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Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy

An origin of replication and conditional technology, applied in the direction of recombinant DNA technology, microorganism-based methods, botanical equipment and methods, etc., to achieve the effect of improving cell penetration or distribution ability, and good bioavailability in vivo

Inactive Publication Date: 2008-05-21
AVENTIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available technologies are not entirely satisfactory
[0008] On the one hand, maintaining the risk of transmission in the body

Method used

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  • Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy
  • Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy
  • Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Example 1: Construction of XAC-1pir and pir116 host strains by homologous recombination

[0183] The strain used was E. coli strain XAC-1 (Normanly et al., 1980). The argE gene of this strain advantageously includes a glutamine-53 (CAG) to amber codon (TAG) mutation (Meinnel et al., 1992). The argE gene belongs to the argECBH divergent operon and encodes the arginine biosynthesis enzyme N-acetylornithinase. Therefore XAC-1 cannot synthesize arginine and thus cannot grow in minimal medium. This auxotrophy will be relieved if the strain contains a plasmid that allows expression of the suppressor tRNA. Bacteria carrying this plasmid can thus be selected for by culturing in minimal medium. In order to allow replication of the R6K-derived plasmid, it was necessary to introduce the pir gene into the XAC-1 genome by homologous recombination. The pir gene (wild type or mutated) was introduced into the uidA locus by exchange between the wild type uidA gene and a copy inter...

Embodiment 2

[0203] Example 2: Construction of a plasmid vector from R6X carrying the selectable marker sup Phe

[0204] A vector (pKL2666) containing oriγ from R6K and a kanamycin resistance gene was constructed. The observation of pXL2666 multimers in strain BW19610(pir116)5 (Metcalf et al., 1993) led us to investigate the influence of the cer fragment of ColE1 on this phenomenon. We then introduced the phenylalanine suppressor tRNA (sup Phe) expression cassette into the vector oriγ-Km R -cer (pXL2730). This vector, pXL2760, serves as the basis for constructing a vector that can be used in gene therapy.

[0205] 1) Construction and analysis of vectors containing R6K oriγ and kanamycin resistance genes

[0206] a) construct

[0207] In the first constructed plasmid pXL2666, the kanamycin resistance gene came from pUC4K (Vieira and Messing; 1982), and the replication origin contained in the 417bp EcoRI-BamHI fragment came from the suicide vector pUT-T7pol (Herrero et al., 1990) (Fi...

Embodiment 3

[0217] Example 3: Confirmation that plasmids can be applied to gene therapy by transfecting mouse fibroblasts

[0218] 1) Construction of reporter vector pXL2774

[0219] To test the effectiveness of this system for generating plasmid DNA for gene therapy, we introduced a reporter gene that can be used in eukaryotic cells into pXL2760. We used the gene luc encoding Photinuspyralis luciferase because bioluminescence measurements are very sensitive, linear over a large range, and background noise due to endogenous activity in eukaryotic cells is very low. The Luc gene is controlled using the promoter-enhancer sequence of the human cytomegalovirus early gene (CMV promoter) allowing high level expression. At the 3' end of luc there is an untranslated region derived from the virus SV40 containing a polyadenylation signal (poly(A)+). After intermediate cloning to increase the number of available restriction sites, the "CMV promoter-luc-poly(A)+" cassette was introduced into th...

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PUM

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Abstract

A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

Description

[0001] This case is a divisional application, the parent application number is 200380104184.6 (PCT application number is PCT / US2003 / 032512), the application date is October 14, 2003, and the invention name is "circular DNA molecule with conditional replication origin and its preparation method". technical field [0002] The present invention relates to a novel conditionally replicated DNA molecule, which can be used for gene therapy or recombinant protein production. Hereinafter, the novel DNA molecule according to the present invention is designated as pCOR TM . Background technique [0003] Gene therapy consists in correcting defects or abnormalities by introducing genetic information into affected organs or cells. This information can be introduced in vitro into cells extracted from the organ and then imported into the body; or directly into the target tissue in vivo. As a high molecular weight and negatively charged molecule, DNA has difficulty crossing phospholipid c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/31G01N33/50C12N1/21A61K48/00C07K14/50C07K14/82C12N15/00C12N15/64C12N15/69C12N15/70C12N15/74C12R1/01
CPCC07K14/82C12N15/69C07K14/501A61K48/00C12N15/70C12N15/74A61P21/00A61P25/00A61P25/16A61P25/28A61P31/12A61P31/14A61P31/18A61P35/00A61P7/00A61P7/04A61P9/00A61P9/10C12N1/20C12N15/64
Inventor F·苏布里耶
Owner AVENTIS PHARMA INC
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