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

MSCS 旨在生产用于治疗亨廷顿病的 BDNF 和基因编辑货物

• 批准号: 10443416
• 负责人: Kyle Fink
• 金额: $ 38.89万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443416
• 关键词: Address; Adult; Alleles; Animal Model; Anxiety; Apoptosis; Area; Atrophic; Award; BDNF gene; Binding; Biological Assay; Brain; Brain region; Brain-Derived Neurotrophic Factor; CRISPR/Cas technology; Cell Therapy; Cell secretion; Cells; Chromatin; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Corpus striatum structure; DNA Binding Domain; Data; Disease; Dose; Double-Blind Method; Engineering; Epigenetic Process; Evaluation; Funding; Future; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Transcription; Glycoproteins; Grant; Growth Factor; Guide RNA; Half-Life; Heterochromatin; Human; Huntington Disease; Huntington gene; Immune; Impairment; Induced pluripotent stem cell derived neurons; Injections; Intraventricular; Investigational Drugs; Knowledge; Laboratories; Lead; Length; Link; Liquid substance; Lyssavirus; Mesenchymal; Methods; Mission; Mouse Strains; Mus; National Institute of Neurological Disorders and Stroke; Neurodegenerative Disorders; Neurons; NuRD complex; Pathway interactions; Patients; Phase III Clinical Trials; Positioning Attribute; Precipitation; Precision therapeutics; Production; Proteins; Protocols documentation; Publishing; RNA; Recovery; Repression; Reproducibility; Research; Rodent Model; Route; Safety; Signal Pathway; Signal Transduction; Spinal Cord; Stromal Cells; Techniques; Technology; Testing; Therapeutic; Transcription Repressor; Transgenic Organisms; Tropism; Up-Regulation; Validation; Variant; Vesicle; Vesicular stomatitis Indiana virus; Work; Zinc Fingers; animal data; base; cell type; cellular engineering; cisterna magna; delivery vehicle; design and construction; disease phenotype; effectiveness evaluation; efficacy study; engineered stem cells; excitotoxicity; gene repression; gene therapy; good laboratory practice; implantation; induced pluripotent stem cell; knock-down; mouse model; mutant; nanoengineering; nerve stem cell; nervous system disorder; neurogenesis; neuropathology; neurorestoration; next generation sequencing; novel; novel therapeutics; oxidative damage; phase I trial; preservation; rabies virus glycoprotein G; recruit; restoration; stem; stem cell delivery; therapy development; tool; transcription factor; vector

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.

摘要 亨廷顿病(HD)是一种无法治愈的神经退行性疾病，对疾病的迫切需求尚未得到满足- 修改治疗方法。我们正在开发一种治疗HD的新方法：人骨髓间充质干细胞/基质细胞移植 (MSCs)被设计为分泌脑源性神经营养因子(MSC/BDNF)，这是退变过程中所需的生长因子 大脑的纹状体区域，以及基因编辑货物，以特别减少突变RNA和 蛋白。脑源性神经营养因子在人和先天性巨噬细胞病小鼠中的表达水平较低，而转基因啮齿动物模型中脑源性神经营养因子的表达上调。 HD改善了疾病的表型。由于在纹状体神经病理中的促存活作用，BDNF是一种强大的 神经保护疗法的候选人。我们的实验室还使用一种新的变体开发了有针对性的基因沉默工具 Cas9(xCas9 3.7)，允许广泛的Protspacer邻近基序(PAM)靶向。我们已经演示了这个平台 当与强大的转录抑制因子KRAB融合时，有效地减少了亨廷顿蛋白。我们已经证明了 MSCS是一种出色的交付工具。我们正在测试通过枕大池注射作为脊髓/脑脊液的替代品 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的液体输送。蜂窝 纳米工程xCas9疗法(CellNext)将MSC给药对纹状体的有益效果与 在联合降低突变亨廷顿蛋白水平的同时，BDNF生产的好处。与BDNF通过直接交付不同 向脑内注射载体或注射蛋白质时，MSCs迁移到损伤区域，并有许多 有益的影响。尽管优化的MSCs不会持续超过几个月，但我们假设 脑源性神经营养因子的神经修复作用将超过MSCs的存活时间。这得到了来自我们实验室的动物数据的支持 其他。在我们的双盲疗效研究中，纹状体内注射人骨髓间充质干细胞/脑源性神经营养因子显著降低焦虑和 与赋形剂相比，免疫抑制HD小鼠的神经再生显著增加，存活率增加 治疗后的HD小鼠。我们已经证明，与赋形剂相比，使用MSC/BDNF治疗可以减少纹状体萎缩 经处理的HD小鼠(PMID：26765769)。这种恢复可能是由于促进内源性神经发生的刺激。 骨髓间充质干细胞分泌多种补充性治疗因子，促进BDNF表达。在计划中的研究中， 我们将进行以下研究，以支持向FDA提交研究用新药：在目标1中，我们将评估 XCas9在患者IPSC来源的神经干细胞和神经元中的选择性和持久性。在目标2中，我们将评估剂量和途径 在我们的新型HD小鼠模型中对CellNext进行管理。我们的研究将定义可重复使用的技术和方法， 用于神经退行性变的细胞和基因治疗候选药物评估的良好实验室操作规范水平 精神错乱。我们将在最初阶段将CellNext候选者转移到HD的临床试验中，并帮助其他人 在未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，并将定义 效力分析，以减少突变型亨廷顿蛋白和保存纹状体萎缩为关键读数。