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 core conceptual shift is to treat senescent cells as a distinct delivery problem, not as just hard‑to‑transfect versions of young cells. In senescence, global changes in membrane lipid composition, including phospholipids, sphingolipids, and cholesterol, alter membrane fluidity, curvature, and protein-lipid interactions, while contributing to the senescence-associated secretory phenotype^1,2,3. Senescent cells also exhibit reduced clathrin- and caveolae-mediated receptor endocytosis and altered endocytic routing, driven in part by down‑regulation of amphiphysin‑1 and compensatory changes in caveolin expression, which blunt receptor-mediated uptake and signaling^4,5,6. In parallel, the Rab7-regulated late endosome-lysosome axis and the broader endosomal-autophagic-lysosomal (EAL) network become dysregulated, with Rab7 acting as a master controller of endolysosomal maturation and autophagosome-lysosome fusion, which are perturbed in aging and neurodegeneration⁷.

Senescent cells commonly upregulate multiple components of the lysosomal processing and adaptation system (LYPAS), including TFEB/TFE3-driven lysosomal biogenesis and chaperone-mediated autophagy, thereby increasing lysosomal mass. However, individual lysosomes frequently exhibit elevated luminal pH, membrane damage, and altered proteolytic capacity, contributing to lysosomal dysfunction despite their increased abundance^8,9,10,11. Collectively, these changes can channel internalized material more efficiently into degradative endolysosomal compartments, increasing the likelihood that delivered RNA or protein will be degraded before productive cytosolic release^9,10,11. Reverse transfection directly targets the earliest window when membranes are remodeling, during adhesion, forcing sustained, high‑avidity contact between cells and mRNA-lipid complexes and at least partly bypassing impaired steady‑state endocytosis in fully adherent senescent cells^4,5,6,12.

On the RNA design side, the protocol assumes that in an aging context, a simple cap + tail baseline is insufficient: full nucleoside modification (for example, pseudouridine or N1‑methylpseudouridine plus 5‑methylcytidine) is treated as non‑negotiable to decouple translation from endosomal Toll‑like receptor sensing and type I interferon induction. Foundational work showed that incorporation of modified nucleosides into RNA suppresses activation of TLR3, TLR7, and TLR8 and markedly reduces dendritic cell cytokine production, providing a mechanistic basis for the reduced innate immunogenicity of current nucleoside‑modified mRNA platforms¹³. More recent reviews of mRNA–LNP vaccines and mRNA medicines emphasize the coordinated engineering of the 5′ cap, UTRs, coding sequence, and poly(A) tail to increase stability and translation in vivo while minimizing innate immune activation¹⁴. In particular, poly(A) tail length and architecture have emerged as key determinants of in vitro transcribed mRNA stability and translational output, motivating extended or engineered poly(A) designs in therapeutic mRNA¹⁵.

Layered onto this, the protocol explicitly builds in protection against extracellular and endosomal RNases during complex formation and uptake, together with a defined endosomal‑escape window using a transient lysosomotropic agent such as chloroquine^16,17. Chloroquine has long been used as a functional probe of endosomal barriers and can markedly increase target engagement and silencing by siRNA or antisense oligonucleotides by promoting endosomal membrane disruption and escape of nucleic acid cargo into the cytosol¹⁸. At the same time, live‑cell and super‑resolution imaging of LNP‑delivered mRNA have demonstrated that productive delivery is tightly bottlenecked at rare escape events from early/recycling endosomes, rather than by initial uptake per se, underscoring the importance of deliberately engineering an endosomal‑escape step for mRNA therapeutics¹⁹.

Collectively, these senescence-associated alterations in membrane dynamics, endocytic trafficking, lysosomal processing, and innate immune activation create substantial barriers to efficient mRNA delivery. The reverse-transfection strategy described here was developed to address these limitations by combining optimized mRNA engineering, RNase protection, enhanced endosomal escape, and direct exposure of attaching cells to preformed mRNA-lipid complexes. Although hTERT mRNA is used as the model cargo, the workflow is broadly applicable to the delivery of other large therapeutic mRNA transcripts into senescent and difficult-to-transfect cell populations^20,21,22.

