Telomerase mRNA enhances human skin engraftment for wound healing

Abstract

Deep skin wounds represent a serious condition and frequently require split-thickness skin grafts (STSG) to heal. The application of autologous human-skin-cell-suspension (hSCS) requires less donor skin than STSG without compromising the healing capacity. Impaired function and replicative ability of senescent cutaneous cells in the aging skin affects healing with autologous hSCS. Major determinants of senescence are telomere erosion and DNA damage. Human telomerase reverse transcriptase (hTERT) adds telomeric repeats to the DNA and can protect against DNA damage. Here, we propose and evaluate hTERT mRNA lipid nanoparticles (LNP) for enhancing cellular engraftment and proliferation of hSCS. Transfection with optimized hTERT mRNA LNP system enables delivery and expression of mRNA in-vitro in keratinocytes, fibroblasts, and in hSCS prepared from donors’ skin. Telomerase activity in hSCS is significantly increased. hTERT mRNA LNP enhance the generation of a partial-thickness human skin equivalent in the mouse model, increasing hSCS engraftment (Lamin) and proliferation (Ki67), while reducing cellular senescence (p21) and DNA damage (53BP1).

In conclusion, hTERT mRNA LNP treated hSCS holds promise for enhancing skin regeneration. Improvement of engraftment/ proliferation and mitigation of DNA damage can benefit all preparations of hSCS. This study paves the way for RNA-based cellular therapies aimed at cutaneous conditions.

Graphical Abstract

Human Telomerase Reverse Transcriptase mRNA lipid nanoparticles (hTERT-mRNA LNP) were formulated for improved healing of deep skin wounds using human skin cells suspension (hSCS). Transfection with optimized hTERT-mRNA LNP enables delivery and expression of mRNA in hSCS in-vitro. hTERT-mRNA LNP-treated hSCS enhanced skin regeneration in partial-thickness human-skin equivalent in mice, increasing hSCS engraftment/proliferation, while reducing senescence/ DNA-damage.

1. Introduction

Here, we report a new strategy to enhance the performance of human skin cell suspension (hSCS) in wound healing using a human telomerase reverse transcriptase (hTERT) mRNA nanotherapeutic.

Skin wounds from trauma are prevalent worldwide [1, 2]. For instance, burn injuries annually affect 11 million people globally accounting for >300,000 deaths [3, 4] causing significant socio-economic burden [5, 6] and psychological complications [7, 8]. Deep burn wounds as well as severe chronic wounds can be complex skin injuries that can require surgical intervention for definitive closure. Early surgical interventional strategies such as wound excision and immediate skin grafting substantially enhance wound healing and increase patient survival [9, 10]. Autologous split thickness skin grafts (STSG) are the current standard for treatment of deep partial-thickness and full-thickness burn wounds [11, 12]. To prepare STSG, skin is harvested from an unaffected donor site of the patient and placed on the debrided wound site during surgery [13]. STSG is associated with pain, scarring, pruritis, infection, delayed healing and donor site morbidity [14]. Furthermore, STSG may become non-viable or could be insufficient to cover total wound surface area [15, 16].

A recent approach proven to overcome STSG limitations is preparing Autologous Skin Cell Suspension (ASCS) from a small split-thickness donor area. The procedure uses RECELL^® Autologous Skin Cell Harvesting Device and is performed in a point-of-care setting achieving rapid re-epithelialization of deep wounds. Prior work has shown that autologous hSCS from 1cm² of 0.15-0.20mm-thick donor skin covers 80cm² of wounded area [17]. Clinical data demonstrate that treatment with ASCS is fast, causes less complications at donor skin site [18] and yields comparable functional and aesthetic outcomes as compared to STSG. The subpopulations of cells in harvested autologous hSCS include keratinocytes (~65%) and fibroblasts (~30%).

