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 以及美国1岁以下婴儿的发病率。基因的单基因突变 调节表面活性物质动态平衡，即表面活性蛋白B(SFTPB)、表面活性蛋白C (SFTPC)和三磷酸腺苷结合盒A亚家族成员3(ABCA3)是引起 25%患有严重难治性呼吸衰竭的婴儿出现RDS。标准治疗方案 遗传性肺部疾病仅限于对症治疗和肺移植，这是一种程序 长期生存预后差，并发症发生率高。这些都不能令人满意 结果突显出迫切需要更精确的治疗方法，直接解决遗传问题 RDS背后的像差。在此，我们结合了高度互补的新生儿专业知识 肺部疾病治疗(阿拉帕蒂博士)和非病毒基因传递(沙利文博士) 开发一种非手术方法在出生后早期对肺祖细胞进行基因纠正 肺发展，这是一种广泛使用的战略，旨在防止疾病表现。我们 我将通过两个目标建立这种创新的和翻译相关的方法：目标1.设计 生物相容的、在肺液中稳定的、能够 新生儿AT2细胞的细胞选择性和高效的基因编辑。目标2.设计一种部分液体 用于CRISPR-Cas9传递的通风方法以最大化AT2细胞访问和基因编辑 在新生儿肺模型中的持久性，并展示了这种方法的持久性，广泛的， 在肺上皮细胞中进行安全的非病毒基因编辑。我们的假设是建立在已发表的研究基础上的 证明(I)组蛋白多链基因转移依赖于多链通过 Caveolin-1转运体，一种能够高效地在Caveolin-1中转导的机制- 表达细胞并基于小窝蛋白-1的差异实现精确的细胞靶向 可获得性；和(Ii)CRISPR-Cas9货物经呼吸道输送到充满液体的胎儿肺导致 高效的肺上皮细胞基因编辑。因此，这项工作将发现重要的新 新生儿肺上皮基因同时转移机制的研究进展 建立与多种肺腺癌相关的新的、更具细胞选择性的基因治疗策略 遗传性疾病。研究结果将展示一种新的有效的细胞- 在出生后肺上皮中进行特异和安全的基因编辑，这一策略将使 即使在基础水平的NICU中也是如此，同时与疾病的发生时间保持一致 诊断，并为未来的翻译奠定基础，以实现全新的、更有效的和 遗传性表面活性物质蛋白病的一次性治疗模式。