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Recombinant adeno-associated virus comprising VEGFR Truncated Soluble cDNA and gene therapeutic agent comprising the same

A technology of VEGFR-1 and VEGFR-2, which is applied in the field of recombinant adeno-associated virus containing truncated soluble cDNA of VEGFR and gene therapy agents containing the above-mentioned components, which can solve the problems of no major breakthroughs, etc.

Inactive Publication Date: 2009-05-13
SCT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, although research on polynucleotides is also continuing, no major breakthroughs have been made in research on methods for efficient transfer into the body by viral vectors

Method used

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  • Recombinant adeno-associated virus comprising VEGFR Truncated Soluble cDNA and gene therapeutic agent comprising the same
  • Recombinant adeno-associated virus comprising VEGFR Truncated Soluble cDNA and gene therapeutic agent comprising the same
  • Recombinant adeno-associated virus comprising VEGFR Truncated Soluble cDNA and gene therapeutic agent comprising the same

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Cloning of pAAV plasmids containing genes that inhibit neovascularization

[0046] According to the cloning method, in order to insert VEGF-A (NCBI accession # NM_003376 for human) cDNA into pAAV-FIX cis plasma DNA in the antisense direction using pAAV-FIX cis plasma DNA (US6,093,292), use the antisense of VEGF-A isoform The cDNA replaces the factor IX cDNA gene contained in the pAAV-FIX cis plasmad DNA to create trans-gene constructs of pAAV-AShVEGF-A. In addition, trans-gene constructs of pAAV-AShVEGF-A-IRES-EGFP in which VEGF-Aisoform antisense cDNA was linked to IRES-EGFP were prepared.

[0047] The truncated soluble cDNAs of VEGFR-1 (Flt-1; NCBIaccession #NM_002019 for human) and VEGFR-2 (Kdr / Flk-1; NCBIaccession # AF063658 for human), which are receptors that interact with the three isoforms of VEGF, were also used in the same The method of inserting or replacing pAAV-TShVEGFR-1 and pAAV-TShVEGFR-2 trans-gene constructs. At the same time, two trans-ge...

Embodiment 2

[0086] Embodiment 2: Making the rAAV vector of gene therapy agent for inhibiting neovascularization

[0087] In order to make a recombinant AAV (rAAV) vector for gene therapy, in addition to each pAAV plasmid DNA made in Example 1, the AAV rep-cap plasmid DNA (pAAV-RC plasmid) expressing the AAV rep part and cap part is also needed. , Stratagene Co., USA) and adenovirus helper plasmid (pHelper plasmid, Stratagene Co., USA). After all the above three plasmid DNAs were transfected into HEK293 (human embryonic kidney 293; ATCCCRL-1573) cells and cultured for 96 hours, the HEK293 cells were collected and ultrasonically disrupted, and the recombinant AAV (rAAV) particles were subjected to CsCl density gradient centrifugation for three times. , and then collect the part with RI (Refractive Index) of 1.37~1.41g / ml for purification and separation.

[0088] The titration method of the above-mentioned isolated rAAV particles is as follows: PCR primer [CMV F1 (SEQ ID NO: 14): 5′-GGGCGT ...

Embodiment 3

[0115] Embodiment 3: The treatment of cancer cell line culture and rAAV vector gene therapy agent

[0116] In order to study the anticancer effect of preventing cancer metastasis in vitro, human colorectal cancer cell line (T84: ATCC CCL-248) and human lung cancer cell line (LCSC#1: WO 02 / 061069) and human gastric cancer cell lines (NUGC3, MKN45, MKN74) and other five cancer cell lines. In order to test the anti-cancer therapeutic effect of recombinant rAAV on the above cancer cell lines, 10 branches of the above cancer cell lines were placed on 6 well plates. 5 After cells / well cultured for 24 hours, add a total M.O.I. of 10 5 Various combinations of neovascularization-inhibiting rAAV vectors were cultured for 24 hr. In this example, the negative control (negative control) selects the same cell population that has not been treated with rAAV and the rAAV vector (rAAV-EGFP) that does not insert VEGF antisense and only expresses GFP, and is the same as the recombinant rAAV in ...

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Abstract

The present invention relates to a gene therapeutic agent using truncated soluble cDNA of VEGF receptor protein (VEGFR) and adeno-associated virus(AVV) system, and more particularly, relates to a gene therapeutic agent specific to large intestine cancer, bladder cancer and / or lung cancer, comprising a rAAV vector containing truncated soluble cDNA of VEGFR, and a rAAV vector containing the said vector and VEGF-A anti-sense cDNA. The gene therapeutic agent according to the present invention reduces the growth of tumors by inhibiting the expression and function of VEGF involved in angiogenesis necessary for the proliferation and metastasis of tumors. Thus, the inventive gene therapeutic agent can be effectively used to treat cancer at a gene level.

Description

technical field [0001] The present invention relates to a gene therapy agent utilizing truncated solublecDNA of VEGF receptor protein (VEGFR) and utilizing adeno-associated virus (AAV) system, especially a kind of rAAV vector comprising truncated solublec DNA comprising VEGFR and containing said vector and VEGF -A colorectal cancer, bladder cancer and / or lung cancer specific gene therapy agent of rAAV vector of antisense (antisense) cDNA. Background technique [0002] No matter at home or abroad, cancer mortality is increasing year by year. Worldwide, cancer, infectious diseases and cardiovascular diseases are the leading causes of human death. According to the World Health Report of the World Health Organization (WHO), in 2004, 7,121,00 people, accounting for 12.5% ​​of all deaths, died from cancer. And because of eating habits, environmental changes and life extension, it is estimated that in the next 25 years, the number of cancer-causing people will increase at a rate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/12C12N7/01A61K48/00A61P35/00
CPCC07K14/71C12N2750/14143A61P35/00C12N15/11
Inventor 朴基娘
Owner SCT
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