Alphavirus particles and methods for preparation

• US 7,078,218 B2
• Filed: 12/12/2003
• Issued: 07/18/2006
• Est. Priority Date: 12/13/2002
• Status: Active Grant

• Current Assignee: AlphaVax Incorporated
• Original Assignee: AlphaVax Incorporated
• Inventors: Alterson, Harold, Butler, Kim, Maughan, Maureen F., Smith, Jonathan F., Dryga, Sergey, Rayner, Jon, Kamrud, Kurt
• Primary Examiner(s): Housel, James
• Assistant Examiner(s): SALVOZA, M FRANCO G
• Application Number:
  US10/734,609
• Publication Number:
  US 20040166573A1
• Time in Patent Office: 949 Days
• Field of Search: 424/199.1, 424/218.1, 435/239, 435/461, 435/235.1, 435/320.1, 435/69.3, 435/455, 435/456, 435/457
• US Class Current: 435/235.1
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/36143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2770/36151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2770/36162 by genetic engineering
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

Provided herein are methods for producing alphavirus replicon particles in high yield; replicon RNAs are electroporated into permissive cells, where the cells are at a relatively high density, together with at least one helper nucleic acid providing the necessary functions for packaging. After a growth period in appropriate medium, alphavirus replicon particles are harvested from the surfaces of the cells in which they were produced using a salt wash in which the salt concentration is from about 0.2 to about 5 M sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium acetate, ammonium bicarbonate, among others. After dilution, if necessary, the particles can be purified by a suitable chromatographic technique.

Claims

1. A method for preparing alphaviral replicon particles (ARPS), said method comprising the steps of:

(a) introducing an alphavirus replicon nucleic acid into a host cell, said replicon nucleic acid comprising at least a virus packaging signal and at least one heterologous coding sequence expressible in said alphaviral replicon nucleic acid, wherein said host cell comprises at least one helper function, to produce a modified host cell;

(b) culturing said modified host cell under conditions allowing expression of the at least one helper function, allowing replication of said alphaviral replicon nucleic acid and packaging of said alphaviral replicon nucleic acid to form ARPS, wherein said ARPs are heparin-binding ARPS;

(c) contacting the modified host cells after step (b) with an aqueous solution having an ionic strength of from 0.2 M to 5 M to release the ARPS into the aqueous solution to produce a ARP-containing solution and;

(d) collecting ARPS from the ARP-containing solution of step (c).

2. The method of claim 1, wherein the at least one helper function in the host cell of step (a) is encoded by a nucleic acid sequence stably integrated within the genome of said host cell.

3. The method of claim 1, wherein the at least one helper function in the host cell is introduced on at least one helper nucleic acid which encodes a capsid protein capable of binding said alphaviral replicon nucleic acid, and at least one alphaviral glycoprotein, wherein said alphaviral glycoprotein associates with said alphaviral replicon nucleic acid and said capsid protein, wherein the at least one helper nucleic acid molecule is introduced into the host cell together with said alphaviral replicon nucleic acid.

4. The method of claim 1, wherein the at least one helper function is encoded by at least two helper nucleic acid molecules wherein each of said two helper nucleic acid molecules encodes at least one viral helper function.

5. The method of claim 1, wherein the ionic strength is between 0.5 M and 5 M.

6. The method of claim 1, wherein the at least one helper function is encoded within a DNA molecule.

7. The method of claim 1, wherein the alphavirus replicon nucleic acid is introduced into said host cell by electroporation.

8. The method of claim 7, wherein host cells are present in an electroporation mixture at a concentration from 5×

10⁷ to 5×

10⁸ per mL.

9. The method of claim 3, wherein host cells are present in an electroporation mixture at a concentration from 5×

10⁷ to 1.5×

10⁸ per mL.

10. The method of claim 1, further comprising a cell washing step, prior to step (c) of claim 1.

11. The method of claim 1, wherein the alphavirus is Venezuelan Equine Encephalitis Virus.

12. The method of claim 10, wherein the cell washing solution contains no salt and further comprises deoxyribonuclease.

13. The method of claim 3 or 4, wherein the helper nucleic acid is an uncapped RNA molecule.

14. The method of claim 4, wherein an alphavirus replicon RNA and a first helper RNA molecule and a second RNA helper molecule are present in an electroporation mixture at a ratio of 1:

0.3;

0.3 to 1;

20;

20.

15. The method of claim 14, wherein the ratio is 1:

0.5;

0.5.

16. The method of claim 14, wherein the ratio is 1:

5;

5.

21. The method of claim 1, wherein the alphavirus-permissible cells are Vero cells.

22. The method of claim 1, wherein step (d) is followed by an ion exchange, heparin affinity chromatography, affinity or hydrophobic chromatography step.

23. The method of claim 1, wherein the alphavirus is an attenuated alphavirus.

25. The method claim 1, wherein the salt in the salt wash step is selected from the group consisting of NaCl, KCl, MgCl₂, CaCl₂, NH₄CI, (NH₄)₂SO4, NH₄ acetate and NH₄ bicarbonate.

17. A method of preparing alphavirus replicon particles comprising introducing an alphavirus replicon vector and one or more helper nucleic acid molecules into alphavirus-permissive cells via electroporation, wherein concentration of the alphavirus permissive cells in culture medium during electroporation is from 5×

10⁷ to 5×

10⁸ cells/mL and wherein the concentration of the alphavirus RNA replicon vector added to the cells prior to electroporation is approximately 35 μ

g per mL.

18. The method of claim 17, wherein the electroporation is carried out in an electroporation chamber wherein a gap between electrodes is between 0.4 and 1.0 cm.

19. The method of claim 17, wherein the helper function is encoded within a single DNA helper molecule encoding all alphavirus structural proteins.

20. The method of claim 19, wherein the concentration of the DNA helper molecule is at least 100 μ

g/mL.

24. The method of claim 17, wherein the alphavirus is Venezuelan equine encephalitis virus (VEE).

26. A method for preparing alphaviral replicon particles (ARPS), said method comprising the steps of:

(a) introducing an alphavirus replicon nucleic acid into a host cell, said replicon nucleic acid comprising at least a virus packaging signal and at least one heterologous coding sequence expressible in said alphaviral replicon nucleic acid, wherein said host cell comprises at least one helper function, to produce a modified host cell, wherein said replicon nucleic acid is introduced into the host cell by electroporation of host cells at a concentration of from 5×

10 to 5×

10 cells per milliliter;

(b) culturing said modified host cell under conditions allowing expression of the at least one helper function, allowing replication of said alphaviral replicon nucleic acid and packaging of said alphaviral replicon nucleic acid to form ARPS;

(c) contacting the modified host cells after step (b) with an aqueous solution having an ionic strength of from 0.2 M to 5 M to release the ARPS into the aqueous solution to produce a ARP containing solution and;

(d) collecting ARPs from the ARP-containing solution of step (c).

27. The method of claim 26, wherein the alphavirus is Venezuelan equine encephalitis virus (VEE).

28. The method of claim 26, wherein the helper nucleic acid is an uncapped RNA molecule.