MSCS ENGINEERED TO PRODUCE BDNF AND GENE EDITING CARGO FOR THE TREATMENT OF HUNTINGTON'S DISEASE

摘要 亨廷顿氏病（HD）是一种无法治愈的神经退行性疾病，并且存在对疾病的关键未满足的需求- 修改治疗。我们正在开发一种新的治疗HD的方法：人类间充质干细胞/基质细胞植入 骨髓间充质干细胞（MSC）被工程化以分泌脑源性神经营养因子（MSC/BDNF），这是一种在退行性变中所需的生长因子。 大脑的纹状体区域，沿着基因编辑货物，以特异性地降低突变RNA的产生水平， 蛋白BDNF在患有HD的人类和小鼠中是低水平的，并且BDNF在患有HD的转基因啮齿动物模型的脑中上调。 HD改善了疾病表型。由于在纹状体神经病理学中的促存活作用，BDNF是一种强的神经营养因子。 神经保护疗法的候选人。我们的实验室还开发了靶向基因沉默工具， 7），其允许广泛的原型间隔区邻近基序（PAM）靶向。我们已经展示了这个平台 当与强大的转录抑制因子KRAB融合时，可有效减少亨廷顿蛋白。我们已经证明 MSC是一种优秀的运载工具。我们正在测试通过枕大池注射作为脊髓/CSF的替代品 为未来可能接受我们团队开发的这种细胞和基因治疗产品的患者提供液体输送。蜂窝 纳米工程化的xCas 9疗法（CellNeXT）将MSC施用至纹状体的有益效果与MSC施用至纹状体的有益效果相结合。 BDNF生产的好处，而在组合减少水平的突变亨廷顿蛋白。与BDNF通过直接 当将载体注射或蛋白质施用到脑中时，MSC迁移到损伤区域并具有大量的 有益效果。虽然优化的MSC不会持续超过几个月，我们假设， BDNF的神经恢复作用将比MSC的存活更持久。这得到了我们实验室的动物数据的支持， 他人在我们的双盲疗效研究中，人MSC/BDNF的纹状体内递送显著降低了焦虑， 与溶媒相比，免疫抑制HD小鼠的神经发生显著增加，存活率增加 HD小鼠。我们已经证明，与载体相比，MSC/BDNF治疗减少了纹状体萎缩， 处理的HD小鼠（PMID：26765769）。这种恢复可能是由于刺激内源性神经发生的促进， BDNF的表达，并通过MSC分泌各种互补治疗因子而增强。在计划的研究中， 我们将进行以下研究，以支持向FDA提交的研究性新药申请：在目标1中，我们将评估 xCas 9在患者iPSC衍生的NSC和神经元中的选择性和耐久性。在目标2中，我们将评估 在我们的新型HD小鼠模型中，给予CellNeXT。我们的研究将定义可重复的技术和方法， 药物非临床研究质量管理规范的水平，用于评价用于神经退行性疾病的细胞和基因治疗候选药物 紊乱我们将定位于将CellNeXT候选药物最初用于HD临床试验，并协助其他人 将来使用该产品治疗其他疾病。我们将更好地定义CellNeXT的作用机制，并定义 效力测定，使用突变亨廷顿蛋白的减少和纹状体萎缩的保留作为关键读数。

Abstract Huntington's disease (HD) is a neurodegenerative disorder with no cure, and there is a critical unmet need for disease- modifying treatments. We are developing a novel therapy for HD: implantation of human Mesenchymal Stem/Stromal Cells (MSCs) engineered to secrete Brain-Derived Neurotrophic Factor (MSC/BDNF), a growth factor needed in the degenerating striatal regions of the brain, along with gene editing cargo to specifically reduce levels of production of the mutant RNA and protein. BDNF is low in humans and mice with HD, and up- regulation of BDNF in the brains of transgenic rodent models of HD has ameliorated the disease phenotype. Due to pro-survival effects in striatal neuropathology, BDNF is a strong candidate for neuroprotective therapies. Our labs have also developed targeted gene silencing tools using a novel variant of Cas9 (xCas9 3.7) that allows for broad protospacer adjacent motif (PAM) targeting. We have demonstrated this platform is effective in reducing huntingtin when fused to a powerful transcriptional repressor, KRAB. We have demonstrated that MSCs are an excellent delivery vehicle. We are testing injection via the cisterna magna as a surrogate for spinal cord/CSF fluid delivery in future patients who might receive this cell and gene therapy product developed by our team. Cellular Nanoengineered xCas9 Therapy (CellNeXT) combines the beneficial effects of MSC administration to the striata with the benefits of BDNF production while in combination reduction levels of mutant huntingtin. Unlike BDNF delivery via direct vector injection or protein administration into the brain, MSCs migrate into the areas of damage and have numerous beneficial effects. Although optimized MSCs will not persist longer than several months, we hypothesize that the neurorestorative effects of BDNF will outlast the survival of MSCs. This is supported by animal data from our laboratory and others. In our double-blinded efficacy studies, intrastriatal delivery of human MSC/BDNF significantly reduced anxiety and significantly increased neurogenesis in immune suppressed HD mice, with increased survival, in comparison to vehicle treated HD mice. We have demonstrated that treatment with MSC/BDNF decreased striatal atrophy as compared to vehicle treated HD mice (PMID:26765769). This recovery may be due to the stimulation of endogenous neurogenesis promoted by BDNF and enhanced by the secretion of various complementary therapeutic factors by the MSCs. In the planned studies, we will perform the following studies in support of an investigational new drug filing to the FDA: in Aim 1 we will evaluate the selectivity and durability of xCas9 in patient iPSC-derived NSC and neurons. In Aim 2 we will assess dose and route of administration of CellNeXT in our novel HD mouse model. Our studies will define reproducible techniques and methods, at the level of Good Laboratory Practice, for evaluation of cell and gene therapy candidates to be used in neurodegenerative disorders. We will be positioned to move the CellNeXT candidate into clinical trials for HD initially, and to assist others in using the product for additional disorders in the future. We will better define mechanism of action of CellNeXT and will define potency assays, using reduction of mutant huntingtin and preservation of striatal atrophy as key readouts.