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

总结 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 疾病，包括镰状细胞病和β-地中海贫血。目前的治疗策略是 主要专注于自体患者来源的造血干细胞的离体基因编辑， 干/祖细胞（HSPC），其需要分离患者的HSPC，离体基因编辑， 选择和扩增校正的HSPC，并移植回患者体内。 尽管其初步的成功，这种技术的临床翻译是阻碍了困难， 在HSPC的离体处理中，与骨髓消融相关的风险， 效率，以及个体化细胞治疗的高昂成本。最近的研究 表明HSPCs在成人骨髓中的专门小生境中持续存在。HSPC生态位 位于窦状血管附近，在那里有孔内皮高度 纳米颗粒和病毒载体可渗透。为此，我们建议， 骨髓可以通过CRISPR/cas9原位进行基因编辑。然而，体内CRISPR/cas9基因 由于交付的系统性传播，编辑可能会产生大量脱靶效应 载体和Cas9核酸酶的非特异性活性。最近，我们开发了一种新的 基因编辑平台，将杆状病毒载体与磁性纳米颗粒（MNP- BV）。与传统的病毒载体相比，杆状病毒载体可以广泛地应用于临床， 一系列哺乳动物细胞没有复制。MNP-BV使用外部磁场， 内源性补体系统作为位点特异性转基因递送的开关。在这 项目，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HSPCs。MNP-BV将通过骨内输注给药。我们将 设计磁性靶向方法，提高MNP-BV在骨髓中的滞留 MNP-BV外渗至窦周龛。此外，杆状病毒 载体具有大的DNA装载容量（>38 kb），因此可以递送诱导型cas9或gRNA 靶向特定细胞群的表达盒。我们将设计出一种gRNA， 由HSPC中高度表达的microRNA（miRNAs）激活。核心假设是， 通过结合骨内输注、磁靶向和miRNA介导的翻译后 通过调节，MNP-BV系统可以有效和精确地将HSPCs固定在骨中 骨髓和纠正血液疾病相关的基因突变。的成功 该项目将为开发一种有效和低成本的治疗方法铺平道路， 血液病

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.