# US 10,501,758 B2 · Recombinant baculovirus and method for using the same for preparing recombinant adeno-associated virus vector

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,758 B2
- Document ID: US10501758B2
- Status: Granted
- Kind: B2
- Application number: 15/884,470
- Filed: 2018-01-31
- Published: 2018-06-07
- Granted: 2019-12-10
- Earliest claimed: 2015-09-23
- Assignees: Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences
- Applicants: Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences
- Inventors: Yang Wu; Fuqiang Xu; Xiaobin He
- Web page: https://patentgrep.com/patent/US10501758B2

## CPC

- C12N 15/86: Viral vectors
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- 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 2710/14044: Chimeric viral vector comprising heterologous viral elements for production of another viral vector
- C12N 2710/14143: viral genome or elements thereof as genetic vector
- C12N 2710/14144: Chimeric viral vector comprising heterologous viral elements for production of another viral vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

A recombinant baculovirus, including: an adeno-associated virus Rep gene, an adeno-associated virus Cap gene, and an recombinant adeno-associated virus genome ITR-GOI (gene of interest) flanked by rAAV inverted terminal repeats (ITR). The ITR-GOI includes a 5′ terminal nucleic acid fragment and a 3′ terminal nucleic acid fragment. The ITR-GOI is linked to an expression cassette of the Cap gene and an expression cassette of the Rep gene through the 5′ terminal nucleic acid fragment and the 3′ terminal nucleic acid fragment, respectively.

## Claims

### Claim 1

A recombinant baculovirus, comprising: an adeno-associated virus (AAV) Rep gene, an AAV Cap gene, and a recombinant adeno-associated virus (rAAV) genome ITR-GOI (gene of interest) flanked by AAV inverted terminal repeats (ITR); wherein:
the ITR-GOI comprises a 5′ terminal nucleic acid fragment and a 3′ terminal nucleic acid fragment; and
the ITR-GOI is linked to an expression cassette of the Cap gene and an expression cassette of the Rep gene through the 5′ terminal nucleic acid fragment and the 3′ terminal nucleic acid fragment, respectively.

### Claim 2 (depends on 1)

The recombinant baculovirus of claim 1, wherein the Rep gene has a codon-optimized sequence.

### Claim 3 (depends on 2)

The recombinant baculovirus of claim 2, wherein the Rep gene has the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

### Claim 4 (depends on 1)

The recombinant baculovirus of claim 1, wherein the Cap gene has a codon-optimized sequence.

### Claim 5 (depends on 4)

The recombinant baculovirus of claim 4, wherein the Cap gene has the sequence represented by SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

### Claim 6 (depends on 1)

The recombinant baculovirus of claim 1, wherein each of two ends of the rAAV genome ITR-GOI comprises an inverted terminal repeat (ITR) of the adeno-associated virus genome; and the gene of interest (GOI) is disposed between the two ends of the ITR-GOI.

### Claim 7 (depends on 1)

The recombinant baculovirus of claim 1, wherein the inverted terminal repeat (ITR) has a sequence represented by SEQ ID NO: 7.

### Claim 8 (depends on 1)

The recombinant baculovirus of claim 1, wherein the 5′ terminal nucleic acid fragment and the 3′ terminal nucleic acid fragment have a length of 80-140 bp.

### Claim 9 (depends on 1)

The recombinant baculovirus of claim 1, wherein the 5′ terminal nucleic acid fragment and the 3′ terminal nucleic acid fragment have a sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9.

### Claim 10 (depends on 1)

The recombinant baculovirus of claim 1, wherein the adeno-associated virus is adeno-associated virus serotype 2.

### Claim 11 (depends on 1)

A method for preparing a recombinant adeno-associated vims vector, the method comprising:
(a) constructing a baculovirus by integrating a target gene into a genome of the recombinant baculovirus of claim 1;
(b) infecting an isolated host cell with the baculovirus of (a) to produce a recombinant adeno-associated virus; and
(c) purifying the recombinant adeno-associated virus of (b).

### Claim 12 (depends on 11)

The method of claim 11, wherein in (a), a shuttle vector based on a baculovirus expression system is used.