Novel dual helper plasmid
AssigneeNeuracle Genetics Inc.
InventorsJoo Seok HAN, Hoon Young KONG, Yoon Hyung HWANG
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to a dual helper plasmid for producing a recombinant adeno-associated virus. A double transfection method using the dual helper plasmid of the present disclosure is advantageous over the triple transfection method typically used for production of adeno-associated virus in terms of 1) increased chance of co-transfection, 2) increased productivity of recombinant adeno-associated virus, 3) reduction in cost and time of plasmid production and purification, etc., and thus can be usefully utilized for effective production of a gene therapy agent.
Claims26
A dual helper plasmid comprising a rep-cap gene and one or more additional gene selected from an E2a gene, E4 gene, and VA RNA gene, wherein the rep-cap gene is in a clockwise direction (from 5′ to 3′) or in a counterclockwise direction (from 3′ to 5′).
2-17. (canceled)
A dual helper plasmid comprising a regulatory component, wherein the regulatory component comprises (from 5′ to 3′):
- (1) (i) an E2a gene, which comprises the sequence set forth in SEQ ID NO:
- 34;
- (ii) an E4 gene, which comprises the sequence set forth in SEQ ID NO:
- 35;
- (iii) a VA RNA gene, which comprises the sequence set forth in SEQ ID NO:
- 36;
- (iv) a cap gene, which comprises the sequence set forth in any one of SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, or SEQ ID NO:
- 33;
- and
- (v) a rep gene, which comprises the sequence set forth in SEQ ID NO:
- 29;
- or
- (2) (i) an E2a gene, which comprises the sequence set forth in SEQ ID NO:
- 34;
- (ii) an E4 gene, which comprises the sequence set forth in SEQ ID NO:
- 35;
- (iii) a VA RNA gene, which comprises the sequence set forth in SEQ ID NO:
- 36;
- (iv) a rep gene, which comprises the sequence set forth in SEQ ID NO:
- 29;
- and
- (v) a cap gene, which comprises the sequence set forth in any one of SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, or SEQ ID NO:
- 33.
19-21. (canceled)
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26-29. (canceled)
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38-41. (canceled)
43-44. (canceled)
48-50. (canceled)
A method of producing a protein, comprising contacting a cell with a dual helper plasmid which comprises an E2a gene, E4 gene, VA RNA gene, and rep-cap gene, wherein the E2a, E4, and the VA RNA genes are linked sequentially, and wherein (i) the rep-cap gene is located between the 5′-terminal of the E2a gene and the 3′-terminal of the VA RNA gene in a clockwise direction (from 5′ to 3′);
- or (ii) the rep-cap gene is located between the 5′-terminal of the E2a gene and the 3′-terminal of the VA RNA gene in a counterclockwise direction (from 3′ to 5′).
Dependencies are inferred from the claim text.