Results

Overcoming Low DNA Quantity:

The DNA fragments from A to M, ordered from TWIST, were delivered with blunt ends and in a limited amount of 1000 ng, which was insufficient for direct restriction. To overcome this, the fragments were first ligated into the pJET1.2/blunt plasmid to allow for amplification. The resulting plasmid was then digested with NEB restriction enzymes BamHI HF and XhoI and analyzed on a 1% agarose gel. Two distinct bands were observed: one corresponding to the plasmid backbone and the other to the insert, at the expected size. The insert band was excised and purified from the gel. Finally, the purified insert was ligated into the pcDNA3.1 plasmid, pre-digested with the same restriction enzymes, to achieve directional cloning compatible with the RFC 10 standard.

Further analyis of our methods

Sequencing

Sequencing was performed for positive samples. Extracted DNA was amplified using the Applied Biosystems^TM BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Fisher Scientific). A total of 20 PCR reactions were set up, two per sample, using the CMV forward and BGH reverse primers.

PCR products were purified using OPTIMA DTR^TM 96-Well Plates (EdgeBio) prior to automated Sanger sequencing. Obtained sequences were aligned with the expected sequences using Blast. Sequence identity ranged from 92% to 100% for all constructs.

All plasmids that showed the correct sequence were amplified through transformation of E. coli DH5α cells followed by plasmid purification.

A deeper analysis of the alignment readings can be found in the following file: pCDNA3.1_sequencing_results.pdf

The Spy System

To assess the efficacy of our RING-bait strategy in human fibroblasts, we initially considered using HGPS-affected cells transfected with our RING-progerin interaction-fusion protein. However, to gain more reliable insights into the degradation dynamics of progerin, we opted for the Spy System, which facilitates protein-protein interactions between molecules fused with SpyTag and SpyCatcher. This two-part system allowed the expression of SpyTagged progerin in MRC-5 fibroblasts, enabling the formation of SpyTag-SpyCatcher specific progerin-RING complexes.

The SpyTag/SpyCatcher system originates from the Streptococcus pyogenes FbaB protein, a fibronectin-binding protein and virulence factor. The CnaB2 domain of FbaB forms a stable isopeptide bond between lysine K31 and aspartic acid D117, catalyzed by glutamate E77. By splitting the domain into SpyTag (13 amino acids, containing Asp) and SpyCatcher (138 amino acids, containing Lys and Glu), a covalent bond is reconstituted when the two fragments are combined. This system is highly specific, demonstrating high affinity (≈0.2 µM) and low intrinsic reactivity unless the residues are aligned appropriately. The SpyTag can be incorporated at any position on a protein, including the N- or C-terminus or internally, provided the position is surface-exposed. We selected this system for its precision and one-to-one binding stoichiometry, which mirrors the architecture of our designed chimeric protein. [1]

A comparison between HGPS and healthy fibroblasts cell lines

Cell Lines Used

After defining the experimental strategy, the next step was to identify suitable cell models for testing the constructs. To this end, we established a collaboration with Prof.ssa Giovanna Lattanzi of the National Research Council (CNR) in Bologna, a leading expert in laminopathies. Through her laboratory, we obtained two human fibroblast lines, cc00366s and cc00500s.

The cc00366s line derives from a patient affected by Hutchinson–Gilford Progeria Syndrome (HGPS), while cc00500s corresponds to a healthy control. From their arrival in the laboratory, both lines were amplified to obtain a sufficient number of cells for subsequent experimental applications.

To assess morphological differences between healthy and progeria-affected cells, two T75 flasks were observed under bright-field microscopy: one containing fibroblasts derived from a healthy donor, and the other from a Hutchinson–Gilford Progeria Syndrome (HGPS) patient.

These morphological alterations reflect the nuclear deformation and cytoskeletal instability characteristic of progeria-affected cells. Even under standard culture conditions, the phenotypic differences between the two fibroblast lines are evident, visually confirming the impact of progerin accumulation on cellular structure and organization.

Although both lines were successfully expanded, they exhibited slow proliferation rates, with extended doubling times that made them less suitable for repeated transfection assays or large-scale functional studies. To perform the first experimental phase, we therefore adopted an alternative fibroblast line already available in the laboratory: MRC-5 human lung fibroblasts.

The MRC-5 line consists of diploid, non-transformed fibroblasts derived from human fetal lung tissue. These adherent cells display the typical spindle-shaped morphology and form organized monolayers under standard culture conditions. Their doubling time ranges from 40 to 48 hours in serum-supplemented medium, and they can undergo approximately 42–48 population doublings before entering senescence.

