Baculoviruses with enhanced virion production and a method for the production of baculoviruses
View Patent ↗A method of co-expressing a portion of the VSV G protein gene or a truncated “stem” portion with GP64 and a retrovirus increases the titer of retroviral vectors. A truncated VSV G protein, preferably comprised of a small segment from the C-terminal portion of the ectodomain plus the transmembrane (TM) and cytoplasmic tail (CTD) domains of VSV G, co-expressed with retroviral vectors, enhances the production titers of the retroviral vectors. A preferred embodiment uses a VSV G construct that includes an N-terminal c-Myc epitope plus 42 amino acids from the C-terminal portion of the ectodomain, 20 amino acids from the predicted TM domain, and 29 amino acids from the predicted CTD of the VSV G protein.
1. A method of producing a plurality of viral particles of a retrovirus, comprising the step of delivering a retroviral packaging plasmid, a transgene plasmid, an envelope protein stem construct and an envelope plasmid into a system capable of producing viral particles, wherein the envelope protein stem construct comprises a Rhabdovirus glycoprotein stem construct and wherein the method results in an increased titer compared to delivering the retroviral packaging plasmid, the transgene plasmid, and the envelope plasmid without the envelope protein stem construct.
2. The method of claim 1 , wherein the Rhabdovirus glycoprotein stem construct is a Vesicular Stomatitis Virus G-stem construct.
3. The method of claim 2 , wherein the Vesicular Stomatitis Virus G-stem construct comprises amino acid sequences from each of: a C-terminal portion of an ectodomain; a predicted transmembrane domain; and a predicted cytoplasmic domain.
4. The method of claim 3 , wherein the G stem construct comprises 42 amino acids from the C-terminal portion of the ectodomain; 20 amino acids from the predicted transmembrane domain; and 29 amino acids from the predicted cytoplasmic tail domain.
5. The method of claim 1 , wherein the envelope plasmid is selected from the group consisting of GP64; an F protein; an envelope protein from lymphocytic choriomeningitis virus (LCMV); an envelope protein from an influenza virus; an envelope protein from a paramyxovirus; an envelope protein from a rhabdovirus; an envelope protein from a filovirus; and an envelope protein from Ross River virus (RRV).
6. The method of claim 1 , wherein the retrovirus is selected from the group consisting of feline immunodeficiency virus; murine leukemia virus (MLV); human immunodeficiency virus type 1 (HIV-1); simian immunodeficiency virus (SIV); and equine infectious anemia virus (EIAV).
7. The method of claim 1 , wherein a method of delivery is selected from the group consisting of:
a) co-transfecting the retroviral packaging plasmid, the transgene plasmid, the envelope protein stem construct and the envelope plasmid;
b) generating stable cell lines to express DNA, RNA, or protein from at least one of the retroviral packaging plasmid, the transgene plasmid, the envelope protein stem construct and the envelope plasmid; and
c) a combination of a) and b).
8. A method of producing a plurality of viral particles of a retrovirus, comprising the step of delivering a retroviral packaging plasmid, a transgene plasmid, a G-stem construct and an GP64 envelope plasmid into a system capable of producing viral particles, wherein the G-stem construct comprises a Rhabdovirus glycoprotein stem construct and wherein the method results in an increased titer compared to delivering the retroviral packaging plasmid, the transgene plasmid and the GP64 envelope plasmid without the G-stem construct.
9. The method of claim 8 , wherein the retrovirus is feline immunodeficiency virus.
10. The method of claim 8 , wherein a method of delivery is selected from the group consisting of:
a) co-transfecting the retroviral packaging plasmid, the transgene plasmid, G-stem construct and the GP64 envelope plasmid;
b) generating stable cell lines to express DNA, RNA, or protein from at least one of the retroviral packaging plasmid, the transgene plasmid, the G-stem construct and the GP64 envelope plasmid; and
c) a combination of a) and b).
11. A method of producing a plurality of viral particles of a retrovirus, comprising the step of delivering a plurality of components comprising a retroviral packaging plasmid, a transgene plasmid, an envelope protein stem construct and an envelope plasmid by co-transfecting plasmids, generating stable cell lines, or using other methods to express or otherwise deliver the appropriate proteins, DNA or RNA to produce the viral particles, wherein the envelope protein stem construct comprises a Rhabdovirus glycoprotein stem construct and wherein the method results in an increased titer compared to delivering the retroviral packaging plasmid, the transgene plasmid, and the envelope plasmid without the envelope protein stem construct.
12. The method of claim 11 , wherein the envelope protein stem construct comprises a viral envelope protein stem construct that includes a transmembrane domain, a cytoplasmic domain and a truncated or abbreviated portion of a protein ectodomain.
13. The method of claim 11 , wherein the Rhabdovirus glycoprotein stem construct is a Vesicular Stomatitis Virus G-stem construct.
14. The method of claim 13 , wherein the Vesicular Stomatitis Virus G-stem construct comprises amino acid sequences from each of: a C-terminal portion of an ectodomain; a predicted transmembrane domain; and a predicted cytoplasmic domain.
15. The method of claim 14 , wherein the G stem construct comprises 42 amino acids from the C-terminal portion of the ectodomain; 20 amino acids from the predicted transmembrane domain; and 29 amino acids from the predicted cytoplasmic tail domain.
16. The method of claim 11 , wherein the envelope plasmid is selected from the group consisting of GP64 ; an F protein; an envelope protein from lymphocytic choriomeningitis virus (LCMV); an envelope protein from an influenza virus; an envelope protein from a paramyxovirus; an envelope protein from a rhabdovirus; an envelope protein from a filovirus; and an envelope protein from Ross River virus (RRV).
17. The method of claim 11 , wherein the retrovirus is selected from the group consisting of feline immunodeficiency virus; murine leukemia virus (MLV); human immunodeficiency virus type 1 (HIV-1); simian immunodeficiency virus (SIV); and equine infectious anemia virus (EIAV).
18. The method of claim 11 , wherein the envelope plasmid is an envelope protein from a family of viruses selected from the group consisting of retroviruses, orthomyxoviruses, rhabdoviruses, bunyaviruses, paramyxoviruses, filoviruses, coronaviruses, togaviruses, arenaviruses, bornaviruses, flaviviruses, arteriviruses, poxviruses, baculoviruses, polydnaviruses, ascoviruses, and nimaviruses.
19. The method of claim 11 , wherein a method of delivery is selected from the group consisting of:
a) co-transfecting the retroviral packaging plasmid, the transgene plasmid, the envelope protein stem construct and the envelope plasmid;
b) generating stable cell lines to express DNA, RNA, or protein from at least one of the retroviral packaging plasmid, the transgene plasmid, the envelope protein stem construct and the envelope plasmid; and
c) a combination of a) and b).
20. The method of claim , wherein the Vesicular Stomatitis Virus G-stem construct comprises amino acids 421 to 511 of a Vesicular Stomatitis Virus G protein.
21. The method of claim 8 , wherein the Rhabdovirus glycoprotein stem construct is a Vesicular Stomatitis Virus G-stem construct.
22. The method of claim 21 , wherein the Vesicular Stomatitis Virus G-stem construct comprises amino acids 421 to 511 of a Vesicular Stomatitis Virus G protein.
23. The method of claim 13 , wherein the Vesicular Stomatitis Virus G-stem construct comprises amino acids 421 to 511 of a Vesicular Stomatitis Virus G protein.