Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

Abstract

Traumatic brain injury (TBI) is a leading cause of chronic neurological disability, yet no disease-modifying therapy exists. Emerging evidence indicates that TBI activates cellular aging programs, including telomere erosion and persistent inflammation, that contribute to progressive neurodegeneration. Telomerase reverse transcriptase (TERT) maintains telomere homeostasis and provides cytoprotective effects in the central nervous system but it has not been therapeutically targeted after TBI. Here, we developed an mRNA nanotherapy consisting of mouse TERT mRNA encapsulated in lipid nanoparticles (mTERT-LNPs) and evaluated it in a mouse model of moderate TBI. We first established that TBI transiently disrupts TERT biology, with reduced cortical TERT mRNA and shortened telomeres at 3 days post-injury (dpi), followed by partial recovery by 14 dpi. mTERT-LNPs were well tolerated in vitro and in vivo. Following intravenous delivery in the acute post-injury window, LNPs localized to the injured brain and displayed expected peripheral biodistribution. A single systemic dose increased cortical TERT mRNA and protein and partially restored telomere length at 3 dpi. TERT mRNA delivery significantly reduced Iba1+ microglial activation and suppressed pro-inflammatory cytokines. Systemically, mTERT-LNPs lowered serum C-reactive protein indicating reduced peripheral inflammation, without adverse effects on peripheral organs. Several outcomes showed sex-dependent patterns. Collectively, these data provide the first in vivo evidence that telomerase therapy can modulate telomere biology and neuroinflammation after TBI, supporting mRNA-LNP-mediated TERT restoration as a scalable, mechanistically grounded strategy for disease modification in TBI and related disorders.

Introduction

Traumatic brain injury (TBI) is a major cause of death and long-term disability, affecting ∼69 million people worldwide annually and ∼2.8 million in the United States [1,2]. Beyond the acute insult, TBI is now understood as a chronic disorder that accelerates late-life neurodegeneration [3] and increases the risk for Alzheimer's disease [4], Parkinson's disease, and chronic traumatic encephalopathy [5]. Secondary injury cascades, including blood-brain barrier disruption, axonal degeneration, mitochondrial dysfunction, oxidative stress, and sustained neuroinflammation, drive progressive tissue loss and functional decline [6,7]. Despite intensive clinical and preclinical efforts, no disease-modifying therapy has yet proven effective in improving long-term outcomes of TBI.

Accumulating evidence indicates that TBI also activates cellular aging programs. Telomeres, the repetitive DNA sequences that cap chromosome ends, shorten with cell division, oxidative stress, and inflammation [[8], [9], [10]]. Clinical and experimental studies show that telomere length (TL) is reduced after TBI and may serve as a biomarker of injury severity and outcome [[11], [12], [13], [14], [15], [16]]. For example, repetitive mild TBI in rodent models induces significant telomere shortening within days of injury, coinciding with heightened oxidative stress and neuroinflammation [16]. These findings suggest that telomere erosion is an active component of TBI pathophysiology.

Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, maintains telomere homeostasis, but also exerts non-canonical, telomere-independent functions in the central nervous system (CNS). Beyond its role in telomere extension [17,18], TERT protects neurons from oxidative damage, modulates mitochondrial function, reduces toxic protein accumulation, and dampens senescence-associated inflammation while promoting neurogenesis and cognition in multiple preclinical models [[19], [20], [21], [22], [23], [24]]. Telomerase activation has, therefore, emerged as a promising therapeutic strategy in neurological disease; however, telomerase-targeted interventions have not been evaluated in TBI. Recently, we demonstrated that delivery of human TERT mRNA encapsulated in lipid nanoparticles (LNPs) enhances human skin cell suspension engraftment and proliferation in a humanized mouse wound-healing model, establishing the feasibility and safety of TERT mRNA-LNP therapy in vivo [25]. We also reported that TERT mRNA-LNPs significantly reduced radiation-induced DNA damage in human primary skin cells and tissues, enhanced DNA repair, decreased mitochondrial ROS, and lowered apoptosis, without extending telomere length during the experimental period, indicating a non-canonical role of TERT in accelerating cellular recovery from radiation [26]. mRNA therapeutics provide a flexible platform for the transient expression of therapeutic proteins, including targets that are otherwise considered “undruggable” [27,28]. Advances in in vitro-transcribed (IVT) mRNA chemistry and LNP formulations, highlighted by the success of mRNA-LNP COVID-19 vaccines, have transformed mRNA into a clinically validated modality. Since naked mRNA is rapidly degraded in biological fluids, efficient delivery systems such as LNPs are essential to protect mRNA, promote cellular uptake, and enable controlled in vivo protein production [25,27,28]. Among non-viral vectors, LNPs have shown the most advanced safety and efficacy profile in humans, making them attractive candidates for CNS-targeted gene therapies.

Building on prior work linking TBI to telomere attrition and on our previous demonstration of TERT mRNA-LNPs’ efficacy in a regenerative setting, we hypothesized that transient restoration of TERT expression after TBI could counteract injury-induced telomere shortening and attenuate downstream neuroinflammation. Here, we develop and characterize a mouse TERT (mTERT) mRNA-LNP formulation and test its therapeutic potential in a murine model of moderate TBI. We show that a single intravenous dose of mTERT-LNPs administered shortly after injury enhances cortical TERT expression and dampens microglial activation and pro-inflammatory cytokine production, with sex-dependent effects, while maintaining a favorable systemic safety profile. These findings position TERT mRNA-LNP therapy as a mechanistically grounded, clinically translatable strategy to mitigate secondary injury and potentially modify long-term outcomes after TBI.

