High-efficiency Delivery of Antimicrobial mRNAs into Human Mucosal Epithelial Cells

Oral keratinocytes can protect against and suppress invasive pathogens using two antimicrobial effector systems, calprotectin (S100A8 and S100A9) in the cytosol and LL-37 (also known as cathelicidin antimicrobial peptide, CAMP, CAP-18) in endosomes.

Objective: To selectively augment the expression of calprotectin and LL-37 in mucosal epithelial cells by mRNA delivery.

Method: Human S100A8, S100A9 and LL-37 open reading frames containing Kozak sequences were cloned into pGEM-2bgUTR.64A and pGEM-2bgUTR.150A.  These vectors contain two sequential 3’UTRs of human β-globin gene and 64 poly(A) and 150 poly(A) tails, respectively. The fragments containing all necessary elements were amplified from the vectors and used as templates for in vitro transcription using the mMESSAGE mMACHINE T7 Ultra kit (Ambion).  Optimized mRNAs were transiently delivered into keratinocytes by transfection using TransIT-mRNA transfection reagent.

Result: Using real-time PCR analysis, we showed that levels of these mRNAs peaked at 4h after transfection.  By 40 to 50 h after mRNA delivery, message levels decreased by half.  The S100A8, S100A9 and LL-37 constructs containing 150 poly(A) tail mRNA show a longer half-life than 64 poly(A) mRNA constructs.  Transient delivery of mRNAs into keratinocytes increased the intracellular expression of calprotectin subunits (S100A8 and S100A9) and LL-37 more than 500-fold based upon Western blot and immunochemistry analysis.  Levels of these proteins were maximal at 15 h after mRNA delivery.

Conclusion: Taken together, these results establish high-efficiency mRNA delivery and expression of the antimicrobial effector proteins, calprotectin (S100A8, S100A9) and LL-37, in human keratinocytes.