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

• 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
• 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

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基因编辑对治疗遗传血液学的巨大希望 包括镰状细胞疾病和β-甲性疾病在内的疾病。当前的治疗策略是 首先专注于自体患者造血的体内基因编辑 茎/祖细胞（HSPC），需要分离患者的HSPC，离体基因编辑， 校正的HSPC的选择和扩展，并将其移植回患者。 尽管最初取得了成功，但该技术的临床翻译受到困难的阻碍 在HSPC的离体处理中，与骨髓相关的风险，低植入 效率和禁止的个性化细胞疗法的高成本。最近的研究 表明HSPC在成年骨髓中的专门壁ni中维持。 HSPC利基 位于正弦血管附近，那里的内皮高度高 可渗透到纳米颗粒和病毒载体。为此，我们提出了HSPC 骨髓可以由CRISPR/CAS9原位对基因编辑。但是，体内CRISPR/CAS9基因 由于交付的全身传播，编辑可能会产生重大的脱靶效应 CAS9核酸酶的车辆和非特异性活动。最近，我们开发了一本小说 基因编辑平台将细菌病毒载体与磁性纳米颗粒（MNP- BV）。与常规病毒载体相比，杆状病毒载体可以转导宽 无复制的哺乳动物细胞范围。 MNP-BV使用外部磁场， 内在的完成系统作为特定地点转换交付的开关和偏置。在这个 项目，我们将开发一种基于MNP-BV的基因编辑技术，以精确的基因编辑 骨髓中的HSPC。 MNP-BV将通过骨内输注进行管理。我们将 设计一种磁性靶向方法，以增强MNP-BV在骨髓中的保留 MNP-BV向核苷壁细分市场的渗出。此外，细菌病毒 向量具有较大的DNA载荷能力（> 38 kb），因此可以提供可诱导的Cas9或GRNA 靶向特定细胞群的表达盒。我们将设计只能 在HSPC中高表达的microRNA（miRNA）激活。中心假设是 通过结合骨内输注，磁靶向和miRNA介导的翻译后 调节，MNP-BV系统可以有效，精确地翻译骨骼中的HSPC 骨髓和正确的血液疾病相关基因突变。这个成功 项目将为开发有效和低成本的治疗铺平道路 血液学疾病。