Method for producing enveloped viruses
The invention relates to a process for producing enveloped viruses in a mildly acid medium. The processes of the invention are useful for producing and recovering at a large scale enveloped viruses under conditions observing good manufacturing practice (GMP).
1. A process for producing an enveloped virus comprising cultivating host cells producing said enveloped virus in a mildly acidic culture medium, wherein the enveloped virus is a lentivirus, optionally pseudotyped.
2. The process according to claim 1 , the mildly acidic medium having a pH between 5.8 and 6.2.
3. The process according to claim 1 , the lentivirus being pseudotyped with an envelope protein selected from the VSV-G envelope protein or the GALVTR envelope protein.
4. The process according to claim 1 , the host cell being a HEK293, HEK293T, HEK293FT, Te671, CEM, NIH-3T3, Mpf, or D17 cell.
5. The process according to claim 1 , said process comprising the following steps:
transient transfection of HEK293T cells by means of one or several plasmids coding for the elements required for producing said enveloped vector;
cultivation of said cells in a suitable medium, of which the pH is about 6; and
harvesting the enveloped virus in the culture supernatant.
6. The process according to claim 5 , the cells being transfected by means of four plasmids: one plasmid bearing an expression cassette comprising a lentiviral gagpol gene, one plasmid bearing an expression cassette comprising a lentiviral rev gene, one transfer plasmid comprising an expression cassette of a transgene of interest, comprised between a lentiviral LTR-5′ and a LTR-3′, and one plasmid bearing an expression cassette of envelope glycoprotein(s).
7. The process according to claim 1 , wherein said cells are cultivated in a mildly acid medium 5 to 24 hours after transfection.