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

项目概述：肌肉睫状神经营养因子受体α（CNTFRα）的表达是由：1） 失神经病变53 -55，2）人类失神经疾病56，57，包括ALS 56，和3）所有ALS模型 测试.肌肉CNTFRα敲低抑制运动神经元（MN）轴突再生和运动恢复 在所有ALS模型中，神经损伤53并加速疾病，这表明肌肉CNTFRα诱导是一种 神经保护反应，可以加强治疗ALS。我们在SOD 1G 93 A中增加了肌肉CNTFRα， 用AAV载体（AAV1.1-CNTFRα）治疗ALS小鼠，延长存活时间并增加运动功能（无 副作用），即使在症状发作后很好地开始，也可以说是最有临床前景的 迄今为止的治疗，因为人类ALS直到症状发作后很久才治疗58 -61。我们发现向量衍生的 CNTFRα蛋白转位到MN并增加MN末端，提示其作用机制。这 治疗应该：1）抑制大多数/所有ALS，2）独立于ALS原因工作，3）由于 肌肉表达可以通过批准的人类基因治疗技术增加42 - 47，49，50，如我们使用的那些 这里.最可能参与肌肉CNTFRα抗ALS作用的配体是肌肉心肌营养素样 细胞因子（CLC）。我们同样增加了SOD 1G 93 A小鼠的肌肉CLC，再次延长了存活时间， 效果，使其成为另一种非常有前途的新ALS治疗方法。结合两种治疗方法表明， 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 作为ALS治疗的增强。为了使效果最大化，我们将在SOD 1G 93 A小鼠中进行剂量反应测试：1）a 密码子优化的CNTFRα cDNA，2）具有极大增强的肌肉转导的AAV衣壳（AAV1.1），3） 肌肉特异性启动子（tMCK），4）用于更快和更大表达的自身互补AAV，和5）IV 注射另一种下一代衣壳（AAV 218）用于转导更多肌肉。最好的治疗方法是 进一步检查：1）旋转杆和握力测试，2）qRT-PCR和原位杂交载体- 衍生的CNTFRα RNA，3）HA标签解剖学，以定位载体衍生的CNTFRα蛋白并鉴定潜在的 作用位点，4）多标记免疫组织化学以确定对ALS变性的影响，和5）两种 其他不同的ALS模型（SOD 1G 37 R和TDP-43 Q331 K小鼠），以广泛测试治疗。目标2：优化 并将肌肉CLC增强表征为ALS治疗。我们将完全按照 目的1，除了用CLC代替CNTFRα。目的3：优化和表征组合的CNTFRα和 CLC治疗。肌肉CLC和CNTFRα可能作为MN保护性CLC/CNTFRα复合物释放 使得CLC和CNTFRα增加可进一步增强功效。一个飞行员与1：1的比例， 未优化的CLC和CNTFRα载体在雌性中发现显著增强的作用。我们将测试其他 目标1和2中优化的CNTFRα和CLC处理的比例，以进一步提高雌性效应， 可能增强对雄性的影响。最佳治疗方法将按照目标1和目标2进行充分表征。

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.