Introduction

1. Introduction Parkinson’s disease is a degenerative disorder of the central nervous system characterized by death of the dopaminergic neurons in the substantianigra and reduced levels of dopamine in the striatum (1). Current drugs for Parkinson’s disease do not restore endogenous dopamine levels and cannot regenerate neurons (2). Furthermore, current treatments are unable to attain site-specific delivery resulting in unwanted side effects thus requiring a need for alternative treatments such as gene therapy (3). Genes such as Glial-Derived Neurotropic Factor (GDNF), promote the survival of dopaminergic neuron but cannot cross the blood brain barrier (4). This can be solved by using Adeno-Associated Virus (AAV), a gene transfer vector, to deliver GDNF directly into the brain (5). However, chronic over-expression of GDNF can cause cellular abnormalities (6). A proper regulator, such as a hypoxia regulator, is required to control the basal expression (7). The goal of this experiment was to develop a hypoxia regulated AAV transfer vector that exploits hypoxic conditions as a trigger for dosing GDNF in the brain to combat the effects of Parkinson’s disease. By regulating GDNF in vivo, the negative side effects associated with taking external medications will not be present as the protein is synthesized directly in the brain. Regulation of Gene Expression using Hypoxia Driven Viral VectorsBridget Mais, Pablo Garcia Miranda, Corinna BurgerSenior Honors Thesis, Dept. of Neurology, University of Wisconsin, Madison, WI Results Methods GDNF Expression in Rat Striatum Building the Constructs:Different permutations of hypoxia response elements (HRE’s) and promoters were cloned into AAV vector. We had two categories of constructs: (1) one that had the HRE sequence within the promoter gene JMY, a hypoxia- responsive promoter that regulates expression of Junction Mediating Regulatory Protein. And (2) one where the HRE sequences were adjacent to a minimal promoter such as the cytomegalovirus minimal promoter (mCMV) or the minimal heat shock protein 70 (mHsp70). AAV Table 1: Successful AAV Constructs Each construct contained a promoter, either JMY, mCMV, or mHsp70, and various HRE sequences derived from different sources. The HR12 was a combination of HRE’s from PGK1, Enolase 1, Lactate Dehydrogenase A. Figure 2:GDNF expression in rats with two controls: one group of injected animals that were exposed to normoxia and one group of uninjected animals that were exposed to all conditions. Hypoxia 72h represents the 48 hour hypoxia with 24 hour normoxiagroup. • Discussion • From the in vitro stage, we found a number of vectors inducible under hypoxic conditions. We saw that mCMVwas a stronger promoter than its mHsp70 counterpart and that there was no correlation between the level of expression and the quantity of HRE’s present. • We tested three constructs in vivo that had higher GDNF expression in hypoxia than normoxia from our in vitro results. Unfortunately, the animal stage showed that these constructs were not responsive to hypoxia in vivo as the hypoxic and normal conditions expressed the same levels of GDNF for all trials. • If we repeated the experiment, we would look for a way to reduce the expression in the normal state to make the hypoxia expression more significant. • Overall, this experiment still holds strong merit to make a significant impact in neurodegenerative diseases. Future trials will help pave the way to establish a method to regulate GDNF in vivo to express the gene without the dependence of external drugs. GDNF Expression in Culture Cells Normalized Cell Culture:Screen these DNA constructs in vitro under high and low hypoxia (6% and 10% O2, respectively) and normoxia (21% O2), using a human endothelial kidney cell line (HEK 293). The GDNF expression was measure using an enzyme-linked immunosorbent assay (ELISA) test. Blank Animal Testing:The three optimal vectors were packaged into capsids viruses and injected into the striatum of rats to test these vectors in vivo. The rats were exposed to hypoxia conditions (11%) for 24 hours, 48 hours, and 48 hour hypoxia with 24 hours normoxia. The expression of GDNF in the rat striatium was examined with an ELISA test. • References • Olanow, C.W., Tatton, W.G., 1999 Etiology and pathogenesis of Parkinson’s disease. Annu. Rev. Neurosci. 22, 123-144. • Wijeyekoon R, Barker RA. Cell replacement therapy in Parkinson’s disease. BiochimBiophysActa 2009; 1792:688-702 • Harder, S., H. Bass, and S. Rietbrock. 1995. Concentration-effect relationship of levodopa in patients with Parkinson’s disease. Clin. Pharmacokinet. 29: 253-256. • Deierborg, T., Soulet, D., Roybon, L., Hall, V., and Brundin, P. (2008). Emerging restorative treatments for Parkinson’s disease. Prog. Neurobiol. 85, 407-432. • P.E. Monahan, R.J. Samulski, Mol. Med. Today 6 (2000) 433-440. • Kirk, D., Rosenblad, C., Bjorklund, A., and Mandel, R.J. (2000). Long-Term rAAV-mediated gene transfer of GEND in the rat Parkinson’s model: Intrastriatal but not intranigral transduction promotes functional regeneration in the lesionednigrostriatal system. J. Neurosci. 20, 4686-4700. • Dachs GU, Patterson AV, Firth HD, Ratcliffe PJ, Townsend KM, Stratford IJ, Harris AL. Target gene expression to hypoxic tumor cells. Nat Med 1997; 3:515-520. Figure 1: A number of constructs show robust induction under hypoxia conditions when compared to normoxia conditions. Three clones were selected to test in vivo (asterisk) Acknowledgements A special thanks to the members of the Burger Lab for all their patience and help throughout this project. In particular, thank you Pablo Garcia Miranda. Without you, none of this would be possible. Thank you for all your dedication, diligence, and kindness. The field of science would be nothing without people like you.