Aging and accompanying conditions (e.g., smoking) negatively affect wound healing [19]. In the aged population skin integrity, functionality and regenerative potential are impaired [20] and aged skin cells undergo cellular senescence [21]. In addition to changes in the DNA structure and functionality, senescent skin cells are characterized by an increased secretion of proinflammatory cytokines and production of reactive oxygen species (ROS). Consequently, wound healing is compromised [22, 23]. Older individuals have difficulty in wound healing, due to impaired function and replicative capacity of senescent cutaneous cells caused in part by telomere erosion, DNA damage and accumulated oxidative stress [24]. Telomeres are the guanine-rich DNA repeats located at the ends of chromosomes [25], protecting against DNA degradation, fusion events and loss of genetic information due to the end replication problem [26]. Telomere shortening is implicated in the loss of the regenerative capacity of cells and is a feature of aging [27, 28].

Human telomerase reverse transcriptase (hTERT) adds telomeric repeats to the DNA[29] and has non-canonical functions, such as genomic DNA damage repair, and protection against oxidative stress [30]. In our previous studies in aged human cells, introduction of hTERT messenger RNA (mRNA) enabled transient expression of hTERT, which restored telomere length, cell replicative capacity, cell morphology and functions, and reduced DNA damage markers [31-34]. mRNA nanotherapeutics are an emerging class of drugs. Due to chemical and enzymatic instability of naked mRNA in the physiological fluids, in vivo mRNA administration necessitates a stable and efficient delivery carrier. Lipid nanoparticles (LNP) are clinically used for mRNA delivery, including in mRNA LNP vaccines against COVID-19 [34, 35].

In the current work we aimed to evaluate hTERT mRNA LNP based therapy for enhancing engraftment and proliferative capacity human skin cell suspension (hSCS), prepared with the RECELL^® Device, using a partial-thickness wound healing model in immunocompromised mice. Three mRNA LNP formulations were designed and assessed in vitro to optimize the delivery of hTERT and reporter gene (enhanced green fluorescent protein, EGFP) mRNA in hSCS isolated from donors’ skin. Further, uptake and mRNA expression studies with the best performing system were carried out in the major cell populations in hSCS, namely, keratinocytes and fibroblasts. In vivo studies further confirmed the ability of hTERT mRNA LNP to enhance the capacity of hSCS to develop human epidermal layer in vivo by increasing proliferation and reducing senescence markers.

2. Results

2.1. Optimization of LNP for mRNA Transfection in vitro

Six LNP formulations containing EGFP mRNA with three cationic/ionizable lipids (DOTAP, MC3 and SM-102) and different helper lipids as specified in Methods were designed. LNP were formulated with >91% mRNA encapsulation efficacy, sizes in the range of 72.2±4.2 to 109.8±1.1 nm, and mostly neutral zeta potential (Fig. 1). Following the optimization of cellular uptake and mRNA expression, hTERT mRNA DOTAP-A system was also formulated with the characteristics summarized in Fig. 1.

Figure 1:

mRNA LNP characterization. Formulations A and B containing DOTAP, MC3 and SM-102 GFP mRNA LNP and Formulation A containing DOTAP hTERT mRNA were designed and characterized. Formulations A of DOTAP, MC3 and SM-102 LNP were prepared at 49:49:2 molar ratio of DOPE, cationic/ionizable lipid and DMG-PEG2000. Formulations B of DOTAP, MC3 and SM-102 LNP were prepared at 8:1.5:38.5:52 molar ratio of DSPC, DMG-PEG2000, cholesterol and cationic/ionizable lipid (DOTAP, Dlin-MC3-DMA or SM-102). A. Size distribution of the systems by Dynamic Light Scattering. B. Summary of physico-chemical characteristics of the mRNA LNP systems used in the study.

