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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/141—MicroRNAs, miRNAs
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

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.
• Adeno-associated Virus is a small and helper dependent virus. It was discovered in the 1960s as a contaminant in adenovirus (a cold causing virus) preparations. Its growth in cells is dependent on the presence of adenovirus and, therefore, it was named as adeno-associated virus. AAV vectors have emerged as an effective platform for in vivo gene transfer. However, 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 AAV3B, AAV4 and AAV5 have been identified and are disclosed herein that possess useful tissue targeting properties.
• variants of AAV3B are provided that are useful for transducing cells, such as, human hepatocytes (e.g., present in liver tissue) and others.
• variants of AAV4 and AAV5 are provided that are useful for targeting cells of the central nervous system (CNS), cardiopulmonary tissue, ocular tissue, and other tissues.
• CNS central nervous system
• the variant AAVs described herein target tissue other than the tissue targeted by their corresponding wild- type AAVs.
• AAV3B variants target cells of the central nervous system (CNS) or the heart.
• AAV4 variants target cells of the liver or kidney.
• AAV5 variants target cells of the CNS, liver, spleen or heart.
• the disclosure in some aspects provides an isolated nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NO: 1 to 47 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: 98 to 100.
• 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: 51 to 61.
• the isolated AAV capsid protein comprises a sequence selected from the group consisting of: SEQ ID NOs: 51 to 61, 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: 98 is replaced with a conservative substitution.
• the isolated AAV capsid protein comprises a sequence selected from the group consisting of: SEQ ID NOs: 62 to 67, 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: 99 is replaced with a conservative substitution.
• the isolated AAV capsid protein comprises a sequence selected from the group consisting of: SEQ ID NOs: 68 to 97, 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: 100 is replaced with a conservative substitution.
• composition 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.
• a host cell contains a nucleic acid that comprises a coding sequence selected from the group consisting of: SEQ ID NO: 1 to 47 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.
• the at least one transgene encodes a small interfering nucleic acid.
• 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 skeletal muscle, heart, liver, pancreas, brain or lung. In some embodiments, 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. In certain embodiments, 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 at least one transgene is a protein coding gene.
• the at least one transgene encodes a small interfering nucleic acid.
• the at least one transgene encodes a reporter molecule.
• the small interfering nucleic acid is a miRNA.
• the small interfering nucleic acid is a miRNA sponge or TuD RNA that inhibits the activity of at least one miRNA in the animal.
• the miRNA is expressed in a cell of the target tissue
• the target tissue is skeletal muscle, heart, liver, pancreas, brain or lung.
• 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, 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, or a cardiac Troponin T (cTnT) promoter.
• TSG liver- specific thyroxin binding globulin
• PY pancreatic polypeptide
• PPY pancreatic polypeptide
• Syn synapsin-1
• MCK creatine kinase
• DES mammalian desmin
• a-MHC a-myosin heavy chain
• cTnT cardiac Troponin T
• 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: 1 to 47.
• 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, heart, skeletal muscle, brain and pancreas tissue targeting capabilities. These tissues are associated with a broad spectrum of human diseases including a variety of metabolic, cardiovascular and diabetic diseases.
• 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 rAAV has a AAV capsid having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 51 to 97.
• 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 a 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 which is optionally a reporter enzyme, such as Beta-Galactosidase or a Fluorescent protein, such as GFP.
• 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 heart, liver, skeletal muscle, brain or pancreas tissue, in some embodiments.
• the target tissue of an AAV is gonad, diaphragm, heart, stomach, liver, spleen, pancreas, or kidney.
• the rAAV may transduce many different types of tissue, such as muscle fibers, squamous epithelial cells, renal proximal or distal convoluted tubular cells, mucosa gland cells, blood vessel endothelial cells, or smooth muscle cells.
• 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, 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, or a cardiac Troponin T (cTnT) promoter.
• TSG liver- specific thyroxin binding globulin
• PY pancreatic polypeptide
• PPY pancreatic polypeptide
• Syn synapsin-1
• MCK creatine kinase
• DES mammalian desmin
• a-MHC a-myosin heavy chain
• cTnT cardiac Troponin
• 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
• FIG. 1 depicts an alignment of AAV 3B variant capsid sequences
• FIG. 2 depicts an alignment of AAV 4 variant capsid sequences
• FIG. 3 depicts an alignment of AAV 5 variant capsid sequences
• FIG. 4 depicts results of recombinant AAV transduction assays.
• 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.
• 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. Methods for Discovering AA Vs
• 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 proviral 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.
• 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 reverse transcription
• 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 non-human primate tissues using the methods disclosed herein. Nucleic acids encoding capsid proteins of these novel AAVs have been discovered in both viral genomic DNA and mRNA isolated from the non-human primate tissues. Nucleic acid and protein sequences as well as other information regarding the AAVs are set forth in Tables 1 and 2 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 1 to 47 as well as any nucleic acid having a sequence with substantial homology thereto.
