215. Dimerizer-Regulated AADC Expression with Two AAV Vectors in 6-OHDA Lesioned Rats

Abstract

Recombinant AAV vectors containing the dimerizer-inducible system of transcriptional activation provide a promising strategy for regulated control of therapeutic gene expression in the CNS. To test the feasibility of applying this approach to our current treatment for Parkinson's disease, we explored regulation of expression of the enzyme Aromatic L-Amino Acid Decarboxylase (AADC) in rat striatum by means of a regulated AAV vector system. Expression of the AADC transgene was made dependent on reconstitution of a functional transcription factor (TF) by the dimerizer rapamycin. Two vectors were constructed: 1) AAV-CMV-TF and 2) AAV-Z12-hAADC. We evaluated them in vivo in a rodent model of Parkinson's disease. A 1:1 combination (3 × 1010 vg of each vector) was infused ipsilaterally into unilaterally 6-OHDA-lesioned rats. Induction of transgene expression was achieved via intraperitoneal administration of rapamycin at designated time points. Since increased expression of AADC in the lesioned striatum correlates with increased conversion of L-dopa to dopamine, we would expect to see altered behavioral rotational response to 5mg/kg L-dopa. This parameter was evaluated six times during the course of the experiment. The rotational response to L-dopa in the vector-infused (+) rapamycin group was significantly increased (P<0.003, one-way ANOVA) above both control groups (vector-infused (–) rapamycin and excipient-infused animals). There was no significant difference between the two control groups, suggesting that expression of AADC in the absence of induction was not high enough to elicit behavioral response. Seven weeks following intrastriatal infusion rats were sacrificed and tissue was processed for immunostaining. Serial sections of fixed brain tissue were stained for AADC. We quantitated the spread volume and positive cell number for AADC transgene-derived immunostaining by stereology for each animal. The vector-infused (+) rapamycin group had the greatest number of AADC positive cells (75,825+/−30,506 cells), followed by a lower number of positive cells (31,000+/−25,812 cells) in the vector-infused (–) rapamycin group (P<0.01, one-way ANOVA), and no detectable expression in the excipient-infused control group. Intensity of staining appeared much lower in the non-induced vector-treated rats compared to those that received rapamycin. In summary, this study demonstrated robust behavioral response of parkinsonian rats to AAV-mediated AADC treatment that could be regulated using the rapamycin system. A low level of AADC transgene expression was observed in the absence of rapamycin, however, it was not significant enough to elicit behavioral response. Recombinant AAV vectors controlled by rapamycin or rapamycin analogs show promise as candidates for CNS therapies in which regulation of the transgene may be important.