Self-Assembling Peptide Nanoparticles for in vivo Genome Editor Delivery to Hematopoietic Stem Cells

项目摘要 镰状细胞病和输血依赖？地中海贫血是全世界最常见的单基因疾病，每年影响全球约40万新生儿，并使预期寿命缩短数十年。正在进行的临床试验已经确定了基因组编辑作为这些疾病的治疗策略的潜力。虽然有希望，但造血干细胞和祖细胞（HSPC）的基因组编辑目前需要资源密集型的离体过程，限制了公平获得潜在的治疗性干预。用于将基因组编辑器体内递送至HSPC的载体可以规避该问题，但是报道的HSPC递送系统受到载体免疫原性、低编辑效率和缺乏细胞靶向特异性的限制。 作为一种自组装的人源蛋白质聚合物，其易于掺入靶向结构域，弹性蛋白样多肽（ELP）已被建立用于纳米颗粒介导的药物递送;然而，尚未定义ELP介导的基因组编辑器递送的方法。所提出的工作的目的是产生ELP纳米颗粒，用于将mRNA编码的基因组编辑器递送到体内造血干细胞。在目标1中，ELP结构域将被优化用于递送靶向管家基因HPRT 1的Cas9 mRNA和单向导RNA（sgRNA）。将筛选ELP文库以鉴定用于细胞系中货物络合、释放和细胞内递送的最佳结构域设计。在目标2中，将鉴定能够体外基因组编辑器递送至HSPC的HSPC特异性单克隆抗体（mAb）。针对在鼠HSPC上富集的标志物的抗体将以不同的化合价缀合至ELP纳米颗粒以促进HSPC特异性摄取。将在从Ai 9-SauSpyCas 9小鼠分离的HSPC中评估Cas9的细胞特异性递送，这使得能够荧光检测编辑活性。在目的3中，将确定mAb标记的ELP纳米颗粒用于体内基因组编辑器递送至HSPC的效用。将通过对加载有独特DNA条形码的纳米颗粒进行合并筛选来评价mAb标记的ELP纳米颗粒在野生型小鼠中的生物分布和HSPC嗜性。将评估从生物分布研究鉴定的铅纳米颗粒制剂在Ai 9-SauSpyCas 9小鼠中的Cas9递送效率。将定义动员HSPC至外周血对纳米颗粒生物分布和HSPC编辑效率的影响。这些研究将建立用于HSPC体内基因组编辑的非病毒递送载体，从而能够治疗各种遗传性血液病。通过定义控制核酸复合和ELP纳米颗粒递送的关键物理化学原理，这项工作也将为建立ELP作为核酸递送到其他细胞和组织的平台奠定基础。

Project Summary Sickle cell disease and transfusion-dependent ?-thalassemia are the most common monogenic diseases worldwide, affecting ~400,000 newborns per year globally and shortening life expectancy by decades. Ongoing clinical trials have established the potential of genome editing as a curative strategy for these disorders. While promising, genome editing of hematopoietic stem and progenitor cells (HSPCs) currently requires resource-intensive ex vivo processes, limiting equitable access to a potentially curative intervention. Vectors for in vivo delivery of genome editors to HSPCs could circumvent this issue, but reported HSPC delivery systems are limited by vector immunogenicity, low editing efficiencies, and a lack of cell targeting specificity. As a self-assembling, human-derived protein polymer amenable to facile incorporation of targeting domains, elastin-like polypeptides (ELPs) have been established for nanoparticle-mediated drug delivery; however, an approach for ELP-mediated genome editor delivery has not been defined. The objective of the proposed work is to generate ELP nanoparticles for the delivery of mRNA-encoded genome editors to hematopoietic stem cells in vivo. In Aim 1, ELPs domain will be optimized for delivery of Cas9 mRNA and single guide RNA (sgRNA) targeting housekeeping gene HPRT1. A library of ELPs will be screened to identify an optimal domain design for cargo complexation, release, and intracellular delivery in cell lines. In Aim 2, HSPC specific monoclonal antibodies (mAbs) that enable in vitro genome editor delivery to HSPCs will be identified. Antibodies against markers enriched on murine HSPCs will be conjugated to ELP nanoparticles with varying valency to promote HSPC specific uptake. Cell-specific delivery of Cas9 will be evaluated in HSPCs isolated from Ai9-SauSpyCas9 mice, which enable fluorescent detection of editing activity. In Aim 3, the utility of mAb-labeled ELP nanoparticles for genome editor delivery to HSPCs in vivo will be determined. The biodistribution and HSPC tropism of mAb-labeled ELP nanoparticles in wild type mice will be evaluated by conducting pooled screens of nanoparticles loaded with unique DNA barcodes. The lead nanoparticle formulation identified from biodistribution studies will be assessed for Cas9 delivery efficiency in Ai9-SauSpyCas9 mice. The effects of mobilizing HSPCs to the peripheral blood on both nanoparticle biodistribution and HSPC editing efficiency will be defined. These studies will establish a non-viral delivery vector for in vivo genome editing of HSPCs, enabling the treatment of a wide variety of inherited hematologic disorders. By defining critical physicochemical principles that govern nucleic acid complexation and delivery by ELP nanoparticles, this work will also form a foundation for establishing ELPs as a platform for nucleic acid delivery to other cells and tissues.