Prenatal pulmonary cell gene editing to cure monogenic lung diseases

项目总结 先天性单基因肺部疾病，包括囊性纤维化、表面活性蛋白紊乱和α-1 抗胰蛋白酶缺乏症(A1ATD)，可导致围产期呼吸衰竭、死亡或慢性肺部疾病。尽管 医学进步，治疗选择有限，往往集中于治疗疾病并发症，并达到顶峰 许多患者需要进行肺移植。因此，迫切需要针对单基因的新疗法。 肺部疾病。许多导致肺部疾病的单基因突变是众所周知的，可以在出生前诊断出来， 而且，通常情况下，一种突变导致了大多数病例。G-→A突变(Glu342Lys，PIZ等位基因) SERPINA1基因占A1ATD突变的90%，并导致严重疾病，增加了患A1ATD的风险 发展为慢性阻塞性肺疾病。CRISPR基因编辑技术的进展提供了一种 前所未有的机会永久纠正单基因肺部疾病的致病突变 只需一次治疗。尽管令人鼓舞，但针对成人其他器官的体内CRISPR基因编辑研究 人类疾病的小鼠模型突出了出生后方法的局限性，包括低水平的 同源定向修复(HDR)由不可及和非增殖性靶细胞和成熟免疫引起 障碍。这些障碍在出生后的肺中更加令人望而生畏，因为肺是一个具有免疫和生理功能的屏障器官 只有1%的上皮祖细胞(大多数肺部疾病的靶细胞群)存在障碍 在动态平衡状态下骑车。在子宫内进行基因编辑有可能克服这些障碍并治疗围产儿 致命性疾病在不可逆转的病理发作之前。胎儿具有免疫耐受性和祖细胞 包括肺在内的多个器官的细胞高度增殖，在发育过程中可以接触到。这个 这项建议的目的是建立产前肺基因编辑的可行性，并使用产前基因编辑 将小鼠A1ATD模型作为单基因肺部疾病的模型。我们的中心假设是 产前肺细胞基因编辑比产后编辑更有效率，而产前基因编辑不会 对编辑后的肺祖细胞的命运有不利影响。我们假设产前人类发育迟缓可以提供 A1ATD患者循环α-1抗胰蛋白酶蛋白治疗水平与肺细胞基因校正 老鼠模型。我们的假设是基于我们发表的数据，证明了有效的肝脏和肺脏 通过产前CRISPR-非同源末端连接(NHEJ)进行的上皮细胞编辑。为了达到我们的目标，我们将 追求以下目标：1)评估正常和新生儿损伤状态下的产前肺细胞基因编辑， 2)评估产前以肺为靶点的HDR，并与出生后HDR进行比较；3)产前纠正A1ATD 并将其与出生后的HDR进行比较。我们的研究在肺部的产前计时和靶向方面具有创新性。 用于治疗性基因编辑。这项工作的重大贡献将是为一个-- 这是一种治疗A1ATD的长期注射疗法，适用于其他单基因肺部疾病。

PROJECT SUMMARY Congenital monogenic lung diseases, including cystic fibrosis, surfactant protein disorders, and alpha-1 antitrypsin deficiency (A1ATD), can cause perinatal respiratory failure and death or chronic lung disease. Despite medical advances, therapy options are limited, often focused on treating disease complications, and culminating in the need for a lung transplant for many patients. Thus, there is a critical need for novel therapies for monogenic lung diseases. Many monogenic lung disease-causing mutations are well known, can be diagnosed before birth, and, often, one mutation is responsible for the majority of cases. A G→A mutation (Glu342Lys, the PiZ allele) in the SERPINA1 gene accounts for 90% of A1ATD mutations and results in severe disease, increasing the risk of developing chronic obstructive pulmonary disease. Advances in CRISPR gene editing technology provide an unprecedented opportunity to permanently correct disease-causing mutations in monogenic lung diseases after a single treatment. Although encouraging, in vivo CRISPR gene editing studies targeting other organs in adult mouse models of human diseases highlight limitations to the postnatal approach including low-levels of homology directed repair (HDR) due to inaccessible and nonproliferative target cells and a mature immune barrier. These obstacles are even more daunting in the postnatal lung, a barrier organ with immune and physical barriers in which only 1% of epithelial progenitor cells, the target cell population for most lung diseases, are cycling at homeostasis. In utero gene editing has the potential to overcome these barriers and treat perinatal lethal diseases before the onset of irreversible pathology. The fetus is immunologically tolerant and progenitor cells of multiple organs, including the lung, are highly proliferative and accessible during development. The objective of this proposal is to establish the feasibility of prenatal lung gene editing and use prenatal gene editing to treat a mouse A1ATD model as a model for monogenic lung diseases. Our central hypotheses are that prenatal pulmonary cell gene editing is more efficient than postnatal editing and prenatal gene editing will not have a detrimental effect on edited pulmonary progenitor cell fate. We hypothesize that prenatal HDR can provide therapeutic levels of circulating alpha-1 antitrypsin protein and pulmonary cell gene correction in the A1ATD mouse model. Our hypotheses are based on our published data demonstrating efficient liver and pulmonary epithelial cell editing via prenatal CRISPR-nonhomologous end joining (NHEJ). To attain our objective, we will pursue the following aims: 1) evaluate prenatal pulmonary cell gene editing in normal and neonatal injury states, 2) evaluate prenatal HDR targeting the lung and compare it to postnatal HDR, and 3) correct A1ATD by prenatal HDR and compare it to postnatal HDR. Our research is innovative in the prenatal timing and targeting of the lung for therapeutic gene editing. The significant contribution of this work will be to provide the foundation for a one- shot, long-term therapy that cures A1ATD and is applicable to other monogenic lung diseases.