544. Targeted Gene Delivery To the Enteric Nervous System: α-Synuclein Impairs Colonic Motility

The enteric nervous system (ENS) is a division of the autonomic nervous system that coordinates the control of gastrointestinal (GI) function. Several diseases are associated with abnormalities of the ENS, for example, GI dysfunction is one of the most common non-motor complaints associated with Parkinson's disease (PD). GI pathology affects virtually every level of the GI tract, primarily presenting as decreased colonic motility. Recent studies have demonstrated that the protein, α-synuclein (α-syn), forms large inclusion within neurons of the ENS, suggesting that α-syn pathology within the ENS may play a role in the etiology of GI dysfunction in PD. To determine if α-syn expression within neurons of the ENS is sufficient to produce GI dysfunction, we aimed to overexpress human wildtype α-syn within the ENS using direct injections of adeno associated virus (AAV) to the descending colon. However, as there have been no focused investigations into the targeted transduction of the ENS using AAV, we first examined the efficiency and tropism of transduction using several AAV serotypes. Rats received direct injections (6 × 5ml) of either AAV 1, 2, 5, 6, 8 or 9 expressing green fluorescent protein (GFP) into the descending colon. Transduction occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction, with AAV9 primarily transducing neurons, and AAV6 transducing neurons as well as enteric glia. Transduction efficiency scaled with titer and time, to plateau at 4-weeks post injection at a titer of 6.25×1012 viral genomes (vg)/ml. Following characterization of AAV transduction in the ENS, it was determined that AAV9 demonstrated the highest levels of neuronal transduction, and was selected to deliver the α-syn transgene to the ENS. Adult male rats received direct injections (6 × 5ml at 1×1013vg/ml) of AAV9 expressing either human wildtype α-syn or a GFP control transgene into the descending colon. Four weeks post-surgery, colonic motility was assayed by quantifying the time necessary to transit and excrete a bead through 5cm of the descending colon. Animals that received AAV-α-syn had significantly impaired colonic motility, with a mean colonic transit time of 171.5 ± 43.4 minutes compared to animals that received AAV-GFP with a mean colonic transit time of 15 ± 6.8 minutes. Here we present a thorough characterization of the transduction profile of AAV in the ENS following direct injection to the descending colon. Further, using AAV to deliver human α-syn to neurons of the ENS, colonic motility was significantly decreased.