Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules

Abstract

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:
 (i) a self-inactivating transfer vector comprising: multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and   (ii) a helper construct which lacks a 5′ LTR, wherein said 5′ LTR has been replaced with a heterologous promoter, said helper construct further comprising: a lentiviral env nucleic acid sequence containing a deletion, wherein said deleted env nucleic acid sequence does not produce functional env protein; a packaging signal containing a deletion, wherein said deleted packaging signal is nonfunctional.   
     
     
         2 . The lentiviral transfer system of  claim 1 , wherein in the transfer vector, one or more of said regulatory nucleic acid sequences comprises a general promoter. 
     
     
         3 . The lentiviral transfer system of  claim 2 , wherein said general promoter is CMV-IE promoter. 
     
     
         4 . The lentiviral transfer system of  claim 1 , wherein in the transfer vector, one or more of said regulatory nucleic acid sequences comprises a cell or tissue-specific promoter. 
     
     
         5 . The lentiviral transfer system of  claim 4 , wherein said cell or tissue-specific promoter is selected from the group consisting of: TSTA promoter, mesothelin promoter, hPSA promoter, hCCKAR promoter, hAFP promoter, and hNSE promoter. 
     
     
         6 . The lentiviral transfer system of  claim 1 , wherein in the transfer vector, a translation initiation site is located between gene units. 
     
     
         7 . The lentiviral transfer system of  claim 6 , wherein the translation initiation site is internal ribosome entry site (IRES). 
     
     
         8 . The lentiviral transfer system of  claim 1 , wherein the heterologous nucleic acid sequence encodes an RNAi or a polypeptide. 
     
     
         9 . The lentiviral transfer system of  claim 8 , wherein the polypeptide or RNAi inhibits expression or activity of a gene or protein that contributes to progression of cancer. 
     
     
         10 . The lentiviral transfer system of  claim 8 , wherein the polypeptide or RNAi inhibits expression or activity of a tumor promoting gene or protein. 
     
     
         11 . The lentiviral transfer system of  claim 8 , wherein the polypeptide or RNAi inhibits expression or activity of a growth factor, growth factor receptor, angiogenic factor, angiogenic factor receptor, cell cycle regulator, apoptosis-inducing molecule, or cell adhesion molecule. 
     
     
         12 . The lentiviral transfer system of  claim 8  wherein the polypeptide or RNAi inhibits the expression or activity of a vascular endothelial growth factor, a vascular endothelial growth factor receptor, epidermal growth factor receptor, hTR, hTERT, papillomavirus E6, papillomavirus E7, BCR-abl, CEACAM6, MMP9, or a cathepsin. 
     
     
         13 . The lentiviral transfer system of  claim 8 , wherein said RNAi is targeted to Bcl-2, AEC-1, Myc or K-ras. 
     
     
         14 . The lentiviral transfer system of  claim 8 , wherein said polypeptide is P53 protein. 
     
     
         15 . The lentiviral transfer system of  claim 8 , wherein said heterologous nucleic acid sequence encodes the P53 protein and RNAi is targeted to Bcl-2. 
     
     
         16 . The lentiviral transfer system of  claim 1 , wherein in the transfer vector, a sequence encoding a cell or tissue-specific enzyme cleavage site is located between one or more gene units, wherein cleavage at the site occurs within a fused polypeptide that is expressed by the heterologous nucleic acid sequences of the gene units. 
     
     
         17 . The lentiviral transfer system of  claim 16 , wherein said cell or tissue-specific enzyme cleavage site is a protease 2A cleavage site, a presecretory protein signal peptidase cleavage site, or a pancreatic prechymotrypsinogen cleavage site. 
     
     
         18 . The lentiviral transfer system of  claim 16 , wherein one of the gene units is an intercellular trafficking signal. 
     
     
         19 . The lentiviral transfer system of  claim 18  wherein the intercellular trafficking signal is a membrane-penetrating protein or a fragment thereof. 
     
     
         20 . The lentiviral transfer system of  claim 18  wherein the membrane-penetrating protein is a plant or bacterial protein toxin. 
     
     
         21 . The lentiviral transfer system of  claim 18  wherein the membrane-penetrating protein is a viral protein. 
     
     
         22 . The lentiviral transfer system of  claim 21 , wherein said trafficking signal is derived from herpesvirus VP22 or HIV-Tat. 
     
     
         23 . The lentiviral transfer system of  claim 22 , wherein said trafficking signal is the HIV-Tat eleven amino acid transduction sequence. 
     
     
         24 . The lentiviral transfer system of  claim 21 , wherein said herpesvirus is HSV1. 
     
