New biotechnology to inhibit microRNA activity and novel applications for craniofacial and dental research

Objectives: A new plasmid-based microRNA inhibitor system (PMIS) effectively inhibits microRNA (miR) activity in cells and mice. To develop a new method for the inhibition of miRs with high efficacy, non-toxicity and highly specific.
Methods: We generated a novel RNA-based hairpin molecule carrying an anti-sense miR seed sequence that effectively knocks down endogenous miRs and mitigates their effects on messenger RNA in the cell. By using a native RNA-based molecule for miR knockdown, the PMIS platform avoids many of the traditional pitfalls associated with miR inhibition using synthetic oligonucleotides.
Results: The PMIS approach is more specific and stable, effective at a lower dose, more cost-effective and more importantly it is not toxic to living tissues, in contrast with many traditional miR inhibition strategies. We have generated miR knockdown mice that show defects in craniofacial and tooth development. Mice with the PMIS in the Rosa26 loci are activated with specific Cre lines or can be constitutively expressed during development. miR inhibition in mice reveals a role for miRs in many developmental pathways and cellular processes. When combined with a bioinformatics approach the PMIS can determine new miR targets and biological processes. The PMIS can be delivered using viral vectors, nanoparticles and lipid-based systems to effectively knockdown miR activity. We have used the PMIS to reprogram cells, define new developmental processes and inhibit cancer cell growth.
Conclusions: The PMIS is a new tool that is highly effective for inhibiting miR activity in vivo and in vitro. Importantly, the non-toxic nature of the PMIS molecule makes it promising platform for the delivery of miR inhibiting effects that could have potential as a treatment of human diseases and genetic defects, something that has proven difficult for traditional oligonucleotide approaches to miR inhibition.