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

项目摘要 遗传性疾病，如β-地中海贫血和囊性纤维化，是有吸引力的基因组工程目标，因为 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)在相关基因组位置进行校正；例如，工程 包括CRISPR/Cas9系统在内的核酸酶已经显示出希望并进入临床试验。替代非- 基于核酸酶的三链形成肽核酸(PNAS)在体内也取得了成功。PNA没有 内源性核酸酶活性和激活内源性DNA修复活性时，与邻近的 并与含有正确序列的供体DNA链共同传递。PNA介导的基因 编辑通过核苷酸切除修复(NER)和HR途径发生，并表现出较低的脱靶效应。而当 这些编辑技术到目前为止是成功的，但在翻译到 诊所。能高效包裹基因的安全高效递送载体的研制 编辑剂和与疾病相关的靶细胞/组织对于这些的发展是必要的 治疗学。这项研究的目标是进一步推动基因组工程技术的转化。 通过将可生物降解的聚(胺-共酯)(PACE)开发成高效的聚合物载体 将基因编辑剂包裹并输送到骨髓和肺中的靶细胞 全身静脉(IV)给药。PACE在结构上是多样化的，允许我们生成 载体，并鉴定靶向骨髓或肺的组合物。在前期工作中，我们观察到 使用PACE高效封装和传递基因编辑剂。此外，具体的速度公式 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：a 开发阶段(UG3)和示范阶段(UH3)。在UG3阶段，PACE聚合物的文库 将合成独特的特性，并测试其包裹PNA-和 基于CRISPR/Cas9的编辑试剂，将它们传递到靶细胞，并促进在体外和在 活着。将使用创新的高通量单细胞RNA来量化细胞类型的靶向和编辑 测序(scRNA-seq)屏幕。针对骨髓和肺的候选配方将被给予 分别建立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UH3阶段，候选配方和 基因编辑试剂将扩大规模，以适应大型动物研究，主要是在猪身上，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，使用基因编辑的量化措施，以及疾病改善，使 迈向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 交通工具，进一步翻译由基因突变引起的疾病的基因编辑疗法。

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.