Rate-limiting steps in retrovirus production and gene transfer

We undertook studies to understand the rate limiting steps of production of recombinant retrovirus and develop strategies to increase the titer and purity of retroviral preparations for gene therapy. We found that synthesis and encapsidation of viral mRNA is a limiting step in production of infectious retrovirus. Moreover, increasing the level of mRNA expression increased the potency of retroviral preparations. In addition, we obtained highly purified retrovirus by immobilizing retroviral particles on recombinant fibronectin or poly-L-lysine. After dialysis and ultrafiltration, the titer and transduction efficiency of the purified virus increased by 20 and 10 times, respectively. Our results provide an insight into the limiting step of virus assembly and suggest ways to improve the titer and purity of retroviral preparations for gene therapy.