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 approximately 30 min and reactions containing larger templates (approximately 4 kb) for 3–4 h.
2. DNase treatment
   1. Add 1 µL of RNase-free DNase I directly to the IVT reaction mixture.
   2. Incubate the reaction at 37 °C for 15 min.
      NOTE: DNase treatment should be performed directly in the IVT reaction without prior purification of the synthesized mRNA.
3. mRNA purification
   1. Add 115 µL of nuclease-free H₂O to dilute the reaction mixture.
   2. Purify the mRNA using an RNA cleanup kit according to the manufacturer’s instructions.
   3. Elute the purified mRNA in 20–30 µL of nuclease-free H₂O.
   4. Measure mRNA purity by spectrophotometry and verify RNA integrity by electrophoresis on a 1.0–1.5% denaturing agarose gel or a low-percentage polyacrylamide gel.
4. Poly(A) tailing
   1. Prepare a 50 µL poly(A)-tailing reaction according to Table 2. Mix the reaction components gently and incubate at 37 °C for 30–60 min.
      NOTE: Adjust the incubation time according to the amount of input RNA.
   2. Terminate the reaction by adding 2 µL of 0.5 M EDTA (pH 8.0).
5. Final purification and quality control
   1. Purify the poly(A)-tailed mRNA using an RNA cleanup kit according to the manufacturer’s instructions.
   2. Elute the purified mRNA in 20–30 µL of nuclease-free H₂O.
   3. Assess mRNA quality before proceeding to transfection
      1. Measure mRNA concentration at 260 nm and determine purity using the A260/A280 and A260/A230 ratios. Use mRNA preparations with A260/A280 values of approximately 2.0 and A260/A230 values greater than 2.0.
      2. Evaluate mRNA integrity using gel electrophoresis or an RNA analysis system according to the manufacturer’s instructions.
      3. Confirm poly(A) tail length by RT-PCR or Bioanalyzer analysis. Verify that the poly(A) tail length is between 120 and 200 nucleotides.
      4. Aliquot the purified mRNA and store the aliquots at −80 °C to minimize freeze-thaw cycles.

2. Reverse transfection

1. Day −1: Pre-Transfection preparation
   1. Verify the senescent phenotype
      1. Perform senescence-associated β-galactosidase (SA-β-gal) staining on a representative cell sample. Verify that more than 70% of cells are positive.
      2. Confirm expression of senescence markers (p16 and p21) by qRT-PCR or immunofluorescence.
      3. Determine cell viability and verify that viability exceeds 90%.
   2. Prepare reagents
      1. Pre-warm complete culture medium to 37 °C.
      2. Pre-warm phosphate-buffered saline (PBS) to 37 °C.
      3. Prepare fresh Opti-MEM I medium.
         NOTE: Do not add RNase inhibitor to Opti-MEM I before use.
2. Day 0: Transfection day
   NOTE: Allow approximately 3 h from reagent preparation to incubation of transfected cells.
   1. hTERT mRNA preparation
      1. Thaw hTERT mRNA on ice. Measure the mRNA concentration and use preparations with concentrations between 0.5 and 1.0 µg/µL.
      2. Dilute the desired amount of hTERT mRNA in Opti-MEM I according to the culture format used (Table 3). Add RNase inhibitor to a final concentration of 0.5–1.0 U/µL and mix gently.
      3. Maintain the diluted mRNA on ice until complex formation.
         NOTE: Add RNase inhibitor immediately before use. Do not store mRNA with an RNase inhibitor, as its activity decreases after freezing.
   2. Transfection reagent preparation
      1. Combine 6.0 µL of transfection reagent with 100 µL of Opti-MEM I in a sterile RNase-free tube. Mix gently by pipetting.
      2. Incubate the mixture at room temperature for 5 min.
         NOTE: For large hTERT mRNA constructs (~5 kb), optimize reagent-to-mRNA ratios between 0.4 and 0.6 µL reagent per 100 ng mRNA.
   3. mRNA-Lipid complex formation
      1. Add the diluted hTERT mRNA solution (100 µL) to the diluted transfection reagent solution (106 µL). Mix gently by pipetting 5–6 times.
      2. Incubate the mixture at room temperature for 15–20 min.
      3. Do not exceed 30 min of complexation.
      4. Verify successful complex formation by the appearance of a slightly cloudy solution.
         NOTE: Perform cell preparation during the complexation period.
   4. Cell preparation for reverse transfection
      1. Aspirate the culture medium from senescent cell cultures.
      2. Wash the cells once with pre-warmed PBS (2 mL per well of a 6-well plate).
      3. Add 500 µL of 0.05% trypsin-EDTA per well.
      4. Incubate the cells at 37 °C for 3–5 min.
      5. Neutralize the trypsin with 3 mL of complete medium containing serum.
      6. Gently triturate the cells to obtain a single-cell suspension.
      7. Determine cell number using a hemocytometer or automated cell counter.
      8. Prepare a suspension containing 2.0–2.5 × 10⁵ cells per well in complete medium.
         NOTE: Avoid prolonged trypsinization because senescent cells are highly susceptible to mechanical stress. Maintain cells in suspension; do not pre-plate prior to reverse transfection.
   5. Reverse transfection
      1. Culture plate preparation
         1. Add 500 µL of 0.01% poly-L-lysine to each well of a 6-well plate and incubate for 30 min at room temperature.
         2. Aspirate the coating solution immediately before use.
         3. Add 200 µL of the hTERT mRNA-lipid complex to the center of each well.
         4. Incubate the plate at 37 °C and 5% CO₂ for 30 min.
            NOTE: Perform plate coating in parallel with complex formation and cell preparation.
      2. Cell suspension preparation
         1. Suspend 2.0–2.5 × 10⁵ cells in 1800 µL of antibiotic-free complete medium.
         2. Gently resuspend the cells to obtain a uniform single-cell suspension.
            NOTE: Use antibiotic-free medium during transfection.
      3. Cell seeding onto the complexes
         1. Add 1800 µL of the cell suspension directly onto the preformed hTERT mRNA-lipid complexes.
         2. Gently tilt the plate 2–3 times in each direction to distribute the cells evenly.
            NOTE: Do not pipette the suspension after cell addition, as this may disrupt mRNA-lipid complexes.
      4. Cell incubation
         1. Incubate the cells at 37 °C in a humidified incubator containing 5% CO₂ for 6–8 h.
         2. Monitor cell attachment by light microscopy 2–3 h after transfection.
         3. Verify that cells are evenly distributed and beginning to attach to the culture surface.
            NOTE: An excessive number of floating cells or cellular debris may indicate transfection-associated cytotoxicity.

