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Virus vectors and methods of making and administering the same

a virus and parvovirus technology, applied in the field of virus vectors, can solve the problems of inability to address the problem of packaging b19 genomes into altered capsids, inefficiency of vector transduction and packaging constraints, and recent light on obstacles to the application of raav vectors

Inactive Publication Date: 2006-08-24
THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015] These and other aspects of the invention are set forth in more detail in the description of the invention below.

Problems solved by technology

While these studies evaluated capsid structure and assembly, they did not address the issue of packaging B19 genomes into the altered capsids.
While each of these factors remains valid, some obstacles to the application of rAAV vectors have recently come to light.
These include inefficiency of vector transduction and packaging constraints.
With respect to the efficiency of vector transduction, several recent studies have shown great promise in terms of duration of transgene expression in vivo; however, there has been a shortfall in the efficiency of transduction, which was unexpected based on previous results in vitro (Flotte et al., (1993) Proc. Nat. Acad. Sci.

Method used

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  • Virus vectors and methods of making and administering the same
  • Virus vectors and methods of making and administering the same
  • Virus vectors and methods of making and administering the same

Examples

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Effect test

example 1

AAV Vectors

[0195] All production of AAV vectors used in these investigations utilized the vector production scheme as described in Ferrari et al., (1997) Nature Med. 3:1295 and Xiao et al., (1998) J. Virology 72:2224. Utilizing a transient transfection procedure, rAAV devoid of adenovirus has been generated. Id. This protocol utilizes an adenovirus DNA genome that has been incapacitated for viral replication and late gene expression. The mini Ad plasmid while unable to replicate and produce progeny, is still viable for adenovirus gene expression in 293 cells. Using this construct, the AAV packaging strategy involving new AAV helper plasmid (pAAV / Ad ACG) and AAV vector DNA (sub 201) has been successfully complemented (Samulski et al., (1989) J of Virology 63:3822). This new construct typically generates rAAV of 107-109 / 10 cm dish of 293 cells (Xiao et al., (1998) J. Virology 72:2224). Efficient gene delivery is observed in muscle, brain and liver with these vectors in the complete a...

example 2

Cells and Viruses

[0196] Human 293 and HeLa cells were maintained at 37° C. with 5% CO2 saturation in 10% fetal bovine serum (Hyclone) in Dulbecco's modified Eagles medium (Gibco BRL), with streptomycin and penicillin (Lineberger Comprehensive. Cancer Center, Chapel Hill, N.C.). Four×106293 cells were plated the day before transfection onto a 10 cm plate. Cells were transfected by both calcium phosphate (Gibco BRL) or Superfection (Qiagene) according to manufacturers specifications. The insertional mutant packaging plasmids, described below, were transfected along with pAB11 containing the CMV driven Lac Z gene with a nuclear localization signal. For each transfection the same amount of packaging plasmid (12 μg) and pAB11 (8 μg) were used for each 10 cm plate. For each transfection an additional plate was used containing the transgene plasmid only to assess transformation efficiencies. After transfection the cells were infected with helper virus Ad5 d / 309 at an MOI of 5, and 48 hour...

example 3

Construction of AAV Packaging Plasmids

[0198] The capsid domain of pAAV / Ad was cloned into pBS+ (Stratagene) using Hind III, resulting in pAV2Cap. Partial digestion of pAV2Cap using the restriction enzymes Hae III, Nla IV, and Rsa I and gel purification of the unit length DNA fragment resulted in the isolation of the starting material for cloning. The aminoglycoside 3′-phosphotransferase gene, conferring kanamycin resistance (kanr), from pUC4K (Pharmacia) digested with Sal I was flanked by linkers containing Nae I and Eco RV sites, a Sal I overhang at one end and an Eco RI overhang at the other end (top 5′-MTTCGCCGGCGATATC-3′, SEQ ID NO:6, bottom 5′-TCGAGATATCGCCGGC-3′, SEQ ID NO:7). This fragment was cloned into the Eco RI site of pBluescript SK+ (Stratagene). Digestion with Nae I released the kanr gene, and this fragment was ligated into the pAV2Cap partials. The resulting plasmids were screened for insertion into the capsid domain and, then digested with Eco RV to remove the kanr...

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Abstract

The present invention provides genetically-engineered parvovirus capsids and viruses designed to introduce a heterologous gene into a target cell. The parvoviruses of the invention provide a repertoire of vectors with altered antigenic properties, packaging capabilities, and / or cellular tropisms as compared with current AAV vectors.

Description

RELATED APPLICATION INFORMATION [0001] This application is a divisional of co-pending U.S. patent application Ser. No. 10 / 205,942, filed Jul. 26, 2002, which is a divisional of U.S. patent application Ser. No. 09 / 438,268, filed Nov. 10,1999 (now U.S. Pat. No. 6,491,907), which claims the benefit of U.S. Provisional Application Ser. No. 60 / 107,840, filed Nov. 10,1998, and Ser. No. 60 / 123,651, filed Mar. 10, 1999, which are incorporated herein by reference in their entireties.STATEMENT OF FEDERAL SUPPORT [0002] This invention was made, in part, with government support under grant numbers DK42701 and 5-32938 0-110 from the National Institutes of Health. The United States government has certain rights to this invention.FIELD OF THE INVENTION [0003] The present invention relates to virus vectors, in particular, modified parvovirus vectors and methods of making and administering the same. BACKGROUND [0004] Parvoviruses are small, single-stranded, non-enveloped DNA viruses between twenty t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/861C12N7/00A61K35/76C12N15/09A61K38/00A61K39/00A61P3/00A61P3/10A61P7/00A61P11/00A61P25/00A61P25/08A61P25/14A61P25/16A61P25/28A61P27/02A61P31/18A61P35/00C07K14/015C07K19/00C12N1/11C12N1/15C12N1/19C12N1/21C12N5/10C12N7/01C12N15/864
CPCA61K39/00A61K48/00A61K2039/525C12N15/86C12N2750/14043C12N2750/14122C12N2750/14143C12N2750/14145C12N2750/14222C12N2750/14243C12N2750/14245C12N2810/40C12N2810/405C12N2810/60C12N2810/854Y10S977/804A61P1/18A61P11/00A61P21/02A61P21/04A61P25/00A61P25/08A61P25/14A61P25/16A61P25/28A61P27/02A61P3/00A61P31/18A61P35/00A61P37/04A61P7/00A61P7/04A61P3/10Y02A50/30
Inventor RABINOWITZ, JOSEPHSAMULSKI, RICHARDXIAO, WEIDONG
Owner THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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