Top > Search of International Patents > Retrograde transport viral vector system having envelope comprising fused glycoprotein

Retrograde transport viral vector system having envelope comprising fused glycoprotein

Foreign code F120007070
File No. A301-01WO
Posted date Dec 10, 2012
Country EPO
Application number 10834471
Gazette No. 2508599
Gazette No. 2508599
Date of filing Nov 11, 2010
Gazette Date Oct 10, 2012
Gazette Date Apr 20, 2016
International application number JP2010070136
International publication number WO2011068019
Date of international filing Nov 11, 2010
Date of international publication Jun 9, 2011
Priority data
  • P2009-274156 (Dec 2, 2009) JP
  • 2010JP070136 (Nov 11, 2010) WO
Title Retrograde transport viral vector system having envelope comprising fused glycoprotein
Abstract 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).
Scope of claims [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.
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • KOBAYASHI KAZUTO
IPC(International Patent Classification)
Reference ( R and D project ) (In Japanese)CREST 脳の機能発達と学習メカニズムの解明 領域
Please contact us by E-mail or facsimile if you have any interests on this patent.

PAGE TOP

close
close
close
close
close
close