NEW YORK (GenomeWeb) – New data published this month by researchers from Regulus Therapeutics and academic collaborators show that long-term therapeutic silencing of microRNA-33 could result in a number of deleterious effects in mice including fatty liver and elevated triglyceride levels.
Notably, the report comes just months after Regulus and partner AstraZeneca announced they were dropping their miR-33 program in atherosclerosis. At the time, Regulus CSO Neil Gibson said that the decision stemmed from undisclosed challenges facing the effort
In their paper, which appeared in EMBO Molecular Medicine, the scientists called their findings "unexpected," and wrote that they highlight the importance of testing chronic miRNA inhibition strategies in non-human primates before moving them into human studies.
Interestingly, Regulus' data do not jibe with the results of a monkey study conducted by collaborators of Santaris Pharma, which is developing its own miR-33 inhibitor and found that silencing the miRNA did not cause adverse effects.
For some time, various academic groups have been investigating the therapeutic potential of silencing miR-33, which has been shown to control genes involved in regulating cellular cholesterol export, fatty acid oxidation, insulin signaling, and glucose production.
For instance, in 2010 a team from Massachusetts General Hospital published a study showing that miR-33 can affect cholesterol homeostasis by working with sterol regulatory element-binding proteins (SREBPs), which are key transcription regulators of cholesterol synthesis and uptake genes. In 2011, Santaris took a license to the miR-33 intellectual property generated at Mass General.
Given that levels of circulating high-density lipoprotein cholesterol (HDL-C) and increased reverse cholesterol transport were found to have a strong inverse correlation with atherosclerotic vascular disease, miR-33 became a target of interest for influencing the progression and regression of atherosclerosis, according to the EMBO Molecular Medicine report.
Regulus found that blocking miR-33 promoted the clearance of excess cholesterol as well as the regression of atherosclerosis in mouse models with established atherosclerotic plaques. As described in a paper appearing in the Journal of Clinical Investigation in 2011, company scientists and collaborators at NYU Langone Medical Center found that treating mice with a miR-33 antagonist increased HDL-C, enhanced reverse cholesterol transport to the plasma, liver, and feces, and reduced plaque size and lipid content.
Based on these and other data, Regulus added a miR-33 inhibitor for atherosclerosis to its pipeline. In 2012, the program became part of a broad alliance with AstraZeneca.
Earlier this year, however, Regulus disclosed that the companies had opted not to pursue miR-33, offering few details about the reasoning behind the move other than Gibson citing "certain technical challenges" and "increasing complexity in the competitive landscape for atherosclerosis."
This month's EMBO Molecular Medicine publication appears to offer some additional insights.
While previous studies tested the effects of 2' fluoro/methoxyethyl phosphorothioate-backbone-modified anti-miR-33 oligonucleotides in mice receiving either high-fat or normal diets over four weeks, in the latest experiments treatment was extended to 12 weeks.
Prolonged silencing of miR-33 was found to result in hepatic lipid accumulation and increased plasma triglyceride levels in mice fed the high-fat diet. Further, hepatic expression of genes involved in fatty acid synthesis was increased with treatment.
"Proteomic analysis demonstrates that long-term anti-miR-33 therapy results in a pronounced upregulation of major urinary proteins that can make up 5 percent of the total RNA transcripts in the male murine liver," according to the EMBO Molecular Medicine paper.
Meanwhile, nuclear transcription Y subunit gamma (NFYC) was markedly increased in mice administered with miR-33 antisense oligos compared to control mice. NFYC is a target of miR-33 and a member of the three NF-Y subunits required for DNA binding and full transcriptional activation of SREBP-responsive genes.
"Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia," the researchers concluded.
In their paper, Regulus and its collaborators pointed to two studies conducted by other groups — including one led by Santaris and Mass General researchers — examining the effects of miR-33 knockdown in non-human primates and finding that inhibition of the miRNA did not have any adverse effects, including liver toxicity as assessed by measuring plasma transaminase levels.
Importantly, in the Santaris/Mass General study, animals were treated for over 100 days.
Despite these conflicting reports, "the adverse effects reported in miR-33-deficient mice and in this study raise awareness that long-term inhibition of miR-33 might cause adverse effects," according to the EMBO Molecular Medicine paper.
The findings also "open new questions about how miR-33 regulates lipid and glucose metabolism at the organismal level," Regulus and its collaborators wrote. "Further studies will be important for elucidating the molecular mechanism and tissue specificity by which miR-33 controls cholesterol, fatty acid, and glucose metabolism."
Officials from Regulus did not return requests for additional comment.