3. Cytotoxicity mitigation

1. Reduce the transfection exposure period to 48 h if cell loss exceeds 20%.
2. Supplement cultures with 10 µM ROCK inhibitor Y-27632 during the transfection period to improve cell survival.
3. After 6–8 h of transfection, carefully aspirate the transfection medium while minimizing disturbance of partially attached cells. Leave approximately 100 µL of residual medium in each well.
4. Add 1 mL of fresh pre-warmed complete growth medium supplemented with 15% FBS to each well.
5. Add chloroquine to a final concentration of 10–20 µM immediately after medium replacement.
6. Incubate the cultures for an additional 2 h at 37 °C in a humidified incubator containing 5% CO₂.
7. Replace the chloroquine-containing medium with fresh complete growth medium.
8. Return the cultures to the incubator for continued growth.
   NOTE: Monitor cell morphology and viability throughout the recovery period. Excessive cell detachment or debris may indicate transfection-associated cytotoxicity.

4. Post-transfection analysis

1. Assess TERT protein expression 24–48 h after transfection.
2. Measure TERT protein levels by western blotting or immunofluorescence staining according to standard laboratory procedures.
3. Measure telomerase activity during the same interval using an appropriate telomerase activity assay.
   NOTE: Telomerase activity typically declines after 48 h because of degradation of the transiently delivered hTERT mRNA.
4. Assess telomere length 48–72 h after transfection using monochrome multiplex quantitative PCR (MMqPCR) or an equivalent telomere-length analysis method.
   NOTE: Detectable telomere elongation may be observed following successful hTERT mRNA delivery.
5. Evaluate senescence-associated and rejuvenation-associated phenotypes 7 days after transfection
   1. Measure senescence-associated β-galactosidase (SA-β-gal) activity.
   2. Measure p16 and p21 expression levels.
   3. Assess Ki67 expression.
   4. Examine cellular morphology by light microscopy.
      NOTE: Compare treated cultures with untreated or mock-transfected senescent controls to assess changes in senescence-associated phenotypes and proliferative characteristics.

5. Repeated dosing protocol (Optional)

1. Perform the initial transfection as described in Sections 2–4.
2. Repeat transfections according to the following schedule: Day 0: First transfection; Day 5: Second transfection; Day 10–12: Third transfection.
3. Maintain cells as adherent cultures and do not trypsinize before subsequent transfections.
4. Prepare fresh hTERT mRNA–lipid complexes as described in Sections 2.2.1–2.2.3.
5. Aspirate the culture medium from each well.
6. Add 200 µL of hTERT mRNA–lipid complexes directly to each well.
7. Add 800 µL of fresh complete F-10 medium immediately after complex addition.
8. Incubate the cells for 4–6 h at 37 °C in a humidified incubator containing 5% CO₂.
9. Replace the transfection medium with fresh complete medium.
   NOTE: Limit treatment to a maximum of three transfection cycles to minimize cumulative transfection-associated cytotoxicity.