Materials and methods

Synthesis of messenger RNA and LNPs

mTERT mRNA was synthesized by the Houston Methodist Research Institute (HMRI) RNA Core via IVT, with pseudouridine added to the nucleotide mix to enhance translation and reduce the inflammatory response caused by the mRNA, as previously described [29]. Firefly luciferase (Luc) mRNA was purchased from TriLink Biotechnologies (cat. #L-7602). mRNA-LNP formulations were generated using a NanoAssemblr Benchtop (Precision Nanosystems). Briefly, LNPs were formulated using a molar ratio of 8:1.5:38.5:52 for Distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids, cat. #850365), 1,2-Dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000, Avanti Polar Lipids, cat. #880151P), cholesterol (Millipore Sigma, cat. #3667), and Dlin-MC3-DMA (MedChemExpress, cat. #HY-112251), respectively. For in vitro cellular uptake and mRNA expression studies, LNPs were formulated with Cy3-labeled lipid (0.5% w/w of total lipid) and encapsulated Green Fluorescent Protein (EGFP) mRNA. The Cy3-labeled lipid (Lissamine™ Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) was purchased from Invitrogen, and EGFP mRNA was obtained from TriLink Biotechnology. For all in vivo (biodistribution and efficacy) studies, LNPs were fluorescently labeled with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-Cyanine 5.5 (Avanti Research, cat. #810336) at 0.5% w/w of total lipid to enable fluorescence-based tracking. The lipids were dissolved in ethanol, and the mRNA in citrate buffer (100 mM, pH 5.0). The aqueous and ethanolic phases were further mixed at a 3:1 ratio at a flow rate of 10 mL min⁻¹. Subsequently, dialysis was performed in PBS at 4 °C for at least 12 h to eliminate residual ethanol and unbound mRNA [25]. The size, polydispersity index (PDI), and zeta potential of the LNPs were measured using dynamic light scattering (DLS) with a Zetasizer NanoZS (Malvern Instruments). The mRNA encapsulation efficiency (EE) in the LNPs was measured using RiboGreen Assay (Fisher Scientific, cat. #R11490) based on the manufacturer's protocol. Both encapsulated and unencapsulated mRNA were measured. The total mRNA (encapsulated + unencapsulated) was measured following LNPs digestion with 1% Triton X-100 to release the RNA's contents. EE was calculated using the formula: EE% = encapsulated mRNA x 100/total mRNA.

In vitro viability in neuronal cell culture

N2A (Neuro-2A, cat. # CCL-131) murine cell line was obtained from ATCC (US). The cells were cultured with Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin. In vitro biocompatibility of Luc mRNA-LNPs and mTERT mRNA-LNPs was determined using the WST-1 assay. Cells were seeded at a density of 3000 cells per well in 96-well plates and incubated overnight. Subsequently, they were treated with mRNA-LNPs at 0, 0.5, 1, and 2 μg/mL of mRNA, followed by incubation for 24 and 48 h (n = 5). WST-1 assay reagent (Millipore Sigma, cat. #12352200) was added at the end of the incubation period, and the plates were incubated for 2.5 h. Absorbance was measured at 490 nm, and the values were normalized to the untreated control.

LNP cellular uptake and mRNA expression kinetics live in vitro analysis

LNP uptake and mRNA expression were assessed for 48 h by live fluorescence microscopy. The lipids were labeled with Cy3 lipid as described above for LNP uptake and loaded with reporter EGFP mRNA for assessment of mRNA expression. N2A cells were seeded in 96-well clear flat-bottom plates (Falcon) and left overnight to attach in the incubator at 37 °C and 5% CO₂. Media was removed, and fresh media was added with the LNP at 0.2, 0.5, 1, or 2 μg GFP mRNA/mL dilution. Using Incucyte S3®, five images per well were acquired from three replicates hourly for 48 h with a 10× objective. Green acquisition time was 300 ms, and red acquisition time was 400 ms. Images were analyzed by Incucyte Live-Cell Analysis Systems software, and fluorescence was quantified.

Mice and TBI model

6-month-old male and female C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME, US) were housed at the HMRI animal facility. The mice were kept under a 12 h light-dark cycle and provided ad libitum access to food and water. All animal experiments adhered to approved protocol as specified by the Institutional Animal Care and Use Committee (IACUC) at HMRI. These procedures were conducted in accordance with established institutional guidelines and regulations. All mice were anesthetized with isoflurane during surgeries, starting at 3% for induction and maintained at 1.5–2%. We used an electromagnetic Impact One stereotaxic impactor (Leica Biosystems, Buffalo Grove, IL, USA) to induce moderate TBI in the left hemisphere of mice, targeting the primary motor and somatosensory cortices [[30], [31], [32]]. The impact site was located 2 mm lateral and 2 mm posterior to Bregma using a flat impact tip with a diameter of 3 mm. The impact was delivered at a velocity of 3.2 m/s and a depth of 1.5 mm.

Biodistribution of mRNA-LNPs using IVIS

Brain accumulation and biodistribution of mRNA-LNPs were assessed in male and female mice with TBI using an in vivo imaging system (IVIS). For these studies, Cy5.5-labeled LNPs containing Luc-mRNA were used to track the LNP distribution and protein expression. Mice received an intravenous injection of either Luc-Cy5.5-LNPs or mTERT-Cy5.5-LNPs 30 min post-injury, with a dose of 1–3 mg/kg of mRNA. 24 h after TBI, mice treated with Luc-Cy5.5-LNPs were intraperitoneally injected with 150 mg/kg luciferin immediately before IVIS analysis (PerkinElmer IVIS). Mice were then euthanized and their brain, heart, lungs, spleen, liver, kidneys, and blood were collected for imaging. Fluorescence (ex = 640 nm, em = 720 nm) and bioluminescence were measured. Data analysis was performed using the Living Image software.

