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.

项目摘要镰状细胞疾病和依赖输血依赖性？ - 丘陵症是全球最常见的单基因疾病，每年全球约有400,000名新生儿，几十年来预期寿命缩短。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愈策略的潜力。尽管有希望，但目前需要资源密集型的离体过程，造血茎和祖细胞（HSPC）的基因组编辑（HSPCS）限制了公平地获得潜在的治愈性干预措施。体内基因组编辑器向HSPC的载体可能会避免此问题，但据报道，HSPC递送系统受到矢量免疫原性，较低的编辑效率以及缺乏细胞靶向特异性的限制。作为一种自组装，人类衍生的蛋白聚合物，可容纳靶向结构域的便捷掺入，已经为纳米粒子介导的药物递送而建立了弹性蛋白样的多肽（ELP）。但是，尚未定义用于ELP介导的基因组编辑器传递的方法。拟议的工作的目的是生成ELP纳米颗粒，用于在体内将mRNA编码的基因组编辑者传递到造血干细胞。在AIM 1中，将优化ELPS结构域，以递送CAS9 mRNA和靶向管家基因HPRT1的单个指南RNA（SGRNA）。将筛选ELP的库，以识别用于货物络合，释放和细胞内递送的最佳域设计。在AIM 2中，将确定能够在体外基因组编辑器传递到HSPC的HSPC特异性单克隆抗体（MAB）。富含鼠HSPC的标记物的抗体将与具有不同价值的ELP纳米颗粒相结合，以促进HSPC特异性摄取。将在从AI9-Sauspycas9小鼠中分离出的HSPC中评估CAS9的细胞特异性递送，从而使荧光检测编辑活性。在AIM 3中，将确定将MAB标记的ELP纳米颗粒用于基因组编辑器在体内递送到HSPCS的效用。通过进行装有独特的DNA条形码的纳米颗粒的合并筛选，可以评估野生型小鼠中mAb标记的ELP纳米颗粒的生物分布和HSPC向流。从生物分布研究中确定的铅纳米颗粒制剂将在AI9-Sauspycas9小鼠中的CAS9递送效率进行评估。动员HSPC对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，从而可以治疗多种遗传性血液学疾病。通过定义关键的物理化学原理，该原理控制核酸络合并通过ELP纳米颗粒递送，这项工作还将为建立ELP作为核酸递送到其他细胞和组织的平台的基础。