295. Intrathecal Delivery of rAAV9-ABCD1 by Osmotic Pump in a Mouse Model of Adrenomyeloneuropathy

Abstract

Adrenomyeloneuropathy (AMN) is a debilitating neurological disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. The Abcd1-/- mouse develops a phenotype similar to AMN, manifesting spinal cord axon degeneration as well as peripheral neuropathy due to affected dorsal root ganglion neurons (DRGs). We previously reported successful transduction of central nervous system cells in vitro and in vivo using recombinant adeno-associated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene. Unfortunately, intravenous delivery in young mice was associated with cardiac toxicity due to transgene overexpression. We therefore set out to optimize delivery to the spinal cord while minimizing systemic leakage using an intrathecal osmotic pump. Self complementary AAV9 GFP(scAAV9GFP) and rAAV9 encoding ABCD1 (rAAV9-ABCD1) were delivered to Abcd1-/- mice intrathecally (IT) either by bolus over a 2min duration or by osmotic pump over 24h duration with PBS injection as sham control. Two weeks after injection, mice were sacrificed and perfused with 4% PFA. Tissues were then collected, sectioned and stained for immunofluorescence analysis. scAAV9-GFP delivered IT by osmotic pump led to widespread expression across CNS-relevant cell types and DRGs in a dose-dependent manner. Spinal cord and DRG had higher expression compared with brain, but GFP expression was also detected in peripheral organs (liver, heart and adrenal gland), with highest expression seen at 3X1011GC. A similar distribution pattern of ABCD1 protein was detected after rAAV9-ABCD1 intrathecal pump delivery. In general, higher doses (2X1011GC and 1X1011GC) led to more expression in CNS and peripheral organs compared with a lower dose (0.5X1011GC). However, widespread expression of ABCD1 across CNS was even detected after direct intrathecal bolus injection of 0.5X1011GC. Importantly, the same dose delivered by pump led to higher expression in brain and spinal cord far from the injection site and comparatively less leakage to peripheral organs compared with bolus injection. Preliminary experiments delivering rAAV9-ABCD1 at 0.5X1011GC by intracerebroventricular approach suggest behavioral improvement in the Abcd1-/- mouse despite localized expression in brain. We therefore anticipate even better performance at this dose using the outlined intrathecal pump delivery. We conclude that rAAV9-mediated ABCD1 gene transfer via intrathecal osmotic pump leads to more uniform and widespread gene delivery to CNS with reduced leakage into the systemic circulation compared with intrathecal bolus injection.