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Retroviral vectors containing direct repeats of homologous sequences constitute a powerful in vivo model system to analyze reverse transcriptase (RT) template-switching events. Directly repeated sequences in retroviral genomes are unstable and frequently deleted from the final integrated provirus due to RT's low template affinity and processivity. We developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors containing 701-bp direct repeats composed of segments of the herpes simplex virus-1 thymidine kinase gene (HTK). After one cycle of retroviral replication, we demonstrated functional reconstitution of the HTK suicide gene at a frequency of 57% when the direct repeats were adjacent to one another, and 91% when the direct repeats flanked the MLV packaging signal (Ψ). This demonstrated that direct repeat deletions do occur in MLV at high frequencies, similar to spleen necrosis virus. Deleting Ψ from the final provirus made these vectors safer for gene therapy as they could not be efficiently mobilized by a replication competent retrovirus. Furthermore, we demonstrated that the high frequency deletion of direct repeats could be used to efficiently excise the drug resistance gene. One retroviral vector containing a direct repeat deleted the neomycin expression cassette with its translational control region at >99% efficiency. We also determined the effect of distance between direct repeats on the frequency of deletion by separating the 701-bp HTK direct repeats by spacer fragments of various lengths (0.1 to 3.5 kb). After one replication cycle, all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing <1,500 bp between homologous sequences exhibited lower frequencies of deletion (37–82%). Further analysis showed that RT switched templates at a greater frequency within the 5′ versus the 3′ regions of the downstream direct repeat, and this frequency doubled when Ψ was between the direct repeats. However, Ψ did not increase the rate of template switching for shorter direct repeats. These results indicate that linear distance between homologous sequences increases the rate of RT template switching, and suggest that duplex formation between nascent DNA and homologous template sequences 3′ of RT promote template switching.