Nanoparticles-based Delivery of gene edIting Tools in hematopoietic Stem cells to treat Beta-hemoglobinopathies in vivo

Objetivo

Beta-hemoglobinopathies aBeta-hemoglobinopathies are the most common genetic diseases worldwide. Current gene therapies rely on ex vivo treatment of isolated hematopoietic stem cells (HSCs) with CRISPR/Cas9, followed by reinfusion in the patient. However, HSCs are highly sensitive to DNA double-strand breaks (DSBs) that can lead to apoptosis and generate large genomic rearrangements. Furthermore, current CRISPR/Cas9 therapeutic protocols involve complex HSC manipulations that reduce HSC fitness and can lead to cytotoxicity (e.g. because of the electroporation of the gene editing components). This is particularly true for HSCs from β-hemoglobinopathy patients, as they are more susceptible to electroporation-based gene editing procedures. In the ongoing DITSB ERC project, we developed efficacious and safe treatment strategies for β-hemoglobinopathies based on precise base editing (rather than DSB-induced DNA repair). However, toxicity due to the delivery method (i.e. electroporation) was still observed. In NDITSB, we will use lipid nanoparticles (LNPs) to deliver the editing components to human HSCs, with the goal of achieving a safe and reproducible transfection efficiency. Cutting-edge LNP formulations will be first screened in vitro in patients’ HSCs to select the safest and most efficacious particles that will be subsequently tested in an in vivo xenotransplantation model to demonstrate the targeting and correction of long-term HSCs. Importantly, we will evaluate the effect of LNPs in patients’ HSCs, as our studies highlighted disease-specific responses to gene editing. Therefore, the use of LNPs could represent an alternative and less toxic delivery system of the gene editing components. This work will pave the way for the development of an in vivo gene therapy strategy based on the intravenous injection of LNPs targeting HSCs in situ. This will simplify the gene therapy protocol and reduce the costs, thus allowing a widespread use of this therapeutic approach.