Precision base editing for the treatment of motor neuron diseases

运动神经元疾病（mnd），如脊髓性肌萎缩症（SMA）和肌萎缩侧索硬化症（ALS）是一类进行性神经退行性疾病，通常由运动神经元特别脆弱的轻微遗传异常引起。因为这些疾病都是罕见的，分子病理是如此多样化和未解决的，很难设想为每个人量身定制的治疗干预，或者一种单一的治疗方法来有效地治疗它们。目前的基因组编辑工具很容易编程，以针对离散的基因组位点和受影响的组织在很大程度上是相似的。因此，针对每种突变的mnd的通用基因组编辑治疗策略可以满足这一迫切需求。碱基编辑工具理论上可以纠正任何C:G>T:A和A:T>G:C转换，但决定碱基编辑效率和精度的因素尚未完全了解，并且在给定位点上的编辑结果往往是不可预测的。为了在大量基因座上开发碱基编辑校正策略，通过大量碱基编辑器变异体进行筛选——目前>10脱氨酶和>15 Cas蛋白的任何排列，以及对每个靶标的sgRNA组合进行计数——是难以实现的。为了促进碱基编辑策略的设计，需要清楚地了解Cas蛋白、脱氨酶和编辑结果的序列决定因素。我们打算开发一个通用的工作流程，为MND snp设计有效的碱基编辑策略，并将这些工具交付给MND受影响的组织。SMA是一种单基因MND，具有明确的遗传原因，动物模型包含人类致病基因，忠实地概括了SMA患者的疾病表型。成功开发一种有效且安全的SMA基因组编辑治疗方法，将有助于开发针对其他遗传性mmd的类似基因组编辑治疗方法，包括某些形式的家族性ALS。在本项目中，我们将(1)建立一个碱基编辑的计算预测模型，以方便设计有效的碱基编辑策略；(2)制定方案，有效地将碱基编辑疗法传递到小鼠疾病相关组织，以实现基因组编辑；(3)利用该管道优化一种碱基编辑疗法，以挽救小鼠的SMA，并开发针对其他MND致病突变的基因组编辑疗法，首先关注北美最常见的导致ALS的单点突变SOD1A4V。我将利用布罗德研究所的世界级基因组学环境和斯坦利精神病学研究中心的aav递送专业知识来实现这些目标，并培养技能，帮助我作为一名独立研究者继续这项工作。通过导师会议和拨款写作和数据可视化课程，我将提高我的科学沟通技能，以便我可以作为教员成功竞争额外的NIH R01和R21资金。通过参加会议和发表我的工作，我打算确立自己在治疗性基因组编辑领域的领导者地位。

Motor neuron diseases (MNDs) such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are a class of progressive neurodegeneration disorders, often caused by minor genetic abnormalities to which motor neurons are particularly vulnerable. Because each of these diseases is rare and the molecular pathologies so diverse and unresolved, it is hard to envision a therapeutic intervention tailored to each individually, or a single treatment to effectively treat them all. Current genome editing tools are easily programmable to target discrete genomic loci and affected tissues of MNDs are largely similar. Thus, a general genome editing therapeutic strategy for MNDs fitted to each mutation could meet this urgent need. Base editing tools can theoretically correct any C:G>T:A and A:T>G:C transition, yet the factors that determine efficiency and precision of base editing are not fully understood, and editing outcomes at a given locus are frequently unpredictable. For the development of base editing correction strategies at a great number of loci, screening through a multitude of base editor variants – currently any permutation of >10 deaminase enzymes and >15 Cas proteins, and counting – and sgRNA combinations for every target is prohibitive. A clear understanding of Cas protein, deaminase, and sequence determinants of editing outcomes is needed to facilitate the design of base editing strategies. We intend to develop a general workflow to design effective base editing strategies for causal MND SNPs and deliver these tools to MND affected tissues. SMA is a monogenic MND with a well-defined genetic cause, and animal models harboring the human causal gene that faithfully recapitulate disease phenotypes of SMA patients. Successful development of an effective and safe SMA genome editing treatment will assist the development of similar genome editing therapeutics for other genetic MNDs, including some forms of familial ALS. In this project we will (1) create a computational predictive model of base editing to facilitate the design of effective base editing strategies; (2) develop protocols to efficiently deliver base editing therapeutics to disease relevant tissues in mice to enable genome editing; and (3) use this pipeline to optimize a base editing therapeutic to rescue SMA in mice, and develop genome editing therapeutics for other causal MND mutations, focusing first on the most common single point mutation causal to ALS in North America, SOD1A4V. I will take advantage of the world-class genomics environment of the Broad Institute and the expertise in AAV-delivery at the Stanley Center for Psychiatric Research to realize these goals and develop skills that will help me to continue this work as an independent investigator. Through mentorship meetings and courses on grant-writing and data visualization I will improve my science-communication skills so that I may compete successfully for additional NIH R01 and R21 funding as a faculty member. By attending conferences and publishing my work I intend to establish myself as leader in the field of therapeutic genome editing for MND.