Telomere length (TL) analysis

We assessed TL using a quantitative real-time PCR (qRT-PCR) assay kit for relative mouse telomere length (ScienCell Research Laboratories, cat. #M8908) according to the manufacturer's instructions. Before the assay, we isolated genomic DNA from the injured brain sites of each mouse using the TRIzol Reagent (Invitrogen, cat. #15596026) protocol. We performed two qPCR reactions for each genomic DNA sample using telomere and single-copy reference (SCR) primers, along with TaqGreen qPCR master mix. The SCR primer set was used as a reference for data normalization. The PCR cycling protocol was set up as follows: an initial denaturation step at 95 °C for 10 min, followed by 32 cycles of denaturation at 95 °C for 20 s, annealing at 52 °C for 20 s, and extension at 72 °C for 45 s. The relative TL was determined by calculating the T/S ratio, which indicates the amplification of the telomere product (T) relative to the single-copy reference gene (S). The relative T/S ratio was computed using the 2^−ΔΔCt method, which compares the variation of each DNA sample's T/S ratio to a reference sample [33]. Measurements were performed in triplicate for all samples.

RNA extraction and qPCR

Total RNA was extracted from injured brain tissue using TRIzol™ Reagent (Invitrogen, cat. #15596026) following the manufacturer's recommended protocol. One μg of isolated RNA from each sample was used as a template and reverse-transcribed into complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad, cat. #1708891) with the following thermal cycle conditions: 25 °C for 5 min, 46 °C for 20 min, and 95 °C for 1 min. Relative gene expression was normalized to the housekeeping control gene, β-actin. cDNAs were amplified using SsoAdvanced Universal SYBR Green Supermix with the CFX384 Touch Real-Time PCR Detection System (BioRad), and the relative differences in gene expression were determined using the comparative threshold cycle (2^−ΔΔCt) method.

Sequences of the primers used are as follows: Tert: F:5′-TCTCTATGAATGAGAGCAGC-3′ and R:5′-TATAGCACCTGTCACCAATC-3'; TNF-α: F:5′-CTATGTCTCAGCCTCTTCTC-3′ and R:5′-CATTTGGGAACTTCTCATCC-3'; IL-1β: F:5′-GGATGATGATGATAACCTGC-3′ and R:5′-CATGGAGAATATCACTTGTTGG-3'; IL-6: F:5′-AAGAAATGATGGATGCTACC-3′ and R:5′-GAGTTTCTGTATCTCTCTGAAG-3'; IL-18: F:5′-AAATGGAGACCTGGAATCAG-3′ and R:5′-CCTCTTACTTCACTGTCTTTG-3'; TGF-β: F:5′-GGATACCAACTATTGCTTCAG-3′ and R:5′-TGTCCAGGCTCCAAATATAG-3'; IL-10: F:5′-CAGGACTTTAAGGGTTACTTG-3′ and R:5′-ATTTTCACAGGGGAGAAATC-3'; and β-Actin: F:5′-GATGTATGAAGGCTTTGGTC-3′ and R:5′-TGTGCACTTTTATTGGTCTC-3'.

Western blot

Serum was obtained by centrifuging the blood samples collected from each mouse at 4000 rpm for 20 min at 4 °C. Next, serum samples (1:5 v/v) were combined with 4x Laemmli sample buffer (Bio-Rad, cat. #1610747) and incubated at 100 °C for 5 min. The samples were subjected to SDS-PAGE with a 4%–15% gradient for protein separation and electro-transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, cat. #1620177). The membranes were blocked in 5% w/v skim milk powder in PBS-Tween 20 (PBS-T) buffer for 1 h at room temperature. The expression level of C-reactive protein (CRP) was detected using a specific CRP antibody (Proteintech, cat. #66250-1-Ig) and the corresponding HRP-conjugated secondary antibody. Immunoblots were visualized using Clarity Western ECL (Bio-Rad, cat. #1705061) in a ChemiDoc MP imaging system (Bio-Rad) and quantified with a densitometer using ImageJ.

MDA analysis

Malondialdehyde (MDA), the end product of lipid peroxidation, was determined in serum using the MDA assay kit (Abcam, cat. #ab118970) according to the manufacturer's instructions. The color reaction was measured at 540 nm. The MDA levels were expressed as mM.

Brain tissue preparation and fluorescent in situ hybridization with immunofluorescence labeling

Brain samples were fixed in 4% paraformaldehyde overnight, then transferred to 30% sucrose for additional processing. Using a cryostat (Epredia Cryostar NX50, Fisher Scientific), the brains were sectioned into 16 μm slices. Sections from the frontal cortex through the dorsal hippocampus were collected at coronal planes. These sections were either mounted directly onto gelatin-coated glass slides (Superfrost Plus, Fisher Scientific, cat. #12-550-15) and kept at −80 °C or kept in a free-floating cryoprotective solution containing 30% sucrose, 1% polyvinylpyrrolidone, 30% ethylene glycol, and 0.01 M PBS until they were used. Fluorescent in situ hybridization (FISH) was performed according to the manufacturer's instructions using the RNAscope™ 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics, cat. #322350), as previously described [30]. Brain tissue sections were dehydrated through a series of ethanol concentrations at 50%, 70%, and twice in 100% ethanol for 5 min each. Next, they were boiled for 2 min in pretreatment 2 solution. Finally, the slides were incubated in pretreatment solution 3 (protease IV) for 30 min before hybridization. For hybridization, sections were incubated at 40 °C for 2 h with the specific target probe: Mus musculus TERT (Advanced Cell Diagnostics, cat. #313441). Additionally, the negative control probe (Advanced Cell Diagnostics, cat. #310043) and the positive control probe (Advanced Cell Diagnostics, cat. #313911) were applied and hybridized for 2 h at 40 °C. The amplification steps were performed according to the manufacturer's instructions.