The efficiency of all the LNP systems and Lipofectamine control in mRNA delivery into hSCS and mRNA expression was assessed. Cells isolated from donors’ human skin were incubated with fluorescent EGFP mRNA LNP at 1 and 2 μg/mL of mRNA. To assess LNP uptake, fluorescent lipid (red signal) was incorporated into the LNP structure and to evaluate mRNA translation, the expression of reporter EGFP mRNA (green signal) was monitored. LNP uptake and mRNA translation were assessed for 24 hours and quantified. DOTAP-A formulation enabled a prompt uptake and efficient mRNA expression in skin cells as compared to other systems tested. Figure 2 and Supplementary Figure 2 present data with 1μg/mL mRNA and Supplementary Fig. 1 with 2μg/mL. mRNA expression (Fig. 2a and 2b) and LNP uptake (Fig. 2c and 2d) were measured over a 24h period. DOTAP-A LNP, containing DOPE and DMG-PEG2000 were taken up by hSCS as early as 2h post treatment and a robust expression of EGFP was observed, as compared to all the other systems tested at both concentrations of mRNA. Normalized per cell GFP signal (Supplementary Figure 3) at 24h for DOTAP-A LNP was significantly higher (more than 3 times) than for any other tested system, with the order of DOTAP-A>DOTAP-B>SM102-A>MC3A>SM102-B.

Figure 2. In vitro studies for optimization of LNP delivery in human skin suspension cells (hSCS) isolated from a donor skin.

(a, b) EGFP mRNA expression (green) and (c,d) LNP uptake (red) of six LNP systems and Lipofectamine. Formulations A of DOTAP, MC3 and SM-102 LNP were prepared at 49:49:2 molar ratio of DOPE, cationic/ionizable lipid and DMG-PEG2000. Formulations B of DOTAP, MC3 and SM-102 LNP were prepared at 8:1.5:38.5:52 molar ratio of DSPC, DMG-PEG2000, cholesterol and cationic/ionizable lipid (DOTAP, Dlin-MC3-DMA or SM-102). Kinetics of mRNA expression (a) and LNP uptake (c) over 24 hours as quantified by Incucyte software, n=4. Micrographs (b, d) acquired by Incucyte Live Cell Imaging at 4 and 24 hours after transfection of 1 μg mRNA/mL. Scale bar= 400μm

Since it was previously shown that hSCS mainly contains keratinocytes (65%) and fibroblasts (30%) [36, 37], although small fractions of other cells such as immune cells, melanocytes and endothelial cells are present, it was important to assess the uptake of the optimized formulation in major hSCS cell populations. As shown in Figure 3 and Supplementary Figure 3, both cell populations have efficiently taken up DOTAP-A LNP and yielding in EGFP expression as early as 2 hours. Based on the slope of the LNP uptake and mRNA expression plot, the systems were taken up faster in keratinocytes as compared to fibroblasts. Similar trend was observed for mRNA expression

Figure 3. In vitro studies in the cultured major cell populations in hSCS, fibroblasts and keratinocytes, with the optimized LNP (DOTAP-A).

DOTAP-A system (49:49:2 molar ratio of DOPE, DOTAP and DMG-PEG2000) was tested at two concentrations equivalent to 0.5 and 1 μg/mL mRNA. LNP were fluorescently labeled with rhodamine (red) and EGFP mRNA (green) was employed as a reporter for mRNA expression. The studies were carried out for 1-48 h on Incucyte Live Cell Imaging system and the data was quantified. (a) LNP uptake in fibroblasts; (b) LNP uptake in keratinocytes; (c) mRNA GFP expression in fibroblasts; (d) mRNA GFP expression in keratinocytes; (e) Micrographs acquired by Incucyte Live Cell Imaging at 2 and 24 hours after transfection of 1 μg mRNA/mL. n=4. Scale bar= 400μm.

We further performed TRAP assay with hSCS treated with hTERT mRNA DOTAP-A LNP (Fig. 4). The optimized system augmented telomerase activity in hSCS in a dose dependent manner (0.25-1 μg/mL mRNA) at 1, 2 and 24 h post-treatment (Fig. 4 a, b). In comparison, hSCS transfected with EGFP mRNA LNP did not increase telomerase activity (Fig. 4b).

