Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
The present application discloses a lentiviral transfer system which includes: (i) a self-inactivating transfer vector comprising: multiple gene units, wherein each gene unit includes a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and (ii) a helper construct which lacks a 5′ LTR, wherein the 5′ LTR has been replaced with a heterologous promoter, in which the helper construct further comprises: a lentiviral env nucleic acid sequence containing a deletion, wherein the deleted env nucleic acid sequence does not produce functional env protein; and a packaging signal contains a deletion, wherein the deleted packaging signal is nonfunctional.
1. A lentiviral transfer system comprising:
a self-inactivating transfer vector comprising:
(a) multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and
(b) a mammalian insulator sequence and splice acceptor and donor sites,
wherein the self-inactivating transfer vector is free of wPRE (wood-chuck hepatitis virus post-transcriptional element).
2. The lentiviral transfer system of claim 1 , further comprising an envelope construct for providing a functional env protein.
3. The lentiviral transfer system of claim 1 , wherein the multiple gene units comprise a first gene unit operably linked to a first regulatory nucleic acid sequence and a second gene unit operably linked to a second regulatory nucleic acid sequence.
4. The lentiviral transfer system of claim 3 , wherein at least one of the first gene unit or the second gene unit encodes a trafficking signal.
5. The lentiviral transfer system of claim 1 , wherein the regulatory nucleic acid sequence comprises a cell-specific or tissue-specific promoter.
6. The lentiviral transfer system of claim 5 , wherein the cell or tissue-specific promoter is selected from: TSTA promoter, mesothelin promoter, hPSA promoter, hCCKAR promoter, hAFP promoter, and hNSE promoter.
7. The lentiviral transfer system of claim 6 , wherein the heterologous nucleic acid sequence encodes at least one RNAi agent or at least one polypeptide that inhibits expression of Bcl-2.
8. A pharmaceutical composition comprising a lentiviral particle for gene transfer, said lentiviral particle produced using a lentiviral transfer system comprising:
a self-inactivating transfer vector comprising:
(a) multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and
(b) a mammalian insulator sequence and splice acceptor and donor sites,
wherein the self-inactivating vector is free of wPRE (wood-chuck hepatitis virus post-transcriptional element).
9. The pharmaceutical composition of claim 8 , further comprising an envelope construct for providing a functional env protein.
10. The pharmaceutical composition of claim 8 , wherein the multiple gene units comprise a first gene unit operably linked to a first regulatory nucleic acid sequence and a second gene unit operably linked to a second regulatory nucleic acid sequence.
11. The pharmaceutical composition of claim 10 , wherein at least one of the first gene unit or the second gene unit encodes a trafficking signal.
12. The pharmaceutical composition of claim 8 , wherein the regulatory nucleic acid sequence comprises a cell-specific promoter or tissue-specific promoter.
13. The pharmaceutical composition of claim 8 , wherein the pharmaceutical composition further comprises a chemotherapeutic agent or a steroid agent.
14. The pharmaceutical composition of claim 8 , wherein the heterologous nucleic acid sequence encodes at least one RNAi agent or at least one polypeptide that inhibits expression of Bcl-2.