Creation and correction of Spinal Muscular Atrophy in the pig

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种常染色体隐性遗传疾病，是婴儿死亡的最常见遗传原因。SMA是由运动神经元存活基因1（SMN 1）的缺失或突变以及SMN 2基因的保留引起的，这导致运动神经元的SMN水平不足。在SMA小鼠中通过血管系统或经由鞘内（CSF）递送引入scAAV 9-SMN导致表型的显著校正。鞘内递送的优点是病毒滴度较低， 可用于校正，从而简化了scAAV 9-SMN的生产。虽然已经在猪和猴中研究了运动神经元的转导效率，但尚未研究在大型动物模型中纠正SMA表型的能力。这是由于目前没有SMA的大型动物模型。SMA的大型动物模型可用于解决运动神经元中高SMN水平的重要性、获得临床益处所需的SMN转导效率以及何时需要诱导SMN以获得临床益处。因此，我们提出在SMA的临床相关大型动物猪模型中优化scAAV 9-SMN向运动神经元的递送，并开发用于在该模型中治疗SMA的基因治疗方案。特别是，我们将解决转导效率，包括重复给药，以及何时必须引入SMN来纠正SMA猪模型。 我们将通过使用scAAV 9- shRNA：SMN敲低减少所需神经元中的SMN来创建猪SMA模型。该shRNA对猪SMN具有特异性，并且不影响人SMN的水平。通过鞘内递送表达SMN和GFP的scAAV 9 shRNA构建体来递送shRNA。在5日龄仔猪中进行注射，将使用EMG、临床表现和斜坡试验评价受影响的猪。GFP可用于跟踪运动神经元转导的效率，EMG可用于跟踪功能性运动神经元损失的进展。然后，我们将在疾病的各个阶段引入scAAV 9-SMN，包括症状前、表型的第一个迹象和后肢严重虚弱的后期阶段。此外，我们将确定是否必须在CMAP和MUNE降低之前恢复SMN水平，这反过来将表明SMA患者何时需要恢复SMN以获得最大临床获益。这些实验将设定SMA中所有SMN诱导治疗的参数，并允许确定何时引入治疗剂。

DESCRIPTION (provided by applicant): 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.