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Gene transfer-mediated angiogenesis therapy

A vascular, transgenic technology, applied in the field of gene therapy

Inactive Publication Date: 2004-11-03
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If this is not possible, the transgene will be expressed systemically and problems will ensue

Method used

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  • Gene transfer-mediated angiogenesis therapy
  • Gene transfer-mediated angiogenesis therapy
  • Gene transfer-mediated angiogenesis therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Adenovirus Constructs

[0122] A helper-independent replication-deficient human adenovirus 5 system was used with the target genes LacZ and FGF-5. The full-length cDNA of human FGF-5 is a 1.1 kb ECOR1 fragment from plasmid pLTR122E (Zhen, et al, Mol. Cell. Biol., 8:3487, 1988), which includes a 981 bp gene open reading frame, which was cloned into Multiple cloning site for plasmid ACCMVPLPA containing the CMV promoter and SV40 polyadenylation signal and part of the flanking adenovirus sequence from which the E1A and E1B genes (essential for viral replication) have been deleted. This plasmid was co-transfected (lipofection) into 293 cells with the JM17 plasmid, which contains the entire genome of human adenovirus 5 and an additional 4.3 kb insert, which makes pJM17 too large to be packaged. Homologous salvage recombination forms an adenoviral vector containing the transgene lacking the E1A / E1B sequence. Although these recombinants were inca...

Embodiment 2

[0123] Example 2: Adult murine cardiomyocytes in cell culture

[0124] Adult rat cardiomyocytes can be prepared by Langendorf perfusion according to standard methods using perfusate containing collagenase. The rod-shaped cells were cultured on a laminin-coated plate for 24 hours, and infected with the adenovirus encoding β-galactosidase obtained in Example 1 above at a multiplictity of 1:1. Glutaraldehyde fixation and incubation with X-gal. In good agreement with the above results, 70-90% of adult rat cardiomyocytes were infected with recombinant adenovirus and expressed β-galactosidase, and no cytotoxicity was observed when the multiplicity of infection was 1-2:1.

Embodiment 3

[0125] Example 3: In vivo porcine myocardium

[0126] According to the method of Example 1, the adenovirus vector encoding β-galactosidase obtained in Example 1 was propagated in compatible 293 cells, and purified by CsCl gradient ultracentrifugation until the final virus titer was 1.5×10 10 virus particles. Thoracotomy was performed on an anesthetized, ventilated 40 kg pig, a 26-gauge butterfly needle was inserted into the middle of the left anterior descending coronary artery (LAD), and a 2 ml volume of vehicle (1.5 ×10 10 virions), the chest was sutured to allow the animal to recover. Animals were sacrificed on the fourth day after vehicle injection, hearts were fixed with glutaraldehyde, incubated with X-gal for 16.5 hours after sectioning, and tissues were counterstained with eosin after embedding and sectioning.

[0127] Microscopic observation of tissue sections (full-thickness tissue sections of the LAD bed 96 hours after intracoronary injection of ...

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PUM

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Abstract

PCT No. PCT / US96 / 02631 Sec. 371 Date Dec. 29, 1997 Sec. 102(e) Date Dec. 29, 1997 PCT Filed Feb. 27, 1996 PCT Pub. No. WO96 / 26742 PCT Pub. Date Sep. 6, 1996The transgene-inserted replication-deficit adenovirus vector is effectively used in in vivo gene therapy for peripheral vascular disease and heart disease, including myocardial ischemia, by a single intra-femoral artery or intracoronary injection directly conducted deeply in the lumen of the one or both femoral or coronary arteries (or graft vessels) in an amount sufficient for transfecting cells in a desired region.

Description

[0001] This application is a divisional application of an invention application with an application date of February 27, 1996, an application number of 96190363.5, and an invention title of "gene transfer-mediated angiogenesis therapy". [0002] Referenced related applications [0003] This application is a continuation-in-part of US Application Serial No. 08 / 485,472, filed June 7, 1995, which is a continuation-in-part of US Application Serial No. 08 / 396,207, filed February 28, 1995. [0004] Statement on Officially Funded Research [0005] This invention was made with government support under grant numbers HL 0281201 and HL 1768218, with grants from the National Institutes of Health. The government has certain rights in this invention. technical field [0006] The present invention relates to gene therapy, and more particularly to virus-mediated gene therapy and other forms of gene therapy and certain adenoviral cons...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/08A61K35/76A61K38/00A61K38/18A61K38/19A61K38/27C12N15/09A61K48/00A61P9/00A61P9/08A61P9/10C07K14/50C12N7/00C12N15/861
CPCC12N15/86C07K14/50A61K38/1866C12N2710/10343A61K48/00C12N2830/008A61K38/00A61P9/00A61P9/08A61P9/10
Inventor H·K·哈蒙德弗兰克·J·乔达诺沃尔夫冈·H·迪尔曼
Owner RGT UNIV OF CALIFORNIA
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