(170d) Engineering Megakaryocyte-Derived Microparticles for Gene Delivery

Cell-derived microparticles (MPs) or microvesicles are small particles (100-1000 nm) shed from the plasma membrane from various types of cells, such as hematopoietic cells, endothelial cells, and embryonic stem cells (Cocucci et al., 2009). MPs play an important role in cell-to-cell communication by carrying and transferring various signaling molecules to target cells, such as RNAs, proteins or lipids. MPs have several key characteristics, and notably target specificity, biocompatibility and immunological inertness, that make them a vehicle for potential cargo delivery to desirable cell targets for therapeutic applications.

Megakaryocytic MPs (MkMPs) are the most abundant MPs in blood circulation, and were shown by our group to induce and promote the megakaryocytic (Mk) differentiation of human hematopoietic stem and progenitor cells (HSPC) (Jiang et at., 2014). We have recently shown that MkMPs have exquisite specificity in targeting only HSPCs, and preferably the more primitive HSCs. In this study, we aim to engineer MkMPs as vectors for delivering nucleic acids to HSPCs. We largely employed CHRF cell-derived MPs (CMPs) as a model MP in lieu of MkMPs, since CMPs can be produced readily and in large quantities, and are able to induce and promote Mk differentiation of HSPCs as well as MkMPs can. CHRF cells, a human megakaryoblastic cell line, have been validated as an excellent model of Mk differentiation of human cells. However, we also used primary human megakaryocytes and MkMPs when appropriate.

First, we desired to reduce, if not completely eliminate, the endogenous nucleic-acids (notably RNAs as most MPs contain no DNA) in CMPs so that we can load them with desirable cargo. We developed an RNase-treatment protocol so that the amount to RNA in CMPs decreased by 40 to 60 percent, and this was sufficient to abolish the function of CMPs in inducing and promoting Mk differentiation of HSPCs. This indicated that our RNase treatment protocol was effective in eliminating most of the endogenous RNA load. Then, we aimed to develop a reliable protocol for loading of desired cargo (and notably plasmid DNA (pDNA)) into CMPs. We examined two different methods for loading: endogenous (cargo loaded into parent cells before CMP generation) and exogenous (cargos loaded directly into CMPs). In endogenous loading, pDNA cargo was loaded into parent cells (both CHRF cells but also primary megakaryocytic cells were examined) by transfection, and MPs were collected after 48 hrs. In exogenous loading, pDNA cargo was directly loaded into MPs by electroporation at various conditions. The loading efficiency was quantitated by either measuring the fluorescent positive MPs if fluorescent-labeled pDNA was loaded, or quantitating pDNA amount from MPs and confirmed with DNA gel electrophoresis. We are detail our studies to optimize the loading efficacy and quantitate the efficiency of cargo delivery by CMPs or MkMPs to HSPCs.

Cocucci, E., G. Racchetti, and J. Meldolesi. 2009. Shedding microvesicles: artefacts no more. Trends Cell Biol. 19:43-51.

Jiang, J., D.S. Woulfe, and E.T. Papoutsakis. 2014. Shear enhances thrombopoiesis and production of megakaryocytic (Mk) microparticles that induce Mk differentiation of stem cells. Blood.

Acknowledgements: This work was supported by a State of Delaware Bioscience Center for Advanced Technology (CAT) grant (# 15A01570).