Thanks to their moderate transfection efficiency, MRC-5 fibroblasts allow only a fraction of cells to express the introduced constructs. This condition prevents excessive accumulation of reactive metabolites and stress factors that would otherwise occur in highly transfected cultures, potentially affecting neighboring cells and leading to widespread toxicity. Maintaining a partially transfected population therefore enables a more physiological response and makes it easier to observe potential recovery effects or compensatory mechanisms within the culture.

MRC-5 fibroblasts are particularly suited for short-term assays, such as the evaluation of morphological alterations, oxidative stress, and viability changes associated with progerin expression, while their limited lifespan and sensitivity to nutrient depletion and culture stress make them less suitable for long-term experiments.

The choice to use fibroblast lines rather than immortalized cell models was guided by both biological and translational considerations. Since the HGPS-derived cells obtained from Prof.ssa Lattanzi are fibroblasts, maintaining the same cell type ensures experimental continuity and phenotypic comparability. Moreover, fibroblasts are among the most affected tissues in Progeria, where nuclear envelope defects and cytoskeletal disorganization are most evident, making them a biologically relevant and disease-representative model.

Overall, MRC-5 fibroblasts provided a reliable and physiologically relevant system for studying the cellular effects of progerin accumulation, validating construct expression, and laying the groundwork for future optimization in more easily transfectable cell models such as HEK293T.

Unexpected Culture Loss and Impact on Experimental Timeline

During the second half of August, a technical failure in the CO₂ delivery system of the cell culture incubators led to a complete loss of all our fibroblast cultures. As a result, we were left without viable cells from mid-August until the second week of September.
During this period, the team focused exclusively on cloning procedures and plasmid amplification, which had already been initiated in the preceding weeks. Although this allowed us to advance the molecular part of the project, the incident significantly delayed the experimental phase.
Because of the time constraints imposed by the Wiki Freeze deadline, we were only able to perform each cellular assay once, without the opportunity to repeat or optimize the experiments. This limitation prevented us from obtaining more robust and statistically consolidated data, even though the results provided valuable qualitative insights for future repetitions.

Details on the experimental rationale and control design are provided in the following section (Experiments).

Fluorescence Microscopy

To visualize the expression and localization of the fluorescently tagged constructs, MRC-5 fibroblasts were analyzed by fluorescence microscopy on Day 3 (single transfections) and Day 4 (double transfections).
Fluorescence was acquired at an excitation wavelength of 488 nm and emission at 525 nm in the FITC channel. Images were taken from cultures transfected as part of other experiments, and not from newly transfected samples. All the constructs are cloned into the same pcDNA3.1 backbone.

The following constructs were evaluated:

• SpyTag-mEGFP-NLS-SpyTag: visualization of mEGFP expression;
• RING-NLS-mEGFP: visualization of RING expression through mEGFP fusion;
• progerin-mEGFP: visualization of progerin expression through mEGFP fusion;
• SpyTag-progerin + mEGFP-SpyCatcher: visualization of progerin localization through forced interaction (SpyTag/Catcher system) with mEGFP

Fluorescence microscopy allowed the visualization of mEGFP expression, acting as a first visual control.

SpyTag-mEGFP-NLS-SpyTag visualization

RING-NLS-mEGFP visualization

mEGFP-progerin visualization

Progerin-Spytag + mEGFP-SpyCatcher visualization

Fluorescence microscopy confirmed that all tested constructs were correctly expressed in MRC-5 fibroblasts, though with very low transfection efficiency. This limitation was particularly evident in double transfection experiments, where the probability of cells simultaneously expressing both constructs was extremely low. As a result, fluorescence patterns and subsequent readouts, such as those for degradation or interaction assays, are likely affected by the low transfection efficiency, reducing the statistical robustness of the observations. Future experiments will focus on optimizing transfection conditions or employing alternative cell lines with higher transfectability to ensure more reliable expression and localization analyses.

Western Blot Analysis – Progerin Expression

To confirm the successful expression of progerin, we performed a Western blot analysis on lysates obtained from MRC-5 fibroblasts transfected with the HA-progerin construct. Cells were collected on Day 3, corresponding to 48 hours after transfection. All the constructs are cloned into the same pcDNA3.1 backbone.

