A NOVEL HUMAN AMPHOTROPIC PACKAGING CELL-LINE - HIGH-TITER, COMPLEMENT RESISTANCE, AND IMPROVED SAFETY

Successful retroviral-mediated gene therapy will depend on safe, effic ient packaging cell lines for vector particle production. Existing pac kaging lines for murine leukemia virus (MLV)-based vectors are predomi nantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we scre ened genomic DNA from eight cell lines. DNA from the human 293 cell li ne did not cross-hybridize with MLV sequences, and these cells were ab le to secrete Gag particles after transfection. We derived a stable am photropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately fro m a heterologous (non-MLV) promoter, to maximally reduce homology betw een packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. I n addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivat ed by human serum. Thus, the packaging cells we describe are as effici ent and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in viv o gene delivery. (C) 1996 Academic Press. Inc.