O.8 Intrathecal delivery of AAV9 vectors to model and rescue a large animal model of SMA

Abstract

Spinal muscular atrophy (SMA) is the leading genetic cause of death in infants and results in the loss of motoneurons (MNs). Gene transfer based on systemic delivery of scAAV9 viral vector expressing human SMN (hSMN) has successfully rescued severe SMA mice and lead to the development of clinical trials. We have previously demonstrated that intrathecal delivery of scAAV9 in the pig results in efficient transduction of MNs and thus is an alternative to intravenous delivery. We now propose (1) to create a large animal model of SMA by knocking down SMN in MNs with a scAAV9 vector expressing shRNA (2) to correct the SMA phenotype using a second injection of scAAV9 vector expressing hSMN. We injected 5-day old piglets with a single intrathecal dose of scAAV9-shRNA (6.5×10e12vg/kg, n = 6). A few weeks following injection, piglets developed symptoms resembling SMA (abnormal gait and posture) with hind limbs being particularly affected. The phenotype progressed rapidly to a general weakness with posterior paralysis. EMG analysis showed evidence of an active denervation process. We also observed a decrease in both the compound muscle action potential amplitude and the estimated number of MNs. This is remarkably similar to what is observed in SMA patients. Next, we investigated correction of the SMA phenotype. 5-day old piglets (n = 4) received both scAAV9-shRNA (6.5×10e12 vg/kg) and scAAVV9-hSMN (8×10e12 vg/kg) with a 24hrs interval between injections. Two piglets showed no phenotype whereas the other two developed weakness that did not progress. Importantly, no EMG abnormality was observed. We have demonstrated that knockdown of SMN in the pig results in a phenotype displaying the key features of SMA. Furthermore we showed that early reintroduction of SMN significantly alters the phenotype. We are now determining whether later reintroduction of SMN at the first sign of symptoms has a therapeutic benefit as this more closely mimics what will occur in a human clinical trial.