Lung selective CRISPR delivery for treatment of genetic surfactant disease

肺部选择性 CRISPR 递送治疗遗传性表面活性物质疾病

• 批准号: 10610428
• 负责人: Deepthi Alapati
• 金额: $ 22.05万
• 依托单位: ALFRED I. DU PONT HOSP FOR CHILDREN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610428
• 关键词: 1 year old; Address; Adult; Alveolar; Binding; Biophysics; CRISPR/Cas technology; Cause of Death; Cell Fraction; Cells; Cessation of life; Chromosome Mapping; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complication; Confocal Microscopy; DNA Sequence Alteration; Development; Disease; Distal; Engineering; Environment; Epithelial Cells; Epithelium; Family; Fetal Lung; Flow Cytometry; Future; Gene Delivery; Gene Transfer; Genes; Genetic; Genetic Diseases; Histones; Homeostasis; Immune; Infant; Inhalation; Life; Liquid Ventilation; Liquid substance; Lung; Lung Transplantation; Lung diseases; Mediating; Modeling; Morbidity - disease rate; Multidrug Resistance-Associated Proteins; Mus; Mutation; Natural regeneration; Neonatal; Neonatal Respiratory Distress; Operative Surgical Procedures; Organ; Outcome; Outcome Study; Palliative Care; Partial Liquid Ventilation; Peptides; Perflubron; Perinatal mortality demographics; Phenotype; Probability; Procedures; Prognosis; Proteins; Publishing; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Protein C; Pulmonary alveolar structure; Reagent; Refractory; Regimen; Reporter; Respiratory Failure; Techniques; Therapeutic; Trachea; Transfection; Translations; United States; Up-Regulation; Work; airway epithelium; alveolar epithelium; base editing; biomaterial compatibility; caveolin 1; design; disease diagnosis; epithelial stem cell; gene correction; gene therapy; innovation; lung development; lung failure; member; mouse model; nanocarrier; neonatal human; neonate; non-viral gene delivery; novel strategies; post-transplant; postnatal; prenatal; prevent; real-time images; respiratory; respiratory distress syndrome; stem cell proliferation; stem cells; surfactant; symptom treatment; therapeutic target; uptake; ventilation

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.

新生儿呼吸窘迫综合征（RDS）是最常见的呼吸系统死亡原因 美国 1 岁以下婴儿的发病率和基因单基因突变。 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 (ABCA3) 是 25% 患有严重难治性呼吸衰竭的婴儿出现 RDS 标准治疗方案。 对于遗传性肺病，仅限于对症治疗和肺移植，这是一种手术 长期生存预后差，并发症发生率高。 结果凸显了迫切需要更精确的疗法来直接解决遗传问题 在此，我们结合了新生儿方面高度互补的专业知识。 肺部疾病治疗（Alapati 博士）和非病毒基因传递（Sullivan 博士）是必要的 开发一种非手术方法，在产后早期对肺祖细胞进行基因纠正 肺部发育，一种旨在预防疾病表现的广泛使用的策略。 将通过两个目标建立这种创新且与转化相关的方法： 目标 1. 设计 非病毒纳米载体（“多聚体”）具有生物相容性，在肺液中稳定，并且能够 对新生儿 AT2 细胞进行细胞选择性和高效基因编辑 目标 2. 设计部分液体。 用于 CRISPR-Cas9 输送的通气方法可最大限度地提高 AT2 细胞的接触和基因编辑能力 新生儿肺模型中的持久性，并证明这种方法可以持久、广泛、 我们的假设是基于我们已发表的研究。 证明 (i) 组蛋白多聚体基因转移取决于多聚体通过 Caveolin-1 转运蛋白，一种在 Caveolin-1 中实现高效转染的机制 表达细胞并允许根据 Caveolin-1 的差异进行精确的细胞“靶向” 可用性；以及 (ii) 将 CRISPR-Cas9 货物气道输送至充满液体的胎儿肺部，从而导致 因此，这项工作将揭示有效的肺上皮细胞基因编辑。 有关新生儿肺上皮基因转移机制的信息，同时 建立与多种肺部相关的新的、更具细胞选择性的基因治疗策略 该研究成果将展示一种有效的细胞传递新平台。 对出生后肺上皮进行特异性且安全的基因编辑，这一策略将能够广泛实现 即使在基础级 NICU 中也能使用，同时与疾病发生时间保持一致 诊断，并为未来转化为全新的、更有效的、更 遗传性表面活性蛋白疾病的一次性治疗模式。