Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.

心脏损伤后，心肌细胞会丢失并且不会再生。取而代之的是，成纤维细胞侵入死亡组织，在那里它们会产生疤痕，从而降低心脏功能。我们和其他人已经证明，由单个慢病毒/逆转录病毒在体内传递的特定 miRNA（miR 组合）或转录因子（GMT）的组合可以将成纤维细胞转化为心肌细胞并改善心脏功能。然而，由于 miR 组合或 GMT 的传递效率低，效果相对温和。我们假设通过优化交付可以提高效率。在第一个例子中，我们开发了一个多顺反子系统，以从单个构建体中表达 miR 组合的所有四种 miRNA。多顺反子构建体中每个 miRNA 的顺序导致了不同水平的 miRNA 表达。导致 miR 组合的四种 miRNA 中每一种表达水平相同的组合显示出最高的重编程效率。通过直接靶向成纤维细胞可以实现进一步的效率。几种 AAV 血清型的筛选表明 AAV1 显示出对心脏成纤维细胞的嗜性。将多顺反子表达与 AAV1 递送相结合，可在体内将心脏成纤维细胞重编程为心肌细胞。