Precise in vivo gene editing of HSPC for the treatment of genetic hematologic diseases

HSPC体内精准基因编辑治疗遗传性血液病

基本信息

批准号：
10548540
负责人：
Sheng Tong
金额：
$ 22.95万
依托单位：
UNIVERSITY OF KENTUCKY
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：
2023-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10548540
关键词：
Adult Allogenic Autologous Autologous Transplantation Back Basic Science Binding Sites Blood Vessels Bone Marrow Bone Marrow Cells Bone Marrow Purging CRISPR/Cas technology Cell Therapy Cells Chromosome Mapping Clinical Treatment Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Sequence DNA Sequence Alteration DNA cassette Disease Endothelium Engraftment Extravasation Face Femur Future Gene Delivery Gene Mutation Genes Genetic Guide RNA Hematological Disease Hematopoiesis Hematopoietic Hematopoietic stem cells Hemoglobinopathies Human Immunologics In Situ Infusion procedures Insect Viruses Magnetic nanoparticles Magnetism Mammalian Cell Mediating Messenger RNA Methods MicroRNAs Mus Mutation Myelogenous Nanotechnology Patients Permeability Population Post-Translational Regulation Regulator Genes Regulatory Element Risk Sickle Cell Anemia Site Specificity System Techniques Therapeutic Toxic effect Transfection Translation Initiation Transplantation Untranslated RNA Viral Vector adaptive immunity beta Globin beta Thalassemia clinical application clinical translation complement system cost curative treatments delivery vehicle design gene correction genotoxicity in vivo intravenous injection lipid nanoparticle mRNA Translation magnetic field mouse model multidisciplinary nanomedicine nanoparticle novel nuclease preclinical study self-renewal stem cell biology stem cells success synthetic biology targeted delivery therapeutic target tool transgene delivery translational potential vector

项目摘要

Summary CRISPR/cas9 gene editing has shown great promise for the treatment of genetic hematologic disorders including sickle cell disease and β-thalassemia. Current therapeutic strategies are primarily focused on ex vivo gene editing of autologous patient-derived hematopoietic stem/progenitor cells (HSPCs), which require isolation of patients’ HSPCs, ex vivo gene editing, selection and expansion of corrected HSPCs, and transplantation back into the patients. Despite its initial success, the clinical translation of this technique is hampered by the difficulties in ex vivo processing of HSPCs, the risks associated with myeloablation, the low engraftment efficiency, and the prohibitively high cost of individualized cell therapy. Recent studies have shown that HSPCs are sustained in specialized niches in the adult bone marrow. HSPC niches are located near the sinusoidal blood vessels, where the fenestrated endothelium is highly permeable to nanoparticles and viral vectors. To this end, we propose that the HSPCs in the bone marrow can be gene-edited by CRISPR/cas9 in situ. However, in vivo CRISPR/cas9 gene editing can have substantial off-target effects due to the systemic dissemination of the delivery vehicles and the non-specific activities of the cas9 nuclease. Recently, we developed a novel gene-editing platform that combines the baculoviral vector with magnetic nanoparticles (MNP- BV). Compared with conventional viral vectors, the baculoviral vector can transduce a broad range of mammalian cells without replication. MNP-BV uses an external magnetic field and the intrinsic complement system as the on- and off-switch for site-specific transgene delivery. In this project, we will develop an MNP-BV-based gene-editing technique for precise gene editing of HSPCs in the bone marrow. MNP-BV will be administrated via intraosseous infusion. We will design a magnetic targeting method to enhance the retention of MNP-BV in the bone marrow and the extravasation of MNP-BV to the perisinusoidal niches. Furthermore, the baculoviral vector has a large DNA loading capacity (>38 kb) and thus can deliver inducible cas9 or gRNA expression cassettes targeting specific cell populations. We will design gRNAs that can only be activated by microRNAs (miRNAs) highly expressed in HSPCs. The central hypothesis is that by combining intraosseous infusion, magnetic targeting, and miRNA-mediated posttranslational regulation, the MNP-BV system can efficiently and precisely transduce HSPCs in the bone marrow and correct hematological diseases-associated gene mutations. The success of this project will pave the way for developing an effective and low-cost cure for a range of hematological diseases.

概括 CRISPR/cas9基因编辑在治疗遗传性血液病方面显示出巨大的前景目前的治疗策略包括镰状细胞病和β-地中海贫血。主要专注于患者自体造血的离体基因编辑干/祖细胞（HSPC），需要分离患者的 HSPC、离体基因编辑、选择和扩增校正后的 HSPC，然后移植回患者体内。尽管取得了初步成功，但该技术的临床转化却因困难而受到阻碍在 HSPC 的离体加工中，与清髓相关的风险、低植入率最近的研究表明，个体化细胞治疗的效率和成本过高。表明 HSPC 在成人骨髓中的特殊生态位中持续存在。位于正弦血管附近，有孔内皮高度为此，我们建议 HSPCs 具有纳米粒子和病毒载体的渗透性。骨髓可以通过 CRISPR/cas9 进行原位基因编辑，但是体内 CRISPR/cas9 基因。由于交付的系统性传播，编辑可能会产生严重的脱靶效应最近，我们开发了一种新型的载体和 cas9 核酸酶的非特异性活性。将杆状病毒载体与磁性纳米颗粒（MNP- 与传统病毒载体相比，杆状病毒载体可以转导广泛的病毒。 MNP-BV 使用外部磁场和复制的哺乳动物细胞范围。内在补体系统作为位点特异性转基因递送的开关。项目中，我们将开发一种基于MNP-BV的基因编辑技术，用于精确的基因编辑骨髓中的 HSPC 将通过骨内输注进行给药。设计一种磁性靶向方法来增强 MNP-BV 在骨髓中的保留以及 MNP-BV 外渗到窦周微环境此外，杆状病毒。载体具有较大的 DNA 负载能力 (>38 kb)，因此可以传递诱导型 cas9 或 gRNA 我们将设计只能针对特定细胞群的表达盒。由 HSPC 中高表达的 microRNA (miRNA) 激活。通过结合骨内输注、磁性靶向和 miRNA 介导的翻译后调节，MNP-BV系统可以高效、精确地转导骨中的HSPC 骨髓和纠正血液疾病相关基因突变的成功。该项目将为开发一种有效且低成本的治疗方法铺平道路血液系统疾病。