Cellular perturbations to enhance precise therapeutic genome editing to treat FTD/ALS

细胞扰动增强精确治疗基因组编辑以治疗 FTD/ALS

• 批准号: 10607752
• 负责人: Bria Macklin
• 金额: $ 6.91万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607752
• 关键词: Address; Adjuvant Therapy; Affect; Alleles; Amyotrophic Lateral Sclerosis; Biological Assay; Biological Process; CRISPR therapeutics; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Core Facility; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Gene; DNA Repair Pathway; DNA Sequence Alteration; DNA-Directed RNA Polymerase; Data; Disease; Dominant-Negative Mutation; Doxycycline; Engineering; Environment; Enzymes; Event; Excision; Exhibits; Frameshift Mutation; Frequencies; Frontotemporal Dementia; Future; Gene Fusion; Gene Targeting; Genes; Genetic; Genetic Diseases; Genetic Materials; Genetic Transcription; Grant; Guide RNA; Impairment; In Vitro; Induced pluripotent stem cell derived neurons; Knock-out; Ligation; Measures; Mentorship; Messenger RNA; Methods; Mutate; Mutation; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Nonhomologous DNA End Joining; Nonsense-Mediated Decay; Outcome; Pathologic; Pathway interactions; Phenotype; Prevalence; Probability; Process; Proteins; Proteomics; Protocols documentation; RNA; Regulation; Research; Role; Small Interfering RNA; Specificity; System; Therapeutic; Time; Tissues; Training; Transgenic Organisms; Virus-like particle; Work; base editing; cell type; comparative; design; differentiation protocol; directed differentiation; disease phenotype; frontotemporal lobar dementia amyotrophic lateral sclerosis; fused in sarcoma; gene repression; in vivo; induced pluripotent stem cell; insertion/deletion mutation; insight; knock-down; mutant; neurogenetics; next generation sequencing; novel virus; overexpression; pharmacologic; postmitotic; repaired; small molecule inhibitor; synergism; targeted treatment; therapeutic candidate; therapeutic genome editing; transcription factor; transcriptomics

PROJECT SUMMARY CRISPR holds great promise toward therapeutically editing pathological mutations in the gene fused in sarcoma (FUS), known to cause 5% of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. The FUS gene is ideal for therapeutic editing, as it is a dominant-negative genetic mutation, allowing for one allele to produce enough of the FUS protein for cells to survive. However, there are several challenges that must be addressed before this promise can be realized, including the inefficient delivery of Cas9 and gRNA into cells and the lack of indel formation after Cas9 cleavage. Towards this, my lab has established a robust neuronal differentiation protocol via doxycycline-inducible transcription factors. With this protocol, I can derive phenotypic neurons from iPSCs that recapitulate disease phenotypes within 7 days13. We have additionally engineered virus-like particles (VLP), that harbor no genetic material, to deliver Cas9 protein and gRNA to neurons12. In this grant, I propose exploring small perturbations within neurons to gain further insights into how CRISPR therapies can be designed for the precise editing of neurodegenerative diseases. I have collected preliminary data with neurons and iPSCs, using the same CRISPR editing strategy, and found that iPSCs and neurons have divergent editing outcomes. Specifically, we used gRNA designed to cleave sequences in the NEFL and B2M genes. In both genes, we found that iPSCs had multiple editing outcomes not present in neurons, commonly large deletions. Editing in neurons frequently resulted in no indel being produced. The few indels that do occur in neurons appear to be almost exclusively small insertions (+1 indel) are produced via mutagenic NHEJ. These small indels are ideal to cause frameshifts and induce nonsense-mediated decay and represent an optimal strategy for therapeutic editing in neurons. I propose studying how iPSC-neurons can be perturbed to increase the prevalence of our desired editing outcome. Using our directed differentiation protocol as well as our VLP delivery system, I will explore how indel frequency and identity are affected by changes in the transcription of the edited gene and the expression and activity of DNA damage proteins. In Aim 1, I will alter the transcription of genes to explore how interactions with RNA polymerase change editing frequency and indel formation. In Aim 2, I will investigate how changing the expression or activity of DNA repair enzymes affects editing outcomes. The insight gained from the work described in this grant can be used to precisely edit neurons to silence pathological genetic mutations, like FUS mutations. Future studies would also apply this editing strategy to other mutated genes in neurons and potentially other post-mitotic cells. The superior training environment provided at the Gladstone Institutes will allow me the opportunity to explore these aims with expert research mentorship, support from core facilities, and beneficial collaborations.

项目摘要 CRISPR在治疗上编辑融合在基因中的病理性突变方面具有很大的前景。 肉瘤（FUS），已知引起5%的肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD） 例FUS基因是治疗性编辑的理想选择，因为它是一种显性负性基因突变， 一个等位基因产生足够的FUS蛋白质供细胞存活。然而，有几个挑战， 在实现这一承诺之前，必须解决一些问题，包括将Cas9和gRNA低效地递送到 细胞和Cas9切割后indel形成的缺乏。为此，我的实验室已经建立了一个强大的神经元 分化方案通过强力霉素诱导的转录因子。通过这个实验，我可以推导出 来自iPSC的神经元在7天内重现疾病表型13。我们还设计了 病毒样颗粒（VLP），不携带遗传物质，将Cas9蛋白和gRNA递送到神经元12。在这 我建议探索神经元内的小扰动，以进一步了解CRISPR疗法是如何实现的。 可以被设计用于神经退行性疾病的精确编辑。 我已经收集了神经元和iPSC的初步数据，使用相同的CRISPR编辑策略， 发现iPSC和神经元具有不同的编辑结果。具体来说，我们使用了设计用于切割 NEFL和B2M基因中的序列。在这两个基因中，我们发现iPSC具有多种编辑结果， 存在于神经元中，通常是大的缺失。神经元中的编辑经常导致不产生indel。 在神经元中确实发生的少数插入缺失似乎几乎完全是产生的小插入（+1插入缺失）。 通过诱变NHEJ。这些小的插入缺失是引起移码和诱导无义介导的衰变的理想选择 并代表了神经元治疗编辑的最佳策略。 我建议研究如何干扰iPSC神经元，以增加我们所期望的编辑的普遍性。 结果。使用我们的定向分化方案以及我们的VLP递送系统，我将探索插入缺失如何在细胞中表达。 频率和同一性受到编辑基因转录和表达变化的影响， DNA损伤蛋白的活性。在目标1中，我将改变基因的转录，以探索如何与 RNA聚合酶改变编辑频率和插入缺失形成。在目标2中，我将研究如何改变 DNA修复酶的表达或活性影响编辑结果。 从这项资助中描述的工作中获得的洞察力可以用于精确编辑神经元以使其沉默 病理性基因突变，如FUS突变。未来的研究也将把这种编辑策略应用于其他领域。 神经元和潜在的其他有丝分裂后细胞中的突变基因。提供的上级培训环境， 格莱斯顿研究所将让我有机会与专家研究导师一起探索这些目标， 核心设施的支持和有益的合作。