Methods to express recombinant proteins from lentiviral vectors

Abstract

Lentivector constructs for expression of recombinant proteins, polypeptides or fragments thereof and methods of making the same are described. The lentivectors typically have a self-processing cleavage sequence between a first and second protein or polypeptide coding sequence allowing for expression of a functional protein or polypeptide under operative control of a single promoter and may further include an additional proteolytic cleavage sequence which provides a means to remove the self-processing cleavage sequence from the expressed protein or polypeptide. The vector constructs find utility in methods relating to enhanced production of biologically active proteins, such as immunoglobulins or fragments thereof in vitro and in vivo.

1 . A lentivector for expression of a recombinant immunoglobulin, comprising:
 in the 5′ to 3′ direction, a promoter operably linked to the coding sequence for a first chain of an immunoglobulin molecule or a fragment thereof, an additional proteolytic cleavage site, a sequence encoding a self-processing cleavage site and the coding sequence for a second chain of an immunoglobulin molecule or fragment thereof, wherein the sequence encoding the self-processing cleavage site is inserted between the coding sequence for the first chain and the coding sequence for the second chain of said immunoglobulin molecule.   
     
     
         2 . The lentivector according to  claim 1 , wherein the sequence encoding the self-processing cleavage site comprises a 2A sequence. 
     
     
         3 . The lentivector according to  claim 2 , wherein the 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence. 
     
     
         4 . The lentivector according to  claim 3 , wherein the 2A sequence encodes an oligopeptide comprising amino acid residues LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2) or EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:9). 
     
     
         5 . The lentivector according to  claim 3 , wherein the coding sequence for the first chain of said immunoglobulin molecule or a fragment thereof encodes an immunoglobulin heavy chain. 
     
     
         6 . The lentivector according to  claim 3 , wherein the coding sequence for the first chain of said immunoglobulin molecule or a fragment thereof encodes an immunoglobulin light chain. 
     
     
         7 . The lentivector according to  claim 5 , wherein the coding sequence is the full length coding sequence of an immunoglobulin heavy chain. 
     
     
         8 . The lentivector according to  claim 6 , wherein the coding sequence is the full length coding sequence of an immunoglobulin light chain. 
     
     
         9 . The lentivector according to  claim 1 , wherein said additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RKRR (SEQ ID NO: 15). 
     
     
         10 . The lentivector according to  claim 3 , wherein the promoter is selected from the group consisting of an elongation factor 1-alpha promoter (EF1 a) promoter, a phosphoglycerate kinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early gene promoter (CMV), a chimeric liver-specific promoter (LSP) a cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a tetracycline responsive promoter (TRE), a transthyretin promoter (TTR), a MND promoter, a simian virus 40 promoter (SV40) and a CK6 promoter. 
     
     
         11 . The lentivector according to  claim 10 , wherein said promoter is a CAG hybrid promoter/enhancer. 
     
     
         12 . The lentivector according to  claim 10 , wherein said promoter is a CMV promoter/enhancer. 
     
     
         13 . The lentivector according to  claim 3 , further comprising a signal sequence. 
     
     
         14 . The lentivector according to  claim 3 , wherein said heavy and light chain immunoglobulin coding sequences are expressed in a substantially equimolar ratio. 
     
     
         15 . The lentivector according to  claim 11 , wherein said lentivector comprises a CAG promoter operably linked to the coding sequence for a first chain of an immunoglobulin molecule, a sequence encoding a self-processing cleavage site and the coding sequence for a second chain of an immunoglobulin molecule, wherein the sequence encoding said self-processing cleavage site is inserted between the coding sequence for the first chain and the coding sequence for the second chain of said immunoglobulin molecule. 
     
     
         16 . A producer cell transduced with the vector of  claim 11 . 
     
     
         17 . A producer cell transduced with the vector of  claim 12 . 
     
     
         18 . A producer cell transduced with the vector of  claim 14 . 
     
     
         19 . A recombinant immunoglobulin molecule produced by a producer cell infected with the lentivector of  claim 11 . 
     
     
         20 . A recombinant immunoglobulin molecule produced by a producer cell infected with the lentivector of  claim 12 . 
     
     
         21 . A recombinant immunoglobulin molecule produced by a producer cell infected with the lentivector of  claim 14 . 
     
     
         22 - 39 . (canceled)