6. Forward transfection protocol (2 µg/mL, 6-well format)

1. Day −1: Cell seeding
   1. Detach senescent or early-senescent fibroblasts using 0.05% trypsin-EDTA.
   2. Neutralize the trypsin with complete culture medium.
   3. Determine cell number and viability.
   4. Seed cells into 6-well plates at a density of 2.0–2.5 × 10⁵ cells per well.
   5. Incubate the cells overnight at 37 °C in a humidified incubator containing 5% CO₂ until they reach approximately 60–80% confluence.
2. Day 0: Preparation of hTERT mRNA–reagent complexes
   1. Thaw an aliquot of nucleoside-modified hTERT mRNA on ice.
   2. Dilute hTERT mRNA in Opti-MEM to a final concentration of 2 µg/mL in a sterile RNase-free tube (Tube A).
   3. Add RNase inhibitor to Tube A to a final concentration of 0.5–1.0 U/µL and mix gently.
   4. Dilute the transfection reagent in Opti-MEM in a second RNase-free tube (Tube B) using 0.4–0.6 µL reagent per 100 ng mRNA.
   5. Add the contents of Tube B dropwise to Tube A while gently mixing.
   6. Incubate the mixture at room temperature for 15–20 min.
3. Day 0: Forward transfection
   1. Confirm that cells are 60–80% confluent and display healthy morphology.
   2. Aspirate the culture medium.
   3. Wash the cells once with 1–2 mL of pre-warmed PBS.
   4. Add 0.5 mL of pre-warmed complete or serum-reduced medium to each well.
   5. Optionally add chloroquine to a final concentration of 10–20 µM.
   6. Add 0.5 mL of the hTERT mRNA–reagent complexes prepared in Step 6.2.6 to each well.
   7. Gently rock the plate to distribute the complexes evenly.
   8. Incubate the cells for 4–6 h at 37 °C in a humidified incubator containing 5% CO₂.
      ​NOTE: Extend the incubation period to 6–8 h for senescent cells if necessary.
   9. Aspirate the transfection medium.
   10. Add 2.0 mL of fresh complete medium to each well.
   11. Perform post-transfection analyses as described in Section 4.
       NOTE: Use the same hTERT mRNA batch, transfection reagent, and nominal dose when comparing forward and reverse transfection conditions.

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 cassette also contained a synthetic enzyme stabilizer sequence (Es) and a synthetic transcription enhancer sequence (TEs) designed to improve T7 polymerase processivity and reduce premature transcription termination. Following cloning downstream of a T7 promoter, in vitro transcription was performed using anti-reverse cap analog (ARCA) and modified nucleotides (m5CTP and ΨUTP), followed by enzymatic poly(A) tailing (approximately 120–200 nt) to generate capped, polyadenylated, high-integrity d2EGFP-T2A-hTERT modRNA (Figure 1A).

Automated electrophoretic RNA analysis confirmed efficient synthesis of the transcript, revealing a predominant RNA species of the expected size with minimal degradation products across multiple input amounts, consistent with successful generation of full-length modRNA (Figure 1B). Following transfection of early-senescent LF1 fibroblasts with 2 µg/mL d2EGFP-T2A-hTERT modRNA, hTERT protein became detectable within 24 h and remained elevated for 48–96 h, indicating sustained translation of the synthetic transcript (Figure 1C). Analysis of the linked d2EGFP reporter demonstrated robust fluorescence following reverse transfection, whereas substantially lower fluorescence was observed following forward transfection (Figure 1D,E). These findings indicate enhanced delivery and expression of hTERT modRNA using the reverse-transfection approach in early-senescent LF1 fibroblasts.

Repeated forward transfection (FT) of early-senescent LF1 fibroblasts with d2EGFP-T2A-hTERT modRNA at 5-day intervals produced a progressive increase in average telomere length (rT/S) relative to mock-transfected controls, as measured by monochrome multiplex quantitative PCR (MMqPCR) (Figure 2A). The rT/S ratio increased after each FT cycle, with statistical significance improving from *P < 0.05 to **P < 0.01.

Application of the same dosing schedule using reverse transfection (RT) resulted in a more pronounced increase in telomere length. The rT/S ratio increased following each RT cycle and reached highly significant differences relative to mock-transfected controls (*P < 0.05 to ****P < 0.0001) (Figure 2B). Direct comparison of FT and RT after three treatment cycles demonstrated that both approaches significantly increased rT/S values relative to mock-transfected controls; however, RT produced a greater increase than FT under identical dosing and timing conditions (*P < 0.05, ***P < 0.001, ****P < 0.0001) (Figure 2C).

