AptamiR has discovered that inhibition of microRNA-22-3p by several complementary single-stranded antagomirs administered subcutaneously resulted in increased lipid oxidation, mitochondrial activity and energy expenditure. These effects may be mediated through activation of target genes like KDM3A, KDM6B, PPARA, PPARGC1B and SIRT1 involved in lipid catabolism, thermogenesis and glucose homeostasis. In the model of Diet-Induced Obesity in mice of various ages, weekly subcutaneous injections of various microRNA-22-3p antagomirs produced a significant fat mass reduction, but no change of appetite nor body temperature. Insulin sensitivity as well as circulating glucose and cholesterol levels were also improved.
In Silico Results
Prediction of hsa-miR-22-3p conserved pairing to the 3’UTR region of KDM3A and other metabolic/thermogenic genes (TargetScanHuman 7.2 prediction of microRNA targets database).
In Vitro Results
In vitro studies of miR-22-3p inhibition were carried out in primary cultures of human subcutaneous adipocytes, the ultimate targets for an anti-obesity drug (“clinical trial in a dish”).
Next Generation Sequencing after a miR-22-3p antagomir single transfection showed that the expression of the following genes was significantly altered (fold-changes and/or p values) in the presence of a miR-22-3p antagomir: they are CEBPD, CREB1, EIF4EBP2, KDM3A, KDM6B, KLF11, KLF6, LAMC1, MFGE8, NAA20, NCOA1, PPARA, PPARGC1B, PRDX3, RUNX2, SIRT1, TRPM8 and UCP1. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) resource, it appears that the PPAR signaling pathway is activated in human adipocytes in culture in the presence of the miR-22-3p antagomir.:
In Vivo Results
Body weight profile of C57Bl/J6 adult male mice on 10% fat diet alone (saline 10% fat, gray line) or on 60% high fat diet alone (saline 60% fat, black line) or in the presence of a miR-22-3p inhibitor (miR-22 60% brown line) during 8 weeks of treatment, starting at week 14 of age:
Representative pictures of 22-week old C57Bl/J6 adult male mice on normal diet (left picture), 60% high fat diet alone (right picture) or 60% high fat diet on miR-22-3p inhibitor (middle picture) at the end of 8 weeks of treatment. The average body weight per group is represented under each corresponding picture:
H&E staining of various fat tissues of 20-week old C57Bl/J6 adult on 60% high fat diet alone (Saline) or in the presence of the miR-22-3p antagomir APT-110 (APT-110) collected at the end of 6 weeks of treatment started at week 14 of age:
For subscapular brown fat, the smaller size of the cells prevented an accurate measurement of their cell perimeter. Instead, the percentage of fat was calculated. It was 47 ± 3% in the saline group versus 19 ± 1% in the miR-22 antagomir group (p = 0.0004).
We performed a dedicated study exploring the metabolic and energetic effects of our first miR-22-3p antagomir drug candidate (APT-110) in the model of Diet-Induced Obesity in C57BL/6J mice. Administration of the APT-110 miR-22-3p antagomir reduced body weight gain in mice on 60% high fat diet when compared to the vehicle-treated animals. The body weight difference became statistically significant as of the third week on treatment. At the end of 12 weeks on high fat diet (27-week old mice), the mice treated with APT-110 gained significantly less weight (-30%, p = 0.0041) than the mice receiving saline injections. Body composition analysis by NMR at the end of the study (day 82 of treatment) showed that the body weight difference was due to a fat mass difference only (-12% in the APT-110 group, p ≤ 0.001) whereas lean mass was identical in both groups.
Weekly subcutaneous injections of the APT-110 for 12 weeks produced a sustained increase of energy expenditure as early as Day 11 of treatment, a significant fat mass reduction, but no change of appetite nor body temperature. Insulin sensitivity as well as circulating glucose, cholesterol and leptin were also improved. Oral glucose tolerance tests were normalized. There was a dramatic reduction of liver steatosis at completion of the study after 3 months of active treatment. RNA sequencing by NGS revealed an activation of lipid metabolism pathways.
Total energy expenditure measured over 24 hours at different time points in mice receiving saline injections (black columns) or the APT-110 miR-22-3p antagomir (brown columns). Measurements on day 53 were made in the presence of the beta-3 receptor agonist CL316243;
Oral Glucose Tolerance Test (OGTT) during the last 12th week of treatment in mice receiving saline injections (black line) or the APT-110 miR-22-3p inhibitor (brown line):
Homeostatic Model Assessment of Insulin Resistance
(HOMA-IR) during the OGTT (time point -30 min) on the last 12th week of treatment in mice receiving saline injections (black dots) or the APT-110 miR-22-3p inhibitor (brown squares):
Histologic appearance of livers (H&E staining) at the end of 12 weeks of treatment in mice receiving saline injections or the APT-110 miR-22-3p inhibitor:
APT-110 effects on gene expression
RNA-Seq analyses of organs and tissues after 3 months of treatment with the APT-110 miR-22-3 p antagomir showed a general down-regulation of fatty acid biosynthetic pathway genes in the APT-110 treated livers and a general upregulation of fatty acid metabolism genes in the APT-110 treated inguinal fat (log2FC is comparing APT110 to Controls). Of note, the complement and coagulation pathways were downregulated in the APT-110 treated subscapular and inguinal samples.
Is shown below our model of miR-22-3p target genes involved in lipid oxidation, mitochondrial functions, thermogenesis, glucose metabolism, adipocyte differentiation, inflammation and anti-oxidation (One miRNA/Several Targets model):