Figure 4. Telomerase activity in hSCS following treatment with DOTAP-A hTERT mRNA LNP.

(a) TRAP assay for telomerase activity in lysates from hSCS transfected with hTERT mRNA LNP (lane 05), with EGFP mRNA LNP (lane 07), and no treatment control (lane 09). Lysate from iPSCs (induced pluripotent stem cells) was used as a positive control (lane 03). Even number lanes were loaded with heat-killed (HK; 85°C for 10 minutes to inactivate telomerase) lysate set up in parallel for each sample extract (lanes 04, 06, 08, 10). Lane 1 was loaded with PCR amplified positive control template. Lane 2 was loaded with lysis buffer only as a negative control. (b) Time and dose-response analysis of telomerase activity. hSCS were treated with three concentrations (1.00, 0.50, and 0.25 μg mRNA/mL) of hTERT mRNA LNP or EGFP mRNA LNP. Lysates were collected 1, 2 and 24 h post transfection and subjected to TRAP assay to detect telomerase activity. DOTAP-A system contains 49:49:2 molar ratio of DOPE, DOTAP and DMG-PEG2000.

2.2. Humanized Murine Partial Thickness Wound Model for Evaluation of hSCS Engraftment

To evaluate the effect of hTERT mRNA DOTAP-A LNP on hSCS engraftment, a partial thickness wound healing model was developed in immunocompromised mice[38]. The overall flow of the experiment is summarized in Fig. 5a. hSCS was isolated using the RECELL^® Device which includes enzymatic and mechanical processing steps, resulting in a cell suspension containing >50% viable cells, mainly keratinocyte and fibroblasts. A full-thickness wound was created on the back of a Foxn1^nu immunocompromised mice. A grafting dome was surgically inserted to cover the excisional wound bed (Fig. 5b). A human dermal fibroblasts (HDF) suspension combined with fibrin hydrogel was applied to the wound bed to create a synthetic dermis. After this synthetic dermis had solidified, we implanted untreated hSCS, hSCS pre-treated with hTERT mRNA LNP, or hSCS treated with control EGFP mRNA.

Figure 5. Workflow for in vivo studies in humanized skin wound model.

(a) A schematic of the workflow. (1) Human donor skin is dermatomed to a partial thickness (2mm) and mechanically dissociated and enzymatically digested to generate the human skin cell suspension (hSCS), consisting largely of keratinocytes (brown) and fibroblasts (green), together with other cell types found in human skin (blue). (2) Human dermal fibroblasts (HDF) suspension and fibrin hydrogel (FH) are prepared separately; (3) 1cm surgical incision is made on the upper dorsal region and a silicon dome is inserted; (4) hSCS are exposed to mRNA LNP; (5) FH is added to the area under the dome followed by HDF to generate a synthetic dermis. (6) hSCS treated with mRNA LNP are added to the dome. (7) The animals are maintained for 7 days and sacrificed. (8) Wound bed is harvested, processed for histological analysis, and imaged. (b) Photographs of the mice undergoing surgical procedure to establish the humanized wound model (steps 3 and 5 in the workflow).

One-week post-surgery, removal of the dome chamber revealed a moist layer covering the wound area, without the formation of eschar (Fig. 6a-d, left panels). The engrafted region and the surrounding skin were removed and processed for tissue sectioning. The H&E shows a cross section view of the harvested wound region, illustrating healing status after 7 days (Fig. 6a-c, middle panel) H&E staining also provides a landmark to map the regions where Lamin+ human skin cells are identified (as shown in the immunofluorescence staining). Immunohistochemical staining (Fig. 6a-c, right panels) indicated engraftment of human skin cells (Lamin⁺) from hSCS and presence of human epithelial cells in the wound region (CK10⁺). No Lamin⁺ human cells or CK10⁺ positive cells were found in wounds treated only with the HDF-fibrin hydrogel.

Figure 6. Evaluation of the hSCS engraftment.

