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Compositions and methods for helper-free production of recombinant adeno-associated viruses

a technology of adenovirus and amplification, which is applied in the direction of dsdna viruses, semiconductor/solid-state device testing/measurement, instruments, etc., can solve the problems of unacceptably high levels of wt aav, slow progress towards establishing aav as a transducer vector for the delivery of dna in the form of a desired transgene, etc., and achieves efficient production

Inactive Publication Date: 2007-01-04
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present invention allows for the efficient production of rAAV containing a desired transgene DNA. Particularly, the present invention provides both compositions and methods which enable the production of a rAAV without producing contaminating re-assembled wt AAV during rAAV production.

Problems solved by technology

However, progress towards establishing AAV as a transducing vector for the delivery of DNA in the form of a desired transgene has been slow for a variety of reasons.
One obstacle to the use of AAV for delivery of DNA has been lack of highly efficient schemes for encapsidation of recombinant genomes and production of infectious virions.
However, in this method co-infection is mandatory and leads to unacceptably high levels of wt AAV resulting from non-homologous recombination and contamination of the rAAV produced with wt AAV.

Method used

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  • Compositions and methods for helper-free production of recombinant adeno-associated viruses
  • Compositions and methods for helper-free production of recombinant adeno-associated viruses

Examples

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example 1

Use of the B-50 Cell Line and Ad / AAV Hybrid Vector for Production of a Helper Independent Cell Line

[0089] A recombinant Ad / AAV hybrid vector is constructed using the methods described in U.S. Pat. No. 5,856,152 except that the E3 gene is deleted and the E1 gene operably linked to and under the control of the RSV or PGK promoter is cloned into the E3 region of the adenovirus genome. The Ad / AAV hybrid vector is packaged as described in U.S. Pat. No. 5,856,152.

[0090] Briefly described, B-50 is a cell which stably expresses AAV type 2 rep and cap genes under the control of the homologous p5 promoter. This cell line is characterized by integration of multiple copies (at least 5 copies) of P5-rep-cap gene cassettes in a concatamer form into the host chromosome. This B-50 cell line was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 on Sep. 18, 1997 under Accession No. CRL-12401 pursuant to the requirements of The Budapest Treaty ...

example 2

Production of rAAV in B50 Cells by Replication Competent Ad-AAV Hybrid Virus

[0093] A rAAVCMVGFP genome in which a Green Fluorescent Protein (GFP)reporter gene is driven by CMV promoter and flanked by AAV2 ITRs was cloned into the E3 region of an Ad5 mutant, sub100r virus. The E2b terminal protein gene of sub100r was disrupted by a 3 bp insertion, rendering a temperature sensitive phenotype. The resulting recombinant Ad-AAV hybrid is a genotypically wild type for E1, E2a, E4 and VARNA genes but its E3 genes are now replaced with a rAAVCMVGFP genome. Thus this second generation Ad-AAV hybrid possesses all essential helper genes and a rAAV genome and, theoretically, a single infection of B50 cells with the virus should lead to rescuing, replicating and packaging of rAAV genomes.

[0094] A. Construction of a Replication-Competent rAd-AAV Hybrid Virus

[0095] A commercially available plasmid construct pAB27 was purchased from Microbix Biosystems (Ontario, Canada). It carries the following...

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Abstract

A method for producing recombinant adeno-associated virus in the absence of contaminating helper virus or wild-type virus involves culturing a mammalian host cell containing an rAd / AAV hybrid virus, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. The rAd / AAV hybrid virus contains a rAAV construct to be packaged into an AAV virion in a backbone containing the adenoviral sequences necessary to express E1a and E1b gene products and to permit replication of the hybrid virus. The method of the invention permits replication of the hybrid virus and production of rAAV virion in this host cell in the absence of a helper virus and obviates a subsequent purification step to purify rAAV from contaminating virus.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a continuation of U.S. patent application Ser. No. 10 / 251,157, filed Sep. 20, 2002, now U.S. Pat. No. 7,022,519, issued on Apr. 4, 2006, which is a continuation of U.S. patent application Ser. No. 09 / 826,510, filed Apr. 5, 2001, now U.S. Pat. No. 6,485,966, issued on Nov. 26, 2002, which is a divisional of U.S. patent application Ser. No. 09 / 404,555, filed Sep. 23, 1999, now U.S. Pat. No. 6,258,595, issued on Jul. 10, 2001, which is a continuation-in-part of International Patent Application PCT / US99 / 05870, filed Mar. 18, 1999, published as International Patent Application Publication No. WO 99 / 47691, published Sep. 23, 1999.BACKGROUND OF THE INVENTION [0002] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/861C12N5/06G01R31/30C12N5/10C12N7/00C12N7/01C12N15/09C12N15/864C12R1/91C12R1/92G01R31/01G01R31/27G01R31/28G01R31/316H01L21/66
CPCC12N15/86C12N2710/10344C12N2750/14143G01R31/316C12N2840/20G01R31/013G01R31/275C12N2750/14151
Inventor GAO, GUANGPINGWILSON, JAMES M.
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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