Poly(amine-co-ester)s for Targeted Delivery In Vivo of Gene Editing Agents to Bone Marrow and Lung

用于将基因编辑剂体内靶向递送至骨髓和肺的聚(胺-共酯)

• 批准号: 10274829
• 负责人: PETER M GLAZER
• 金额: $ 75.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-07 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10274829
• 关键词: Amines; Animal Model; Animals; Binding; Biocompatible Materials; Biodistribution; Biology; Biomedical Engineering; Bone Marrow; CRISPR/Cas technology; Cells; Characteristics; Charge; Clinic; Clinical Trials; Collaborations; Cystic Fibrosis; DNA; DNA Repair; DNA Sequence Alteration; Development; Disease; Disease model; Encapsulated; Esters; Exhibits; Family; Family suidae; Formulation; Frequencies; Gene Delivery; Genes; Genetic Diseases; Genome; Genome engineering; Genomic DNA; Genomics; Glycolates; Goals; Guide RNA; Health; Hematopoietic stem cells; Hereditary Disease; Human; In Vitro; Inherited; Injections; Interdisciplinary Study; Intravenous; Laboratories; Libraries; Lung; Measures; Mediating; Messenger RNA; Methods; MicroRNAs; Modification; Mus; Nanotechnology; Nucleic Acids; Nucleotide Excision Repair; Oligonucleotides; Pathway interactions; Patients; Peptide Nucleic Acids; Phase; Polymers; Process; Protocols documentation; Public Health; Reagent; Research; Research Personnel; Safety; Site; Small Interfering RNA; Somatic Cell; Structure; Surface; Symptoms; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Translations; United States National Institutes of Health; Validation; Work; base; beta Thalassemia; burden of illness; cell type; clinical translation; deep sequencing; design; disease-causing mutation; engineered nucleases; genome editing; homologous recombination; improved; in vivo; innovation; intravenous injection; nanoparticle; nanosized; new technology; nonhuman primate; novel; nuclease; plasmid DNA; programs; repaired; response; scale up; screening; single-cell RNA sequencing; success; targeted delivery; therapeutic gene; therapeutic genome editing

Project Summary Hereditary disorders such as β-thalassemia and cystic fibrosis are attractive targets for genome engineering as these maladies are curable upon correction of the disease-causing mutation. New technologies can catalyze correction at the associated genomic site by homologous recombination (HR); for example, engineered nucleases including CRISPR/Cas9 systems have shown promise and entered clinical trials. Alternative non- nuclease-based triplex-forming peptide nucleic acids (PNAs) have also been successful in vivo. PNAs have no intrinsic nuclease activity and enable activation of endogenous DNA repair activity when bound adjacent to the target site and co-delivered with a donor DNA strand containing the corrected sequence. PNA-mediated gene editing occurs via nucleotide excision repair (NER) and HR pathways and exhibits low off-target effects. While these editing technologies have been successful thus far, important challenges remain before translation to the clinic. The development of safe and effective delivery vehicles that are able to efficiently encapsulate gene editing agents and target disease-relevant cells/tissues is necessary for the advancement of these therapeutics. The goal of this research is to further the translation of genome engineering technologies by developing biodegradable poly(amine-co-ester) (PACE) into polymeric vehicles that efficiently encapsulate and deliver gene editing agents to target cells in the bone marrow and the lung upon systemic intravenous (IV) administration. PACE is structurally diverse, allowing us to generate libraries of vehicles and identify compositions for targeting bone marrow or lung. In preliminary work, we have observed efficient encapsulation and delivery of gene editing agents using PACE. Further, specific PACE formulations have exhibited favorable biodistribution to the bone marrow and lung. The project will proceed in two phases: a development phase (UG3) and a demonstration phase (UH3). In the UG3 phase, a library of PACE polymers with unique characteristics will be synthesized and tested for their ability to encapsulate PNA- and CRISPR/Cas9-based editing reagents, deliver them to target cells, and promote efficient editing in vitro and in vivo. Cell-type targeting and editing will be quantified using an innovative high-throughput single cell RNA sequencing (scRNA-seq) screen. Candidate formulations targeting bone marrow and lung will be administered to murine disease models of β-thalassemia and cystic fibrosis, respectively, to confirm their editing capabilities and determine their ability to ameliorate disease symptoms. In the UH3 phase, candidate formulations and gene editing agents will be scaled up to accommodate large animal studies, primarily in pigs, but also in non- human primates in collaboration with other investigators in the SCGE program. These studies are designed to confirm cell-type targeting, using quantitative measures of gene editing, and disease improvement, enabling key steps towards clinical trials. This interdisciplinary research will yield a platform of targeted delivery vehicles, furthering the translation of gene editing therapeutics for diseases resulting from genetic mutations.

项目摘要 遗传性疾病如β-地中海贫血和囊性纤维化是基因组工程的有吸引力的目标， 这些疾病在纠正致病突变后是可以治愈的。新技术可以催化 通过同源重组（HR）在相关基因组位点进行校正;例如，工程改造 包括CRISPR/Cas9系统的核酸酶已经显示出前景并进入临床试验。备选非 基于核酸酶的三链体形成肽核酸（PNA）在体内也是成功的。PNAS没有 内源性核酸酶活性，并且当与内源性DNA修复活性相邻结合时能够激活内源性DNA修复活性。 靶位点，并与含有校正序列的供体DNA链共递送。PNA介导基因 编辑通过核苷酸切除修复（NER）和HR途径发生，并表现出低脱靶效应。而 迄今为止，这些编辑技术已经取得了成功，但在翻译到 诊所开发安全有效的载体，能够有效地包裹基因， 编辑剂和靶向疾病相关细胞/组织对于这些进展是必要的。 治疗学本研究的目的是进一步翻译基因组工程技术 通过将可生物降解的聚（胺-共-酯）（PACE）开发成聚合物载体， 将基因编辑剂封装并递送到骨髓和肺中的靶细胞， 全身静脉内（IV）给药。PACE在结构上是多样化的，使我们能够生成 载体和鉴定用于靶向骨髓或肺的组合物。在初步工作中，我们观察到 使用PACE的基因编辑剂的有效封装和递送。此外，计算机设备行动伙伴关系的具体提法 在骨髓和肺中表现出良好的生物分布。该项目将分两个阶段进行： 开发阶段（UG 3）和示范阶段（UH 3）。在UG 3阶段，PACE聚合物库 将合成具有独特特征的肽，并测试其封装PNA的能力， 基于CRISPR/Cas9的编辑试剂，将它们递送到靶细胞，并促进体外和体内的有效编辑。 vivo.细胞类型靶向和编辑将使用创新的高通量单细胞RNA进行定量 测序（scRNA-seq）筛选。将给予靶向骨髓和肺的候选制剂 β-地中海贫血和囊性纤维化的小鼠疾病模型，以确认其编辑能力 并确定它们改善疾病症状的能力。在UH 3阶段，候选制剂和 基因编辑剂将扩大规模，以适应大型动物研究，主要是在猪，但也在非 人类灵长类动物与SCGE计划中的其他研究人员合作。这些研究旨在 确认细胞类型靶向，使用基因编辑和疾病改善的定量措施， 临床试验的关键步骤。这种跨学科的研究将产生一个有针对性的交付平台 载体，进一步转化基因编辑疗法治疗基因突变引起的疾病。