The following conditions were analyzed:

• Control (untransfected cells): used to assess background signal and confirm antibody specificity;
• HA-progerin: to verify the expression of the HA-tagged progerin construct using an anti-HA antibody

Western blot results confirmed the expression of HA-progerin, with a distinct band detected at approximately 74 kDa, corresponding to the expected molecular weight of the protein. No signal was observed in the untransfected control, confirming the specificity of the anti-HA antibody and the absence of non-specific background bands.

These results demonstrate that the HA-progerin construct was correctly expressed in MRC-5 fibroblasts and can serve as a reliable reference for subsequent analyses of progerin variants and degradation assays.

alamarBlue^TM Cell Viability assay

To evaluate the effect of progerin expression and its targeted degradation through the RING-SpyCatcher system, cell viability was measured in MRC-5 fibroblasts four days and six days after transfection using the alamarBlue^TM assay. The assay provided a quantitative readout of metabolic activity, reflecting the overall viability of cells transfected with different construct combinations. Each condition was analyzed in eight replicates to ensure statistical robustness, and fluorescence values were normalized as Relative Fluorescence Units (RFU).

Unlike the experimental samples, the control conditions were performed with a different number of replicates.The negative control was measured in duplicate, while the laminA-SpyTag condition was tested in six independent replicates. This difference in replication number should be taken into account when comparing standard errors between conditions, as lower replication increases variability and reduces statistical confidence in the mean values obtained. This difference derives from the limited amount of cells we had in culture. All the constructs are cloned into the same pcDNA3.1 backbone.

The following transfection conditions were tested:

• Control: Untransfected cells, serving as a baseline for cell viability;
• Progerin-SpyTag: To assess the impact of progerin expression tagged with SpyTag on cell viability;
• SpyTag-mEGFP-SpyTag + RING-SpyCatcher: To evaluate whether the degradative activity of RING towards mEGFP affects cell viability;
• Progerin-SpyTag + RING-SpyCatcher: To investigate whether Progerin degradation via the RING-SpyCatcher system restores cell viability to normal levels or improves it;
• LaminA-SpyTag: A lamin A control construct to compare the effects of progerin-induced viability defects with a non-pathological lamin protein

The results at four days revealed a marked decrease in viability in cells expressing Progerin-SpyTag, confirming the cytotoxic effect of progerin accumulation. Mean fluorescence intensity was approximately 250 RFU for progerin-expressing cells, significantly lower than that observed in both the laminA-SpyTag (≈ 550 RFU) and Control (≈ 570 RFU) groups, which displayed comparable viability levels.

Co-expression of progerin-SpyTag and RING-SpyCatcher did not fully restore viability, with fluorescence values remaining close to those of progerin alone, indicating that progerin degradation via the RING-SpyCatcher system was only partially effective or not sufficient to counteract the toxic effects of progerin accumulation at this time point. However, fluorescence microscopy and FACS analysis revealed a low transfection efficiency in MRC-5 fibroblasts, suggesting that the second transfection (Day 2) may not have been fully effective. This limitation could explain the absence of a clear recovery in cell viability, as a significant proportion of cells might not have received both constructs required for efficient progerin degradation.

In contrast, the mEGFP + RING condition, used as a functional control for RING activity, showed no evident reduction in cell viability compared to the control group; however, this observation should be interpreted cautiously, as the low efficiency of the second transfection might also have affected this condition, potentially masking subtle effects of RING expression on cell metabolism.

Overall, these findings demonstrate that progerin expression significantly reduces cell viability at four days, whereas lamin A and mEGFP + RING maintain metabolic activity comparable to untransfected controls. The partial or absent recovery observed in the progerin + RING condition may be linked to the limited transfection efficiency rather than to a complete lack of effect of the RING-SpyCatcher system. These data are consistent with fluorescence microscopy observations showing limited co-expression of the two constructs, and future experiments with optimized transfection conditions will be required to confirm the functional activity of the degradative system.

Cell viability was evaluated again at six days post-transfection using the alamarBlue^TM assay to assess the proliferation progression dependent from progerin expression and the RING-SpyCatcher system.

At this time point, all transfected conditions showed a strong decrease in metabolic activity compared to the negative control, indicating a general reduction in cell viability. Mean fluorescence values ranged between 52–84 RFU for all transfected samples, while the negative control displayed a markedly higher mean value (≈ 320 RFU).

Notably, cells were not provided with a medium change after transfection, which likely contributed to the overall decline in viability observed across all conditions. This lack of medium renewal, combined with prolonged culture stress, may have led to nutrient depletion and waste accumulation, resulting in a widespread loss of metabolic activity independent of transfection type.

