In vivo biological function, biodistribution and targeted gene therapies of hematopoietic stem cells using endogenous and engineered cargo-loaded megakaryocytic membrane vesicles
Date
2023
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
There is a large spectrum of highly pervasive hematological disorders affecting red blood cells (erythrocytes), white blood cells (granulocytes), platelets (thrombocytes) and lymphocytes. Hematopoietic stem and progenitor cells (HSPCs) can differentiate into any of these blood cell types, and directed gene editing of HSPCs can provide therapeutic benefits to patients suffering from a variety of genetic hematological disorders, ranging from immunodeficiencies to thrombocytopenia. For more transient gene therapy, gene expression can be modulated epigenetically using RNA interference through administration small RNAs into the target HSPCs. Thus, by directly administering gene therapeutics into HSPCs, a significant proportion of hematological diseases can potentially be ameliorated. ☐ Previously, our lab has demonstrated that extracellular vesicles (EVs) derived from megakaryocytes (Mks), which are platelet-producing blood cells, can readily interact with and deliver cargo to HSPCs in vitro. In our studies, we demonstrated that these MkEVs also have the propensity to localize to the HSPC-rich murine bone marrow roughly 24-hours after administration in vivo in both wild-type (WT; Balb/c) and immunocompromised (NSGTM) mice. Upon delivery to the bone marrow, we observed robust MkEV-induced megakaryopoiesis (i.e., development of megakaryocytes), which subsequently promoted de novo biosynthesis of platelets. Furthermore, through histology, we confirmed that these MkEVs specifically interact with murine HSPCs and other blood cells in the bone marrow and lungs in vivo, thus enabling EVs to serve as efficacious drug delivery vehicles. Thus, we hypothesized that CRISPR Cas9, siRNA, and other therapeutics can be delivered specifically to HSPCs through receptor-mediated endocytic pathways to HSPCs using both natural (MkEVs) and semi-synthetic membrane vesicles (MkMVs). ☐ Accordingly, we have successfully loaded membrane-wrapped PLGA nanoparticles (NPs) with siRNA for homotypic delivery to HSPCs in vitro and observed effective2downregulation of a characteristic HSPC-specific gene following uptake. Next, we observed that the native HSPC tropism of Mk and CHRF-288 (a megakaryoblastic cell line) MV-wrapped NPs is conserved in vivo in WT mice, as the MV-wrapped NPs preferentially localized to HSPCs within the marrow. We extended this concept by loading fluorescent protein-expressing plasmid DNA into MkEVs and CHRF EVs, which facilitated successful targeted delivery and subsequent expression of the DNA in the HSPCs of treated NSG mice. ☐ Likewise, CRISPR Cas9, a tunable sequence-specific endonuclease capable of performing precise cuts to DNA, was successfully delivered to HSPCs via another membrane-wrapped polymeric system in vitro which exhibited more efficient gene knockout and reduced cytotoxicity in comparison to traditional plasmid-based Cas9 systems. With this system, we also disrupted a key regulatory gene (c-myc) in uncommitted HSPCs, which ultimately induced HSPC differentiation to megakaryocytes. Finally, to showcase the versatility of EVs and MVs, we explored the potential for using SARS-CoV-2 spike (S) protein-expressing megakaryoblastic cells to produce S protein-decorated EVs and MVs, which could function as viral decoys that could prophylactically prime the immune system prior to a COVID-19 infection. As blood-derived membrane vesicles exhibit comparably less immunogenic risk than some other drug delivery vehicles, both unmodified and cargo-loaded megakaryocytic membrane vesicles could potentially serve as safe and effective methods for facilitating HSPC-specific gene editing and modulation in vivo.
Description
Keywords
Extracellular vesicles, Gene therapy, Hematopoietic stem and progenitor cells, Megakaryopoiesis, Targeted drug delivery, Targeted hematological therapies, Blood cells