Production of Trans-Lentiviral Vector with Predictable Safety

Retroviruses use homologous recombination to reverse transcribe a doublestranded copy of DNA from their RNA genome. Safety advancements in retroviral vector design include the development of packaging systems that provide all of the retroviral proteins in trans to a replication-defective gene transfer vector. The packaging component expresses viral structural proteins that are required for assembly of the virus particle and is stripped of cis-acting sequences necessary for efficient transfer and replication of the viral genome (1 ). These cis- acting sequences are retained within the gene transfer vector where they facilitate its encapsidation, reverse transcription, and integration. Nonetheless, this split design does not completely exclude encapsidation of other viral and cellular RNA, including that derived from the viral packaging construct. Copackaging of packaging and vector RNA genomes enables genetic recombination to occur during reverse transcription and thus, the rejoining of viral structural protein-coding sequences and cis-acting sequences of the vector. If recombinants are formed that contain replicative functions it is possible they may propagate and spread to infect other cells. Thus, the inadvertent production of replication-competent retrovirus (RCR) (2 –5 ) constitutes the principal safety concern for the use of retroviral vectors in human clinical protocols (6 –8 ).