These results are consistent with the Total ROS Production Assay, where increased oxidative stress was detected in parallel cultures at the same time point, confirming that cellular stress and decreased metabolic function contributed to the loss of viability at six days.

Overall, the six-day data indicate that while progerin expression exerts cytotoxic effects, the generalized cell death observed here is largely attributable to culture conditions rather than to specific construct effects, emphasizing the need for medium renewal in extended assays to accurately evaluate viability differences over time.

Total ROS Production Assay

To assess potential oxidative stress associated with progerin expression and its possible modulation by the RING-SpyCatcher system, we performed a Total ROS Production Assay in MRC-5 fibroblasts. The assay was conducted six days after seeding, at the same timepoint as the alamarBlue^TM viability assay. Fluorescence intensity was measured as Relative Fluorescence Units (RFU), providing a quantitative indicator of intracellular ROS accumulation. All the constructs are cloned into the same pcDNA3.1 backbone.

The following transfection conditions were tested:

• HA-progerin: To evaluate ROS production due to the presence of progerinl;
• SpyTag-progerin: To determine the effect of progerin tagged with SpyTag on ROS levels;
• mEGFP-progerin: A fusion construct of progerin with mEGFP, used to investigate whether the addition of a fluorescent tag alters ROS production;
• RING-mEGFP: To assess the effect of the RING domain (fused to mEGFP) on ROS production, testing whether the RING domain influences oxidative stress;
• RING-SpyCatcher: To analyze whether the combination of the RING domain with SpyCatcher modifies ROS production, potentially due to the structural impact of SpyCatcher;
• mEGFP-NLS: Used as a control to evaluate whether the mEGFP tag alone contributes to ROS production;
• mEGFP-SpyCatcher: A control to test the effect of the SpyCatcher tag in the context of mEGFP-tagged constructs;
• laminA-SpyTag: A lamin A control construct to compare progerin-induced ROS with a non-pathological lamin protein;
• Negative control: Untransfected cells used to establish baseline ROS production, representing normal ROS levels in cells not subjected to transfection or overexpression

Each condition was analyzed in multiple replicates, and mean RFU values with their respective standard deviations are reported below the chart.

As shown in Chart 3, all transfected conditions exhibited relatively similar fluorescence levels, with progerin-expressing constructs (HA-progerin, progerin-SpyTag, progerin-mEGFP) showing mean RFU values around 0.5, comparable to those of RING and SpyCatcher constructs. The negative control displayed a slightly higher fluorescence value (≈ 0.84 RFU).

At this stage of the experiment, cell viability was markedly reduced across all conditions, indicating that most cells were undergoing late-stage stress or death. Under these conditions, any potential differences in ROS production between constructs were likely masked by the generalized loss of viable cells. The residual fluorescence detected primarily reflects the number of surviving cells, rather than genuine differences in oxidative stress levels. The higher RFU observed in the control wells is therefore consistent with a greater number of viable cells, which naturally produce more ROS as a consequence of normal metabolic activity.

Overall, these results indicate that by six days post-seeding, cell death had leveled out differences in ROS production, resulting in comparable oxidative readouts across all transfected conditions. Future repetitions of this assay should be performed at four days post-transfection, when the alamarBlue^TM viability assay revealed the greatest difference in cell survival between conditions. Performing the ROS test at that timepoint will allow a more accurate assessment of the relationship between progerin expression, oxidative stress, and cell viability.

RING-SpyCatcher Functionality

To evaluate whether RING-SpyCatcher could induce mEGFP degradation in the presence of SpyTag, we performed a flow cytometry (FACS) assay on MRC-5 fibroblasts transfected with different construct combinations. The analysis was conducted on Day 4, following two sequential transfections. Fluorescence was detected using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, collected in the FITC channel. All the constructs are cloned into the same pcDNA3.1 backbone.

The following conditions were tested:

• Negative Control: untransfected cells, used to determine baseline fluorescence and gating parameters;
• SpyTag-mEGFP-SpyTag: single transfection to measure mEGFP expression levels in the absence of RING-SpyCatcher;
• RING-SpyCatcher + SpyTag-GFP-SpyTag: co-transfection to test whether the RING-SpyCatcher system induces mEGFP degradation

Each condition was analyzed in duplicate, and data were acquired from gated populations excluding debris and artifacts.

