened for secreted F.IX expression, EGFP expression, or Luc expression in 1-month longitudinal studies to profile AAV variant- mediated transgene expression.
• Routes of administration for the CNS/brain include peripheral intravascular (IV, to test transduction across the blood-brain barrier),
• intracerebroventricular IBV
• intraparenchymal intraparenchymal
• intrathecal intrathecal
• Administration for retina is performed via subretinal injection.
• IV injections also target the liver.
• transgenes delivered by AAV2, AAV2/3, or AAV8 are sacrificed and harvested for organs. Individual organs are assayed for the presence and abundance of barcoded transgenes by conventional PCR amplification of bulk DNA extracts or cDNA libraries containing transgene message, followed by Illumina sequencing to trace barcoded transgenes enriched in each tissue.
• FIG. 9 outlines the general design strategy for transgene indexing. The abundance and tissue/organ distribution of detectable barcoded transgenes reflects candidate rAAV variant tropism and transduction efficacy of each group. Highly efficacious candidate groups with desirable vector properties are selected.
• Clustered-indexing can be carried out iteratively in multiple rounds of hierarchical selection to reduce the workload.
• Candidate rAAV variants are screened for bio-distribution in non-human primates by a modality similar to the clustered-indexing methodology outlined for mouse studies (FIG. 6B). The transduction efficiencies to target organs via different routes of administration are re-assessed in NHPs to validate rAAV variant profiles observed in precursor mouse studies.
• Immunogenicity prevalence of neutralization antibody in human populations, capacity for genotoxicity, and general aspects of pathogenicity are gauged alongside primary assessments, for example histopathology of multiple tissues and organs to scrutinize T-cell or neutrophil infiltrates, monitoring hepatotoxicity by ALT/AST activity, and analyzing inflammation by examination of histological sections, to determine transduction profiles in non-human primate (NHP) animals.
• NEP non-human primate
• AAV capsid sequences were isolated. Using variant analysis pipelines developed from bioinformatic tools, an additional 263 previously undescribed, high- confidence AAV2 and AAV2/3 hybrid capsid sequence variants were identified. For purposes of comparison, wild-type AAV2 and AAV3 capsid amino acid sequences are described in SEQ ID NOs: 869 and 870, respectively.
• Table 8 Additional unique AAV2 and AAV2/3 hybrid variants (amino acid sequences) identified by SMRT sequencing and bioinformatic s analyses.
• the corresponding DNA sequences are provided for all libraries.
• the nucleic acid sequences for the AAV2 capsid variants correspond to SEQ ID NOs: 1989-2077.
• the nucleic acid sequences for the AAV2/3 capsid variants correspond to SEQ ID NOs: 2078-2251.

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