Gene Therapy Targeting of CNTFRalpha and CLC in Muscle to Treat ALS

项目摘要：肌肉睫状神经营养因子受体α(CNTFRα)表达诱导：1) 去神经损伤53-55，2)人类去神经疾病56，57，包括ALS 56，3)在所有ALS模型中 测试过。肌肉CNTFRα基因敲除后抑制运动神经元轴突再生和运动恢复 在所有的肌萎缩侧索硬化症模型中，神经损伤53和加速疾病，提示肌肉CNTFRα的诱导是一种 可以增强神经保护反应来治疗肌萎缩侧索硬化症。我们在SOD1G93A中增加了肌肉CNTFRα 携带AAV载体(AAV1.1-CNTFRα)的ALS小鼠，延长存活并增加运动功能(未 副作用)，即使在症状出现后很好地开始，这使得它可以说是最有临床前景的 迄今为止的治疗，因为人类肌萎缩侧索硬化症直到症状出现后才得到很好的治疗。我们发现向量派生的 CNTFRα蛋白转位至MN，增加MN末端，提示其作用机制。这 治疗应该：1)抑制大部分/全部肌萎缩侧索硬化症，2)独立于肌萎缩侧索硬化症的原因工作，以及3)可翻译，因为 肌肉表达可以通过批准的人类基因治疗技术来增加42-47，49，50，就像我们使用的那些 这里。肌肉CNTFRα的S抗肌萎缩侧索硬化症作用的最可能的配体是肌肉促心肌营养素 细胞因子(CLC)。我们同样增加了SOD1G93A小鼠的肌肉CLC，再次延长了没有侧翼的存活时间 效果，使其成为另一种非常有前途的ALS治疗新方法。将这两种疗法结合起来表明，这可能 是一种更有效的治疗方法。我们将：目标1：优化和表征肌肉CNTFRα 增强作为ALS的一种治疗方法。为了使效果最大化，我们将在SOD1G93A小鼠中进行剂量反应测试：1)a 密码子优化的CNTFRAAVcDNA2)一个肌肉转导显著增强的α衣壳(AAV1.1)，3)a 肌肉特异性启动子(TMCK)，4)自身互补的AAV，以更快和更大的表达，以及5)IV 注射下一代衣壳蛋白(AAV2I8)以转导更多的肌肉。最好的治疗方法是 进一步检查：1)旋转棒和握力试验，2)qRT-PCR和载体的原位杂交- 3)HA标签解剖以定位载体衍生的αα蛋白并鉴定其潜在的功能 作用部位，4)多标记免疫组织化学以确定对ALS退变的影响，以及5)两个 其他不同的ALS模型(SOD1G37R和TDP-43Q331K小鼠)以广泛测试该治疗方法。目标2：优化 并将肌肉CLC增强作为ALS的治疗方法。我们将进行的实验与 目的1，用CLC而不是CNTFRα。目的3：优化和表征α和CNTFR联合应用 《中图法》治疗。肌肉ClC和CNTFRα可能以MN保护性ClC/CNTFRα复合体的形式释放 因此，CLC和CNTFRα的增加可能会进一步增强疗效。飞行员的比例是1：1 未经优化的CLC和CNTFRα载体在雌性中发现显著增强的效果。我们将测试其他 在目标1和2中优化了CNTFRα和CLC处理的比率，以进一步提高雌性效应和 潜在地增强了对男性的影响。然后，最好的治疗将如目标1和目标2所示得到充分描述。

Project Summary: Muscle ciliary neurotrophic factor receptor α (CNTFRα) expression is induced: 1) by denervating nerve lesion53-55, 2) in human denervating diseases56,57, including ALS56, and 3) in all ALS models tested. Muscle CNTFRα knockdown inhibits motor neuron (MN) axon regeneration and motor recovery after nerve lesion53 and accelerates disease in all the ALS models, suggesting the muscle CNTFRα induction is a neuroprotective response that could be enhanced to treat ALS. We increased muscle CNTFRα in SOD1G93A ALS mice with an AAV vector (AAV1.1-CNTFRα), extending survival and increasing motor function (without side effect) even when started well after symptom onset, making it arguably the most clinically promising treatment to date since human ALS is not treated until well after symptom onset58-61. We find vector-derived CNTFRα protein translocated to MNs and increased MN terminals, suggesting a mechanism of action. This treatment should: 1) inhibit most/all ALS, 2) work independent of ALS causes, and 3) be translatable since muscle expression can be increased with approved human gene therapy techniques42-47,49,50, like those we use here. The ligand most likely involved in muscle CNTFRα's anti-ALS effects is muscle cardiotrophin-like cytokine (CLC). We similarly increased muscle CLC in SOD1G93A mice, again extending survival without side effect, making it another very promising new ALS treatment. Combining the two treatments suggests this could be an even more effective treatment. We will: Aim 1: Optimize and Characterize muscle CNTFRα enhancement as an ALS treatment. To maximize effect, we will dose response test in SOD1G93A mice: 1) a codon optimized CNTFRα cDNA, 2) an AAV capsid with greatly enhanced muscle transduction (AAV1.1), 3) a muscle specific promotor (tMCK), 4) self-complementary AAV for faster and greater expression, and 5) IV injection of another next-gen capsid (AAV2i8) for transduction of more muscles. The best treatment will be further examined with: 1) rotarod and grip strength tests, 2) qRT-PCR and in situ hybridization for vector- derived CNTFRα RNA, 3) HA tag anatomy to localize vector-derived CNTFRα protein and identify potential sites of action, 4) multi-label immunohistochemistry to determine effects on ALS degeneration, and 5) two other diverse ALS models (SOD1G37R and TDP-43Q331K mice) to broadly test the treatment. Aim 2: Optimize and Characterize muscle CLC enhancement as an ALS treatment. We will run experiments exactly as in Aim 1, except with CLC instead of CNTFRα. Aim 3: Optimize and Characterize combined CNTFRα and CLC treatment. Muscle CLC and CNTFRα are likely released as a MN protective CLC/CNTFRα complex such that an increase in both CLC and CNTFRα may further enhance efficacy. A pilot with a 1:1 ratio of the non-optimized CLC and CNTFRα vectors found a substantially enhanced effect in females. We will test other ratios with CNTFRα and CLC treatments optimized in Aims 1 and 2, to further increase the female effect and potentially enhance effect in males. Best treatments will then be fully characterized as in Aims 1 and 2.