Titers of HIV-based vectors encoding shRNAs are reduced by a dicer-dependent mechanism.

Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs.