Precise in Vivo Gene Editing of HSPC for the Treatment of Genetic Hematologic Diseases

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.