Background/Hypothesis/Objective.

Expression of specific genetic information (genes) controls the diversity of the cells forming the human body. Genes can be active ("on") or silenced ("off"). DNA methylation (the addition of a methyl group, "a flag" to a DNA molecule) is the mechanism involved in turning-off certain genes and it is carried out by DNA methyltransferases (DNMTs). Although numerous studies have established a link between abnormal DNA methylation and dangerous conditions as cancer, the causes inducing the abnormal methylation remain unknown. RNAs are essential molecules produced in our body, with multiple biological functions. We previously identified a specific class of RNA interacting with the DNA methyltransferase 1 (DNMT1) and regulating its activity. Based on these findings, we hypothesize that abnormal DNA methylation can be corrected by RNAs. Aptamers are short synthetic RNAs acting as high affinity ligands for selected targets (in our case – DNMT1). Here, we plan to demonstrate that: (1) DNMT1-binding aptamers can be used to reduce globally DNA methylation by blocking DNMT1 activity; and (2) gene-specific DNMT1-binding aptamers can be used to turn-on exclusively targeted genes which are silenced by DNA methylation.

The critical question to be addressed by the proposed project.

Can we repurpose synthetic RNA molecules as global or gene-specific demethylating agents? For the past decades, demethylating agents (Azacitidine and analogs) have been extensively tested to correct abnormal DNA methylation, the hallmark of cancer, and received approval by the U.S. Food and Drug Administration (FDA) for the treatment of Myelodysplastic Syndromes (MDS). However, the high toxicity resulting from the spurious incorporation of these compounds in DNA and RNA molecules has limited their clinical use. Introduction of a tool able to target DNMT1 will provide a platform to modulate DNA methylation with less toxicity and high specificity.

The specific BMF disease to be researched.

MDS are an acquired Bone Marrow Failure syndrome with a risk of progression to Acute Myeloid Leukemia (AML) in approximately 30 percent of the cases. MDS are considered a pre-leukemic condition in which the bone marrow, the organ responsible for the production of all blood cells, is malfunctioning. Abnormal DNA methylation is one of the dominant mechanisms to turn-off genes preventing tumor formation, also known as "Tumor Suppressors," especially during the evolution of MDS to AML. MDS affect primarily elderly and as the population continue to age, the incidence of the disease is expected to grow. Thus, development of less toxic and more targeted hypomethylating treatments is warranted.

Innovative aspects of the proposed project. The major task of this study is to use synthetic RNA molecules, with strong affinity for DNMT1, to correct DNA methylation globally or in loco. The ability to control the expression of individual genes carries immense potential for clinical applications, together with the promise to limit drug high-toxicity and low specificity effects, which have held back MDS treatment. The proposed here innovative strategy aims at correcting abnormal DNA methylation without the limitations of current demethylating agents. The studies proposed in Aim 1 will compare the demethylating efficacy of DNMT1-binding aptamers to conventional demethylating agents, along with the respective molecular and biological effects. Aim 2 will test the demethylating efficacy of modified DNMT1-binding aptamers targeting specific genes, a feature completely lacking in the available today demethylating agents. The ultimate goal of this research is to develop an innovative gene-specific therapeutic platform to block deleterious DNA methylation profile.

The impact of the proposed research might have on the BMF field.

There is much interest in using RNA molecules as therapeutic tools and the proposed approach offers great advantages over the existing hypomethylating-based protocols: (a) high gene specificity; (b) low cytotoxicity; and (c) limited if not "absent" drug based off-target side-effects. Once completed, this project will provide both practical and scientific contribution to the field of BMF: on the one hand, a tool able to modulate DNA methylation will be instrumental in understanding the role of DNA methylation in MDS; on the other, by developing a specific approach to "switch-on" genes, silenced by DNA methylation, this study will offer an alternative therapy to existing hypomethylating agents "non-specific and highly toxic," and greatly improve care of patients with acquired BMF.