Retrograde transport viral vector system having envelope comprising fused glycoprotein

Patent metadata

Field	Value
Publication number	US-9352052-B2
Application number	US-201013511873-A
Country	US
Kind code	B2
Filing date	Nov 11, 2010
Priority date	Dec 2, 2009
Publication date	May 31, 2016
Grant date	May 31, 2016

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).

First claim

What is claimed is:

1. A kit for preparing a retrograde transport viral vector comprising:
(A) a first packaging plasmid containing the gag gene and the pol gene of HIV-1;
(B) a second packaging plasmid containing an accessory gene of HIV-1;
(C) a transfer plasmid containing a target gene; and
(D) an envelope plasmid containing an envelope gene encoding a chimeric envelope protein, wherein the chimeric envelope protein comprises the amino acid sequence of SEQ ID NO: 2.

2. The kit for preparing a viral vector according to claim 1 , wherein the envelope gene is expressed under control of a cytomegalovirus enhancer and an avian β-actin promoter in the envelope plasmid.

3. The kit for preparing a viral vector according to claim 2 , wherein the envelope gene comprises SEQ ID NO 1.

4. A kit for producing a producer cell, comprising the kit for preparing a viral vector according to claim 1 and a host cell.

5. The kit according to claim 4 , wherein the host cell is a HEK293 T-cell.

6. A method of producing a producer cell, comprising:
co-transfecting a host cell with the first and the second packaging plasmids, the transfer plasmid, and the envelope plasmid contained in the kit of claim 1 .

7. The method according to claim 6 , wherein the host cell is a HEK293 T-cell.

8. The method according to claim 6 , wherein the transfection is performed using a calcium phosphate method.

9. A producer cell obtained by the method according to claim 6 .

10. A method of producing a viral vector, comprising:
culturing the producer cell according to claim 9 to form a cell culture with supernantant; and
harvesting a virus particle from the supernatant of the culture.

11. A viral vector possessing a retrograde transportation ability, produced by the method according to claim 10 .

12. A chimeric envelope comprising a fused polypeptide, wherein the fused polypeptide comprises the amino acid sequence of SEQ ID NO: 2, and wherein the chimeric envelope is capable of pseudotyping an HIV-1 lentivirus vector system to produce pseudotyped particles comprising said chimeric envelope such that the pseudotyped virus particles can facilitate retrograde gene transfer into neurons.

13. An isolated nucleic acid encoding the chimeric envelope according to claim 12 .

14. An envelope plasmid comprising the nucleic acid according to claim 13 .

15. A pseudotyped retrovirus particle comprising the chimeric envelope according to claim 12 .

Assignees

• Kobayashi Kazuto
• Japan Science & Tech Agency

Inventors

• Kobayashi Kazuto

Classifications

• A61P25/16

  Anti-Parkinson drugs · CPC title

• A61K48/0075 Primary

  characterised by an aspect of the delivery route, e.g. oral, subcutaneous · CPC title

• C12N2810/6081

  rhabdoviridae, e.g. VSV · CPC title

• C12N2760/20122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2740/16052

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title