The Nobel Prize in Medicine was awarded in early October to Victor Ambros and Gary Ruvkun for discovering how microRNAs (miRNAs) have central roles in gene expression by binding to target mRNAs and inhibiting their translation. Since their discovery in 1993, miRNAs have been found to influence gene expression pathways in cell differentiation, proliferation, and survival. Years of research have been done to understand how genetic pathways are directly influenced by miRNA binding and how these pathways are interrupted when miRNA expression becomes abnormal, leading to disease. However, it has been difficult to develop therapies that use or target miRNAs to restore normal gene expression patterns in diseases such as cancer. Understanding how miRNAs manage to regulate a plethora of pathways, in particular in disease states, is highly desirable for drug discovery, but right now, biotech companies must buckle down and search for miRNA’s targets.

Some companies¹ have tried to use miRNAs as therapeutics — as miRNA mimics and as antimiRs. A miRNA mimic behaves similarly to an endogenous miRNA, restoring or enhancing the function of a miRNA that may be lost or downregulated in a disease. An antimiR is designed to do the opposite — binding to and silencing an endogenous miRNA that is overexpressed. Both miRNA mimics and antimiRs can bind to multiple genes, amplifying either enhancement or silencing of specific signaling pathways. This is a strength for therapeutics aiming to restore normal phenotypes in a complex disease, but the flexibility of binding sites means that off-target effects are near impossible to avoid.

Preclinical studies have yielded some promising results, and a few have even moved into clinical trials, but none has reached phase 3 trials or been approved by the US Food and Drug Administration (FDA) to date. The first miRNA-based cancer therapy that went to a phase 1 clinical trial was MRX34, an miR-34a mimic, in 2013. miR-34a acts as a tumor suppressor, selectively targeting the p53 protein, and its expression is downregulated in many cancers. Mirna Therapeutics halted the trial 3 years later because of severe immune-mediated toxicities and patient deaths². A miRNA mimic to miRNA-29b from Viridian Therapeutics (previously miRagen Therapeutics) looked promising as a treatment for fibrosis, but was discontinued this year after phase 2 trials³. A phase 2 trial of an antimiR of miR-21 by Regulus Therapeutics for treating Alpert syndrome was terminated early in 2022. Two antimiRs targeting miR-122, an important host factor for hepatitis C infection, were also both discontinued in phase 2.

This trend is in many ways opposite to the therapeutic trajectory of small interfering RNA (siRNA), the discovery of which (several years after miRNA) won the Nobel Prize in 2006. Today, there are 6 double-stranded siRNA drugs approved by the FDA, and, over the past few years, more than 50 siRNA-based drugs have entered clinical trials.

miRNA therapies suffer from the major challenges of any other RNA-based therapy — primarily inefficient delivery and toxicity leading to immune responses⁴. But although mRNA vaccines and siRNA drugs have overcome some of these challenges, miRNA has been stuck. A primary reason is that miRNAs have lower specificity compared to an siRNA that is designed to target a single sequence, and functional validation is time-consuming and expensive.

The first step in generating any miRNA therapeutic is finding its target(s). We have improved algorithms for determining miRNA–mRNA binding interactions, large-scale sequencing data, and a massive data repository in MiRBase — all necessary to determine where a therapeutic can bind. But even in MiRBase, the majority of miRNAs are not well annotated and are tough to validate⁵. A miRNA–target binding validated by a crosslinking and immunoprecipitation sequencing (CLIP-seq) assay is not necessarily functionally relevant to gene regulation. Perhaps more functionally relevant are targets identified in miRNA overexpression assays, but it is difficult to distinguish direct miRNA targets from indirect ones. Targets identified in vitro often differ significantly from what is seen in vivo.

For a miRNA mimic that replaces a missing or under-expressed miRNA, target specificity is not the main problem. The main hurdles are finding a miRNA that influences a desired pathway directly and proving that restoring activity relieves the phenotype. Ideally, this miRNA would be influential in multiple stages of a disease or cancer, or in multiple cancers, so that targeting it would have a lasting effect. Researchers have used sequencing data to show that specific miRNAs that influence oncogenes and tumor suppressors are under-expressed in certain cancers, but they also have seen a huge amount of heterogeneity of miRNA expression, even within a single tumor. This heterogeneity is further promoted by hypoxic tumor microenvironments and local inflammation. Additionally, miRNA biogenesis enzymes are downregulated under these conditions, leading to large numbers of abnormally expressed miRNAs. To find a common regulatory miRNA candidate in a cancer or other disease, multiple biopsies need to be taken over the course of disease progression.

For antimiRs, when used as cancer treatments, target specificity is crucial — an antimiR may bind to a desired oncogene, but it may also bind to a tumor suppressor or to targets not involved in cancer. miRNA can bind to mRNA, but also to other miRNAs or noncoding RNAs. Science cannot yet predict all of the cellular pathways that would be influenced, or even all of the possible binding sites. An miRNA therapy would need to avoid targeting genes involved in normal cell homeostasis, and this will require a more complete understanding of the ‘targetomes’ of different miRNAs.

Challenges related to the delivery and toxicity of miRNA therapeutics can be addressed by adapting current methods for siRNA and mRNA delivery. In the first miRNA clinical trial, a lipid nanoparticle was used that had high uptake of miR-34a and a long circulation time but was not targeted to cancer cells. Today, we have targeted tumor delivery, as well as package-free delivery of ligand-conjugated miRNAs⁶ and ways to avoid endosomal sequestration⁷.

Right now, miRNA holds most potential as a diagnostic tool. miRNAs migrate out of cells and into body fluid, where they are protected from degradation by exosomes. It is relatively easy to get sequencing data from circulation to identify miRNAs that are more highly expressed in specific cancers or diseases, or positively or negatively associated with a treatment response. Diagnostic tools for miRNA panels are already available to clinicians.

Understanding the biological roles of miRNAs in disease has been a game-changer for the way we understand abnormal gene regulation, and miRNA biomarkers have a path forward in the clinic. However, until researchers find a way to address the issue of off-target effects with miRNA therapeutics, siRNA or mRNA will remain far ahead as therapeutic options.