(a) Untreated hSCS applied on top of the synthetic dermis, n=6. (b) hSCS pre-treated with EGFP mRNA LNP applied on synthetic dermis, n=6. (c) hSCS pre-treated with hTERT mRNA LNP applied on synthetic dermis n=6. (d) Synthetic dermis alone without hSCS, n=4. The grafting dome was removed at day 7 after the procedure, a representative image from wound bed (left) was processed for histology. Area surrounding the wound region (dashed line) was excised, tissue was cut through the midline and sectioned. Hematoxylin and eosin (H&E) staining (middle) of the entire cross section of the engrafted wound bed plus the surrounding mouse skin. Immunofluorescence staining (right) of human specific Lamin (red) and an early differentiation keratinocyte marker CK10 (green). DAPI stains cell nuclei (blue).

Samples were further immunostained for cell proliferation marker Ki67, cellular senescence marker p21, and DNA damage marker 53BP1. The entire cross-section was scanned by EVOS automated imaging platform, where images were acquired at high magnification (20x) and stitched for high resolution mapping of an entire cross-section (Fig. 7a). Blinded observers quantified all positive stained cells from each wound section (Fig. 7b). hSCS transfected with hTERT mRNA LNP yielded a greater number of engrafted human cells (Lamin⁺) and Ki67⁺ proliferative cells, suggesting that hTERT LNP enhanced human skin cell engraftment and proliferation. In addition, there was a reduction in the number of p21⁺ senescent cells. Since the process of hSCS preparation exerted great chemical and mechanical distress to the isolated skin cells, we examined the engrafted area for the presence 53BP1, a key marker of DNA damage repair. Addition of hTERT mRNA LNP to hSCS showed a trend in reducing 53BP1⁺ cells in the wound region, compared to untreated cells and EGFP mRNA LNP transfected hSCS.

Figure 7. Quantitative analysis of hSCS engraftment.

Wound sections from mice implanted with hSCS treated with hTERT mRNA LNP (n = 6), hSCS treated with EGFP mRNA LNP (n = 6), and untreated hSCS groups were stained for Lamin (human cell marker), Ki67 (proliferation), p21 (cellular senescence), and 53BP1 (DNA damage). (a) Representative immunofluorescence images in skin histological sections. (b) Quantification of biomarker-positive cells of an entire skin cross-section, as assessed by observers blinded to the treatment group; mean ± SD. Images were acquired at high magnification (20x) and stitched for high resolution mapping of an entire 5μ skin wound cross-section for the quantitative analysis. *p<0.05

3. Discussion

In this study our objective was to evaluate the impact of a novel hTERT mRNA therapeutic on skin regeneration. Our promising results indicate that a point-of-care treatment with an mRNA nanotherapeutic can increase telomerase activity in human skin cells. This effect is associated with an increase in human cells in the wound bed and an upregulation of the cellular proliferation marker Ki67. Furthermore, there is a reduction in the senescence marker p21, and the DNA damage marker 53BP1. These studies indicate that mRNA telomerase may be useful in improving cell viability and positively augment wound healing.

Partial and full-thickness skin wounds and other traumatic injuries present a significant clinical challenge [2, 39, 40]. In the case of burns, ASCS is an alternative to traditional STSG. Use of ASCS requires significantly less donor skin and, thereby, reduces complications from the procedure. As a result, ASCS therapy has been found to reduce length of hospital stay and associated costs [41]. Additionally, the ASCS method is performed at the point-of-care and does not require in vitro expansion of the cells. By contrast cultured epidermal/dermal cell culture autografts require 2-3 weeks of ex vivo culture [37]. Although no comparative studies have been conducted to date, decreasing time associated with preparing the cultured autografts directly impacts the total cost of treatment.

