Producer cells for replication competent retroviral vectors
The disclosure provide cell lines and methods for the production of vectors and viral particles useful in gene therapy.
1. A replication competent retrovirus particle produced by a retrovirus producing HT1080 cell line, wherein said HT1080 cell line stably express a recombinant retroviral genome comprising a gag gene, pol gene, env gene, a heterologous polynucleotide, and retroviral psi (Ψ) factor for the assembly of the recombinant retroviral genome, wherein the replication competent retrovirus particle comprises the recombinant retroviral genome, and wherein the HT1080 cell line has been adapted to be grown and continuously passaged in serum free media and in suspension.
2. The replication competent retrovirus particle of claim 1 ,
wherein the replication competent retrovirus comprises:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain;
a cassette comprising an internal ribosome entry site (IRES) or a nucleic acid regulatory domain operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.
3. The replication competent retrovirus particle of claim 2 , wherein the retroviral polynucleotide sequence is from murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Gibbon ape leukemia virus (GALV), murine mammary tumor virus (MuMTV), Rous Sarcoma Virus (RSV), baboon endogenous virus (BEV), or feline virus RD114.
4. The replication competent retrovirus particle of claim 3 , wherein the MLV is an amphotropic MLV.
5. The replication competent retrovirus particle of claim 1 , wherein the retrovirus is a gammaretrovirus.
6. The replication competent retrovirus particle of claim 2 , wherein the nucleic acid regulatory domain is a pol III regulatory domain.
7. The replication competent retrovirus particle of claim 1 , wherein the heterologous polynucleotide encodes a protein binding domain.
8. The replication competent retrovirus particle of claim 7 , wherein the protein binding domain is an antibody, antibody fragment, antibody domain or receptor ligand.
9. The replication competent retrovirus particle of claim 1 , wherein the heterologous polynucleotide encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug.
10. The replication competent retrovirus particle of claim 9 , wherein the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase.
11. The replication competent retrovirus particle of claim 6 , wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.
12. The replication competent retrovirus particle of claim 11 , wherein the inhibitory polynucleotide comprises an RNAi or siRNA sequence and wherein the regulatory nucleic acid domain is a promoter.