Long-term growth analysis further demonstrated that repeated delivery of hTERT modRNA extended replicative lifespan (Figure 2D). Cells receiving one, two, or three treatment cycles exhibited progressively greater cumulative population doublings than mock-transfected controls. Cultures receiving three rounds of hTERT modRNA treatment, particularly by RT, displayed the largest divergence from control growth curves over approximately 250 days (up to ****P < 0.0001). Population doubling calculations are shown in Supplementary File 2.

Early-senescent LF1 fibroblasts transfected with d2EGFP-T2A-hTERT modRNA displayed strong TRAP laddering 48 h after transfection, whereas mock- and eGFP-modRNA-treated cells exhibited only background signal, indicating that telomerase activity originated from exogenous hTERT expression (Figure 3A). Heat pretreatment abolished the laddering pattern, confirming assay specificity (Figure 3B).

Time-course analysis demonstrated that telomerase activity peaked between 24 and 48 h after transfection and subsequently declined, returning to near-baseline levels by 72–96 h (Figure 3C). Increasing the hTERT modRNA concentration from 50 to 3,000 ng/mL produced a corresponding increase in TRAP signal intensity, demonstrating dose-dependent telomerase activation (Figure 3D).

Functional analysis using wild-type, catalytically inactive (CI), and nuclear localization signal-deficient (ΔNLS) hTERT constructs demonstrated that only wild-type hTERT generated robust telomerase activity. Both CI and ΔNLS variants failed to induce comparable activity despite similar delivery conditions (Figure 3E). Consistent with these findings, MMqPCR analysis revealed a marked increase in average telomere length (rT/S ≈ 1.4) only in cells receiving wild-type hTERT modRNA. Cells receiving CI-hTERT, ΔNLS-hTERT, or mock treatment remained near baseline levels (rT/S ≈ 0.3; ****P < 0.0001) (Figure 3F).

Three sequential rounds of reverse transfection with hTERT modRNA at 5-day intervals significantly reduced the proportion of senescence-associated β-galactosidase-positive cells. The percentage of positive cells decreased from approximately 85% in mock-treated cultures to approximately 40% in hTERT-treated cultures 15 days after the final transfection (***P < 0.001) (Figure 4A).

Confocal immunostaining revealed increased Ki67 expression in cells receiving three rounds of hTERT reverse transfection relative to mock-treated controls, indicating increased proliferative activity (Figure 4B). RT-qPCR analysis further demonstrated reduced expression of the senescence-associated markers p16 and p21 following hTERT treatment. Relative to mock-treated early-senescent cells, p16 expression decreased from approximately 2.3-fold to 0.7-fold, and p21 expression decreased from approximately 11-fold to 6-fold (**P < 0.01; ****P < 0.0001) (Figure 4C,D). Supplementary Figure 1 further demonstrates that treatment with chloroquine (CQ) or Y-27632 alone did not significantly alter p16 or p21 expression in mock-transfected cells. In contrast, the combination of CQ and Y-27632 during three rounds of hTERT reverse transfection produced the greatest reduction in both p16 and p21 expression compared with hTERT reverse transfection alone or with either agent individually, whereas single-agent treatment produced intermediate effects. These findings indicate that the combined use of CQ and Y-27632 enhanced the reduction of senescence-associated marker expression following hTERT mRNA reverse transfection. Together, these findings demonstrate that repeated reverse transfection of hTERT mRNA promotes telomere elongation, reduces senescence-associated markers, and enhances proliferative characteristics in early-senescent LF1 fibroblasts.

Figure 1: Design, synthesis, and functional validation of d2EGFP‑T2A‑ hTERT mRNA in early‑senescent LF1 fibroblasts. (A) Schematic design of the hybrid hTERT mRNA construct containing the T2A peptide-linked d2EGFP cassette for simultaneous expression of hTERT and destabilized EGFP. The d2EGFP-T2A-hTERT ORF is flanked by the 3’ UTR of the human β globin gene (HBB) and the synthetic 5’ UTR. The mRNA was synthesized using modified nucleotides, and a poly (A) tail was finally added. (B) Effective synthesis of high-integrity d2EGFP-T2A-hTERT mRNA using in vitro transcription as analyzed by automated electrophoretic RNA analysis. (C) Time-course analysis of hTERT protein expression using an automated capillary-based immunoassay following transfection of 2 µg/mL of d2EGFP-T2A-hTERT mRNA. (D) The effectiveness and expression of d2EGFP-T2A-hTERT mRNA transfection, as assessed by EGFP expression between forward transfection (FT) and reverse transfection (RT), were examined in early senescent primary diploid fibroblasts (LF1). Scale bar = 10 µm. (E) Mean fluorescence intensity (MFI) quantification of eGFP signal after forward or reverse transfection. A total of 20 individual images were analyzed for each condition, encompassing 4 distinct areas from 5 biological replicates. Data represent means ± SD. **P < 0.01; as assessed by the unpaired t-test with Welch's correction. Please click here to view a larger version of this figure.