Immunofluorescence analysis

Brain sections underwent immunohistochemistry, starting with three detailed 5 min washes in PBS containing 0.5% Triton X-100 (PBS-T). To avoid nonspecific binding, sections were incubated for 1 h at room temperature with 5% normal goat serum (NGS) in PBS-T. The next step was an overnight incubation at 4 °C using a solution of 3% NGS in PBS-T, containing primary antibodies: anti-rabbit Iba-1 (Wako, cat. #019-19741) at 1:500 to label microglia and macrophages, and anti-mouse TERT (Novus, cat. #NB100-317) at 1:500. The following day, the sections were washed three times for 5 min each in PBS-T and then incubated with the appropriate secondary antibodies, all diluted at 1:1000, for 2 h at room temperature. The sections were rinsed three times with PBS for 5 min each, then stained with a DAPI solution in PBS to label the nuclei. Afterward, they were rinsed thoroughly with distilled water and mounted using Fluoro-Gel and Tris Buffer mounting medium.

Quantitative analysis of immunolabeled images

All histological images were captured by a Slideview VS200 Universal Whole Slide Imaging Scanner (Evident, USA) and a confocal imaging system (Leica Microsystems, Deerfield, IL, USA). We employed unbiased, standardized sampling methods to assess cortical regions exhibiting positive immunoreactivity for quantitative analysis of immunolabeled sections. For proportional area measurements, Iba-1 immunoreactivity was expressed as the proportion of the target region covered by immunohistochemically stained cellular profiles. To quantify the number of Iba-1 and TERT cells in the injured cortex, we counted and imaged an average of four coronal sections from the lesion epicenter (−1.34 to −2.30 mm from Bregma) for each mouse.

Organ paraffin embedding and hematoxylin and eosin staining for in vivo biocompatibility

The heart, lungs, liver, spleen, and kidneys were collected and fixed in 4% paraformaldehyde for 48 h before being transferred to 70% ethanol. They were processed with a Shandon Exelsion ES Tissue Processor and then embedded in paraffin according to the manufacturer's standard procedures. The organs were sectioned into 5 μm-thick slices. These sections underwent two 30 min dehydration steps in 95% ethanol, followed by a 1 h soak in xylene at 60-70 °C, and were then embedded in paraffin for 12 h. Following dehydration, the tissues were stained with hematoxylin for 6 h at 60-70 °C, rinsed with tap water, differentiated with a solution of 10% acetic acid and 85% ethanol diluted in water (twice for 2 min), and washed with tap water.

Statistical analysis

Two-way analysis of variance (ANOVA) analyses were performed to evaluate the effects of sex (male vs. female) and treatment (Luc-LNP vs. mTERT-LNP) groups using Tukey's post hoc tests. A one-way ANOVA followed by Tukey's multiple comparison test was used for biodistribution studies. Immunofluorescence staining data, including Iba-1+ and TERT + cells, were evaluated using two-way ANOVA. Student's t-test was used for the qPCR assay. All mice were randomly assigned to experimental groups, and the experimenters remained blinded to the treatments throughout the study. Data are expressed as the mean with the standard error of the mean (±SEM). Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) for multiple groups, assuming a normal distribution of all data points. Significance levels are indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Results

Design, physicochemical characterization of mRNA-LNP formulations

We generated LNP formulations encapsulating either firefly luciferase mRNA (Luc-LNPs) or mouse TERT mRNA (mTERT-LNPs) and characterized their physicochemical properties (Fig. 1). All three independently prepared batches for each formulation showed high encapsulation efficiency (>95%) by RiboGreen assay (Fig. 1A and B). DLS revealed a narrow size distribution with mean hydrodynamic diameters of ∼90–110 nm and low polydispersity indices (PDI<0.2), indicating a uniform particle population (Fig. 1C and D). Both Luc-LNPs and mTERT-LNPs exhibited slightly negative zeta potential (Fig. 1E and F), consistent with reduced nonspecific protein adsorption and prolonged circulation. Finally, the mTERT-LNP formulation maintained stable RNA content when stored at 4 °C for up to 14 days, with no significant loss in mRNA concentration (Fig. 1G), supporting its suitability for in vivo use.

Fig. 1.

Physicochemical characterization of Luc- and mTERT-LNPs. (A, B) Encapsulation efficiency (%EE) of luciferase mRNA-loaded LNPs (Luc-LNP) and mouse TERT mRNA-loaded LNPs (mTERT-LNP) across three independently prepared batches. (C, D) Hydrodynamic diameter (bars, left y-axis) and polydispersity index (PDI; circles, right y-axis) of Luc-LNP (c) and mTERT-LNP (d) batches, showing uniform particle size (∼100 nm) and low PDI. (E, F) Zeta potential measurements of Luc-LNP (E) and mTERT-LNP (F) for three independent batches, indicating slightly negative surface charge. (G) mTERT-LNP RNA concentration after storage, measured on days 1, 8, and 14, demonstrating preserved RNA content over time. Data are presented as mean ± SEM of independent preparations/experiments. LNP, lipid nanoparticle; Luc, mRNA encoding luciferase; mTERT, mRNA encoding mouse telomerase reverse transcriptase; PDI, polydispersity index.