In the current work, we tested if application of hTERT mRNA in optimized LNP to hSCS can improve cellular functions associated with promoting wound healing in a partial-thickness murine wound model. hTERT adds telomeric repeats to the DNA [29] and has non-canonical functions, such as genomic DNA damage repair, and protection against oxidative stress [30]. These features can be beneficial to the cell, improving viability and proliferation required during wound healing. We have previously shown that telomere erosion can be repaired, and cell functions restored, by transient transfection with hTERT mRNA.

RNA therapeutics require LNP for efficient administration and protection from enzymatic and chemical degradation [42]. Accordingly, we first optimized the LNP system for mRNA delivery to hSCS. To deliver hTERT mRNA to hSCS during the point-of-care procedure, the carrier should enable a prompt and efficient uptake into the skin cells ex vivo pre-application to the wound, and effective mRNA expression. To encapsulate highly anionic mRNA, lipids in LNP should bear positive charge. Clinically, RNAs are administered systemically and contain ionizable (at low pH) lipids [42]. While in vivo parenteral administration of ionizable LNP was proven safer than cationic (constantly charged) lipids [43-45], in topical/ex vivo mRNA applications the latter can be considered. We have formulated six LNP formulations (Fig. 1) and tested their delivery and mRNA expression in hSCS isolated from human donor skin. Our data (Fig. 2) show that out of the six tested systems (Fig. 1) and commercially available transfection reagent, LNP formulated with cationic lipid (DOTAP-A) were promptly taken up by hSCS resulting in high transfection efficiency (Fig. 2). However, other LNP formulations and lipofectamine did not allow for efficient mRNA transfection under the conditions of the hSCS therapy relevant for clinical translation (Fig. 2). This can partially be related to the ability of cationic lipids in LNP to attach to the negatively charged cellular membranes, facilitating cellular uptake and expression. We further demonstrated that optimized EGFP mRNA DOTAP-A LNP were taken up and expressed in the major cell populations in hSCS, keratinocytes (65%) and fibroblasts (30%)[36, 37] (Fig. 3) and hTERT mRNA formulated in DOTAP-A LNP enhanced the telomerase activity in hSCS from donor, as early as one-hour post treatment, and in a dose-dependent fashion (Fig. 4).

An in vivo humanized partial-thickness wound model was used to study human skin cell engraftment after transfection with hTERT mRNA LNP (Fig. 5), modifying previously reported models [38, 46, 47]. Our studies have shown that hSCS engraftment was significantly improved by pre-treatment with hTERT mRNA DOTAP-A LNP as compared to untreated hSCS or hSCS pretreated with EGFP mRNA LNP. The following markers were assessed: Lamin A, Ki67, p21 and 53BP1 (Fig. 7). Lamin A is a marker for nucleoskeleton in human cells [48] and was used as a marker to differentiate between the engrafted hSCS and mouse cells. Treatment with hTERT mRNA LNP doubled the number of human cells (Lamin+) at the 7-day-timepoint as compared to both control groups, indicating that hTERT therapy enhanced engraftment. Since the quantification represents the total number of Lamin+ cells per one transverse 5 μm skin wound section and based on a wound diameter of 1 cm, we can roughly estimate that the total number of human cells engrafted in the wound in hTERT mRNA LNP treated group was 1.20x10⁶, as compared to 0.72x10⁶ and 0.77x10⁶ in EGFP mRNA LNP and untreated control groups, respectively. Next, Ki67 staining confirmed that the number of proliferating cells in the hTERT mRNA LNP treated hSCS was five times higher than in other groups. These data suggest that the expansion of the cells, necessary in wound therapy with autologous hSCS is more efficient after treatment with hTERT mRNA LNP. Cyclin-dependent kinase inhibitor p21 is a cellular senescence marker strongly associated with cell cycle arrest [49]. hTERT mRNA LNP therapy decreased the number of p21-positive cells, indicating a reversal of the senescent phenotype. hTERT also has non-canonical functions not directly related to the telomere elongation. One of these functions is the regulation of DNA damage response [50]. Based on the data from 53BP1 staining, hTERT therapy mitigated DNA damage.

