Abstract
Single-gene murine leukemia virus-based retroviral vectors carrying the G418-resistance gene (neo) under transcriptional control of the long terminal repeat were used to study the effect of selection on long-term vector expression in a murine lymphoid cell line, L691. We used two isogenic vectors carrying either a strong or a weak transcriptional enhancer from low-leukemogenic Akv and high-leukemigenic SL3-3 murine leukemia virus, respectively. Effects of G418 selection were studied at the level of vector-transduced cell populations and at the level of single-vector-transduced cell clones obtained without selection for vector expression.
Selection for vector expression prior to isolation of cell clones changed the range of vector expression levels for cell clones carrying the Akv enhancer, but did not influence the long-term stability of vector expression for the two populations of cell clones.
Cell clones harboring the Akv enhancer, isolated without selection and then subjected to prolonged growth under selective conditions, exhibited no mutations in the enhancer region or major vector rearrangements although showing increased vector expression in some cases.
Our results are discussed in terms of retrovirus-mediated gene transfer strategies employing selection for expression of a selective marker in single-gene or bicistronic vectors with a low- or nonleukemogenic virus-derived backbone.
Overview summary
Retroviral vectors in use for gene therapy are mostly derived from the high-leukemogenic Moloney murine leukemia virus (MLV) harboring a strong transcriptional enhancer. Duch et al. have analyzed vectors derived from two MLVs differing in leukemogenicity and enhancer strength. After selection for vector expression, no difference in expression level and long-term stability of the transduced cells was observed between the two vectors, and selection for expression of vectors with the weaker enhancer did not result in the appearance of alterations in the enhancer structure. These results point to the use of vectors with weak enhancers as a safety feature in ex vivo protocols.
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