Optimized Reverse Transfection Protocol for Telomerase mRNA Delivery to Early-Senescent Human Fibroblasts.

Abstract

Cellular senescence is associated with profound alterations in cellular physiology, including reduced membrane fluidity, impaired endosomal trafficking, diminished endocytic capacity, and increased extracellular RNase activity, all of which hinder efficient mRNA delivery. These barriers have limited the application of RNA-based approaches in senescent cells, particularly for delivering large therapeutic transcripts. This protocol describes an optimized reverse-transfection method for the efficient delivery of modified messenger RNA (mRNA) into senescent human fibroblasts. Although human telomerase reverse transcriptase (hTERT) mRNA was used as the model transcript, the workflow is broadly applicable to other mRNAs. In contrast to conventional transfection methods, in which RNA-lipid complexes are added to the culture medium after cell attachment, reverse transfection deposits the complexes onto the culture surface before cell seeding, enabling direct interaction between attaching cells and transfection complexes. To maximize transfection efficiency, the protocol incorporates nucleoside-modified mRNA containing pseudouridine and 5-methylcytidine, extended poly(A) tails, optimized complex-formation timing, RNase inhibition, transient elevation of endosomal pH with chloroquine, increased cell-seeding density, and extended incubation periods. Using this approach, transfection efficiencies of approximately 50%-80% were achieved in senescent fibroblasts following delivery of a 5 kb hTERT mRNA transcript. Peak telomerase activity was detected 24-48 h after transfection. A single transfection cycle produced measurable telomere elongation, whereas three sequential transfections resulted in substantial but finite telomere extension. Partial reversal of senescence-associated phenotypes was detectable within 72-96 h, including reduced senescence-associated β-galactosidase activity, decreased p16 and p21 expression, restoration of cell morphology, and extension of replicative lifespan. The delivered hTERT mRNA was degraded within 72-96 h, and immortalization was not observed. This protocol provides a practical approach for transient mRNA delivery into senescent cells and may be adaptable to a wide range of cell types and species.