The objective of this research project is to design and test the safety and efficacy of the attenuated hepatitis A strain HM175 (HAV) as a vector for gene transfer into cultured human fetal brain tissue and into the CNS of nonhuman primates. This project involves cloning expression vectors for neurogenes by recombinant techniques and testing their infection efficiency as well as their transcription and translation ability. Studies comparing the tropism of HAV for neuronal and glial elements will be carried out in cultured human fetal spinal cord and brain. Studies have indicated that HAV can efficiently infect cultured human fetal brain tissue without noticeable cytopathic effects. Direct inoculation of HAV into the spinal cord and thalamus of Macaca mulatta did not cause any significant clinical or histologic effects. The HAV negative strand replicative intermediate was detectable by RT-PCR in the brain up to four weeks after direct inoculation. RT-PCR in situ is being developed to determine the cellular location of the HAV negative strand replicative intermediate. Plasmid vectors containing the full length cDNA of HAV are grown in E. coli competent cells (strain DH5alpha) and picomaviral genetic elements, reporter genes and human cDNAs are being inserted into known loci within the HAV genome that do not effect the virus' viability.
