Methods for generating high titer helper-free preparations of released recombinant AAV vectors

a technology of aav vector and aav plasmid, which is applied in the direction of hydrocarbon preparation catalysts, group 5/15 element organic compounds, peptides, etc., can solve the problems of inability to achieve ideal treatment, inability to produce vector byproducts, and inability to treat the subj

Inactive Publication Date: 2005-12-01
ATKINSON EDWARD M +5
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  • Description
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Problems solved by technology

However, they are non-transforming, and are not implicated in the etiology of any human disease.
This approach is not ideal, because the copy number per cell cannot be rigorously controlled and episomal DNA is much more likely to undergo rearrangement, leading to production of vector byproducts.
The presence of significant quantities of infectious helper virus in a preparation of AAV vectors is problematic in that the preparation is intended for use in human administration.
Even the presence of non-replicative helper virus components can cause an unacceptable immunological reaction in the treated subject.

Method used

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  • Methods for generating high titer helper-free preparations of released recombinant AAV vectors
  • Methods for generating high titer helper-free preparations of released recombinant AAV vectors
  • Methods for generating high titer helper-free preparations of released recombinant AAV vectors

Examples

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example 1

Illustrative Production of Recombinant AAV Vector Using a Wild-Type Helper Virus (AD5) and a Temperature-Sensitive Helper Virus (AD TS149)

[0267] This example illustrates the use of a wild-type helper virus (Ad5) and a temperature-sensitive helper virus (Ad ts149) to provide helper functions for the replication of a recombinant AAV vector particle comprising a model therapeutic gene.

[0268] The ptgAAVCF plasmid consists of the left hand AAV2 ITR; a full length cystic fibrosis transmembrane regulator cDNA; a synthetic polyadenylation sequence based on the mouse β-globin polyadenylation sequence; AAV2 sequences downstream of the cap coding sequences; and the right-hand AAV2 ITR in a pBR322 plasmid backbone (Afione et al,, 1996). The pGEM-RS5 packaging plasmid was derived from the pHIVrep plasmid (Antoni et al., 1991) and consists of the U3 and R regions from the HIV-1 LTR; the rep and cap regions from AAV2 including the p19 and p40 promoters; pBR322 and pGEM plasmid sequences for bact...

example 2

Quantitation of RAAV and Adenovirus Titers in Vector Preparations

[0273] Cell lysates from the preceding example were assayed for production of rAAVCF vector by C37 replication assay and analyzed for adenovirus production by slot-blot hybridization.

[0274] HeLa C37 was constructed to allow inducible expression of AAV Rep proteins for rAAV vector replication. Briefly, an AAV Rep / Cap expression cassette consisting of the mouse metallothionein I promoter, AAV2 rep and cap genes and AAV transcription termination site was constructed. Also included in the plasmid was a neomycin resistance gene under the control of the SV40 early promoter, SV40 small T intron and the SV40 polyadenylation signal. HeLa cells were transfected with the plasmid and clones were selected in G418. A panel of clones was screened by a rAAV vector amplification assay. One clone, C37, demonstrated consistent and dose dependent amplification of rAAV vector following transduction and adenovirus infection.

[0275] Detect...

example 3

Optimization of Helper Function to Improve RAAV Production

[0279] This example illustrates various attempts to improve the level of rAAV obtained when using temperature-sensitive helper virus. Increasing infection levels of the helper virus was unhelpful, but adjusting the kinetics was surprisingly effective.

[0280] The effects of increasing multiplicity of infection on vector production was evaluated first. 293-1 cells were infected with either Ad5 at a MOI of 5 or ts149 at various MOI, followed by transient co-transfection with vector and packaging plasmids. After 72 hours, the cells were lysed and assayed for production of rAAVCF vector by C37 vector replication assay and analyzed for adenovirus production by slot-blot hybridization. An additional 96 hour time point was collected for cells infected with ts149 at a MOI of 5.

[0281]FIG. 3 shows the results of the rAAVCF replication assay conducted on cell lysates prepared with ts149 at various MOI. Increasing the MOI of ts149 did n...

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Abstract

This invention provides methods and compositions for producing high titer, substantially purified preparations of recombinant adeno-associated virus (AAV) that can be used as vectors for gene delivery. At the onset of vector production, AAV producer cells of this invention typically comprise one or more AAV packaging genes, an AAV vector comprising a heterologous (i.e. non-AAV) transgene of interest, and a helper virus such as an adenovirus. The AAV vector preparations produced are generally replication incompetent but are capable of mediating delivery of a transgene of interest (such as a therapeutic gene) to any of a wide variety of tissues and cells. The AAV vector preparations produced according to this invention are also substantially free of helper virus as well as helper viral and cellular proteins and other contaminants. The invention described herein provides methods of producing rAAV particles by culturing producer cells under conditions, such as temperature and pH, that promote release of virus. Also provided is a quantitative, high-throughput assay useful in the assessment of viral infectivity and replication, as well as in the screening of agent that affect viral infectivity and / or replication.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a continuation-in-part of U.S. Ser. No. 09 / 142,474, filed Sep. 4, 1998, which was a U.S. National filing under Section 371 (via PCT application PCT / US98 / 18600, filed Sep. 4, 1998), which claims the priority benefit of provisional U.S. Ser. Nos. 60 / 071,733, filed Jan. 16, 1998, and 60 / 084,193, filed Sep. 5, 1997. This application also claims the priority benefit of PCT patent application PCT / US99 / 20524 filed Sep. 7, 1999, designating the U.S., which claims priority to provisional application U.S. Ser. No. 60 / 123,685, filed Mar. 10, 1999 and U.S. Ser. No. 09 / 142,474, filed Sep. 4, 1998. All of these applications are incorporated by reference in their entirety.STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH [0002] This invention was made in part during work supported by a grant from the National Institutes of Health (NIH) R44DK4460. The government may have certain rights in this invention.TECHNICAL FIELD [...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C07C2/32C07D207/32C07D207/34C07D207/50C07D295/30C07D401/14C07D409/14C07F7/08C07F9/572C07F9/58C07F15/04C07K14/015C08F4/659C08F10/00C08F110/02C08F110/14C08F210/16C12N5/10C12N7/00C12N7/02C12N15/861C12N15/864
CPCA61K48/0091C12N2750/14152C07C2531/22C07D207/32C07D207/34C07D207/50C07D295/30C07D401/14C07D409/14C07F7/0812C07F9/5721C07F9/585C07F15/045C07K14/005C12N7/00C12N15/86C12N2750/14122C12N2750/14143C12N2750/14151C07C2/32C07F9/572C07F9/58
Inventor ATKINSON, EDWARD M.FUNG, VICTOR P.WILKINS, PERRY C.TAKEYA, RYAN K.REYNOLDS, THOMAS C.ARANHA, IAN L.
Owner ATKINSON EDWARD M
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