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

Summary

This article presents an optimized reverse transfection protocol for efficient delivery of nucleoside-modified human telomerase reverse transcriptase (hTERT) mRNA (hTERT mRNA) into senescent human fibroblasts. Pre-coating culture surfaces with hTERT mRNA-lipid complexes enhances uptake, enabling telomerase activation, telomere extension, and partial rejuvenation. Cultures ultimately re-enter senescence or crisis without immortalization.

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.

Introduction

The reverse transfection approach represents a critical methodological adaptation for senescent cells that overcomes the fundamental barriers these cells pose to standard transfection methods. Unlike forward transfection, where cells are pre-plated and allowed to adhere before adding transfection complexes, reverse transfection involves adding cells in suspension directly onto pre-formed hTERT mRNA-lipid complexes. This technique dramatically increases transfection efficiency in senescent cells by maximizing exposure to transfection complexes during the critical attachment phase, when membrane remodeling is most active.

The protocol’s....

Protocol

1. In vitro Transcription of hTERT mRNA

1. In vitro Transcription (IVT) with ARCA and ΨUTP
   1. Prepare a 20 µL IVT reaction according to Table 1. Scale the reaction linearly if a larger yield is required.
   2. Combine all reaction components except T7 RNA polymerase in an RNase-free tube. Add T7 RNA polymerase last, mix gently, and briefly centrifuge the tube.
   3. Incubate the reaction at 37 °C for 0.5–4 h.
      NOTE: Adjust the incubation time according to the template length. Incubate reactions containing shorter templates (approximately 500 bp) for approximatel....

Results

The d2EGFP-T2A-hTERT modRNA was engineered as a hybrid open reading frame in which a destabilized EGFP (d2EGFP) reporter was linked to human TERT through a self-cleaving T2A peptide. The complete nucleotide and protein sequences of all constructs are provided in Supplementary File 1. The coding sequence was flanked by a synthetic 5′ untranslated region (UTR) and the 3′ UTR of human β-globin to enhance translational efficiency and mRNA stability (Figure 1A). The expression ca.......

