Rescue of mitochondrial power outages with γPNA-based miR-122 inhibitor

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MicroRNAs (miRNAs) are crucial post-transcriptional regulators that significantly affect gene expression in metazoans through base pairing with target mRNAs. They can traverse cellular and tissue boundaries via extracellular vesicles or as miRNA-Ago complexes to reach distant organs, thereby exerting substantial influences on miRNA activity in both donor and recipient cells. This phenomenon has been demonstrated in various disease models.¹ The role of miR-122, a liver-specific microRNA that is increasingly released by hepatocytes under elevated lipid conditions, has raised concerns about its possible harmful effects on non-hepatic cells.² Endothelial cells (ECs) absorb these microRNAs, and internalized miR-122 subsequently impairs mitochondrial function in ECs, thereby inducing obesity-related vascular dysfunction in response to a high-fat diet. In this issue of Molecular Therapy Nucleic Acids, Gaddam et al.³ demonstrate that a carefully engineered γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (e-γP-122-I), which specifically targets endothelial cells, can restore mitochondrial function by reversing miR-122’s effects and prevent vascular dysfunction in an obesity-associated prediabetes model. These findings underscore the promising potential of γPNA-based miR-122 inhibitor as a targeted therapeutic agent for obesity-related diseases driven by high-fat diets.

Hepatocytes release miRNAs, including miR-122, into the bloodstream in response to metabolic stresses such as lipotoxicity, oxidative stress, and insulin resistance.² The release of miR-122 from stressed liver cells and under metabolic challenges, such as elevated lipid levels, is mediated by the miRNA export regulator protein HuR, a member of the ELAVL family that reversibly binds miR-122 and facilitates its export in stressed hepatic cells.⁴ miR-122 constitutes approximately 70% of liver miRNAs, and its presence in other tissues via extracellular vesicles (EVs) or via Ago-miR-122-complex-mediated transfer plays a crucial role in systemic disease and cell communication.⁵

miR-122 can be transported to tissues, including the vascular endothelium, via extracellular vesicles (EVs), thereby helping maintain vascular health by promoting angiogenesis and tissue repair. In endothelial cells (ECs), miR-122 regulates energy metabolism. This liver-derived miRNA, a metabolic regulator, supports mitochondrial function and energy homeostasis by targeting key factors, including PGC-1α. It influences mitochondrial biogenesis, translation, and ATP production, while promoting a switch toward oxidative metabolism and impacting lipid, glucose, and amino acid pathways.⁶ In non-liver tissues, EV-miR-122 significantly influences metabolic reprogramming, as noted in breast cancer cells, where miR-122 aids tumor cells in conserving glucose.⁷

In their study, Gaddam et al.³ have demonstrated that in obesity and diabetes, excess miR-122 impairs mitochondrial function in the aorta, decreasing oxygen consumption and contributing to diabetic vasculopathy. Therapeutically, agents such as γPNA-based miR-122 inhibitors restore vascular function in prediabetic models without affecting liver or kidney function. miR-122 has been reported earlier to induce EC apoptosis by targeting XIAP or BCL2, which may lead to atherosclerosis.⁵ However, in deep vein thrombosis (DVT), overexpression confers protection to endothelial cells (ECs) against oxidative stress through the p53 pathway. Elevated miR-122 levels also promote endothelial-to-mesenchymal transition (EndMT) by targeting NPAS3, thereby facilitating plaque formation.⁸

miR-122 inhibitors are potential therapeutic agents, primarily antisense oligonucleotides (ASOs) and locked nucleic acids (LNAs), designed to target and sequester liver-specific miR-122. This action decreases miR-122’s ability to stabilize the hepatitis C virus (HCV) genome. Notable examples include Miravirsen (SPC-3649) and RG-101, which have demonstrated significant, dose-dependent reductions in HCV viral load in clinical trials.⁹ Unlike traditional antisense oligonucleotides, the inhibitors used by Gaddam et al.³ employ gamma-modified PNA technology, replacing the standard sugar-phosphate backbone with a neutral pseudo-peptide backbone modified at the gamma position.³ The γPNA structure exhibits exceptional resistance to enzymatic degradation and binds more effectively to the target miR-122 sequence than standard DNA/RNA-based molecules (Figure 1). In the referenced report,³ the authors developed an EC-specific miR-122 inhibitor that modulates mitochondrial function by regulating miR-122-dependent expression of mitochondrial components such as sdha and pkm2. An increase in oxygen consumption rate and a decrease in the electron transport chain complex (ETC) level in the aorta of high-fat-diet mice after γPNA-122-I treatment establish the miR-122’s impact on the ETC function in ECs.³

Figure 1.

