Methods and compositions for increased double stranded rna production

Abstract

The invention provides methods and compositions for improved production of large quantities of unencapsidated double strand RNA (dsRNA) in vivo. The disclosed methods and compositions, comprising co-expression of genes encoding orotate phosporibosyl transferase, bacteriophage coat protein and dsRNA produce a significant improvement over current in vivo methods of producing unencapsidated dsRNA.

We claim: 
     
         1 . A microbial cell comprising
 (1) a coat protein gene encoding a capsid protein, wherein the capsid protein is the capsid protein of bacteriophage MS2, the capsid protein of bacteriophage Qβ, or a fragment thereof, and   (2) a gene encoding a heterologous dsRNA molecule comprising a self-complementary stretch of sequence separated by noncomplementary sequence such that upon hybridization of the complementary sequences a stem-loop structure is formed, and   wherein the stem-loop structure formed has a length exceeding the interior diameter of an MS2 capsid.   
     
     
         2 . The microbial cell of  claim 1 , wherein the capsid protein is encoded by a leviviridae coat protein gene. 
     
     
         3 . The microbial cell of  claim 1 , wherein the capsid protein is encoded by the coat protein gene of bacteriophage MS2 or a fragment thereof. 
     
     
         4 . The microbial cell of  claim 1 , wherein the capsid protein is encoded by the coat protein gene of bacteriophage Qβ or a fragment thereof. 
     
     
         5 . The microbial cell of  claim 1 , wherein the gene encoding the dsRNA and the coat protein gene encoding the capsid protein are expressed from an inducible promoter. 
     
     
         6 . The microbial cell of  claim 5 , wherein the coat protein gene encoding the capsid protein is expressed from a constitutive promoter and the gene encoding the dsRNA is expressed from an inducible promoter. 
     
     
         7 . The microbial cell of  claim 1 , wherein the coat protein gene encoding the capsid protein is expressed prior to or concomitant with the gene encoding the dsRNA. 
     
     
         8 . The microbial cell of  claim 1 , wherein the gene encoding the dsRNA and the coat protein gene encoding the capsid protein are present on one plasmid or extrachromosomal element within the microbial cell. 
     
     
         9 . The microbial cell of  claim 1 , wherein the gene encoding the dsRNA and the coat protein gene encoding the capsid protein are present on separate plasmids or extrachromosomal elements within the microbial cell. 
     
     
         10 . The microbial cell of  claim 1 , wherein one of the genes encoding the dsRNA and the capsid protein are present on a plasmid or extrachromosomal element and the other of the genes encoding the dsRNA and the capsid protein are present on the chromosome of the microbial cell. 
     
     
         11 . The microbial cell of  claim 1 , wherein the gene encoding the dsRNA and the coat protein gene encoding the capsid protein are present on the chromosome of the microbial cell. 
     
     
         12 . The microbial cell of  claim 1 , wherein the dsRNA is an RNAi precursor. 
     
     
         13 . The microbial cell of  claim 1 , wherein the capsid protein comprises a truncated capsid protein. 
     
     
         14 . The truncated capsid protein of  claim 13 , comprising the first 41 amino acids of the capsid protein, the first 35 amino acids of the capsid protein, the first 25 amino acids of the capsid protein, the first 21 amino acids of the capsid protein, or the first 11 amino acids of the capsid protein. 
     
     
         15 . The microbial cell of  claim 1 , wherein the microbial cell is a bacterium or a yeast. 
     
     
         16 . The microbial cell of  claim 1 , wherein the microbial cell is a gram-negative bacterium or a gram-positive bacterium. 
     
     
         17 . The microbial cell of  claim 1 , wherein the microbial cell is a strain of  Escherichia coli , a strain of  Corynebacterium glutamicum , or a strain of  Saccharomyces cerevisiae.    
     
     
         18 . A composition comprising the microbial cell of  claim 1 . 
     
     
         19 . A dosage form comprising the microbial cell of  claim 1 . 
     
     
         20 . Use of the microbial cell of  claim 1  in expressing the heterologous dsRNA.