Design and validation of AAV:miRNA-206 and AAV:miR206-sponge vectors.

<p>(a) The design of AAV vectors encoding either miR-206 or a miR-206-sponge (see Methods for details). (b–c) To confirm function, miR-206 and miR-206-sponge constructs were tested for ability to regulate the HDAC4 3’ UTR <i>in vitro</i> using C2C12 cells. After 48 hr, cells were lysed and luciferase activity was measured and normalized to β-gal activity. Whilst AAV: miR-206 inhibited HDAC4 3’ UTR activity by 65% (*, p<0.01 vs. control), the AAV: miR-206-sponge increased the activity of the HDAC4 3’ UTR by 60% (*, p<0.05 vs. control, n=3 per group repeated three times). (d) Administration of 1×10⁹ and 1×10¹⁰ vector genomes of AAV: miR-206 <i>in vivo</i> increased miR-206 transcription approximately 20 fold (*, p=0.002 vs. control) and 100 fold (*, p<0.001 vs. control) respectively, n=3 per group repeated three times. (e) AAV: miR-206 was injected into the TA muscles of mice and after 28 days, miR-133b and miR-1 levels were measured by RT-PCR (p=ND, n=8). (f) AAV: miR-206-sponge vector (1×10¹⁰ vector genomes administered) reduced miR-206 transcripts, as determined by RT-PCR (*, p=0.03 vs. control, n= 4 per group). (g) Whilst the AAV: miR-206-sponge vector (1×10¹⁰ vector genomes administered) reduced miR-206 transcripts (*, p=0.04 vs. control), it did not affect expression of miR-133b or miR-150 (N=ND, n=3 per group).