     
         25 . The lentiviral transfer system of  claim 21 , wherein said trafficking signal is a VP22 protein homologue of HSV1 VP22. 
     
     
         26 . The lentiviral transfer system of  claim 25  wherein the VP22 transport signal comprises the C-terminal 34 amino acid sequence of VP22 of HSV1, or a fragment having 80% or greater identity to the terminal 34 amino acid sequence of VP22 of HSV1. 
     
     
         27 . The lentiviral transfer system of  claim 22  wherein the VP22 transport signal comprises one or more of RSASR, RTASR, RSRAR, RTRAR, ATATR, or RSAASR. 
     
     
         28 . The lentiviral transfer system of  claim 1 , wherein expression of the heterologous nucleic acid sequences in the transfer vector inhibits progression of a disease or disorder. 
     
     
         29 . The lentiviral transfer system of  claim 28 , wherein expression of the multiple heterologous nucleic acid sequences synergistically inhibits progression of a disease or disorder. 
     
     
         30 . The lentiviral transfer system of  claim 28 , wherein said disease or disorder is cancer. 
     
     
         31 . The lentiviral transfer system of  claim 28 , wherein said disease or disorder is a genetic disorder. 
     
     
         32 . The lentiviral transfer system of  claim 31 , wherein said genetic disorder is metabolic disorder. 
     
     
         33 . The lentiviral transfer system of  claim 32 , wherein said metabolic disorder is Gaucher's Disease or Fabry's Disease. 
     
     
         34 . The lentiviral transfer system of  claim 28 , wherein said disease or disorder is a neurological disorder. 
     
     
         35 . The lentiviral transfer system of  claim 34 , wherein said neurological disorder is Alzheimer's Disease or Parkinson's Disease. 
     
     
         36 . A method for treating a condition, comprising administering to a patient a lentiviral particle for gene transfer, said lentiviral particle produced using a lentiviral transfer system comprising:
 (i) a transfer vector comprising: multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and   (ii) a helper construct which lacks a 5′ LTR, wherein said 5′ LTR has been replaced with a heterologous promoter, said helper construct further comprising: a lentiviral env nucleic acid sequence containing a deletion, wherein said deleted env nucleic acid sequence does not produce functional env protein; a packaging signal containing a deletion, wherein said deleted packaging signal is nonfunctional.   
     
     
         37 . The method of  claim 36 , wherein said condition is a cancer. 
     
     
         38 . The method of  claim 37 , wherein said cancer is liver cancer, pancreatic cancer, or prostate cancer. 
     
     
         39 . The method according to  claim 37 , wherein the cancer is prostate cancer, and wherein the heterologous nucleic acid sequence encodes a P53 protein and an RNAi targeted for Bcl-2. 
     
     
         40 . The method of  claim 36 , wherein said condition is a genetic disorder. 
     
     
         41 . The method of  claim 36 , wherein said condition is a need for cosmetic enhancement. 
     
     
         42 . A pharmaceutical composition comprising a lentiviral particle for gene transfer, said lentiviral particle produced using a lentiviral transfer system comprising:
 (i) a self-inactivating transfer vector comprising: multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and   (ii) a helper construct which lacks a 5′ LTR, wherein said 5′ LTR has been replaced with a heterologous promoter, said helper construct further comprising: a lentiviral env nucleic acid sequence containing a deletion, wherein said deleted env nucleic acid sequence does not produce functional env protein; a packaging signal containing a deletion, wherein said deleted packaging signal is nonfunctional.   
     
     
         43 . The pharmaceutical composition according to  claim 42 , wherein the heterologous nucleic acid sequence encodes a P53 protein and an RNAi targeted for Bcl-2, which expressed both respectively or at the same time. 
     
     
         44 . The pharmaceutical composition of  claim 42 , further comprising a chemotherapeutic agent or a steroid agent. 
     
     
         45 . The pharmaceutical composition of  claim 44 , wherein the steroid agent is prednisolone, cortisone, corticosterone, or dexamethasone. 
     
     
         46 . A pharmaceutical composition comprising a self inactivating lentiviral transfer vector comprising multiple gene units, wherein each gene unit comprises a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence. 
     
     
         47 . The pharmaceutical composition according to  claim 46 , wherein the heterologous nucleic acid sequence encodes a P53 protein and an RNAi targeted for Bcl-2. 
     
     
         48 . The lentiviral transfer system of  claim 1 , wherein the transfer vector further comprises mammalian insulator sequence and splice acceptor and splice donor sites, and is free of wPRE (wood-chuck hepatitis virus post-transcriptional element) downstream of a cloning site.