3031 – NANOTECHNOLOGY BASED DELIVERY OF SMALL INTERFERING RNA MOLECULES FOR EXPANSION OF HUMAN HEMATOPOIETIC STEM CELLS

Hematopoietic stem cell (HSC) transplantation is a life-saving treatment for both malignant and inherited hematological diseases. Umbilical cord blood (UCB) serves as a widely available source of HSCs; however, low cell numbers remain a limiting barrier for adult transplantations. A safe and efficient method to expand and maintain the number of engraftable blood stem cells ex vivo could greatly increase the proportion of UCB units that can be used for transplantations. From high throughput genetic screens, we have identified several lentivirally delivered shRNA molecules with profound expanding effects on human hematopoietic stem and progenitor cells (HSPCs). Here, we reasoned that the corresponding synthetic siRNAs to these highly potent shRNAs, if delivered transiently, could serve as chemical inducers of ex vivo HSC expansion without the permanent effects that would accompany lentiviral delivery. However, a key challenge would be to find a safe and non-toxic approach to deliver these siRNAs, as standard transfection methods for siRNA show substantial toxicity in primary cells. To this end, we recently established a nanotechnology-based protocol that enables gentle delivery of biomolecules through tube-like structures directly into the cytoplasm of HSCs in a completely non-toxic manner. We optimized this method for delivery of siRNA molecules into UCB derived HSPCs and tested a number of siRNAs converted from the shRNAs identified in our previous screens. In particular one siRNA molecule (si31) strongly enhanced the propagation of CD34+CD90+ HSPCs over 5 days of in vitro culture (4-fold compared to control). We are currently performing more detailed functional tests of the expanded cells both in vitro and in vivo. If successful, our approach has the potential to become a new method for HSC expansion, which may be relevant for both research and clinical applications.