Isolation Of Novel AAV'S And Uses Thereof

• US 20100186103A1
• Filed: 01/12/2010
• Published: 07/22/2010
• Est. Priority Date: 05/28/2008
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Gao, Guangping, Zamore, Phillip D.
• Granted Patent: US 9,217,155 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• CPC Class Codes: A01K 2207/05 Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
• CPC Class Codes: A01K 2217/058 due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2015/8527 for producing animal models, e.g. for tests or diseases
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2710/10342 virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 2750/14143 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14145 : Special targeting system for viral vectors
• CPC Class Codes: C12N 2820/002 : inducible or controllable
• CPC Class Codes: C12N 2840/007 : cell or tissue specific
• CPC Class Codes: C12N 2840/102 : inhibiting translation
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The invention in some aspects relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells. In some aspects, the invention provides kits and methods for producing somatic transgenic animal models using recombinant AAV (rAAV) to an animal having at least one transgene that expresses a small interfering nucleic acid or at least one binding site for a miRNA.

Claims

1. A method for generating a somatic transgenic animal model comprising administering a recombinant Adeno-Associated Virus (rAAV) to an animal, wherein the rAAV infects cells of a target tissue of the animal, and wherein the rAAV comprises at least one transgene that expresses a small interfering nucleic acid.

2. The method of claim 1, wherein the small interfering nucleic acid is an miRNA.

3. The method of claim 1, wherein the small interfering nucleic acid is an miRNA sponge that inhibits the activity of at least one miRNA in the animal.

4. The method of claim 1, 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 the target tissue, by hybridizing to the binding site.

7. The method of claim 1, wherein the rAAV has a capsid from an AAV serotype selected from:

AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and variants thereof.

8. The method of claim 1, wherein the rAAV has an Inverted Terminal Repeat (ITR) sequence from an AAV serotype selected from:

AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

9. The method of claim 1, wherein the target tissue is gonad, diaphragm, heart, stomach, liver, spleen, pancreas, or kidney.

10. The method of claim 1, wherein the rAAV transduces muscle fibers, squamous epithelial cells, renal proximal or distal convoluted tubular cells, mucosa gland cells, blood vessel endothelial cells, or smooth muscle cells.

11. The method of claim 1, wherein the administering is at a dose of 10¹⁰, 10¹¹, 10¹², or 10¹³ genome copies.

12. The method of claim 1, wherein the administering is performed intravenously.

13. The method of claim 1, wherein the animal is a mammal, optionally wherein the mammal is selected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, a non-human primate.

14. The method of claim 1, wherein the transgene causes a pathological state in the animal.

15. The method of claim 14, further comprising administering a putative therapeutic agent to the somatic transgenic animal model to determine the effect of the putative therapeutic agent on the pathological state in the animal.

17. A somatic transgenic animal produced by the method of claim 1.

5. A method for generating a somatic transgenic animal model comprising administering a recombinant Adeno-Associated Virus (rAAV) to an 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.

6. The method of claim 5, wherein the transgene comprises a tissue specific promoter or inducible promoter.

16. The method of claim 5, wherein the transgene expresses a reporter gene.

18. A kit for producing a rAAV that generates a somatic transgenic animal that expresses a transgene in a target tissue, comprising:

at least one container housing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene that expresses a small interfering nucleic acid and/or 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 of the transcript,

at least one container housing a rAAV packaging component, and

instructions for constructing and packaging the rAAV, wherein the rAAV transduces cells of the target tissue.

19. The kit of claim 18, wherein a rAAV packaging component comprises:

a host cell expressing at least one rep gene and/or at least one cap gene, wherein the at least one cap gene encodes a capsid protein from an AAV serotype that binds to cells of the target tissue.

20. The kit of claim 19, wherein the AAV serotype is selected from:

AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and variants thereof.