Methods for producing minus-strand RNA viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken β-actin promoter
The present invention provides methods for producing a minus-strand RNA viral vector, which comprise using a promoter comprising a cytomegalovirus enhancer and a chicken β-actin promoter, to induce the transcription of the genome RNA of a minus-strand RNA viral vector and the expression of minus-strand RNA viral proteins that form a ribonucleoprotein with the genome RNA. The methods of the present invention enable high efficiency production of highly safe minus-strand RNA viral vectors. The methods of the present invention are particularly useful for producing minus-strand RNA viral vectors that are deficient in envelope-constituting protein genes.
1. A method for producing a Sendai virus vector, which comprises the steps Of:
expressing a bacteriophage RNA polymerase-encoding DNA under the direct control of a cytomegalovirus enhancer and chicken β-actin promoter-comprising promoter in a virus-producing cell selected from the group consisting of a 293T cell and a BHK-21 cell;
transcribing with the RNA polymerase, a DNA that encodes the Sendai virus genome RNA or the complementary strand thereof, and that is operably linked with a recognition sequence of the RNA polymerase in the virus-producing cell;
and expressing Sendai virus N (nucleocapsid), P (phospho), and L (large) proteins that form a ribonucleoprotein with the genome RNA under the direct control of the cytomegalovirus enhancer and chicken β-actin promoter-comprising promoter in the virus-producing cell, wherein said method produces at least 1000-fold more of said Sendai virus vector than a method where said cytomegalovirus enhancer and chicken β-actin promoter-comprising promoter is replaced by a cytomegalovirus (CMV) promoter.
2. The method of claim 1 , wherein the RNA polymerase-encoding DNA is expressed episomally in the virus-producing cell.
3. The method of claim 1 , wherein the RNA polymerase-encoding DNA is expressed from a chromosome in the virus-producing cell.
4. The method of claim 1 , wherein the bacteriophage is selected from the group consisting of SP6 phage, T3 phage, and T7 phage.
5. The method of claim 1 , wherein the genome RNA or the complementary strand thereof lacks one or more genes encoding an envelope-constituting protein, and wherein the method further comprises the step of expressing a DNA encoding an envelope-constituting protein in the cell.