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Retrograde transport viral vector system having envelope comprising fused glycoprotein

外国特許コード F120007070
整理番号 A301-01WO
掲載日 2012年12月10日
出願国 欧州特許庁(EPO)
出願番号 10834471
公報番号 2508599
公報番号 2508599
出願日 平成22年11月11日(2010.11.11)
公報発行日 平成24年10月10日(2012.10.10)
公報発行日 平成28年4月20日(2016.4.20)
国際出願番号 JP2010070136
国際公開番号 WO2011068019
国際出願日 平成22年11月11日(2010.11.11)
国際公開日 平成23年6月9日(2011.6.9)
優先権データ
  • 特願2009-274156 (2009.12.2) JP
  • 2010JP070136 (2010.11.11) WO
発明の名称 (英語) Retrograde transport viral vector system having envelope comprising fused glycoprotein
発明の概要(英語) Provided is a lentiviral vector system which sustains a high-frequency retrograde transportation ability in animal brain and has a higher titer.
A kit for preparing a retrograde transport viral vector, which comprises: (1) a packaging plasmid containing the gag gene and pol gene of HIV-1; (2) a packaging plasmid containing an accessory gene of HIV-1; (3) a transfer plasmid containing a target gene; and (4) an envelope plasmid containing, as an envelope gene, a gene encoding a fused polypeptide comprising the extracellular domain of rabies virus glycoprotein (RV-G), the transmembrane domain of rabies virus glycoprotein (RV-G) or vesicular stomatitis virus glycoprotein (VSV-G) and the intracellular domain of vesicular stomatitis virus glycoprotein (VSV-G).
特許請求の範囲(英語) [claim1]
1. A kit for preparing a retrograde transport viral vector comprising: (A) a packaging plasmid containing the gag gene and the pol gene of HIV-1; (B) a packaging plasmid containing an accessory gene of HIV-1; (C) a transfer plasmid containing a target gene; and (D) an envelope plasmid containing, as an envelope gene, a gene encoding a fused polypeptide comprising an extracellular domain of rabies virus glycoprotein (RV-G), a transmembrane domain of rabies virus glycoprotein (RV-G) or vesicular stomatitis virus glycoprotein (VSV-G) and an intracellular domain of vesicular stomatitis virus glycoprotein (VSV-G); preferably wherein the fused polypeptide has the amino acid sequence shown in SEQ ID NO 2 or SEQ ID NO 6, and the packaging plasmid (1) is pCAGkGP1.1 R, and the packaging plasmid (2) is pCAG4-RTR2, and the transfer plasmid is pCL20c-MSCV-X where "X" represents the target gene;
more preferably wherein the envelope gene is expressed under control of a cytomegalovirus enhancer and an avian beta -actin promoter in the envelope plasmid;
even more preferably wherein a base sequence of the gene encoding the fused polypeptide is shown in SEQ ID NO 1 or SEQ ID NO 5.
[claim2]
2. The kit for preparing a viral vector according to Claim 1, wherein the target gene is a human gene, preferably wherein the target gene is a gene for treating a cranial nerve disease.
[claim3]
3. A kit for producing a producer cell, comprising the kit for preparing a viral vector according to Claims 1 or 2 and a host cell, preferably wherein an infected cell is a HEK293 T-cell.
[claim4]
4. A method of producing a producer cell, comprising co-transfecting the infected cell with the packaging plasmid, the transfer plasmid and the envelope plasmid contained in the kit for preparing a viral vector according to Claims 1 or 2;
preferably wherein the infected cell is a HEK293 T-cell;
more preferably wherein the transfection is performed using the calcium phosphate method.
[claim5]
5. The producer cell obtained by the method according to Claim 4.
[claim6]
6. A method of producing a viral vector, comprising: culturing the producer cell according to Claim 5 and harvesting a virus particle from the supernatant of the culture.
[claim7]
7. A viral vector possessing a retrograde transportation ability, produced by the method according to Claim 6.
[claim8]
8. A viral vector as claimed in claim 7 for use in a method of gene transfer, the method comprising: infecting a nerve terminal of an animal with the viral vector; introducing the viral vector into a cell body of the nerve at a target region in a brain by retrograde transportation of the viral vector through an axon of the nerve; and expressing the target gene in the cell body;
preferably wherein the nerve terminal is located in striatum, and the target region in the brain is a region in the brain center which projects to striatum;
more preferably wherein: (a) the region in the brain center which projects to striatum is primary motor cortex, primary somatosensory cortex, parafascicular nucleus of thalamus and/or substantia nigra pars compacta; or (b) the region in the brain center which projects to ventral striatum (nucleus accumbens) is piriform cortex, subiculum, amygdala basolateral nucleus, anterior paraventricular nucleus, mediodorsal nucleus of thalamus and/or lateral hypothalamus.
[claim9]
9. A viral vector as claimed in Claim 8, wherein the animal is a mammal, preferably wherein the mammal is a primate, more preferably wherein the primate is a human.
[claim10]
10. An agent for gene therapy, containing the viral vector according to Claim 7 as an active ingredient.
[claim11]
11. A viral vector as claimed in claim 8 or 9, wherein the target gene introduced by the method of gene transfer is integrated into the chromosome of a cell at the target region to be expressed, and wherein the method is a method of gene therapy for a brain disease, preferably wherein the brain disease is Parkinson's disease.
[claim12]
12. A viral vector as claimed in claim 11, wherein the method of gene therapy comprises administering the agent for gene therapy according to Claim 10 to a patient.
[claim13]
13. An envelope for pseudotyping a lentiviral vector, comprising a fused polypeptide comprising an extracellular domain and a transmembrane domain of rabies virus glycoprotein (RV-G), and an intracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), preferably wherein the lentiviral vector is HIV-1 lentivirus.
[claim14]
14. A gene encoding the envelope comprising the fused polypeptide according to Claim 13.
[claim15]
15. An envelope plasmid containing the gene according to Claim 14.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • KOBAYASHI KAZUTO
国際特許分類(IPC)
指定国 Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
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