Acceleration Of Wound Healing With Phd2- And Mir210-targeting Oligonucleotides
Anne Dallas1, Artem Trotsyuk2, Heini Ilves1, Melanie Rodrigues1, Aleksander White3, Geoffrey Gurtner2, Brian H. Johnston1.
1SomaGenics, Inc., Santa Cruz, CA, USA, 2Stanford University, Stanford, CA, USA, 3LayerBio, Medford, CA, USA.
Background. In chronic diabetic wounds, the normal response to hypoxia is impaired and many cellular processes involved in wound healing are hindered. The hypoxia regulator HIF-1α activates factors that promote cellular motility and proliferation, new vessel formation, and re-epithelialization. PHD2 negatively regulates HIF-1α activity under normoxia. HIF-1α also upregulates microRNA-210, which in turn regulates certain factors in ways that are antagonistic to wound repair. Methods. We identified a highly potent short synthetic hairpin RNA (sshRNA) that inhibits expression of PHD2 and an antisense oligonucleotide (antimiR) that inhibits miR-210. Both oligonucleotides were chemically modified for biostability. To assess activity and delivery in diabetic mice, PHD2-targeting sshRNAs and antimiR-210 were formulated using layer-by-layer (LbL) technology, in which RNAs are incorporated into a multilayer coating on a Tegaderm mesh. This coating gradually degrades under physiological conditions, releasing the RNAs. Formulated treatments were applied to full-thickness excisional wounds in db/db mice. Results. Transfection of our sshRNA silenced PHD2 transcripts, stabilized HIF-1α and, in combination with the antimiR-210, increased mobility of cultured keratinocytes. Wounds treated with a single application of PHD2 sshRNA or antimiR-210 closed 4 days faster than untreated wounds, and wounds treated with both oligonucleotides closed 4.75 days faster. Cellular uptake was confirmed using fluorescent sshRNA. Markers for neovascularization and cell proliferation (CD31 and Ki67) were increased in the wound area following treatment, and VEGF increased in sshRNA-treated wounds. Conclusions. Our results suggest that silencing of PHD2 and miR-210 either together or separately by localized delivery of sshRNAs and antimiRs is a promising approach for the treatment of chronic wounds.
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