Quantitative analysis of transgene protein, mRNA, and vector DNA followinginjection of an adenoviral vector harboring glial cell line-derived neurotrophic factor into the primate caudate nucleus

Gene therapy for neurodegenerative diseases relies on stable expression of a vector-mediated transgene in the human central nervous system (CNS). In n onhuman primate CNS, transgene expression has been primarily assessed using descriptive histological methods. Here, we quantified the expression of a human glial cell line-derived neurotrophic factor (hGDNF) transgene using a n ELISA specific for hGDNF protein and real-time quantitative RT-PCR and PC R for hGDNF mRNA and vector DNA, respectively. Transgene expression was ass essed 1 week after injection of an E1-, E3-deleted adenovirus harboring hGD NF into the caudate nucleus of St. Kitts green monkey. We found that 57-147 million and 116-771 million copies of hGDNF mRNA and vector DNA, respectiv ely, were present per 10,000 copies of the beta -actin gene. In the same si tes, 40-152 pg of hGDNF protein per milligram of tissue was measured. Compa risons of these measures among monkeys demonstrated variable vector DNA and protein levels, but consistent mRNA levels at one-third of the level of ve ctor DNA. This suggests that local responses to the vector play a role in t he level of transgene expression and that high levels of vector DNA do not necessarily predict a high level of transgene protein. However, the results of this study do show that neuroprotective levels of GDNF transgene expres sion can be achieved following injection of an adenoviral vector into nonhu man primate caudate. Moreover, these assays provide quantitative methods fo r evaluating and comparing viral vectors in primate CNS.