Miragen Therapeutics’ top official last week confirmed that post-myocardial infarction remodeling has joined chronic heart failure as the first two indications the microRNA drugs startup intends to pursue.
Speaking at the Center for Business Intelligence’s Executing on the Promise of RNAi conference, held last week in Cambridge, Mass., Miragen President and CEO Bill Marshall also presented preclinical data from the new program, while stressing the importance of conducting preliminary studies in normal animals before proceeding into disease models.
According to Marshall, the new effort focuses on inhibiting miR-15, an miRNA that regulates “a whole family of pro-survival factors” including cyclin D2, the over-expression of which has been shown to reduce the area of post-MI tissue death in transgenic animals.
“We consistently see up-regulation of miR-15 [and family members miR-195 and miR-16] in diseased versus normal [tissue], suggesting that the up-regulation is inhibiting the expression of pro-survival factors” and leads to an increase in cardiomyocyte death, he told RNAi News in a follow-up interview this week. “Finding that we can de-repress the pro-survival factors is pretty exciting, so we decided to move that forward.”
During his CBI presentation, Marshall said that Miragen and its collaborators found that a single, 80 mg/kg injection of a miR-15 antagonist into normal mice triggered “very potent down-regulation” of the miRNA, while a mismatch control sequence had no effect.
The researchers then moved onto mice whose left coronary artery had been banded in order to trigger an infarction. The animals were treated with the miR-15 antagonist the next day and analyzed three weeks later using high-resolution ultrasound to measure fractional shortening, which Marshall noted is “the closest equivalent to left ventricular ejection fraction.”
In treated animals, the team observed a roughly 60 percent improvement in fractional shortening compared with controls, Marshall said. A direct analysis of the animals’ hearts revealed about a 40 percent reduction in infarct size in those receiving the miR-15 antagonist.
Treated animals also experienced “a significant improvement in hypertrophy,” he noted.
“You get a significant up-regulation of [miR-15] when you induce the myocardial infarction, and we were able to not only blunt that up-regulation, but take it lower than the levels we observed” in normal mice, Marshall told RNAi News.
Marshall declined to offer a developmental timeline for the miR-15 program. However, he said that Miragen is planning to conduct “a follow-on, independent study in rodents” to confirm these findings.
Once the company has optimized a lead drug candidate, additional rodent experiments will be performed “to show that we can get the same effect as with the validating molecule,” he added. “Then, we will take that into rodent toxicology … [and] reach out to the FDA … to go over in detail what our plans are and what they would like to see.”
At the same time, Miragen is continuing to advance its chronic heart failure program, which focuses on miR-208. As previously reported by RNAi News, the company set its sights on this miRNA in part because of research conducted by Eric Olson, a Miragen co-founder and University of Texas Southwestern Medical Center researcher (see RNAi News, 5/15/2008).
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In a review article published in 2007, Olson noted that a key characteristic of cardiac hypertrophy, which frequently leads to heart failure, is “the re-activation of a set of fetal cardiac genes, including those encoding … fetal isoforms of contractile proteins such as beta-myosin heavy chain.”
“The consequences of fetal gene expression on cardiac function and remodeling are not completely understood, but the up-regulation of beta-MHC, a slow ATPase, and down-regulation of alpha-MHC, a fast-contracting ATPase, in response to stress has been implicated in the diminution of cardiac function,” according to the review.
That same year, Olson reported in Science that miR-208, a heart-specific miRNA that is encoded by an intron of the alpha-MHC gene, is required for cardiomyocyte hypertrophy, fibrosis, and the expression of beta-MHC in response to stress and hypothyroidism.
Marshall said this week that Miragen has found that a single, 80 mg/kg dose of a miR-208 antagonist can significantly inhibit the miRNA’s expression for up to 60 days. Additionally, the treatment raised levels of alpha-MHC while decreasing levels of beta-MHC, and greatly reduced myocyte hypertrophy.
During his presentation last week, Marshall also noted that Miragen has adopted the “vitally important” strategy of thoroughly validating its targets in normal animals before beginning work in models of disease. Doing so, he told RNAi News this week, is “one of the most important aspects” of the company’s miRNA drug development.
“If you look back at the literature in antisense, ribozymes, [or] siRNAs … people tend to want to move quickly towards a disease model,” he said. Using the antisense field as an example, Marshall said that there are many examples in the literature of people moving drug candidates into a disease model “even without understanding whether the [oligo] was capable of inhibiting the expression of the specific gene.”
While these studies sometimes resulted in an apparent therapeutic benefit, such as inhibition of tumor growth, the results were often later found to be due to non-specific effects
“It’s a requirement, from my perspective, that we go into a normal animal, we demonstrate that we can hit the target in the tissue, [and then do the experiment] with a control oligonucleotide,” he said.
In addition, Miragen typically examines the impact of an antagonist on three or four other miRNAs that don’t share homology with the target, “just to make sure we’re not messing with something more generally,” Marshall said.
Demonstrating that a drug candidate is working through the intended mechanism is something that has become a major issue for the RNAi and miRNA fields recently.
Late last year, researchers from Tekmira Pharmaceuticals published data in Human Gene Therapy showing that previously demonstrated antiviral effects of siRNAs targeting influenza in a mouse model were actually due to immune responses triggered by the duplexes, rather than by the RNAi mechanism (see RNAi News, 9/5/2008).
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“The potential influence of siRNA-mediated immune responses on key readouts of therapeutic efficacy is a critical consideration when designing and interpreting in vivo RNAi studies,” the Tekmira team wrote. “However, surprisingly few of the reported studies have adequately tested or controlled for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.”
Earlier in the year, researchers from the University of Kentucky reported in Nature that all siRNAs, regardless of sequence, suppress neovascularization (see RNAi News, 3/27/2008).
The lesson is not lost on Marshall, a veteran of the RNAi field who held various positions at Dharmacon and most recently served as vice president of technology and development at Thermo Fisher Scientific.
“From a process perspective, you end up spending some time up front doing these studies that you probably could have done in a [disease] model, but I think it’s so worthwhile to know you can affect the target before you trudge forward into the disease model,” he said. “These molecules, historically, can cause some artifactual outcomes, and people end up chasing [these] down well into the clinic. I want to make sure we’re on mechanism.”