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Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

A manufacturing process for a replication-selective adenovirus vector that has enhanced lytic capacity is described. Vector Ad- KD3, described in Doronin et al (2000), is produced in the qualified adherent A549 human lung carcinoma cell line. Monolayer cultures in 10-tier cell stacks were initially grown in the recommended serum containing media. To facilitate downstream purification by reducing protein (serum) burden, EXCELL|[trade]| 293 serum-free, phenol red- free media was used as the virus propagation/cell growth media from this point forward. Development studies demonstrated equivalent virus productivity compared to standard growth media, however, the majority of the infectious virus remained cell-associated up to 72 hours post infection. Furthermore, replication kinetic studies demonstrated peak virus productivity at approximately 72 hours post infection. At the peak time point, infected cells were lysed directly with a controlled volume of buffer containing detergent. The clarified cell lysate served as the feedstream for a previously reported three-step column chromatography purification method (Jendrek et al, BioProcessing Journal, May/June 2005). Following purification, the vector was sterile filtered and vialed. The combined advantages of the switch to EXCELL|[trade]| 293 media for virus propagation and determination of peak virus replication time point resulted in an increased net yield of purified vector.