• 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 1 to 47, but that does not encode a protein having an amino acid sequence as set forth in any one of SEQ ID NOs 98 to 100.
• 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 51 to 98 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 98 to 100, but that does not have an amino acid sequence as set forth in any one of SEQ ID NOs 98 to 100.
• “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties.
• the term “substantial homology” 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 which 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 which 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. Tables of corresponding amino acids among various AAV variants are provided in Tables 4-6.
• homology can be determined by hybridization of polynucleotides under conditions which 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 which 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 an AAV capsid protein is a nucleic acid having a sequence selected from the group consisting of: SEQ ID NO: 1 to 47.
• a fragment of an isolated nucleic acid encoding a AAV capsid sequence may be useful for constructing a nucleic acid encoding a desired capsid sequence. Fragments may be of any appropriate length. In some embodiments, a fragment (portion) of an isolated nucleic acid encoding a 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.
• 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: 98 to 100.
• 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 3B, AAV4 or AAV5).
• 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 3B, AAV4 or AAV5). In some embodiments, 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 3B, AAV4 or AAV5).
• 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 3B, AAV4 or AAV5).
• a nucleic sequence encoding an AAV variant e.g. , SEQ ID NO: 92 may comprise 3 amino acid variants compared with a known AAV serotype (e.g. , AAV5).
• a recombinant cap sequence may be constructed having one or more of the 3amino 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 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 51-97, or a protein having substantial homology thereto.
• Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are
• the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein (e.g. , a nucleic acid having a sequence as set forth in any one of SEQ ID NOs 1 to 47) 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 AAV3B,
• AAV3B is known to efficiently transduce human hepatocytes ⁇ e.g., liver tissue). It is also known that AAV3B efficiently transduces cancerous human hepatocytes. Accordingly, in some embodiments, the AAV3B variants described herein may be useful for delivering gene therapy to cancerous and normal human hepatocytes.
• AAV4 and AAV5 are known to target tissue of the central nervous system (CNS), cardiopulmonary tissue and the eye. Accordingly, in some embodiments, the AAV4 and AAV5 variants described herein may be useful for delivering gene therapy to the CNS, cardiopulmonary tissue or the eye.
• the AAV3B, AAV4 and AAV5 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 AAV3B, AAV4 and AAV5 variants described herein may have a tissue tropism useful for delivering gene therapy to additional tissue types that are not targeted by wild- type AAV3B, AAV4 or AAV5. In some embodiments, AAV3B variants described herein ⁇ e.g., CBr-7.4, CBr-7.5, CBr-7.8) may be useful for delivering gene therapy to the central nervous system (CNS). In some
• AAV4 variants described herein may be useful for targeting cells of the kidney or cells of the liver.
• AAV5 variants described herein may be useful for targeting gene therapy to the liver, spleen, heart or brain.
• Non-limiting examples of AAV variants and their target tissues may be found in Table 1.
• 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.
• CNS- related disorders include Parkinson' s Disease, Lysosomal Storage Disease, Ischemia, Neuropathic Pain, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), and
• CD Canavan disease
• 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. Accordingly, in some embodiments, AAV variants described herein may be useful for treatment of pulmonary disease. As used herein 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, pulmonary vascular disease, Respiratory Syncytial Virus (RSV), sarcoidosis, Severe Acute Respiratory Syndrome (SARS), silicosis, sleep apnea, Sudden Infant Death Syndrome (SIDS), and tuberculosis.
• RSV Respiratory Syncytial Virus
• SARS Severe Acute Respiratory Syndrome
• SIDS Sudden Infant Death Syndrome
• 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 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 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, Meso American 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
• AAV variants described herein may be useful for delivering gene therapy to ocular tissue. 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.
• a cardiovascular 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, macula edema, glaucoma, retinitis pigmentosa and eye 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 an 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 membrane.
• 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 which has been transfected. Thus, a "host cell” as used herein may refer to a cell which 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) which provides helper functions to the AAV.
• the vector providing helper functions may provide adenovirus functions, including, e.g., Ela, Elb, E2a, 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. , which 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 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, required to initiate the specific transcription of a 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.
• inhibitory RNA e.g. , shRNA, miRNA, miRNA inhibitor
• an isolated capsid gene can be used to construct and package recombinant AAVs, using methods well known in the art, to determine functional characteristics associated with the capsid protein encoded by the gene.
• 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. ).
• 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.
• 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, which 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 which 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 rAAVs of the present disclosure are pseudotyped rAAVs.
• 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 repeats (ITRs) of one AAV serotype and an capsid protein of a different AAV serotype.
• ITRs inverted terminal repeats
• 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 which 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 contain 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.