In vitro biocompatibility and kinetics of LNP uptake and mRNA expression in neuronal cells

To evaluate in vitro biocompatibility, we exposed N2a murine neural cells to increasing concentrations of Luc-LNPs or mTERT-LNPs (0.5–2 μg/mL mRNA). Neither formulation affected cell viability at 24 h or 48 h compared with untreated controls (Fig. 2A–D). The kinetics of mRNA-LNP uptake and mRNA expression were assessed in N2A cells (Fig. 2E–J). For this study, the LNP were formulated with Cy3 lipid and EGFP mRNA to allow kinetic live-cell tracking with Incucyte®, and the cells were incubated with various doses of EGFP-LNPs (mRNA concentrations of 0–2 μg/mL). The images and quantitative plots show an efficient dose- and time-dependent LNP uptake (Fig. 2G, H, I) and EGFP expression (Fig. 2E and F J) in N2A cells in the time range of 0–48 h.

Fig. 2.

In vitro biocompatibility of Luc- and mTERT-LNPs and LNP uptake/mRNA expression in neuronal cells. (A-D) In vitro cytocompatibility of Luc-LNP (A, C) and mTERT-LNP (B, D) in cultured cells at the indicated mRNA concentrations (0.5–2 μg/mL) compared with untreated control, assessed at 24 h (E, F) and 48 h (G, H). No appreciable reduction in cell viability is observed at any dose or time point. (E-J) uptake of Cy3 lipid-labeled LNP (E, G, I) and expression of EGFP (F, H, J) delivered by mRNA-LNPs in N2A cells. Representative images show high LNP uptake (E, G) and efficient expression of encapsulated mRNA (F, H). There is a time- and dose-dependent kinetics of LNP uptake (I) and EGFP expression (J) in the time frame of 0–48 h. Data are presented as mean ± SEM of independent preparations/experiments. LNP, lipid nanoparticle; Luc, mRNA encoding luciferase; mTERT, mRNA encoding mouse telomerase reverse transcriptase; EGFP, mRNA encoding Green Fluorescent Protein.

Biodistribution of mRNA-LNPs in the brain and peripheral organs

We next evaluated the biodistribution of mRNA-LNPs using ex vivo IVIS imaging 24 h after intravenous administration. Mice received either Luc-LNPs or Cy5.5-labeled mTERT-LNPs (Cy5.5-mTERT-LNP). Luc-LNPs-treated animals displayed robust bioluminescent signal in the spleen and the injured brain, with minimal signal detected in the heart, lungs, kidneys, liver, or blood (Fig. 3A and B). Cy5.5-LNP-mTERT showed a similar pattern of brain localization but, as expected for fluorescently labeled LNPs, also accumulated prominently in the clearance organs, including spleen, kidneys, and liver, with negligible signal in heart, lungs, or blood (Fig. 3C and D). In both formulations, qualitative inspection suggested modest sex-dependent differences in organ signal intensity, with females tending to show higher signal intensity in the brain and spleen. Overall, these data confirm that IV-administered mRNA-LNPs accumulate in the injured brain and enable protein translation, while exhibiting a typical peripheral biodistribution profile for LNPs.

Fig. 3.

Biodistribution of systemically administered mRNA-LNPs in the injured brain and peripheral organs. (A) Quantification of bioluminescent signal (radiance, photons/s/cm²/sr) after intravenous administration of luciferase mRNA-loaded LNPs (Luc-LNP) in ex vivo organs collected from male (dark blue bars, square symbols) and female mice (light blue bars, square symbols) subjected to TBI. Organs analyzed include the brain, heart, lungs, spleen, kidneys, liver, and blood. (B) Representative ex vivo IVIS images of brain and peripheral organs from male and female mice receiving Luc-LNPs, showing luminescent signal overlaid on grayscale images. Color scale indicates relative luminescence intensity. (C) Quantification of epi-fluorescent signal (radiance, photons/s/cm²/sr) in ex vivo organs from male (dark red bars, square symbols) and female (light red bars, circle symbols) mice after intravenous administration of Cy5.5-labeled mTERT-LNPs (Cy5.5-LNP-mTERT). (D) Representative ex vivo IVIS epi-fluorescence images of brain and peripheral organs from male and female Cy5.5-LNP-mTERT-treated mice. Color scale indicates relative fluorescence intensity. Data are presented as mean ± SEM. LNP, lipid nanoparticle; Luc, mRNA encoding luciferase; mTERT, mRNA encoding mouse telomerase reverse transcriptase.

