Spinal muscular atrophy (SMA) is an autosomal recessive disorder which is the most common genetic cause of infant death. SMA is caused by loss or mutation of survival motor neuron 1 gene (SMN1) and retention of the SMN2 gene, which leads to insufficient levels of SMN for motor neurons. Introduction of scAAV9-SMN either through the vascular system or via intrathecal (CSF) delivery in SMA mice results in a marked correction of the phenotype. The advantage of intrathecal delivery is the lower titer of virus that can be used for correction, thus simplifying the production of scAAV9-SMN. While the efficiency of transduction of motor neurons has been investigated in pig and in monkeys, the ability to correct a SMA phenotype in a large animal model has not been investigated. This is due to fact that there is no large animal model of SMA currently available. A large animal model of SMA can be used to address the importance of high SMN levels in motor neurons, the efficiency of SMN transduction required to obtain a clinical benefit, and when SMN needs to be induced to obtain clinical benefit. We therefore propose to optimize scAAV9-SMN delivery to motor neurons in a clinically relevant large animal porcine model of SMA and develop gene therapy protocols for the treatment of SMA in this model. In particular, we will address transduction efficiency including repeat administration and when SMN must be introduced to correct a pig model of SMA. We will create the pig SMA model by reducing SMN in the required neurons using scAAV9- shRNA: SMN knockdown. The shRNA is specific for pig SMN and does not affect the levels of human SMN. The shRNA is delivered via intrathecal delivery of a scAAV9shRNA construct that expresses SMN and GFP. Injection is performed in 5-day-old piglets and EMG, clinical presentation, and a ramp test will be used to evaluate the affected pig. The GFP can be used to follow the efficiency of motor neuron transduction and EMG to follow the progression of functional motor neuron loss. We will then introduce scAAV9-SMN at various stages of the disease including pre-symptomatic, at the first signs of phenotype, and later stages when the hind limbs are severely weak. Furthermore, we will determine if restoration of SMN levels must occur prior to CMAP and MUNE reduction, which will in turn indicate when SMN needs to be restored in SMA patients to obtain the greatest clinical benefit. These experiments will set the parameters for all SMN induction therapies in SMA and allow determination of when to introduce a therapeutic agent.
Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by deficiency of the survival motor neuron (SMN) protein and is the most common genetic cause of death in infants. Self- complementary adeno associated virus 9 (scAAV9), a gene therapy vector, carrying a construct capable of producing SMN (scAAV9-SMN), can restore SMN to the required cell types and as thus can correct SMA in mice. In order to develop this therapeutic further we are developing a pig model of SMA that can be rescued with scAAV9-SMN. We wish to determine the required distribution and timing of administration of this therapeutic agent in a large animal prior to the start of human clinical trials. Since the ig is similar in size and has a closed brain blood barrier at birth like infants, these studies will directly inform the design of future human clinical trials in SMA.