Geminiviral vector for expression of rituximab

Abstract

A single vector or multiple separate vectors that contain two or more non-competing replicons for transient expression of the heavy and light chains of Rituximab in Nicotiana benthamiana leaves is described. The correct assembly of these subunit proteins into functional oligomeric structures to optimize the expression is also described. This system advances plant transient expression technology by eliminating the need for non-competing viruses, and thus, enhances the realistic commercial application of the multi-replicon single vector system for producing Rituximab in plant cells.

1 - 20 . (canceled) 
     
     
         21 . A vector system comprising:
 (a) a first vector comprising a long intergenic region (LIR) of a gem inivirus genome, followed by a single replicon comprising two nucleic acid segments followed by a short intergenic region (SIR) of a gem inivirus genome, followed by a LIR, wherein the two nucleic acid segments comprise
 (i) a nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a heavy chain of Ritixumab, and a 3′ terminator; and 
 (ii) a nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a light chain of Ritixumab, and a 3′ terminator; and 
   (b) a second vector comprising one or more nucleic acid segments that comprise nucleic acids encoding Rep and RepA proteins of a geminivirus genome and at least one promoter.   
     
     
         22 . The vector system of claim  1 , wherein the at least one promoter of the second vector comprises a duplicate enhancer, wherein the promoter drives increased RepA expression relative to native viral RepA expression. 
     
     
         23 . The vector system of claim  1 , wherein the first vector comprises a single replicon viral vector for co-expression of the heavy and light chains of Rituximab selected from the group consisting of pBYR17p-MRtx, pBYR17p1-MRtx, pBYR17p2-MRtx, pBYR17p3-MRtx, and pBYR17b1-MRtx. 
     
     
         24 . A vector system for transient expression of the heavy and light chains of Rituximab, the system comprising,
 a first vector comprising a first replicon comprising a first nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a heavy chain of Ritixumab, and a 3′ terminator;   a second vector comprising a second replicon comprising a second nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a light chain of Ritixumab, and a 3′ terminator,   wherein the first and second replicons are non-competing.   
     
     
         25 . The vector system of claim  4 , wherein the first replicon further comprises a short intergenic region (SIR) of a gem inivirus genome following the 3′ terminator, and two long intergenic regions (LIR) of a gem inivirus genome, wherein the first nucleic acid segment is flanked at each end by a LIR; and
 the second replicon further comprises a short intergenic region (SIR) of a geminivirus genome following the 3′ terminator and two long intergenic regions (LIR) of a gem inivirus genome flanking the second nucleic acid segment, wherein the second nucleic acid segment is flanked at each end by a LIR. 
 
     
     
         26 . The vector system of claim  4 , further comprising at least one nucleic acid sequence encoding Rep and RepA proteins of a gem inivirus genome and at least one additional promoter driving RepA expression. 
     
     
         27 . The vector system of claim  6 , wherein the first vector, or the second vector, or both the first and second vector each comprises the at least one nucleic acid sequence encoding Rep and RepA proteins. 
     
     
         28 . The vector system of claim  6 , wherein the at least one additional promoter driving RepA expression comprises a duplicate enhancer, wherein the promoter drives increased RepA expression relative to native viral RepA expression. 
     
     
         29 . The vector system of claim  4 , further comprising a nucleic acid sequence that comprises a gene-silencing inhibitor. 
     
     
         30 . The vector system of claim  4 , wherein at least one of the 3′ terminator of the first vector and the 3′ terminator of the second vector comprises a sequence encoding a tobacco extensin gene terminator region. 
     
     
         31 . A vector system comprising two replicons, the vector system comprising;
 (a) a first vector comprising a first replicon comprising a first nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a first chain of Rituximab, a 3′ terminator, and a short intergenic region (SIR) of a geminivirus genome, wherein the first nucleic acid segment is flanked at the 5′ end and the 3′ end by a long intergenic region (LIR) of a gem inivirus genome;   (b) a second vector comprising a second replicon comprising a second nucleic acid segment comprising at least one promoter, a 5′UTR, a region encoding a second chain of Rituximab, and a 3′ terminator, and a short intergenic region (SIR) of a gem inivirus genome, wherein the second nucleic acid segment is flanked at the 5′ end and the 3′ end by a long intergenic region (LIR) of a geminivirus genome; and   (c) one or more nucleic acid segments that comprises a nucleic acid encoding Rep and RepA proteins of a geminivirus genome and a promoter driving RepA expression;   wherein the first and second replicons are non-competing.   
     
     
         32 . The vector system of claim  11 , wherein the first vector, or the second vector, or both the first and second vector each comprises the at least one nucleic acid sequence encoding Rep and RepA proteins. 
     
     
         33 . The vector system of claim  11 , further comprising a nucleic acid sequence that comprises a gene-silencing inhibitor. 
     
     
         34 . A method of producing Rituximab in a plant cell comprising: (a) introducing the vector system of claim  1  into a plant cell; and (b) maintaining the cell for a time and under conditions sufficient to allow expression of the first and second replicons. 
     
     
         35 . The method of claim  14 , further comprising maintaining the cell for a time and under conditions sufficient to allow full assembly of the heavy and the light chains of Rituximab. 
     
     
         36 . A method of producing Rituximab in a plant cell comprising: (a) introducing the vector system of claim  4  into a plant cell; and (b) maintaining the cell for a time and under conditions sufficient to allow expression of the first and second replicons. 
     
     
         37 . The method of claim  16 , further comprising maintaining the cell for a time and under conditions sufficient to allow full assembly of the first and the second chains of Rituximab. 
     
     
         38 . The method of claim  17 , wherein the first vector is pBYR9-MRtxG and the second vector is pBYR9-MRtxK. 
     
     
         39 . A method of producing Rituximab in a plant cell comprising: (a) introducing the vector system of claim  11  into a plant cell; and (b) maintaining the cell for a time and under conditions sufficient to allow expression of the first and second replicons. 
     
     
         40 . The method of claim  19 , further comprising maintaining the cell for a time and under conditions sufficient to allow full assembly of the first and the second chains of Rituximab. 
     
     
         41 . The method of claim  20 , wherein the first vector is pBYR9-MRtxG and the second vector is pBYR9-MRtxK.