Virus-like particles useful as a vector for delivering nucleic acid

• US 6,420,160 B1
• Filed: 04/29/1999
• Issued: 07/16/2002
• Est. Priority Date: 06/04/1996
• Status: Expired due to Fees

• Current Assignee: Pasteur Merieux Serums et Vaccins SA (Sanofi )
• Original Assignee: Pasteur Merieux Serums et Vaccins SA (Sanofi )
• Inventors: Bloch, Marie-Aline
• Primary Examiner(s): Clark, Deborah J. R.
• Assistant Examiner(s): CHEN, SHIN LIN
• Application Number:
  US09/194,927
• Time in Patent Office: 1,174 Days
• Field of Search: 435/320.1, 435/235, 435/456, 435/455, 435/239, 424/93.2, 424/93.21, 424/93.6, 424/192.1, 424/204.1
• US Class Current: 435/239
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/20023 Virus like particles [VLP]

Abstract

The present invention relates to the use of virus-like particles (VLP'"'"'s) of papillomavirus for preparing vector pseudoviruses useful for transferring genetic material into target cells of an organism

Claims

1. A noninfectious virus-like particle (VLP) which comprises:

(i) a capsid defining an internal space and consisting of at least a portion of the L₁ protein of a papillomavirus that has the ability to self-assemble into a VLP, and

(ii) a nucleic acid molecule contained in the said internal space;

the nucleic acid molecule being different from the genome of a papillomavirus at least in that it lacks all or part of the regions of the said genome coding for wild-type late proteins.

2. The virus-like particle according to claim 1, in which the capsid consists of said at least a portion of the L₁ protein in the L₁-E₇ chimeric protein state.

3. The virus-like particle according to claim 1, in which the capsid consists of said at least a portion of the L₁ protein of a human papillomavirus (HPV).

4. The virus-like particle according to claim 3, in which the capsid consists of said at least a portion of the L₁ protein of a type 1, 6, 10, 11, 16, 18, 31, 33, 35 or 45 human papillomavirus.

5. The virus-like particle according to claim 4, in which the capsid consists of said at least a portion of the L₁ protein of an HPV-16, -18, -31, -33, -35 or -45 initially isolated from a benign lesion.

6. The virus-like particle according to claim 5, in which the capsid consists of said at least a portion of the L₁ protein of an HPV-16 having an amino acid sequence which comprises at position 202 an amino acid other than histidine.

7. The virus-like particle according to claim 6, in which the capsid consists of said at least a portion of the L₁ protein of an HPV-16 having an amino acid sequence which comprises at position 202 an aspartic acid or glutamic acid residue.

8. The virus-like particle according to claim 5, wherein said benign lesion is condyloma acuminatum or cervical dysplasia.

9. The virus-like particle according to claim 1, in which the capsid, in addition, consists of at least a portion of the L₂ protein of a papillomavirus that has the ability to self assemble into a VLP.

10. The virus-like particle according to claim 9, in which the capsid in addition, consists of said at least a portion of the L₂ protein in the L₂-E7 chimera state.

11. The virus-like particle according to claim 1, in which the nucleic acid molecule comprises a region coding for a protein of interest.

12. The virus-like particle according to claim 11, in which the nucleic acid molecule is DNA and comprises a region coding for a protein of interest placed under the control of a promoter which promotes transcription in mammalian cells.

13. The virus-like particle according to claim 12, in which the DNA molecule comprises, in addition, a papillomavirus E₂ protein binding site of formula ACCN₆MT in which N is A, G, C or T and M is G or T.

14. The virus-like particle according to claim 12, in which the DNA molecule comprises, in addition, all or part of the long control region (LCR) of the genome of a papillomavirus.

15. The virus-like particle according to claim 11, in which the nucleic acid molecule is at most 8 kbp.

16. The virus-like particle according to claim 11, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from cytokines and secondary molecules facilitating cellular recognition by the helper T cells.

17. The virus-like particle according to claim 11, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from tumour-associated antigens.

18. The virus-like particle according to claim 11, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from parasitic, bacterial or viral antigens.

19. The virus-like particle according to claim 18, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from the E₁ and E₂ proteins and the nononcogenic forms of the E₆ and E₇ proteins of a papillomavirus.

20. The virus-like particle according to claim 19, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from the E₁ and E₂ proteins and the nononcogenic forms of the E₆ and E₇ proteins of a papillomavirus of the HPV-16, -18, -31, -33, -35 or -45 type.

21. The virus-like particle according to claim 19 or 20, in which the nucleic acid molecule comprises at least one region coding for a protein of interest selected from the E₁ and E₂ proteins and the nononcogenic forms of the E₆ and E₇ proteins of a papillomavirus of a type different from the one from which the capsid protein or protines are derived.

22. The virus-like particle according claim 10, in which the nucleic acid molecule comprises at least one region coding for a protein of interest in the therapy of genetic diseases.

23. A virus-like particle as a medicament according to claim 1.

24. A method of preparing virus-like particles according to claim 1, wherein a nucleic acid molecule different from the genome of a papillomavirus at least in that it lacks all or part of the regions of the said genome coding for the wild-type late proteins is mixed with said at least a portion of the L₁ protein of a papillomavirus in dissociated form, in the presence of a calcium salt and the said virus-like particles are recovered from the mixture.

25. The method of preparation according to claim 24, wherein all or part of the E₂ protein of a papillomavirus is mixed with the nucleic acid molecule and with the L₁ protein.

26. The method of preparation according to claim 25, wherein the papillomavirus E₂ protein deleted for its N-terminal part is added at the time of mixing.

27. The method of preparation according to claim 24, wherein prior to the mixing step,

(i) said at least a portion of the L₁ protein is expressed by the recombinant routes

(ii) the empty virus-like particles are recovered and

(iii) the empty virus-like particles are treated with a reducing agent and/or with a calcium-ion chelating agent in order to obtain said L₁ protein in dissociated form.

28. The method of preparation according to claim 27, in which said at least a portion of the L₁ protein is expressed by the recombinant route in insect cells infected with a baculovirus into whose genome a DNA fragment is inserted which codes for said L₁ protein placed under the control of an appropriate promoter.

29. The method of preparation according to claim 28, wherein said preparation further comprises at least a portion of the L₂ protein that has the ability to self assemble into a VLP to be expressed by the recombinant route in insect cells infected with a baculovirus into whose genome a DNA fragment is inserted which codes for said L₂ protein placed under the control of an appropriate promoter.

30. The method of preparation according to claim 27, wherein step (i) further comprises at least a portion of the L₂ protein that has the ability to self assemble into a VLP to be expressed by the recombinant route and wherein step (iii) further comprises said L₂ protein in dissociated form.