# US 8,784,799 B2 · Duplexed parvovirus vectors

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- Number: US 8,784,799 B2
- Document ID: US8784799B2
- Status: Granted
- Kind: B2
- Application number: 13/751,819
- Filed: 2013-01-28
- Published: 2013-09-26
- Granted: 2014-07-22
- Earliest claimed: 2000-06-01
- Assignees: The University of North Carolina at Chapel Hill
- Applicants: The University of North Carolina at Chapel Hill
- Inventors: Richard Jude Samulski; Douglas M. McCarty
- Web page: https://patentgrep.com/patent/US8784799B2

## CPC

- C12N 15/864: Parvoviral vectors {, e.g. parvovirus, densovirus}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/12: Antivirals
- A61P 33/00: Antiparasitic agents
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2750/14121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention provides duplexed parvovirus vector genomes that are capable under appropriate conditions of forming a double-stranded molecule by intrastrand base-pairing. Also provided are duplexed parvovirus particles comprising the vector genome. Further disclosed are templates and methods for producing the duplexed vector genomes and duplexed parvovirus particles of the invention. Methods of administering these reagents to a cell or subject are also described. Preferably, the parvovirus capsid is an AAV capsid. It is further preferred that the vector genome comprises AAV terminal repeat sequences.

## Claims

### Claim 1

An adeno-associated virus (AAV) particle comprising:
an AAV capsid; and
a vector genome comprising in the 5′ to 3′ direction:
(i) a 5′ AAV terminal repeat;
(ii) a first heterologous nucleotide sequence;
(iii) an AAV inverted terminal repeat that forms a hairpin structure, does not comprise a functional AAV terminal resolution site (trs), and is resolved by Rep protein to a lesser extent as compared with the 5′ AAV terminal repeat and a 3′ AAV terminal repeat;
(iv) a separate heterologous nucleotide sequence; and
(v) the 3′ AAV terminal repeat;
wherein the first and the separate heterologous nucleotide sequences are essentially self-complementary and form a double-stranded nucleic acid sequence that encodes a factor IX polypeptide.

### Claim 2 (depends on 1)

The AAV particle of claim 1, wherein the factor IX polypeptide is a human factor IX polypeptide.

### Claim 3 (depends on 1)

The AAV particle of claim 1, wherein the AAV inverted terminal repeat of (iii) comprises a deletion of the trs sequence 5′-AGTTGG-3′ and all of the D element.

### Claim 4 (depends on 1)

The AAV particle of claim 1, wherein the AAV capsid is an AAV2 capsid.

### Claim 5 (depends on 1)

The AAV particle of claim 1, wherein the AAV capsid is an AAV5 capsid.

### Claim 6 (depends on 1)

A pharmaceutical formulation comprising a plurality of the AAV particles of claim 1 in a pharmaceutically acceptable carrier.

### Claim 7 (depends on 1)

A method of delivering the vector genome to a mammalian cell, comprising contacting the mammalian cell with the AAV particle according to claim 1 under conditions sufficient for the AAV particle to enter the mammalian cell.

### Claim 8 (depends on 7)

The method of claim 7 wherein the mammalian cell is a human cell.

### Claim 9 (depends on 7)

The method of claim 7, wherein the mammalian cell is a liver cell.

### Claim 10

An AAV particle comprising:
an AAV capsid; and
a vector genome comprising in the 5′ to 3′ direction:
(i) a 5′ AAV terminal repeat;
(ii) a first heterologous nucleotide sequence;
(iii) an AAV terminal repeat modified such that it does not comprise a functional AAV trs and such that resolution by Rep protein is reduced as compared with the 5′ AAV terminal repeat and a 3′ AAV terminal repeat;
(iv) a separate heterologous nucleotide sequence; and
(v) the 3′ AAV terminal repeat;
wherein the first and the separate heterologous nucleotide sequences are essentially self-complementary and may form a hairpin structure, and form a double-stranded nucleic acid sequence that encodes a factor IX polypeptide.

### Claim 11 (depends on 10)

The AAV particle of claim 10, wherein the factor IX polypeptide is a human factor IX polypeptide.

### Claim 12 (depends on 10)

The AAV particle of claim 10, wherein the AAV terminal repeat of (iii) comprises a deletion of the trs sequence 5′-AGTTGG-3′ and all of the D element of the AAV terminal repeat.

### Claim 13 (depends on 10)

The AAV particle of claim 10, wherein the AAV capsid is an AAV2 capsid.

### Claim 14 (depends on 10)

The AAV particle of claim 10, wherein the AAV capsid is an AAV5 capsid.

### Claim 15 (depends on 10)

A pharmaceutical formulation comprising a plurality of the AAV particles of claim 10 in a pharmaceutically acceptable carrier.

### Claim 16 (depends on 10)

A method of delivering the vector genome to a mammalian cell, comprising contacting the mammalian cell with the AAV particle according to claim 10 under conditions sufficient for the AAV particle to enter the mammalian cell.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mammalian cell is a human cell.

### Claim 18 (depends on 16)

The method of claim 16, wherein the mammalian cell is a liver cell.