Figure 2: Telomere length dynamics and proliferative lifespan after repeated forward versus reverse hTERT mRNA transfection in early‑senescent LF1 fibroblasts. (A) The average telomere lengths (rT/S) were determined using Telomere MMqPCR. Early senescent fibroblasts were forward transfected (FT) with d2EGFP-T2A-hTERT mRNA three times in a row at 5-day intervals beginning at Population Doubling (PD) #76. Error bar indicates SEM; n = 5 biological replicates; *P < 0.05, **P < 0.01 compared with cells treated only with mock. (B) Similar to (A), rT/S were evaluated when early senescent cells were repeatedly transfected (RT) with d2EGFP-T2A-hTERT mRNA three times in a row at 5-day intervals beginning at Population Doubling (PD) #76. The error bar represents SEM; n = 7 biological replicates; *P < 0.05, ****P < 0.0001 compared with cells treated solely with mock. (C) The average telomere length (rT/S) comparison after 3 forward and reverse transfections in succession at 5-day intervals, or mock only. Error bar represents SEM; n = 10 biological replicates; *P < 0.05, ***P < 0.001, ****P < 0.0001. (D) Growth curves of LF1 fibroblasts transfected with 2 µg/ml d2EGFP-T2A-hTERT mRNA or mock only, once, twice, or three times in succession at 5-day intervals starting at PD# 76. Black arrows indicate treatment timings. The growth curves were replicated three times, with each population being cultivated in triplicate. 3 technical replicates per biologic sample, data represent means ± SD. *P < 0.05, **P < 0.01; ****P < 0.0001, as assessed by Brown-Forsythe and Welch’s ANOVA test Please click here to view a larger version of this figure.

Figure 3: Catalytically active, nuclear‑localized hTERT mRNA drives a transient, dose‑dependent surge in telomerase activity that alone is sufficient to promote measurable telomere extension in early‑senescent LF1 fibroblasts. (A) Early-senescence cells were transfected with mock, eGFP, or d2EGFP-T2A-hTERT mRNA. 48 h post-transfection, cell extract was prepared from 20,000 cells and subjected to the TRAP assay. Reaction products were run on a native PAGE and stained with SYBR Green. (B) Heat-mediated telomerase inactivation as measured using the TRAP assay. (C) Analysis of telomerase activity over time in early-senescent LF1 cells following hTERT mRNA transfection showed that activity returned to baseline within 72 h, indicating transient enzyme activation. (D) Early senescent LF1 cells received varying amounts of d2EGFP-T2A-hTERT mRNA (0–3,000 ng/mL), showing increased telomerase activity proportional to the dose. (E) Delivery of 2 µg/ml TERT increased telomerase activity in early senescent LF1 cells. ΔNLS partially augmented telomerase activity without inducing telomere elongation, as it operates in the cytoplasm but does not translocate to the nucleus, whereas CI TERT mRNA did not elicit this effect, signifying distinct functional results. (F) Effect of functional TERT, nuclear localization signal-deficient (ΔNLS), and catalytically inactive (CI) TERT reverse transfection on the average telomere lengths (rT/S) of early senescent LF1 cells, indicating the importance of nuclear-localized catalytically active TERT on telomere extension. Error bar represents SD; n = 7 biological replicates; ****P < 0.0001 compared with cells treated only with mocks, as assessed by one-way ANOVA with Welch correction. RTA%-Relative telomerase activity (% of control) is noted at the bottom of every lane. The ratio of intensity levels of TRAP sample ladders and ITAS band (internal control- IC) was calculated by the following formula for each lane: Activity _normalized = Ladder intensity/ IC intensity and each sample was normalized to the positive control as a percentage (positive control; H1299 2,500 cells) using following formula: Relative activity (%) =Activity _normalized (sample)/ Activity _normalized (reference). Background subtraction was applied in each step. Please click here to view a larger version of this figure.

