Breeding of retroviruses by DNA shuffling for improved stability and processing yields

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and conce ntration. To isolate viruses that are resistant to these manufacturing proc esses, we performed breeding of six ecotropic murine leukemia virus (MLV) s trains by DNA shuffling. The envelope regions were shuffled to generate a r ecombinant library of 5 x 10(8) replication-competent retroviruses, This li brary was subjected to the concentration process three consecutive times, w ith amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of th e parental viruses by 30- to 100-fold. The envelopes of these resistant vir uses differed in DNA and protein sequence, and all were complex chimeras de rived from multiple parents. These studies demonstrate the utility of DNA s huffling in breeding viral strains with improved characteristics for gene t herapy.