Diffron C, a Novel Oligonucleotide Based Gene-Silencing Agent, Induces Terminal Differentiation in Primary Murine Cells
Authors: Ichim Christine Victoria , Dervovic Dzana , Koos David Raymond , Wells Richard A. 
Published: October 2019

Abstract
Myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML) are closely related diseases of haematopoietic stem cells (HSCs), characterized by ineffective terminal differentiation, effacement of normal blood cell production and accumulation of neoplastic cells, known as blasts, in the bone marrow, peripheral blood, and other tissues. In the past ten years it has become apparent that cancers are not composed of uniform populations of cancer cells. Instead, the great majority of the cells making up a cancer have very limited ability to undergo cell division, or have completely lost the ability to divide - only a minority of cells, known as cancer stem cells, retain the ability to divide in an unlimited way. To identify potential therapeutic targets, we studied the molecular signature of leukemia stem cells identifying the orphan nuclear receptor NR2F6 as a potential therapeutic target for differentiation therapy for MDS and AML. We've previously shown that NR2F6 is over expressed in patients with myelodysplastic syndromes and acute myeloid leukemia. In vitro, overexpression of NR2F6 inhibits differentiation of cell lines and primary bone marrow cells. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model lead to a condition that resembled myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. While overexpression of NR2F6 in 100% transduced bone marrow grafts that were sorted by flow cytometry prior to transplantation lead to a rapidly fatal erythroleukemia that was characterized by a block in terminal differentiation, pancytopenia and profound anemia. Furthermore, silencing of NR2F6 expression in human and mouse-leukemia cell lines causes terminal differentiation and death by apoptosis. This forms the basis of the therapeutic concept that we wished to develop further, by identifying gene-silencing methods that could be advanced to preclinical development for the treatment of myelodysplastic syndrome and acute leukemia.
The discovery of the role of NR2F6 in leukemogenesis and the induction of differentiation of leukemia cell lines following silencing of NR2F6 expression suggests that this is a logical therapeutic target for differentiation therapy using gene silencing technology. Hence, we wished to show that silencing of NR2F6 using DiffronC, an oligonucleotide based gene-silencing technology, in primary mouse hematopoietic cells promoted terminal differentiation. Herein, we show that while DiffronC did not significantly reduce the number of colony forming units, it did significantly increase colony size. This was especially striking in cultures grown under erythroid conditions, where we observed a 3.21 increase in the number of cells per colony. We then showed that DiffronC reduced the clonal longevity of bone marrow cells: In replating experiments we observed a significant decrease in secondary colonies in cells treated with NR2F6 shRNA. Furthermore, DiffronC resulted in a drastic reduction in KSL stem and progenitor cells after six days of ex vivo culture, and caused a dramatic decline in lineage negative cells, and a concurrent increase in cells expressing the myeloid markers CD11b and Gr-1, suggesting that they had differentiated into cells of the neutrophil lineage. This was confirmed by examination of the cytomorphology of the bone marrow cultures. Taken together, these results establish that NR2F6 is a negative regulator of terminal differentiation of the hematopoietic lineage and lays the foundation for use of gene silencing technology using DiffronC as differentiation therapy for diseases with blocked differentiation such as leukemia and myelodysplastic syndromes.

Disclosures
Ichim: Entest BioMedical Inc: Employment, Equity Ownership, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wells:Alexion: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.