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

摘要 CRISPR/Cas9基因编辑在遗传性血液病的治疗中显示出巨大的前景 疾病包括镰状细胞病和β-地中海贫血。目前的治疗策略是 主要关注自体患者来源的造血细胞的体外基因编辑 干细胞/祖细胞(HSPC)，这需要分离患者的HSPC，体外基因编辑， 选择和扩增矫正的HSPC，并将其移植回患者体内。 尽管这项技术最初取得了成功，但它的临床翻译受到了困难的阻碍。 在体外处理HSPC时，与骨髓清除术相关的风险、低植入率 效率，以及个体化细胞治疗令人望而却步的高昂成本。最近的研究表明 研究表明，HSPC在成人骨髓中的特殊壁龛中持续存在。HSPC利基市场 位于窦状血管附近，有窗孔的内皮细胞高度集中。 对纳米粒子和病毒载体具有渗透性。为此，我们建议 骨髓可以用CRISPR/Cas9进行原位基因编辑。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偏离目标的效果 载体和Cas9核酸酶的非特异性活性。最近，我们开发了一部小说 将杆状病毒载体与磁性纳米颗粒(MNP-)相结合的基因编辑平台 BV)。与常规病毒载体相比，杆状病毒载体可广泛转导 没有复制的哺乳动物细胞的范围。MNP-BV使用外部磁场， 固有的补体系统作为定点转基因传递的开关。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HSPC。MNP-BV将通过骨内输液给药。我们会 设计一种磁靶向方法提高MNP-BV在骨髓中的滞留 MNP-BV外渗至窦周壁龛。此外，杆状病毒 载体具有很大的DNA载量(~gt；38kb)，因此可以传递可诱导的Cas9或gRNA 以特定细胞群体为靶点的表达盒。我们将设计出只能 由在HSPC中高表达的microRNAs(MiRNAs)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系统可以高效和准确地转导骨骼中的HSPC 骨髓和纠正血液病相关基因突变。这件事的成功 该项目将为开发一种有效和低成本的治疗一系列 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.