4. Conclusion

In conclusion, our study demonstrates that telomerase therapy enhances engraftment, proliferative capacity, and mitigates DNA damage of hSCS in a partial thickness humanized murine wound model. Future development will be necessary to realize the full potential of this mRNA therapy to enhance wound healing. Additionally, this work establishes proof of concept of a point-of-care methodology that could potentially be extended to other RNA-based cellular therapies.

5. Experimental Section

Sources (suppliers, abbreviations) and catalogue numbers for Materials and reagents used in this study are listed in Supplementary Table 1.

5.1. Messenger RNA (mRNA) Lipid Nanoparticles (LNP) Design and Characterization:

Human TERT (NM_198253.2) and EGFP mRNA were synthesized by Houston Methodist Research Institute (HMRI) RNA Core by in vitro transcription (IVT) with pseudouridine added to the nucleotide mix to reduce inflammatory activation and to improve translation, as previously described [31]. mRNA was purified by filtration and assessed for integrity using a TapeStation (Agilent). The mRNA LNP were formulated using the NanoAssemblr Benchtop (Precision Nanosystems). Six systems were tested. Formulations A of DOTAP, MC3 and SM-102 LNP were prepared at 49:49:2 molar ratio of DOPE, cationic/ionizable lipid and DMG-PEG2000. Formulations B of DOTAP, MC3 and SM-102 LNP were prepared at 8:1.5:38.5:52 molar ratio of DSPC, DMG-PEG2000, cholesterol and cationic/ionizable lipid (DOTAP, Dlin-MC3-DMA or SM-102). The lipids were dissolved in ethanol and mRNA in citric buffer (100 mM, pH 5.0) and mixed at 3:1 aqueous: ethanol flow rate at 10 mL/min followed by dialysis in PBS at 4°C for at least 8 hours. Fluorescently tagged LNP were prepared with Rhodamine B DHPE (0.5% w/w of total lipid). LNP diameter, polydispersity index (PDI) and zeta potential were measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern Instruments). Six measurements were performed. mRNA encapsulation efficiency in LNP was measured by RiboGreen Assay.

5.2. hSCS Preparation:

De-identified adult human skin samples (from individuals age 57 to 63) were purchased from BIOIVT or procured under the Houston Methodist Research Institute (HMRI) Institutional Review Board approved protocol with patient/family consent (IRB #PR000027413). Tissues were processed immediately or stored in 4°C for up to 48 hours. Split thickness human skin (0.15-0.20 mm) comprised of epidermal and partial dermal layers was isolated from a donor skin using an electric dermatome (#88710100, Zimmer Biomet, Warsaw, IN). hSCS were prepared from split thickness skin using RECELL Autologous Cell Harvesting Device (AVITA Medical) following the manufacturer’s instructions. Cell suspension was filtered through a 100 μM cell strainer and collected for in vitro culture or for grafting in the murine humanized wound model. In general, >50% cell viability was achieved in the procedure.

5.3. Cell Culture:

Normal human neonatal primary epidermal keratinocytes (#PCS-200-010, ATCC) were grown in Dermal Cell Basal Medium (#PCS-200-030, ATCC) and supplemented with the Keratinocyte Growth Kit. Normal adult human dermal fibroblasts (#PCS-201-012, ATCC) were cultured in DMEM media with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C and 5% CO₂.

5.4. Assessment of Cellular Uptake and mRNA Expression of LNP mRNA in vitro:

Cellular uptake of mRNA LNP and mRNA expression kinetics of all the formulations as compared to Lipofectamine in hSCS were assessed for 0-24 h by real-time quantitative live-cell imaging fluorescence microscopy using Incucyte S3 (Essen Bioscience). EGFP mRNA LNP labeled with Rhodamine B lipid were used. Lipofectamine was used as a commercially available transfection reagent control. LNP and Lipofectamine were added at concentration equivalent to 1 μg mRNA/mL. Images (5/well, n=3) were acquired at 0-48 h (10x objective). Fluorescent signal was quantified by Incucyte Live-Cell Analysis software.

