TOXICITY OF REPLICATION-DEFECTIVE ADENOVIRAL RECOMBINANTS IN DISSOCIATED CULTURES OF NERVOUS-TISSUE

Replication-defective human type 5 adenoviral recombinants (AVR) are v ery efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. lit vi tro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecule(s). in vivo, in addition, an immune a ttack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conduc ted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichi a coil lacZ (E. coil lacZ) gene under control of either the very effic ient cytomegalovirus enhancer/promoter or the fast muscle troponin I p romoter, which is not active in these cells. Two factors contributed t o loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 E3)-deleted replication-incompetent AVR at high titers [greater than or equal to 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduc tion efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100- 1000). Despite the efficacy of adenoviral vectors in introducing forei gn genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentration s of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic. (C) 1996 Academic Press, Inc.