Lung selective CRISPR delivery for treatment of genetic surfactant disease

新生儿呼吸窘迫综合征（RDS）是最常见的呼吸道死亡原因 和美国1岁以下婴儿的发病率。单基因突变 调节表面活性剂稳态，即表面活性剂蛋白B（SFTPB）、表面活性剂蛋白C SFTPC和ATP结合盒亚家族A成员3（ABCA 3）是ATP结合的致病驱动因子。 25%的婴儿发生RDS，伴有严重难治性呼吸衰竭。标准治疗方案 对于遗传性肺病，仅限于对症治疗和肺移植， 长期生存预后差，并发症发生率高。这些不满意的 结果凸显了对直接解决遗传问题的更精确疗法的迫切需求 呼吸窘迫综合征的潜在畸变。在此，我们将联合收割机高度互补的专业知识， 肺部疾病治疗（Alapati博士）和非病毒基因递送（Sullivan博士）， 开发一种非手术方法，在出生后早期对肺祖细胞进行遗传校正 肺发育，一个广泛使用的策略，旨在防止疾病的表现。我们 将通过两个目标建立这种创新和预防相关的方法：目标1。设计 非病毒纳米载体（“聚合复合物”），其是生物相容的，在肺液中稳定，并且能够 在新生儿AT 2细胞中进行细胞选择性和有效的基因编辑。目标2.设计一种部分液体 CRISPR-Cas9递送的通气方法，以最大化AT 2细胞访问和基因编辑 持久性的新生儿肺模型，并证明这种方法的持久性，广泛， 和肺上皮中安全的非病毒基因编辑。我们的假设是建立在我们发表的研究 证明了（i）组蛋白复合物基因转移取决于复合物通过细胞的摄取， 小窝蛋白-1转运蛋白，一种能够高效转染小窝蛋白-1的机制， 表达细胞并允许基于小窝蛋白-1的差异的精确细胞“靶向” （ii）CRISPR-Cas9货物到充满液体的胎儿肺中的气道递送导致 高效的肺上皮细胞基因编辑。这项工作将揭示重要的新 新生儿肺上皮基因转移机制的信息，同时 建立新的，更具细胞选择性的基因治疗策略， 遗传疾病该研究结果将展示一种新的有效的细胞- 在出生后的肺上皮中进行特异性和安全的基因编辑，这是一种能够广泛应用的策略。 即使在基础水平的NICU中也能使用，同时与疾病发生的时间保持一致 诊断，并为未来的翻译奠定基础，从根本上新的，更有效的， 遗传性表面活性蛋白疾病的一次性治疗模式。

Neonatal respiratory distress syndrome (RDS) is the most common respiratory cause of death and morbidity in infants <1 year of age in the United States. Monogenic mutations in genes regulating surfactant homeostasis, namely surfactant protein B (SFTPB), surfactant protein C (SFTPC), and ATP binding cassette subfamily A member 3 (ABCA3), are causative drivers of RDS in 25% of infants with severe refractory respiratory failure. Standard therapeutic regimens for genetic lung disease are limited to symptomatic treatments and lung transplant, a procedure with poor prognosis for long-term survival and high complication rates. These unsatisfactory outcomes highlight the pressing need for more precise therapies that directly address the genetic aberrations underlying RDS. Herein, we combine highly complementary expertise in neonatal lung disease treatment (Dr. Alapati) and non-viral gene delivery (Dr. Sullivan) necessary to develop a non-surgical approach to genetically correct lung progenitor cells during early postnatal lung development, a widely accessible strategy designed to prevent disease manifestation. We will establish this innovative and translationally-relevant approach via two aims: Aim 1. Design non-viral nanocarriers (‘polyplexes’) that are biocompatible, stable in lung fluids, and capable of cell-selective and efficient gene editing in neonatal AT2 cells. Aim 2. Engineer a partial-liquid ventilation approach for CRISPR-Cas9 delivery to maximize AT2 cell access and gene editing persistence in models of neonatal lung, and demonstrate this approach for durable, widespread, and safe non-viral gene editing in lung epithelium. Our hypothesis is built on our published studies demonstrating that (i) histone polyplex gene transfer hinges upon polyplex uptake via the caveolin-1 transporter, a mechanism that enables highly efficient transfection in caveolin-1- expressing cells and permits precise cell ‘targeting’ based upon differences in caveolin-1 availability; and (ii) airway delivery of CRISPR-Cas9 cargo into fluid filled fetal lungs results in efficient pulmonary epithelial cell gene editing. This work will thus uncover important new information on neonatal pulmonary epithelial gene transfer mechanisms while simultaneously establishing new, more cell-selective gene therapy strategies relevant to a variety of pulmonary genetic disorders. The study outcome will demonstrate a new delivery platform for effective, cell- specific, and safe gene editing in postnatal lung epithelium, a strategy that would enable wide usage even in basic-level NICUs, while simultaneously aligning with the timing of disease diagnosis, and lay groundwork for future translation to fundamentally new, more effective, and one-shot treatment modes for genetic surfactant protein diseases.