Nature Biotechnology
18, 1279 - 1282 (2000)
doi:10.1038/82391
Breeding of retroviruses by DNA shuffling for improved stability and
processing yieldsSharon K. Powell1, 2, Michele A. Kaloss1, Anne Pinkstaff1, Rebecca McKee1, Irina Burimski1, Michael Pensiero1, Edward Otto1, Willem P.C. Stemmer3
& Nay-Wei Soong31
Genetic Therapy Inc. A Novartis Company,
9 W. Watkins Mill Road, Gaithersburg, MD
20878. 2
Present address: Avigen, 1201 Harbor
Bay Parkway, Alameda, CA 94502. 3
Maxygen Inc., 515 Galveston Drive,
Redwood City, CA 94063.
Correspondence should be addressed to Edward Otto
ed.otto@pharma.novartis.comDNA shufflingretroviral vectorsstress resistanceultracentrifugationManufacturing of retroviral vectors for gene therapy is complicated by
the sensitivity of these viruses to stress forces during purification and
concentration. To isolate viruses that are resistant to these manufacturing
processes, we performed breeding of six ecotropic murine leukemia virus (MLV)
strains by DNA shuffling. The envelope regions were shuffled to generate a
recombinant library of 5  106 replication-competent
retroviruses. This library was subjected to the concentration process three
consecutive times, with 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 the parental viruses by 30- to 100-fold. The envelopes of these
resistant viruses differed in DNA and protein sequence, and all were complex
chimeras derived from multiple parents. These studies demonstrate the utility
of DNA shuffling in breeding viral strains with improved characteristics for
gene therapy.
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