Figure 4: Optimized hTERT mRNA reverse transfection partially reverses senescence‑associated phenotypes and restores proliferative markers in early‑senescent LF1 fibroblasts. (A) Representative images of β-gal-expressing early senescent LF1 cells after modified hTERT mRNA transfection 3 times in succession at 5-day intervals. Scale bar = 100 µm. β-gal expression was analyzed 15 days after the last transfection. Experiments were performed three times with >100 cells per sample scored manually. (B) Representative confocal (20x magnification) images of early senescent LF1 cells, stained with proliferation marker Ki67. Scale bar = 100 µm. Significant increase in Ki-67 expression after 3 rounds of reverse transfection with hTERT. The percentage of Ki67-positive nuclei significantly increases after 3× hTERT (RT). The graph represents the mean ± SD of the Ki67-positive nuclear count, normalized to the Mock-transfected condition. For each condition, a total of 18 images were examined, 6 distinct fields were selected from each of the biological replicates, for a total of n = 3 biological replicates. ****P < 0.0001. (C) Senescence marker expression after 3× hTERT-mediated partial rejuvenation as assessed by RT-qPCR (n = 6). All data represent means ± SD. **P < 0.01; ****P < 0.0001, as assessed by the unpaired t-test with Welch's correction. Please click here to view a larger version of this figure.

Component	Volume/Final Concentration
PCR template DNA	200 –1000 ng
10× IVT Buffer	2 μL
ATP (100 mM)	2 μL (10 mM final)
GTP (100 mM)	1.5 μL (7.5 mM final)
CTP (100 mM)	2 μL (10 mM final)
m1Ψ-5′-TP (100 mM)	2 μL (10 mM final) (You can increase the pseudo-UTP incorporation by incorporating normal UTP at a 1:4-2:2 ratio (m1Ψ-5′-TP:UTP).)
ARCA (10 mM)	4 μL (2 mM final)
RNase Inhibitor (40 U/μL)	0.5 μL (20 U)
T7 RNA Polymerase (50 U/μL)	2 μL (100 U)
Nuclease-free H₂O	20 μL

Table 1: Composition of the in vitro transcription (IVT) reaction for synthesis of nucleoside-modified hTERT mRNA. The reaction components and final amounts used for the ARCA-capped IVT synthesis are listed.

Component	Volume/Final Concentration
Purified mRNA up to 5 μg	≤20 μL
10× Poly(A) Tailing Buffer	5 μL
ATP (10 mM	5 μL (1 mM final) (Although it's already in the buffer, the extra addition helps the poly A stabilization.)
Poly(A) Polymerase (5 U/μL)	1 μL (5 U)
Nuclease-free H₂O	To total 50 μL

Table 2: Composition of the poly(A)-tailing reaction used for post-transcriptional modification of hTERT mRNA. Reagent volumes and reaction conditions for the generation of a 120–200 nucleotide poly(A) tail are shown.

Parameter	12-Well Format	6-Well Format
Growth Surface Area	~3.8 cm²	~9.5 cm²
Media per Well	1.0 mL	2.0 mL
Cell Seeding Number	5× 104–6.5× 104 Cells/ml	2.0–2.5 × 10⁵
Average Cell number (At 100% confluency)	5.7× 104–7.2× 104 Cells/ml	2.9–3 × 10⁵
mRNA transfection Amount	750 –1000ng	2000–3500 ng
mRNA transfection agent	1.5 – 3.0 µL	4.75 – 7 µL
Final volume of mRNA + transfection agent in Opti-MEM	50 µL	200 µL

Table 3: Recommended transfection parameters for hTERT mRNA delivery in 6-well and 12-well culture formats. Cell-seeding densities, mRNA amounts, transfection reagent volumes, and complex-formation volumes used for forward and reverse transfection experiments are summarized.

Supplementary Figure 1: Effect of chloroquine and Y-27632 on senescence-associated p16 and p21 expression following repeated hTERT mRNA reverse transfection. (A) Quantitative RT-PCR analysis of p16 mRNA expression in mock-treated and 3× hTERT reverse-transfected (RT) LF1 fibroblasts following treatment with chloroquine (CQ), Y-27632, or their combination, as indicated. (B) Quantitative RT-PCR analysis of p21 mRNA expression under the same experimental conditions. Transcript levels were normalized to RpL13a and expressed relative to young control cells (PD#12). Individual data points represent biological replicates. Data represent means ± SD from n = 2 biological replicates, with 5 technical replicates per biological replicate. Statistical significance is indicated in the figure; ns, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001, as determined by Brown–Forsythe and Welch's ANOVA. Abbreviations: CQ, chloroquine; RT, reverse transfection; Y-27632, Rho-associated protein kinase (ROCK) inhibitor.Please click here to download this file.

Supplementary File 1: DNA and protein sequences of hTERT expression constructs.Please click here to download this file.

Supplementary File 2: Population doubling (PD) calculation. Population doublings (PD) were calculated using the formula PD = log₂ (Nf/N₀), where N₀ is the number of cells seeded, and Nf is the number of viable cells harvested. Cumulative PD was determined by summing PD values across passages.Please click here to download this file.

