Engineered extracellular vesicles for In Vivo gene therapy in sickle cell disease

Abstract Sickle Cell Disease (SCD) is a genetic blood disorder that affects over 100, 000 persons in the U. S. and 20 million people worldwide. Sickled red blood cells (RBCs) tend to clump together, leading to vessel blockages known as vaso-oclusive crises (VOCs). VOCs can be extremely painful and significantly impact the quality of life for SCD patients. According to the Sickle Cell World Assessment Survey, patients experience an average of 5. 3 VOCs per year, with 33% requiring hospitalization. Additionally, they miss over 1 day of work per week and face an estimated lifetime burden of 1. 7 million in medical costs. In 2023, the FDA approved two gene therapies for SCD: Casgevy and Lyfgenia. While promising, these therapies are prohibitively expensive (over 2 million) and require intensive procedures, such as hematopoietic stem progenitor cell (HSPC) isolation and myeloablative conditioning, as well as lengthy hospitalization. Here, we present an alternative therapeutic strategy to address the limitations of the current gene therapies. We have developed a cost-effective in vivo gene therapy delivery system using engineered extracellular vesicles (EVs). Extracellular vesicles are membrane limited particles secreted by cells. They are used as a mode of intercellular communication and can transport various biological agents between cells, including proteins, RNAs and DNAs. EVs are an attractive delivery vehicle due to their ability to avoid host clearance and travel into difficult to reach areas such as the bone marrow. Our EVs are designed to efficiently and selectively deliver CRISPR-Cas9 ribonucleoprotein (RNP) complexes for BCL11A gene silencing to HSPCs, particularly those with erythroid lineage bias, for effective in vivo gene therapy. To maximize the loading efficiency of Cas9 RNP complexes into our EVs, we generated EV-producing cells to overexpress Cas9 bound to CD63 via a photocleavable linker (PhoCl). Since CD63 is highly expressed on EV membranes, the fusion protein CD63-PhoCl-Cas9 facilitates the enrichment of Cas9 RNP complexes into EVs. This is superior to other conventional methods, including electroporation which is currently used in Casgevy. Furthermore, the use of PhoCl enables the release of RNP complexes into the cytoplasm of EV-recipient cells. Our confocal imaging analysis showed that Cas9 is highly enriched in endosomes and multivesicular bodies (MVBs), the intracellular compartments where EVs are formed, in our EV-producing cells engineered to express CD63-PhoCl-Cas9. To maximize the delivery efficiency to HSPCs for gene therapy, we generated EVs expressing stem cell factor (SCF), the ligand of the c-KIT receptor. SCF exists in two isoforms: a soluble form and a transmembrane form. Given that c-KIT is highly expressed on HSPCs with erythroid lineage bias, by expressing the transmembrane SCF on EVs, we can significantly enhance the efficiency of EV-mediated delivery to c-KIT+ HSPCs. We engineered HEK293T cells to overexpress the transmembrane SCF and confirmed SCF localization to the plasma membrane with confocal microscopy. Western blot analysis confirmed that EVs isolated from our SCF overexpressed cells also expressed SCF. To investigate the c-KIT targeting efficacy of our SCF-EVs, we compared the uptake levels between cells with varying c-KIT expression. We compared Jurkat and HEL cells, which had 21% and 98% c-KIT expression respectively when quantified using flow cytometry. We treated Jurkat and HEL cells with GFP+-SCF-EVs. Both cell lines showed a non-zero, time dependent increase in EV uptake. Notably, HEL cells had a greater than twofold increase in SCF-EVs compared to Jurkat cells (p=0. 0153). To further explore the rate of SCF-EV uptake by c-KIT+ cells, we generated 3 c-KIT knockdown (KD) cell lines of HEL using shRNA, all of which had reduced median c-KIT fluorescence intensity by over 50% (p0. 0001). We used the KD clones to confirm that a higher c-KIT expression enhances SCF-EV uptake. Our therapeutic strategy represents a new paradigm for the targeted, in vivo delivery of CRISPR-Cas9 RNP complexes to HSPCs. Our innovative approach has the potential to serve as a foundational technology for a safer, more accessible, and non-viral gene therapy applicable to a broad range of genetic disorders. The successful completion of this project will generate critical proof-of-concept data and enable us to pursue further translational development for in vivo gene therapy for SCD.