Extended Data Fig. 3: Downregulation of miR-199a target genes in transduced heart tissue and organ distribution of the AAV6-miR-199a vector.

a, Real-time PCR quantification of both strands of miR-199a in AAV6-control- and AAV6-miR-199a-injected pig hearts (n = 4 and n = 10, respectively) normalized to endogenous 5S rRNA. Data are mean ± s.e.m. b, mRNA levels of predicted and annotated target genes of miR-199a in AAV6-control- and AAV6-miR-199a-treated pig hearts (n = 4 per group) one month after MI and viral transduction. Data are mean ± s.e.m.; *P < 0.05 versus AAV6-control; two-sided t-test. c–e, Predicted target sites of miR-199a-3p in the 3′-untranslated region (UTR) sequences of swine cofilin-2, TAOK1 and βTRC according to TargetScan release 7.2. All three of these genes are verified to be direct targets of this miRNA in rodents; the corresponding 3′ UTR target sites for cofilin-2 and TAOK1 are conserved in swine; for βTRC, two alternative target sites in swine are shown. Other miR-199a-3p target genes originally identified in mice (in particular, homer1 and Clic5^11,29) are not conserved in the swine genome. In the pig genome, βTRC also has an additional predicted target sequence for miR-199a-5p, which is indicated. f, Predicted target site of miR-199a-5p in the 3′ UTR of pig HIF1A mRNA. g, Quantification of viral genomes in the indicated organs one month after intracardiac injection of AAV6-miR199a. Data are expressed as fold change over liver levels after normalization for cellular DNA content using 18S DNA as a reference (mean ± s.e.m., n = 4 per group). The levels of viral DNA in the myocardium of the injected animals were more than 18 times higher than in the liver and more than 40 times higher than in other organs (spleen, kidney and lung). h, Levels of miR-199a-3p RNA in the indicated organs one month after intracardiac injection of AAV6-miR-199a. Data are shown as fold change over endogenous miRNA levels in the liver of control animals after normalization for cellular 5S rRNA (n = 4 per group). Data are mean ± s.e.m. The amount of hsa-miR-199a-3p RNA was not elevated in any analysed organ, except for the heart. No overt signs of pathology, including hyper-proliferation (assessed by Ki67 staining) were observed.