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

Dorothy A. Kozlowski, Eric Bremer, D. Eugene Redmond, David George, Beth Larson, Martha C. Bohn*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Gene therapy for neurodegenerative diseases relies on stable expression of a vector-mediated transgene in the human central nervous system (CNS). In nonhuman 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 an ELISA specific for hGDNF protein and real-time quantitative RT-PCR and PCR for hGDNF mRNA and vector DNA, respectively. Transgene expression was assessed 1 week after injection of an E1-, E3-deleted adenovirus harboring hGDNF 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, respectively, were present per 10,000 copies of the β-actin gene. In the same sites, 40-152 pg of hGDNF protein per milligram of tissue was measured. Comparisons of these measures among monkeys demonstrated variable vector DNA and protein levels, but consistent mRNA levels at one-third of the level of vector DNA. This suggests that local responses to the vector play a role in the 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 expression can be achieved following injection of an adenoviral vector into nonhuman primate caudate. Moreover, these assays provide quantitative methods for evaluating and comparing viral vectors in primate CNS.

Original languageEnglish (US)
Pages (from-to)256-261
Number of pages6
JournalMolecular Therapy
Volume3
Issue number2
DOIs
StatePublished - 2001

Funding

The authors thank Ianina Filipovich for excellent histological assistance; Dr. Bronwen Connor, Dr. Michael Hoane, and Dr. Xiaolong He for their assistance in processing tissue for quantitative assays; and Richard Anderson, Dr. Beverly Davidson, and the vector core at the University of Iowa for the production of the viral vectors. We also thank the research staff at the St. Kitts Biomedical Research Foundation, St. Kitts, for their assistance in carrying out the experiments in monkeys. This project was supported by a grant from NIMH, MH 57958; the Medical Research Institute Council of Children’s Memorial Hospital (M.C.B.); the Falk Foundation (E.B.); and Research Scientist Award MH00643-20 (D.E.R.) and partial support was received from the Carver Foundation (Vector Core). The original AdGDNF was a gift from Genetic Therapy/Novartis.

Keywords

  • Dopamine neurons
  • Gene therapy
  • Neurotrophic factor
  • Nigrostriatal system
  • Parkinson's disease
  • Real time RT-PCR

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

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