Development of hematomir technologies for the hematopoietic stem cells ex vivo expanion

Hematopoietic stem cells (HSCs) transplantation is the primary therapeutic modality used to treat many hematopoietic disorders. It based on the capability of a small quantity of HSCs to repopulate whole blood lineages. Along with limited availability of suitable donor, the need for sufficient number of donor HSCs are still challenging in clinical relevance. This has been addressed via ex vivo HSC expansion albeit with restricted success and development of alternative strategy that could improve HSC expansion is required. To this end, we examined about 200 genes that are specifically knockout in HSCs in vivo and determined the loss-of-function phenotypes of gene. 57 of these genes increased at least one and half fold stem cell pool or cell cycle activity. Besides, eight seed sequences of miRNAs using prediction algorithms to target 3' UTR of these hematopoietic factors were determined. We aimed to develop synthetic RNA interference (RNAi) based technologies, namely HematoMiRs, targeting of HSC quiescence factors that could be utilized for the expansion of HSCs ex vivo. Here, we found that HematoMiRs targeting over 50 factors involved in HSC quiescence induced robust ex vivo murine and human HSCs expansion. Ex vivo expanded HSCs with HematoMiR-5 treatment revealed enhanced HSC entry into cell cycle and increased cycling through down-regulation of negative cell cycle regulators without causing HSC senescence. HematoMiR-5 treatment led to sustained HSCs self-renewal ability and no increased change in apoptosis rate of HSCs. HematoMiR-5 expanded HSCs transplantation demonstrated successful engraftment and repopulation capacity in the recipient animals. HematoMiR-5 directed silencing of hematopoietic factors did not result in excessive ROS production and down-regulated several oxidative stress related gene expression. HematoMiR-2 and HematoMiR-5 treated murine HSPCs reduced DNA damage during the time course of their actively cycling. Furthermore, different combinatorial treatments of selected HematoMiRs mixture determined for robust ex vivo HSC expansion. Our results suggest that HematoMiRs technology provides HSC ex vivo expansion through the sequence-dependent regulation of HSC quiescence modulators. This project was funded by ICGEB (CRP/TUR15‐02_EC).