Toxicity of Replication-Defective Adenoviral Recombinants in Dissociated Cultures of Nervous Tissue

Abstract

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neuronsin vitroandin vivo;however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time.In vitro,this may be due to direct toxicity of the protein product of the transgene or adenoviral molecule(s).In vivo,in addition, an immune attack 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 conducted in primary neural cultures. Cultures of dissociated murine spinal cord–dorsal root ganglia were exposed to AVR containing theEscherichia coli lacZ(E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1+E3)-deleted replication-incompetent AVR at high titers [≥5×108viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, β-galactosidase, at titers that result in 55–75% transduction efficiency (5×107–5×108viral particles/ml or m.o.i. 100–1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.