Discussion

This study reframes cellular senescence as a distinct delivery problem rather than a minor variation of proliferative physiology, and develops a telomerase mRNA transfection strategy tailored to the biophysical and immunological constraints of the senescent state. Senescent cells exhibit rigidified, cholesterol-enriched membranes and reduced endocytic activity, creating a substantial barrier to efficient nucleic acid delivery. By adopting a reverse-transfection approach, this protocol exploits the transient adhesion peri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ham's F-10 Nutrient Mix	Thermo Fisher	11550043	Cell culture media.
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog	New England Biolab	S1411L	For most RNAs the cap structure increases stability, decreases susceptibility to exonuclease degradation, and promotes the formation of mRNA initiation complexes.
5-Methyl-Cytidine-5´-Triphosphate (5-Methyl-CTP)	New England Biolab	N0432S	Modified NTPs are commonly used for reduction of immunogenicity for in vitro transcription RNA.
6 Well Plate	Corning	351146	To grow and treat medium-to-large volumes of adherent or suspension cells in independent experimental conditions.
Anti-Ki67 antibody (SP6)	abcam	ab16667	Ki67 is mainly expressed in proliferating cells.
Cellular Senescence Assay Kit (SA-beta-gal Staining)	cellbiolabs	CBA-230	Identification of senescent cells with the SA-β-gal staining assay
DNA LoBind 0.5ml Tubes	Eppendorf	022431005	For efficiently recovering DNA, RNA without any loss
DNA LoBind 1.5ml Tubes	Eppendorf	22431021	For efficiently recovering DNA, RNA without any loss
DNase I, RNase-free (1 U/μL)	Thermo Fisher	EN0521	DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA.
E. coli Poly(A) Polymerase	New England Biolab	M0276S	In Escherichia coli, Poly(A) Polymerase I (PAP I) primarily functions as a catalyst for the template-independent addition of adenosine monophosphate (AMP) residues to the 3' end of RNA molecules.
Fetal Bovine Serum	Thermo Fisher	A5256701	Cell culture Seum supplement
HiScribe T7 ARCA mRNA Kit	New England Biolab	E2065S	The HiScribe T7 ARCA mRNA Kit (with tailing) is designed for quick production of ARCA capped and poly(A) tailed mRNA in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) using T7 RNA Polymerase.
HiScribe T7 mRNA Kit with CleanCap Reagent AG	New England Biolab	E2080S	the HiScribe T7 mRNA Kits with CleanCap Reagent AG , enables the quick and streamlined production of one or many transcripts with typical yields of ≥90 μg per reaction,
LongAmp Hot Start Taq DNA Polymerase	New England Biolab	M0534S	LongAmp Hot Start Taq DNA Polymerase offers exceptional performance with long amplicons.
M-MuLV Reverse Transcriptase	New England Biolab	M0253L	M-MuLV Reverse Transcriptase synthesizes a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template.
Monarch Spin RNA Cleanup Kit (50 μg)	New England Biolab	T2040L	The main function of the Monarch® Spin RNA Cleanup Kit (50 μg) is to purify and concentrate up to 50 μg of high-quality RNA from enzymatic reactions or extraction mixtures.
NEBuilder HiFi DNA Assembly Master Mix	New England Biolab	E2621L	NEBuilder HiFi DNA Assembly Master Mix allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It has utility for the synthetic biology community, as well as in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format.
Opti-MEM -Reduced Serum Medium	Thermo Fisher	31985070	To maximize cell transfection efficiency during lipid-payload complex formation. It serves as an optimized, chemically defined environment that keeps mammalian cells highly viable while reducing the need for standard serum supplementation
PBS, pH 7.4	Thermo Fisher	10010023	The primary functional use of Phosphate-Buffered Saline (PBS) at pH 7.4 is to maintain a stable pH and osmotic balance for mammalian cells and biological molecules.
PCR Strips	Thermo Fisher	AB0776	The primary functional use of PCR strip tubes (usually 8-strip or 12-strip format) is to hold small-volume liquid reactions during high-throughput thermal cycling.
Poly-L-Lysine Hydrobromide	Millipore Sigma	P4707-50ML	The primary functional use of Poly-L-Lysine (PLL) Hydrobromide for cell attachment is to coat negatively charged solid surfaces (like glass or plastic) to create a highly adhesive, positively charged substrate.
Pseudouridine-5´-Triphosphate (Pseudo-UTP- ΨUTP)	New England Biolab	N0433S	Pseudouridine-5´-Triphosphate (Pseudo-UTP) are commonly used for reduction of immunogenicity for in vitro transcription RNA.
Ribonucleotide Solution Set	New England Biolab	N0450L	Four separate solutions of ATP, GTP, CTP and UTP
ROCK inhibitor Y-27632 (Dihydrochloride)	Stem cell technologies	72302	Y-27632 is a cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Y-27632 inhibits both ROCK1 (Ki = 220 nM) and ROCK2 (Ki = 300 nM) by competing with ATP for binding to the catalytic site
RT-PCR Grade Water	Thermo Fisher	AM9935	The primary functional use of RT-PCR Grade Water (often labeled as Nuclease-Free Water) is to serve as the ultra-pure solvent for sensitive enzymatic assays where any contamination would degrade the sample or inhibit amplification.
SUPERase·In RNase Inhibitor	Thermo Fisher	AM2694	SUPERase In RNase Inhibitor is a protein-based inhibitor of non-human origin that noncovalently binds and inhibits the most common and troublesome RNases, including RNase A, B, C, 1, and T1.
SYBR Green I Nucleic Acid Gel Stain	Thermo Fisher	S7563	The primary functional use of SYBR™ Green I Nucleic Acid Gel Stain is to visualize and quantify double-stranded DNA (dsDNA) in agarose or polyacrylamide gels.
TransIT-mRNA Transfection Kit	Mirusbio/ Millipore Sigma	MIR 2250	A high efficiency, low toxicity transfection reagent for large RNA
TRAPeze Telomerase Detection Kit	Millipore Sigma	S7700	The TRAP (Telomeric Repeat Amplification Protocol) assay is a widely used and sensitive PCR-based method for detecting and measuring telomerase activity in mammalian cells and tissue samples. It involves three main steps: extension of telomeric repeats by telomerase, amplification of these products using PCR, and subsequent detection.
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher	25300054	To detach adherent mammalian cells from culture vessels during routine cell passaging (subculturing) and harvesting.