TBI induces transient TERT/telomere dysfunction and is rescued by mTERT-LNPs

We first examined whether TBI alters endogenous TERT expression. Cortical tissue from sham-injured mice and TBI mice at 3 or 14 dpi was analyzed by qPCR. TERT mRNA levels were significantly reduced at 3 dpi and partially recovered by 14 dpi (Fig. 4B). We then evaluated whether TBI affected telomere length. Telomere length, measured as the telomere-to-single-copy gene (T/S) ratio, was lower in mice at 3 dpi, but showed partial recovery by 14 dpi (Fig. 4C). RNAscope confirmed a reduction of Tert mRNA signal in peri-contusional cortex at 3 dpi in both male and female mice relative to controls (Fig. 4D), indicating an early window of TERT insufficiency after TBI. To test whether exogenous TERT mRNA could restore Tert expression in this window, 6-months-old male and female C57BL/6 mice received a single retro-orbital injection of mTERT-LNPs or control Luc-LNPs shortly after TBI induction (Fig. 4A). At 3 dpi, RNAscope quantification showed a robust increase in cortical Tert mRNA signal in mTERT-LNPs-treated mice compared with Luc-LNPs controls in both sexes (Fig. 4E–G). qPCR corroborated this finding, demonstrating significantly higher TERT mRNA levels in the mTERT-LNP group (Fig. 4F). We next assessed whether TERT restoration impacted telomere length. Telomere length tended to be higher in mTERT-LNPs-treated mice compared with Luc-LNPs controls at 3 dpi, with a similar pattern in males and females (Fig. 4H). Consistent with enhanced transcript levels, immunohistochemistry revealed a significant increase in the number of TERT+ cells in peri-contusional cortex following mTERT-LNP treatment in both sexes (Fig. 4I and J). Together, these data demonstrate that systemically delivered mTERT-LNPs effectively increase cortical TERT expression in the injured brain and partially restore telomere homeostasis.

Fig. 4.

Experimental design and TERT mRNA-LNP-mediated restoration of TERT expression and telomere length after TBI. (A) Schematic of the TERT mRNA-LNP system and in vivo study design. TERT or luciferase mRNA is encapsulated in Cy5.5-labeled LNPs and administered intravenously to 6-month-old male and female C57BL/6 mice subjected to controlled cortical impact TBI (day 0). Mice undergo IVIS imaging for mRNA-LNP biodistribution and protein expression on day 1 and neuropathologic and blood analyses on day 3. (B) Endogenous brain TERT mRNA levels were assessed by qPCR at baseline (control), 3 days post-injury (dpi), and 14 dpi, demonstrating an early decrease in TERT expression with partial recovery over time. (C) The telomere length (measured by the telomere-to-single-copy gene (T/S) ratio) decreased at 3 dpi and showed partial recovery by 14 dpi. (D) Representative RNAscope images of Tert mRNA (red) with DAPI nuclear counterstain (blue) in peri-contusional cortex from male and female mice at control and 3 dpi, illustrating reduced Tert signal after TBI. Scale bar, 50 μm. (E) Quantification of RNAscope Tert mRNA signal (% area) in peri-contusional cortex from male and female mice treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert) and analyzed at 3 dpi, showing robust enhancement of Tert mRNA with TERT mRNA-LNP treatment. (F) qPCR analysis of cortical Tert mRNA levels (fold change) in Luc versus Tert groups at 3 dpi, confirming increased Tert transcript levels after TERT mRNA-LNP delivery. (G) Representative RNAscope images of Tert mRNA (red) with DAPI (blue) in peri-contusional cortex of male and female mice receiving Luc or mTERT LNPs. Scale bar, 50 μm. (H) Telomere length in peri-contusional cortex measured by qPCR (T/S ratio) at 3 dpi, showing partial restoration of telomere length in Tert-treated animals relative to Luc controls, in both sexes. (I) Quantification of TERT protein + cells per field in peri-contusional cortex by immunohistochemistry (IHC), demonstrating increased TERT protein expression in Tert versus Luc groups at 3 dpi. (J) Representative IHC images of TERT protein (red) with DAPI (blue) in cortex from male and female Luc- and Tert-treated mice. Scale bar = 50 μm. Data points represent individual mice; bars show mean ± SEM. Statistical significance is indicated by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

mTERT-LNP treatment attenuates microglial activation and pro-inflammatory cytokine expression

To determine whether TERT restoration modulates neuroinflammation, we quantified microglial activation in the peri-contusional cortex at 3 dpi. Immunohistochemistry for Iba-1 revealed that mTERT-LNP treatment significantly reduced the number of Iba-1+ cells in both male and female mice compared with Luc-LNP controls (Fig. 5A). A similar trend was observed for Iba-1+ area, particularly in males (Fig. 5B). Representative images illustrate reduced microglial density and a less activated morphology in mTERT-LNPs-treated brains (Fig. 5C). We then analyzed inflammatory gene expression by qPCR. Cortical mRNA levels of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were significantly lower in mTERT-LNPs-treated mice than in Luc-LNPs controls, while IL-18 was unchanged (Fig. 5D–F, H). In contrast, expression of the anti-inflammatory cytokines TGF-β and IL-10 showed a modest, non-significant increase (Fig. 5G–I). These data indicate that mTERT-LNP therapy selectively suppresses key components of the acute pro-inflammatory response after TBI while preserving or slightly promoting anti-inflammatory pathways.

Fig. 5.

TERT mRNA-LNP treatment attenuates microglial activation and pro-inflammatory cytokine expression after TBI. (A, B) Quantification of microglial activation in peri-contusional cortex at 3 days post-injury (dpi) in male and female mice treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert). Iba-1+ cells per field (A) and Iba-1+ area (%) (B) were measured by immunohistochemistry, showing a significant reduction in Iba-1+ cell number with Tert treatment in both sexes, with a trend toward reduced Iba-1+ area. (C) Representative confocal images of Iba-1 immunofluorescence (green) with DAPI nuclear counterstain (blue) in peri-contusional cortex from male and female Luc- and Tert-treated mice at 3 dpi. Top panels show low-magnification views; bottom panels show higher-magnification insets highlighting microglial morphology. (D-I) qPCR analysis of cortical cytokine mRNA expression at 3 dpi comparing Luc and Tert groups: IL-1β (D), TNF-α (E), IL-6 (F), TGF-β (G), IL-18 (H), and IL-10 (I). Tert treatment significantly reduced IL-1β, TNF-α, and IL-6 transcripts, with no significant changes in TGF-β, IL-18, or IL-10. Data points represent individual mice; bars indicate mean ± SEM. Statistical significance is denoted as ∗p < 0.05, ∗∗p < 0.01.

