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Adeno-associated viruses and uses thereof

a technology of adenovirus and adenovirus, applied in the field of adenovirus, can solve the problems of modulating the efficiency of transduction and persistence, raav vectors may not be useful, and little is known about, so as to increase the abundance and stability, increase the stability of plasmid-based vectors, and increase the abundance

Inactive Publication Date: 2008-07-10
UNIV OF IOWA RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a recombinant adeno-associated virus (rAAV) vector that includes a nucleic acid segment formed by the juxtaposition of sequences in the inverted terminal repeats (ITRs) of rAAV. This vector has been found to have increased persistence and stability in eukaryotic cells, leading to long-term expression of the vector-based genes. The vector can be used for gene delivery in various therapeutic applications, including blood disorders, neurological disorders, and muscle disorders. The vector can also be used to identify and isolate proteins that bind to the ITR sequences present in the vector.

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.
In addition, due to limitations in rAAV vector packaging capacity, a rAAV vector may not be useful if large regulatory elements are needed to control transgene expression.

Method used

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  • Adeno-associated viruses and uses thereof
  • Adeno-associated viruses and uses thereof
  • Adeno-associated viruses and uses thereof

Examples

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example 1

Materials and Methods

[0132]Construction of rAAV Shuttle Vector.

[0133]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

[0155]Production of rAAV Shuttle Vector.

[0156]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

[0175]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.

[0176]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

CLAIM OF PRIORITY[0001]This application is a continuation under 37 CFR 1.53(b) of U.S. patent application Ser. No. 09 / 684,554 filed Oct. 6, 2000, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application Ser. No. 60 / 158,209, filed Oct. 7, 1999, which applications are incorporated herein by reference.CO-PENDING APPLICATION[0002]This invention is related to the following invention which is assigned to the same assignee as the present invention:[0003]U.S. application Ser. No. 09 / 276,625, filed Mar. 25, 1999, titled “ADENO-ASSOCIATED VIRUS VECTORS” (now U.S. Pat. No. 6,436,392, issued Aug. 20, 2002).STATEMENT OF GOVERNMENT RIGHTS[0004]This invention was made at least in part with a grant from the Government of the United States of America (grant HL51887 from the National Institutes of Health). The Government may have certain rights in the invention.BACKGROUND OF THE INVENTION[0005]Adeno-associated virus (AAV) is a non-pathogenic parvovirus with a single-stranded DN...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N7/00C12P21/00C12N15/63C12N1/00
CPCA61K48/00C12N2800/108C12N2750/14143C12N15/86A61P43/00
Inventor ENGELHARDT, JOHN F.DUAN, DONGSHENGYAN, ZIYING
Owner UNIV OF IOWA RES FOUND
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