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 period during which senescent cells undergo membrane remodeling and exhibit increased membrane plasticity, thereby synchronizing cell-complex contact with a period of enhanced uptake potential^3,4. Representative results demonstrate that this strategy improves hTERT modRNA delivery and expression compared with conventional forward transfection, leading to greater telomere elongation and more pronounced rejuvenation-associated phenotypes.

A central feature of the method is a multilayered mRNA engineering strategy designed to address three major barriers in senescent cells: elevated innate immune signaling, increased RNA degradation, and impaired translational capacity^23,24,25. Incorporation of pseudouridine and 5-methylcytidine reduces activation of TLR3, TLR7/8, and RIG-I signaling pathways while simultaneously enhancing transcript stability and translational efficiency^13,26. ARCA capping and poly(A) tail optimization further improve transcript stability and translational output, enabling robust but transient hTERT expression. Consistent with previous studies, these modifications support efficient telomerase expression while minimizing innate immune activation associated with exogenous RNA delivery.

The protocol also addresses the RNase-rich extracellular environment characteristic of senescent cultures²⁷. RNA protection is enhanced through nucleoside modification, lipid complex formation, and immediate pre-transfection addition of RNase inhibitor. In addition, the timing of reverse transfection is adjusted to the slower attachment kinetics of senescent cells, allowing prolonged interaction between cells and mRNA-lipid complexes during cell adhesion. These modifications collectively improve the probability of successful cargo uptake in a cell population that is otherwise difficult to transfect.

Endosomal escape represents a second critical bottleneck for mRNA delivery in senescent cells. Senescent cells exhibit increased lysosomal content and enhanced trafficking of endocytosed cargo toward degradative compartments^17,28. Transient exposure to chloroquine during the early post-transfection period improves functional delivery by promoting endosomal escape and reducing lysosomal degradation of internalized mRNA. Although the optimal dose and exposure duration may vary among cell types, the results presented here indicate that enhancing endosomal escape substantially improves delivery efficiency in senescent fibroblasts.

At the functional level, repeated hTERT modRNA administration produced cumulative increases in relative telomere length, extended replicative lifespan, and reduced multiple senescence-associated phenotypes. Cells receiving repeated hTERT treatment exhibited reduced SA-β-gal activity, decreased p16 and p21 expression, increased Ki67 expression, and enhanced proliferative capacity relative to mock-transfected controls. Importantly, telomerase activity remained transient, returning to near-baseline levels within several days after transfection. This transient expression profile avoids permanent genetic modification while still providing sufficient telomerase activity to promote measurable biological effects. It should be noted, however, that relative telomere length measurements obtained by MMqPCR do not directly quantify absolute telomere extension. Future studies incorporating terminal restriction fragment analysis, quantitative fluorescence in situ hybridization, Flow-FISH, or TeSLA methodologies will provide a more precise assessment of telomere-length dynamics following hTERT mRNA delivery.

Several limitations warrant consideration. Senescent cell populations are inherently heterogeneous and may vary in their responsiveness to mRNA delivery depending on the method of senescence induction, donor source, and culture history. Consequently, transfection efficiency and rejuvenation outcomes may differ between experimental systems. Furthermore, telomerase reactivation primarily addresses telomere-dependent aspects of cellular aging and does not directly correct all hallmarks of senescence. Aging-associated alterations such as epigenetic drift, mitochondrial dysfunction, altered proteostasis, and chronic inflammatory signaling may persist despite successful telomere elongation^29,30. Therefore, this protocol should be viewed as a targeted tool for studying and partially reversing telomere-driven senescence rather than as a comprehensive rejuvenation strategy.

The parameters described in this protocol are intended as practical guidelines for senescent human cells and were developed through iterative optimization informed by published studies and experimental experience. Factors including cell density, transfection reagent-to-mRNA ratio, complexation time, and endosomal escape conditions may require adjustment for individual cell types and experimental settings. Investigators should therefore consider the reported conditions as a starting point for optimization rather than universally applicable endpoints.

In summary, this protocol provides a reproducible framework for delivering modified hTERT mRNA to senescent human cells using a reverse-transfection strategy optimized for the physiological characteristics of senescence. The approach enables transient telomerase reactivation, measurable telomere elongation, extension of replicative lifespan, and partial reversal of senescence-associated phenotypes. Future studies should evaluate the applicability of this strategy to additional senescent cell types, investigate combination approaches with complementary rejuvenation interventions, and explore translation to in vivo delivery platforms. As such, this method provides a valuable tool for investigating telomere biology, cellular rejuvenation, and mechanisms of senescence in aging research.

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.