mTERT-LNP therapy reduces peripheral inflammation and oxidative stress and is well tolerated systemically

To investigate systemic effects of TERT mRNA therapy, we measured circulating markers of inflammation and oxidative stress at 3 dpi. Western blot analysis showed that serum CRP levels were significantly reduced in mTERT-LNP-treated male mice relative to Luc-LNP controls, with a similar but less pronounced trend in females (Fig. 6A and B). In parallel, serum MDA, a marker of lipid peroxidation, was modestly decreased by mTERT-LNPs in both sexes, reaching statistical significance when comparing mTERT-LNP-treated females to Luc-LNP-treated males (Fig. 6C). These findings suggest that mTERT-LNPs dampen not only central but also peripheral inflammatory and oxidative responses to TBI.

Fig. 6.

TERT mRNA-LNPs reduce systemic inflammation and oxidative stress without inducing peripheral organ toxicity. (A) Representative Western blots of serum C-reactive protein (CRP) from male and female mice at 3 days post-injury (dpi) treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert). Stain-free total protein is shown as a loading control. (B) Densitometric quantification of CRP levels (arbitrary units) normalized to total protein demonstrates a significant reduction in circulating CRP in Tert-versus Luc-treated mice, particularly in males. (C) Serum malondialdehyde (MDA) concentrations, a marker of lipid peroxidation and oxidative stress, showing decreased MDA in Tert-treated animals at 3 dpi.

(D-H) Representative hematoxylin and eosin (H&E)-stained sections of spleen (D), kidney (E), lung (F), heart (G), and liver (H) from male and female Luc- and Tert-treated mice at 3 dpi. No treatment-related histopathologic abnormalities are observed in any organ, indicating that TERT mRNA-LNP administration is well tolerated systemically. Scale bar in (H), 100 μm (applies to all histological panels). Data points represent individual mice; bars indicate mean ± SEM. Statistical significance: ∗p < 0.05, ∗∗∗p < 0.001.

To assess safety, we performed histopathologic analysis of the spleen, kidney, lung, heart, and liver. Hematoxylin and eosin-stained sections from mTERT-LNP-treated mice showed no treatment-related abnormalities or tissue damage compared with Luc-LNP controls in either sex (Fig. 6D–H). Consistent with this, no differences in body weight or gross behavior were observed between treatment groups (data not shown). Collectively, these results indicate that mTERT-LNP therapy exerts anti-inflammatory and antioxidant effects while maintaining a favorable systemic safety profile in vivo.

Discussion

We demonstrate that systemic delivery of telomerase mRNA via LNPs is a viable and well-tolerated strategy to modulate early secondary injury mechanisms following TBI. A single administration during the acute post-injury window resulted in robust cortical TERT expression, attenuation of microglial activation, and reduction of pro-inflammatory cytokines, alongside decreased systemic markers of inflammation and oxidative stress. These findings establish telomerase restoration as a tractable therapeutic axis in neurotrauma. To our knowledge, this is the first demonstration that mRNA-LNP-mediated telomerase modulation can attenuate acute secondary injury mechanisms after TBI.

This work extends prior advances in LNP technologies into the field of neurotrauma. Our formulations exhibited physicochemical properties consistent with those of state-of-the-art, clinically validated LNP systems [34]. Both Luc-LNPs and mTERT-LNPs exhibited >95% encapsulation efficiency, narrow size distributions with an average diameter of ∼100 nm with low PDI, mildly negative zeta potentials, and they remained stable for at least two weeks. In vitro, neither formulation impaired N2A cell viability over a range of therapeutically relevant mRNA doses. These data, together with the absence of histopathologic abnormalities in spleen, kidney, lung, heart, or liver, and stable body weight in vivo, support the overall biocompatibility of the platform [35].

In vitro studies confirmed time- and dose-dependent LNP uptake and protein expression kinetics in neuronal cells. Biodistribution studies confirmed brain delivery following systemic administration, while also highlighting a key translational consideration: tissue accumulation of LNPs does not necessarily predict functional mRNA translation. Intravenously administered mRNA-LNPs reached the injured brain while following a typical LNP clearance pattern [36]. Luc-LNPs generated a strong bioluminescent signal in the brain and spleen, indicating efficient protein expression, whereas Cy5.5-labeled mTERT-LNPs showed fluorescence in the brain, spleen, kidneys, and liver, which are organs enriched in reticuloendothelial and filtration functions [35]. It is possible that at 24 h following administration, a fraction of the Cy5.5 signal corresponded to degradation products of LNP cleared through the kidneys and liver.

Our group has previously explored lipid-based nanocarriers as tools for diagnosis and therapy in TBI. We developed leukocyte-inspired “leukosomes,” biomimetic lipid nanoparticles that incorporate leukocyte membrane proteins into a liposomal shell and demonstrated that, after systemic administration, these particles preferentially home to inflamed vasculature in the injured brain, with minimal accumulation in uninjured cortex [37]. In a mouse CCI model, leukosomes enabled noninvasive in vivo imaging of nanoparticle trafficking to the lesion. They revealed a characteristic distribution across the spleen and other clearance organs, providing a blueprint for systemic nanocarrier delivery in TBI. More recently, we extended our LNP work to mRNA cargos, showing that hTERT mRNA-LNPs enhance telomerase activity, proliferation, and engraftment of human skin cell suspensions in a wound model, establishing the feasibility and safety of transient telomerase mRNA delivery in vivo [25]. We have also recently demonstrated that hTERT mRNA-LNPs protect from radiation-induced DNA damage in the skin [26]. Building on these platforms, the current study represents the first application of TERT-mRNA-LNPs directly in a neurotrauma setting. It complements ongoing efforts in our group to adapt LNP formulations for the delivery of genome-editing and other RNA therapeutics to the injured brain.