To further assess the delivery to the major cell populations, present in hSCS [36], the uptake of the optimized formulation (DOTAP containing Formulation A) was assessed in cultured keratinocytes and fibroblasts. The cells were seeded at 5,000 cells/well and hSCS at 50,000 cells/well in a 96-wells clear flat bottom plates (Falcon) and LNP were added at concentration equivalent to 0.5 or 1 μg mRNA/mL.

5.5. Telomerase Activity Detection:

For Telomeric Repeat Amplification Protocol (TRAP) assay (TRAPeze Telomerase Detection Kit, Millipore Sigma) total cell lysates were collected from hSCS with CHAPS lysis buffer, followed by DC Protein Assay. Samples were diluted to equal protein concentration (0.03 μg/μL). For heat-kill (HK) control, each sample was subjected to 85°C for 10 minutes to inactivate telomerase. Following 40 cycles of PCR amplification, TRAP products were resolved by gel electrophoresis. Quantification of telomerase activity in each sample was normalized to HK control.

5.6. In vivo Assessment of Healing in Humanized Wound Model:

Athymic immunodeficient mice (Foxn1^nu, Strain #:002019, Jackson Laboratory) 8-12 weeks old were used in accordance with protocols approved by the HMRI Institutional Animal Use and Care Committee (IACUC #ISO00006024). To create the humanized full-thickness wound skin model, under isoflurane anesthesia a grafting polymeric dome (3D printed in house, d_outer=24mm; d_inner=14mm; h=10mm with two 2mm holes for gas exchange) was inserted in 1-cm vertical surgical incision on the back of the animals as described previously [38]. Fibroblast-fibrin hydrogel (200 μL) (10⁶ fibroblasts, 1 mg fibrinogen, 0.4 U thrombin, 2.6 mM CaCl₂, 1% FBS in DMEM) was placed into the grafting dome following by 200 μL of hSCS (10⁶ cells) untreated or pre-treated with mRNA LNP.

5.7. Histological Analysis:

One week after grafting, mice were euthanized, the domes removed, and the engraftment area and surrounding skin excised, cut into two halves, fixed in 10% formalin, paraffin embedded, sectioned (5μm) and immunostained by the HMRI Pathology Core. Sections were stained with hematoxylin and eosin (H&E) for tissue morphology or immunostained for Lamin, Ki67, p21, or 53BP1 (Supplementary Table 2). Slides were preserved with VECTASHIELD with DAPI and imaged by EVOS-FL-Auto-Imaging System (Life Technologies, Carlsbad, CA). The entire section of each wound tissue was scanned. Positive staining was quantified manually by two observers blinded to the treatment group, using ImageJ image processing software (National Institute of Health).

5.8. Analysis and statistics:

For size, PDI and zeta potential analysis at least six measurements were performed, and the data is presented as Mean ± S.D. Cellular uptake and mRNA expression, as well as label-free cell count data was analyzed using Incucyte Live-Cell Analysis software (Sartorius, USA) for fluorescent intensity in the red (LNP) and green (EGFP) spectra and cell count. At least three independent replicates were analyzed. The data is presented as Mean ± S.E.M. For the in vivo efficacy studies, the data was plotted using GraphPad Prism 8 Software (GraphPad; San Diego, CA, US), which was also used for statistical analysis (n=4-6). The data is presented as mean ± SEM, and results were considered significant if the *p-values were < 0.05.

Abbreviations:

Abbreviations for materials used in LNP production and their sources are provided in supplementary Table 1.

EGFP

enhanced green fluorescent protein

hSCS

human skin cell suspension

hTERT

human telomerase reverse transcriptase

LNP

lipid nanoparticles

mRNA

messenger RNA

STSG

split-thickness skin grafts

TRAP

Telomeric Repeat Amplification Protocol

Data availability statement:

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.