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

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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.