A central mechanistic observation is that TBI induces a transient disruption of telomere/TERT homeostasis. We found that endogenous TERT mRNA levels in the cortex were significantly reduced at 3 days post-injury, with partial recovery by 14 days, and that TL (T/S ratio) was similarly decreased acutely and then rebounded. These findings are consistent with reports that TBI and other brain insults accelerate telomere shortening and that shorter telomeres correlate with poorer clinical outcomes and persistent symptom burden [15]. Our data extend this work by identifying a discrete early window of TERT insufficiency in the injured brain, providing a rational temporal target for telomerase-based interventions.

Within this window, mTERT-LNP treatment robustly increased TERT mRNA and protein in peri-contusional cortex, accompanied by partial restoration of TL. These effects were observed in both sexes, with a tendency toward greater telomere rescue in females, echoing prior work where pharmacologic telomerase activation preferentially increased TERT expression in female TBI rats [38]. TERT appears to act primarily on secondary injury pathways, such as inflammation and oxidative stress, rather than reversing established tissue loss. This distinction aligns with its known roles in cellular stress responses and mitochondrial function rather than classical neuroprotection alone.

Neuroinflammation is a central driver of progressive pathology after TBI and a key determinant of long-term neurological outcomes [39]. mTERT-LNP treatment significantly reduced microglial activation and suppressed pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), while modestly enhancing anti-inflammatory mediators (IL-10, TGF-β), indicating a shift toward a more regulated immune milieu. These findings align with prior studies demonstrating that telomerase activation can dampen inflammatory signaling and improve tissue resilience across multiple models [[40], [41], [42]]. A study reported that the TERT activator compound (TAC) increased TERT transcription, decreased the production of the proinflammatory cytokines IL-1β and IL-6, and promoted adult neurogenesis while also reducing neuroinflammation in the hippocampus of aged mice [24]. Importantly, systemic administration of a TERT-activator compound restored hippocampal TERT levels, reduced the same pro-inflammatory cytokines, attenuated microglial reactivity, and ultimately improved cognitive performance [43]. Together, our data support a model in which restoring TERT expression in the injured brain disrupts non-canonical TERT functions, thereby limiting the acute neuroimmune response that seeds long-term neurodegeneration.

Importantly, the observed effects were consistent across sexes, although subtle sex-dependent differences in telomere restoration and systemic markers warrant further investigation.

In addition to central effects, mTERT-LNPs reduced circulating CRP supporting a broader impact on systemic inflammatory pathways [44]. Given the established links between telomere attrition, systemic inflammation, and neurodegeneration, these findings suggest that telomerase-based interventions may exert multi-compartment benefits relevant to both acute injury and chronic disease progression.

The safety profile of mTERT-LNPs was favorable, with no detectable organ toxicity or behavioral abnormalities. The transient nature of mRNA-driven TERT expression mitigates concerns about sustained telomerase activation and the oncogenic risk of immortalization [42,[45], [46], [47]]; however, long-term safety and repeated-dosing paradigms will require careful evaluation in future studies.

Several limitations should be acknowledged. The present study focuses on a single dose and early time point, and does not assess functional outcomes such as cognition or behavior. In addition, while we demonstrate effects on telomere length and inflammatory signaling, the relative contribution of telomere-dependent versus telomere-independent (e.g., mitochondrial or DNA damage response) mechanisms remains to be defined. Finally, further optimization of dosing, delivery efficiency, and comparative evaluation with alternative RNA or nanoparticle platforms will be necessary to support clinical translation.

Future studies should extend these findings to subacute and chronic phases of injury, incorporate functional outcome measures, and define sex-specific responses. Combination approaches integrating telomerase mRNA with complementary neuroprotective or immunomodulatory strategies may further enhance therapeutic efficacy. Given the convergence of telomere dysfunction and chronic inflammation across neurodegenerative disorders, this platform may have broader applicability beyond TBI.

Overall, this study provides the first in vivo evidence that telomerase mRNA delivery via LNP mitigates acute secondary injury processes after TBI. Systemic administration of mTERT-LNPs restores TERT expression, partially normalizes telomere homeostasis, and suppresses neuroinflammatory and systemic stress responses, while maintaining a favorable safety profile.

These findings position telomerase restoration as a mechanistically grounded and clinically relevant therapeutic strategy for TBI. In the context of established mRNA-LNP technologies, this approach offers a scalable and translational pathway to target telomere- and inflammation-driven pathology in neurotrauma and related neurodegenerative conditions.

Author contributions

B.G., J.P.C. and S.V. initiated, designed, planned, and oversaw all study aspects. G.K., M.H., A.T., H.F., T.E., A.M., M.B., P.P., K.C., B.G. and S.V. performed the experimental work and data analysis, and G.K. and S.V. drafted the manuscript. All authors reviewed and edited the final version of the manuscript.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

J.P.C. is an inventor on patents owned by Stanford University and Houston Methodist Hospital related to the use of mRNA telomerase for the treatment of senescence. J.P.C. is a co-founder of ChromexBio, a company that aims to develop the telomerase technology. The rest of the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.