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

用于体内基因组编辑器递送至造血干细胞的自组装肽纳米颗粒

基本信息

批准号：
10605021
负责人：
Feyisayo Ronald Eweje
金额：
$ 5.27万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10605021
关键词：
Affect Antibodies Bar Codes Behavior Biodistribution Biological Assay C-terminal C57BL/6 Mouse Cell Line Cell Separation Cells Characteristics Charge Clinical Trials Complementary RNA Complex Cytoplasm DNA DNA cassette Detection Development Dideoxy Chain Termination DNA Sequencing Disease Drug Delivery Systems Dyes Elastin Electrophoretic Mobility Shift Assay Encapsulated Endosomes Engineering Engraftment Ensure Equity Exclusion Flow Cytometry Formulation Foundations Gene Delivery Genome Guide RNA HPRT1 gene Harvest Hematological Disease Hematopoietic stem cells Hemoglobinopathies Housekeeping Gene Human In Vitro Inherited Investigation Label Lead Length Libraries Life Expectancy Mediating Mendelian disorder Messenger RNA Monoclonal Antibodies Mus Newborn Infant Nucleic Acids Oligonucleotides Organ Particle Size Pathologic Peptides Peripheral Phase Polymers Population Prevalence Procedures Process Property Proteins RNA RNA Binding Recombinants Reporting Research Research Proposals Resources Scheme Series Sickle Cell Anemia Sorting Specificity Structure Surface System Thalassemia Tissues Transfusion Tropism Wild Type Mouse Work beta Thalassemia biomaterial compatibility biophysical properties burden of illness conditioning cost curative treatments delivery vehicle density design fluorescence imaging genetic variant genome editing immunogenicity in vivo intravenous administration low and middle-income countries mRNA delivery mortality mouse model nanoparticle nanoparticle delivery next generation sequencing novel nucleic acid delivery nucleic acid-based therapeutics peripheral blood polypeptide receptor binding repaired self assembly small molecule sortase stem cell delivery stoichiometry therapeutic genome editing tissue fixing uptake vector

项目摘要

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.

项目概要镰状细胞病和输血依赖性β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。尽管前景广阔，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，限制了潜在治疗干预的公平获取。用于将基因组编辑器体内递送至 HSPC 的载体可以规避这个问题，但报道的 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，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，ELP 介导的基因组编辑器传递方法尚未确定。这项工作的目标是生成 ELP 纳米粒子，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 结构域将针对 Cas9 mRNA 和靶向管家基因 HPRT1 的单引导 RNA (sgRNA) 的递送进行优化。将筛选 ELP 库，以确定细胞系中货物络合、释放和细胞内递送的最佳域设计。在目标 2 中，将鉴定能够将体外基因组编辑器递送至 HSPC 的 HSPC 特异性单克隆抗体 (mAb)。针对小鼠 HSPC 上富集的标记物的抗体将与不同价态的 ELP 纳米颗粒缀合，以促进 HSPC 特异性摄取。 Cas9 的细胞特异性递送将在从 Ai9-SauSpyCas9 小鼠中分离的 HSPC 中进行评估，这使得能够荧光检测编辑活性。在目标 3 中，将确定 mAb 标记的 ELP 纳米颗粒在体内将基因组编辑器递送至 HSPC 的效用。将通过对装载有独特 DNA 条形码的纳米颗粒进行汇总筛选来评估野生型小鼠中 mAb 标记的 ELP 纳米颗粒的生物分布和 HSPC 趋向性。将评估从生物分布研究中确定的先导纳米颗粒制剂在 Ai9-SauSpyCas9 小鼠中的 Cas9 递送效率。将定义将 HSPC 动员到外周血对纳米颗粒生物分布和 HSPC 编辑效率的影响。这些研究将建立一种用于 HSPC 体内基因组编辑的非病毒递送载体，从而能够治疗多种遗传性血液疾病。通过定义控制 ELP 纳米颗粒核酸络合和递送的关键物理化学原理，这项工作还将为建立 ELP 作为将核酸递送到其他细胞和组织的平台奠定基础。