(157s) Targeted Gene Therapy of Hematopoietic Stem and Progenitor Cells Using Cas9 and siRNA-Loaded Megakaryocytic Membrane Vesicles

There is a large spectrum of highly pervasive hematological disorders affecting red blood cells (erythrocytes), white blood cells (granulocytes), platelets (megakaryocytes) and lymphocytes. Hematopoietic stem and progenitor cells (HSPCs) can differentiate into these blood cell types, and gene editing of HSPCs can provide therapeutic benefits to patients for a variety of genetic hematological disorders, ranging from immunodeficiencies to thrombocytopenia^1-3. For more transient gene therapy, gene expression can be modulated epigenetically using RNA interference through administration small RNAs into the target HSPCs^4,5. Thus, by directly administering gene therapeutics into HSPCs, a significant proportion of hematological diseases can potentially be ameliorated.

Previously, it has been demonstrated that microparticles derived from megakaryocytes (MkMPs) can readily interact with and deliver cargo to HSPCs in vitro⁶. In our current study, we have demonstrated that these MkMPs also have the propensity to localize within murine bone marrow roughly 24-hrs after administration⁷. Furthermore, through histology, we determined that these MkMPs also interact with HSPCs and other blood cells in the bone marrow and lungs in vivo, thus enabling microparticles to serve as efficacious drug delivery vehicles. Thus, it is hypothesized that CRISPR Cas9 and siRNA can be delivered specifically to HSPCs through receptor-mediated endocytic pathways to HSPCs using both natural (microparticles) and semi-synthetic membrane vesicles. We have successfully loaded membrane-wrapped PLGA nanoparticles with siRNA for delivery to HSPCs in vitro and observed effective downregulation of an HSPC-specific gene. Likewise, CRISPR Cas9, a sequence-specific nuclease, was successfully delivered to blood cells (CHRF) via a similar membrane-wrapped polymeric nanoparticle in vitro and exhibited efficient gene knockout and reduced cytotoxicity in comparison to traditional plasmid-based Cas9 systems. Finally, blood-derived membrane vesicles minimize the immunogenic risk associated with other vehicles, potentially establishing cargo-loaded membrane vesicles as a safe and effective method for cell-specific in vivo gene editing and modulation.

REFERENCES:

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