Modified anti-miRs resistant to nucleases inhibit endogenous miRNAs

Both LNA modification and γPNA modification enable antisense molecules to be highly resistant to enzymatic degradation and to bind miRNA with high affinity. Locked nucleic acid (LNA) oligonucleotides incorporate a 2′-O, 4′-C-methylene bridge that stabilizes the ribose in a 3′-endowed (north) conformation, making them a molecule of high thermal stability, while γPNA oligos have fewer off-target interactions and lower cytotoxicity, even at higher concentrations.^3,10 In gamma-modified PNA technology, the standard sugar-phosphate backbone is replaced with a neutral pseudo-peptide backbone at the gamma position.³

The neuropilin-1 (NRP-1) protein is crucial for the entry of the Ago-miRNA complex into ECs. Gaddam and colleagues explained that miR-122’s influence on EC is likely mediated by its internalization via the NRP-1 pathway.³ A non-cell-type-specific miR-122 inhibitor, previously used by the same research group, impacts metabolic and respiratory processes across various tissues in high-fat-diet-fed animals, including ECs.¹⁰ Conversely, the EC-specific miR-122 inhibitor used in the recent work by Gaddam et al.³ offers greater potential by targeting mitochondrial function, improving vascular health, and potentially alleviating prediabetic traits, such as insulin resistance, associated with high-fat-diet-induced obesity. They created a γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (γP-122-I or e-γP-122-I) that alleviates vascular endothelial dysfunction in mice fed a high-fat diet. The diethylene-glycol-containing γP-122-I was shown to counteract the diet-associated increases in blood and aortic miR-122 levels, endothelial dysfunction, and poor glycemic control.¹⁰ Safety evaluations demonstrated that γP-122-I did not affect blood counts or liver and kidney biochemical functions during short-term use. Long-term exposure did not alter adiposity or the histology of the kidney, liver, or heart. Therefore, γP-122-I effectively rescues endothelial dysfunction without notable toxicity, underscoring the potential of γPNA technology to develop safe and efficient miR inhibitors. The study also shows that these inhibitors preserve mitochondrial function in endothelial cells by preventing miR-122-mediated reductions in oxygen consumption and respiration in diabetic models. These compelling findings position miR-122 as a promising therapeutic target and establish a foundation for developing safer, more effective treatments for diabetic vasculopathy (Figure 2).

Figure 2.

Effect of inhibition of imported miR-122 on mitochondrial function of endothelial cells (ECs)

This image shows that upregulation of liver-derived miR-122 in endothelial cells under high-fat-diet conditions impairs mitochondrial function by targeting mitochondrial respiratory complexes, leading to altered oxygen consumption and mitochondrial dysfunction. With γPNA-modified miR-122 inhibitors, the process gets reversed, rescuing mitochondrial function in ECs.³

Nevertheless, challenges remain in determining the optimal concentration of anti-miR-122 molecules, elucidating their long-term effects on the immune response and liver function, and minimizing toxicity in chronic hyperlipidemia, which affects metabolic processes. Additionally, elucidating the molecular mechanisms linking the observed alterations in transcriptomic profiles to miR-122 inhibition or ectopic miR-122 expression—particularly their relationship with metabolic regulation and mitochondrial function in endothelial cells—is complex. The approach outlined paves the way for more detailed investigations into targeted delivery to endothelial cells, reducing off-target effects, and enhancing delivery efficiency. Exploring methods to inhibit miRNA release, transport, or entry pathways, as well as comparative analysis with other classes of miR-122 inhibitors, such as LNA-modified antisense oligomers, will be essential to achieving successful therapeutic and clinical outcomes and represent promising future directions.

The exact molecular pathways through which miR-122 influences mitochondrial function, as well as other possible mechanisms by which miR-122 might affect endothelial cell behavior—such as its regulation of miR-204 expression and activation of TLR-870—remain to be clarified.³ Additionally, the long-term consequences of inhibiting miR-122 on systemic metabolism and vascular health need further research. It is essential to investigate similar methods for regulating mitochondrial function in hepatic cells, where miR-122 is more abundant. Additionally, examining the impact of the γPNA-based miR-122 inhibitor on miRNA turnover and export in the liver and recipient organs is an equally important question to follow.

Acknowledgments

S.N.B. acknowledges the University of Nebraska, USA’s Start-Up Support Grant. The Lieberman Research Award, Department of Anesthesiology, UNMC, supports K.M.

Author contributions

S.N.B. and K.M. conceived the idea, planned, and wrote the commentary.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors have used Grammarly to improve grammar and flow.