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

基本信息

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
项目类别：
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

项目摘要

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在融合的基因中对治疗编辑病理突变有很大的希望肉瘤（FUS），已知会引起5％的肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）案例。 FUS基因是治疗编辑的理想选择，因为它是一个主要的阴性基因突变，允许一个产生足够的FUS蛋白的等位基因生存。但是，有几个挑战必须在实现这一承诺之前解决，包括CAS9和GRNA的效率低下 Cas9裂解后细胞和缺乏胶原形成。在此方面，我的实验室建立了一个强大的神经元通过强力霉素诱导的转录因子分化方案。使用此协议，我可以得出表型来自IPSC的神经元在7天内概括了疾病表型13。我们还设计了没有遗传物质的病毒样颗粒（VLP）将Cas9蛋白和GRNA传递到Neurons1212。在这个格兰特，我建议探索神经元内的小扰动，以进一步了解CRISPR疗法可以设计用于神经退行性疾病的精确编辑。我已经使用相同的CRISPR编辑策略收集了使用神经元和IPSC的初步数据，并且发现IPSC和神经元具有不同的编辑结果。具体来说，我们使用设计的GRNA裂解 NEFL和B2M基因中的序列。在这两个基因中，我们都发现IPSC具有多个编辑结果存在于神经元中，通常是大缺失。神经元中的编辑通常导致没有产生indel。确实发生在神经元中的少数插入似乎几乎是仅插入的小（+1 indel）通过诱变NHEJ。这些小的indels是引起移架并引起废话介导的衰减的理想选择并代表神经元中治疗编辑的最佳策略。我建议研究如何扰动iPSC-神经元以增加我们所需的编辑的流行率结果。使用我们的定向分化协议以及我们的VLP交付系统，我将探讨Indel如何频率和身份受编辑基因转录和表达的变化的影响， DNA损伤蛋白的活性。在AIM 1中，我将改变基因的转录，以探索如何与 RNA聚合酶会改变编辑频率和indel形成。在AIM 2中，我将调查如何改变 DNA修复酶的表达或活性会影响编辑结果。从本赠款中描述的工作中获得的洞察力可用于精确编辑神经元以保持沉默病理性的遗传突变，例如FUS突变。未来的研究还将将此编辑策略应用于其他神经元和可能其他有丝分裂后细胞的突变基因。提供的卓越培训环境 Gladstone Institutes将使我有机会通过专家研究指导，探索这些目标，核心设施的支持和有益的合作。