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型地中海贫血是世界上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。尽管前景看好，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体外过程，限制了获得潜在治愈干预的公平机会。将基因组编辑的载体体内递送到HSPC可以避免这个问题，但已报道的HSPC递送系统受到载体免疫原性、编辑效率低和缺乏细胞靶向性的限制。弹性蛋白样多肽(ELPs)是一种自组装的人源性蛋白质聚合物，易于掺入靶向结构域，已被用于纳米颗粒介导的药物传递；然而，ELP介导的基因组编辑器传递的方法尚未定义。这项拟议工作的目标是产生ELP纳米颗粒，用于将mRNA编码的基因组编辑器运送到体内的造血干细胞。在目标1中，ELPS结构域将被优化，以递送针对管家基因HPRT1的Cas9 mRNA和单引导RNA(SgRNA)。将对ELP文库进行筛选，以确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，将鉴定能够在体外将基因组编辑程序传递给HSPC的HSPC特异性单抗(MAbs)。针对小鼠HSPC标志物的抗体将以不同的价态偶联到ELP纳米颗粒上，以促进HSPC的特异性摄取。Cas9的细胞特异性传递将在从Ai9-SauSpyCas9小鼠中分离的HSPC中进行评估，这使得能够对编辑活动进行荧光检测。在目标3中，将确定单抗标记的ELP纳米颗粒在体内将基因组编辑程序输送到HSPC的效用。MAb标记的ELP纳米颗粒在野生型小鼠体内的生物分布和HSPC趋向性将通过对装载有独特DNA条形码的纳米颗粒进行池筛选来评估。从生物分布研究中确定的铅纳米颗粒配方将在Ai9-SauSpyCas9小鼠身上评估Cas9的输送效率。将定义将HSPC动员到外周血中对纳米颗粒生物分布和HSPC编辑效率的影响。这些研究将建立一种用于HSPC体内基因组编辑的非病毒传递载体，使其能够治疗各种遗传性血液病。通过确定管理核酸络合和ELP纳米颗粒输送的关键物理化学原理，这项工作还将为建立ELP作为将核酸输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.