MicroRNAs, small noncoding RNAs that post-transcriptionally regulate gene expression, have been implicated in the development of diseases, including those affecting the heart. With regard to the heart, a considerable number of oligonucleotide therapeutics are presently in preclinical and early clinical trials. Specific delivery of microRNA therapeutics directly to the target cells and organs in vivo would comprise a breakthrough in developing oligonucleotide therapeutics. However, methodology to evaluate the efficacy of those therapeutics in the myocardium has been inadequate and hinders the development of cardiac nucleic acid-based therapeutics. 

Here, we developed a methodology to assess the efficacy of microRNA therapeutics in vivo at the single-cell level and compared the uptake of those oligonucleotides between healthy and diseased myocardium in mice.

We generated a dual fluorescent microRNA activity sensor to assess anti-miR efficacy quantitatively. The optimized sensor exhibited excellent specificity and sensitivity at the picomolar level. With the help of adeno-associated virus serotype 9 (AAV9), the sensor was delivered to the cardiomyocytes of mice. A mouse model of acute myocardial infarction (MI) was then employed to compare the efficacy of systemically administered anti-miR between diseased and healthy myocardium. Interestingly, no anti-miR uptake by the cardiomyocytes of SHAM mice could be detected. More strikingly, a significant anti-miR efficacy could be detected in cardiomyocytes of the mice of the MI group. Spatial quantification of the sensor at the single cell level revealed a more pronounced uptake of the microRNA therapeutics at the border zone of the diseased myocardium.

The microRNA activity sensor we developed and optimized is supreme to the standard methodology and could be beneficial to assess the efficacy of microRNA therapeutics under development. This study suggests that antisense oligonucleotides are taken up only by injured cardiomyocytes. Novel nucleic acid-based therapeutics targeting strategies should exploit this fact in the diseased myocardium.