• 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 which may be useful in this context are those which 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, or a cardiac Troponin T (cTnT) promoter.
• 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.
• 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 CoA 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, S106P, 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 Disease;
• 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, TGFpi, SFTPA1, SFTPA2, SFTPB, SFTPC, HPS1, HPS 3, HPS4, 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, NPHS1, 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, SIXl, SIX5, FRAS1, 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 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, CAPNS1, 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,
• the instant disclosure relates to an rAAV vector comprising a nucleic acid encoding a gene product associated with a CNS-related disorder.
• genes associated with a CNS-related disorder DRD2, GRIA1, GRIA2,GRIN1, SLC1A1 , SYP, SYT1, CHRNA7, 3Rtau/4rTUS, APP, BAX, BCL-2, GRIKl, GFAP, IL-1, AGER, associated with Alzheimer's Disease; UCH-Ll, SKPl, EGLNl, Nurr-1, BDNF, TrkB,gstml, S106P, associated with Parkinson' s Disease; IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin 1, FTL, TITF- 1, associated with
• FXN associated with Freidrich' s ataxia
• ASPA associated with Canavan' s Disease
• DMD associated with muscular dystrophy
• SMN1 UBE1
• DYNC1H1 associated with spinal muscular atrophy.
• 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 which 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.
• 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 hybridization with a small interfering nucleic acid (e.g.
• 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.
• an small interfering nucleic acid that is substantially complementary to a miRNA is an small interfering nucleic acid that is complementary with 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.
• an small interfering nucleic acid sequence that is substantially complementary to a miRNA is an small interfering nucleic acid sequence that is complementary with the miRNA at, at least, one base.
• 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 (derepression of miRNA targets) in cells will be apparent to one of ordinary skill in the art.
• the cloning capacity of the recombinant RNA vector may limited and a desired coding sequence 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.
• tissue targeting capability e.g., liver, heart, pancreas
• 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 missense 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. Gene Ther. 11 :2315-2329, 2000).
• the striatum e.g., the caudate nucleus or putamen of the striatum
• spinal cord and neuromuscular junction e.g., the caudate nu
• 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.
• 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 among animal and tissue.
• 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.
• 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.
• 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,
• rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously,
• administration modalities as described in U.S. Pat. Nos. 5,543, 158;
• 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 of the 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 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.
• 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.
• 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
• 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 and Related Compositions
• 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.
• the instructions included within the kit may involve methods for detecting a latent
• 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.
• chimpanzee tissues were analyzed for the presence of AAV proviral genomes and cap RNAs first by qPCR and qRT-PCR using a set of primer and probe to target short stretches of the conserved cap sequence.
• Six tissues were evaluated from two chimps for AAV cap sequences. The data indicated that all the tissues harbored AAVs in variable abundances, with the highest copy numbers in liver.
• cDNA clones ⁇ e.g., clones of VPl) were analyzed by sequencing. A total of 24 DNA and 23 RNA clones of VPl were isolated by RT-PCR and PCR and fully sequenced. Interestingly, 30 out of these 47 clones were AAV5- like, of which 14 clones were the products of RT-PCR.
• the phylogenetic analysis segregated the capsid clones into three major groups closely related to AAV3B, AAV4 and AAV5, which are useful for gene delivery to various tissues ⁇ e.g., CNS , renal, splenic, hepatic and cardiac targets).
• Table 1 lists the AAV capsid variants identified.
• Table 2 provides the NCBI Accession numbers of the wild-type AAV serotypes related to the identified variant sequences.
• variant cap cDNA clones with amino acid differences from AAV3B, AAV4 or AAV5 and within themselves were selected for further characterization by BLAST analysis and multiple sequence alignment (Table 3). Amino acid substitutions between variant capsid proteins and wild- type AAV serotypes are summarized in Tables 4-6.
• Recombinant AAVs comprising example capsids (namely, CBr-7.4, CBr-7.5 and CBr-7.8 capsids) were constructed and packaged to contain luciferase expressing vector genome. Titers of rAAVs produced were 1.3 x 10 10 , 1.2 x 10 10 , and 1.2 x 10 10 genomic copies/ml for CBr-7.4, CBr-7.5 and CBr-7.8, respectively. Huh-7.5 hepatoma cells were transduced with the recombinants AAVs and luciferase expression was examined to confirm transduction and expression (FIG. 4). AAV9 and AAV3B were examined as controls. TABLE 1 : SEQUENCES OF NOVEL AAVS
• AAV4 Differences/ Variation Variation / Variation / variant Origin tissue AAV4 /VIM VP2 (137- ) VP3 (197- ) AAV4 0 0 0 0
• AAV5 Origi differences/ Variation Variation / Variation / variant n tissue AAV5 /VIM VP2 (137- ) VP3 (193- )

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