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Adeno-associated virus vectors

a technology of plasmid-based vectors and vectors, which is applied in the field of adeno-associated virus vectors, can solve the problems of modulating the efficiency of transduction and persistence, little is known, etc., and achieves the effects of increasing episomal stability, abundance and stability, and increasing the stability of plasmid-based vectors

Inactive Publication Date: 2009-10-22
UNIV OF IOWA RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The approach enhances the stability and persistence of rAAV vectors, enabling long-term transgene expression and increased transduction efficiency, particularly in muscle cells, and facilitates the delivery of therapeutic genes for various disorders, including blood and neurological conditions, by utilizing circularized intermediates that confer increased episomal stability and abundance.

Problems solved by technology

However, little is known about the mechanisms enabling rAAV vectors to persist in vivo or the identity of cellular factors which may modulate the efficiency of transduction and persistence.

Method used

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  • Adeno-associated virus vectors
  • Adeno-associated virus vectors
  • Adeno-associated virus vectors

Examples

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example 1

Materials and Methods

[0117]Construction of rAAV Shuttle Vector.

[0118]A recombinant AAV shuttle vector (AV.GFP3ori) which contained a GFP transgene cassette, bacterial ampicillin resistance gene, and bacterial origin of replication, was generated from a cis-acting plasmid (pCisAV.GFP3ori). Expression of the GFP gene was directed by the CMV promoter / enhancer and SV40 poly-adenylation sequences. pCisAV.GFP3ori was constructed with pSub201 derived ITR elements (Samulski et al., 1987) and the intactness of ITR sequences was confirmed by restriction analysis with SmaI and PvuII, and by sequencing. Recombinant AAV stocks were generated by co-transfection of pCisAV.GFP3ori and pRep / Cap together with co-infection of recombinant Ad.CMVlacZ in 293 cells (Duan et al., 1997). Following transfection of forty 150 mm plates, cells were collected at 72 hours by centrifugation and resuspended in 12 ml of buffer (10 mM Tris pH 8.0). Virus was released from cells by three cycles of freeze / thawing and p...

example 2

Methods

[0140]Production of rAAV Shuttle Vector.

[0141]The cis-acting plasmid (pCisAV.GFP3ori) used for rAAV production was generated by subcloning the Bsp1201 / Not I fragment (743 bp) of the GFP transgene from pEGFP-1 (Clontech) between the CMV enhancer / promoter and SV40polyA by blunt-end ligation. A 2.5 kb cassette containing beta-lactamase and bacterial replication origin from pUC19 was blunt ligated down-stream of GFP reporter cassette. The ITR elements were derived from pSub201.2 The entire plasmid contains a 4.7 kb AAV component flanked by a 2 kb stuffer sequence. The integrity of ITR sequences was confirmed by restriction analysis with SmaI and PvuII, and by direct sequencing using a modified di-deoxy procedure which allowed for complete sequence through both 5′ and 3′ ITRs. Recombinant AAV stocks were generated by co-transfection of pCisAV.GFP3ori and pRep / Cap together with co-infection of recombinant Ad.CMVlacZ in 293 cells. The rAV.GFP3ori virus was subsequently purified thro...

example 3

Evidence for Increased Episomal Persistence of AAV Circular Intermediates in a Model for In Utero Plasmid-Based Gene Therapy

[0159]Persistence of AAV circular intermediates were assessed by injection of plasmid DNA directly into the pronucleus of fertilized Xenopus oocytes. Twenty-five ng of the p81 isolate of AAV circular intermediates was injected at the single cell stage of fertilized Xenopus oocytes. This plasmid was compared to the proviral plasmid pCisAV.GFP3ori, which contains two ITRs separated by stuffer sequence in an alternative confirmation to ITRs in p81. FIG. 13 depicts the persistence of GFP plasmids as assessed by direct fluorescence of GFP. At this state of tadpole development, the fertilized oocyte has expanded from a single cell to approximately 106 cells.

[0160]These studies confirm that AAV circular intermediates (p81) confer a higher level of stability in development Xenopus oocytes than plasmids containing similar transcriptional elements and ITR sequences in an...

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Abstract

The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation under 37 C.F.R. 1.53(b) of U.S. application Ser. No. 11 / 058,751 filed Feb. 15, 2005, which is a continuation under 37 C.F.R. 1.53(b) of U.S. application Ser. No. 10 / 054,665 filed Jan. 22, 2002 (now U.S. Pat. No. 6,897,045), which is a continuation under 37 C.F.R. 1.53(b) of U.S. application Ser. No. 09 / 276,625, filed Mar. 25, 1999 (now U.S. Pat. No. 6,436,392), which is a continuation-in-part application claiming the benefit of U.S. provisional application Ser. No. 60 / 086,166 filed May 20, 1998, the disclosures of which applications are incorporated by reference herein.STATEMENT OF GOVERNMENT RIGHTS[0002]This invention was made with a grant from the Government of the United States of America (Grant No. DK / HL58340 from the National Institutes of Health). The Government may have certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Adeno-associated virus (AAV) is a non-pathogenic parvovirus with...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/00C07H21/04C12N15/63C12N5/10A01K67/027A61K31/711A61K48/00C07K14/47C12N15/00C12N15/10C12N15/64C12N15/861C12N15/864
CPCA01K2217/05A61K48/00A61K48/0008C12N2750/14143C12N15/10C12N15/64C12N15/86C07K14/4712
Inventor ENGELHARDT, JOHN F.DUAN, DONGSHENG
Owner UNIV OF IOWA RES FOUND
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