Data visualization and gating strategy

Data visualization and gating strategy

Each set of plots is composed of three panels:

• Left panel (FSC-A vs SSC-A): represents forward scattering (FSC) versus side scattering (SSC), used to evaluate cell size and internal complexity. A gating region (P1) was defined to select the main cell population and exclude debris, aggregates, or air bubbles;
• Middle panel (B525-A vs SSC-A): displays fluorescence intensity versus side scatter, filtered according to the P1 gate. The fluorescent population (P2) is highlighted in the upper region of the plot, representing cells expressing mEGFP. The corresponding percentage of fluorescent cells within the total population is indicated;
• Right panel (Fluorescence histogram): shows fluorescence intensity distribution (x-axis) against cell count (y-axis) for the gated population. The histogram allows direct comparison of fluorescence magnitude between samples

Results

The negative control (untransfected cells) exhibited only background autofluorescence, confirming the validity of the gating strategy. In the SpyTag-mEGFP-SpyTag condition, a small fluorescent population was detected (~1.8% of total), indicating a low transfection efficiency in MRC-5 fibroblasts. In the RING-SpyCatcher + SpyTag-mEGFP-SpyTag co-transfection, the fluorescent population (~2.1%) and overall fluorescence intensity were comparable to the single mEGFP transfection, with no measurable reduction in mEGFP-positive cells or fluorescence magnitude.

These results suggest that no significant mEGFP degradation was detectable under the present conditions. The very low transfection efficiency likely limited the number of double-transfected cells, preventing clear observation of RING-mediated degradation. Moreover, the fluorescence intensity of mEGFP-positive populations was in the range of 10⁵ RFI, whereas in efficiently transfected cell lines, signals typically reach 10₆–10₇RFI, producing a distinct separation between fluorescent and non-fluorescent populations—an effect not observed here.

Overall, these data indicate that the RING-SpyCatcher system could not be effectively evaluated in this experiment due to low transfection rates in MRC-5 cells. The experiment will be repeated using cell lines (HEK293T) with higher transfection efficiency to confirm the degradative function of RING under optimized conditions.

Western Blot Analysis – progerin Degradation

To evaluate whether the RING-SpyCatcher system could promote progerin degradation, we performed a Western blot on MRC-5 fibroblast lysates collected from cells transfected with various constructs. All samples were detected using the anti-progerin antibody (Diatheva Srl, ANT0046; Rabbit Anti-Human Cleaved-Farnesylated Prelamin A).
Cells were transfected on Day 1 and Day 2 (for double transfections) and harvested on Day 4 for protein extraction.

The following conditions were analyzed:

• Control (untransfected cells): used to verify the absence of background signal;
• Progerin-SpyTag: single transfection to confirm the expression of SpyTag-tagged Progerin;
• Progerin-mEGFP: to evaluate the expression of the mEGFP-fused progerin variant;
• RING-Catcher: used to confirm antibody specificity and ensure no cross-reactivity with RING-containing proteins;
• mEGFP-NLS: used as a negative control for the anti-progerin antibody;
• Progerin-SpyTag + mEGFP: baseline control for progerin expression in a double-transfection setup;
• Progerin-SpyTag + RING-SpyCatcher: experimental condition to test potential Progerin degradation induced by RING-SpyCatcher co-expression.

The resulting Western blot did not show any progerin-specific bands in any of the analyzed conditions. The Thermo Scientific^TM Spectra^TM Multicolor Protein Ladder bands were visible on the right side of the membrane, confirming proper electrophoretic migration and transfer. However, no distinct signal at the expected ~70 kDa band corresponding to progerin was detected in any sample.

This result rendered the assay inconclusive, as it was not possible to evaluate whether RING-SpyCatcher induced progerin degradation. The most plausible explanation is the suboptimal performance of the primary anti-progerin antibody, which had not been validated for Western blot detection.

The antibody was provided by Diatheva Srl within an active collaboration and had been previously validated only for immunofluorescence. As agreed with the company, the present results, whether positive or negative, will be shared to support further antibody validation.

In this assay, the antibody was used at a 1:2000 dilution with a 1-hour incubation, conditions that may have been insufficient for effective detection. Future optimization will include longer incubation times and alternative antibody dilutions, with the goal of establishing a reliable protocol for progerin detection by Western blot and enabling robust evaluation of RING-SpyCatcher–mediated degradation in subsequent experiments.

NanoLuc Binary Technology (NanoBiT Assay)

To extend the validation of our system beyond degradation-focused assays and to directly assess protein–protein interactions between progerin and its designed interactors, we implemented the NanoLuc Binary Technology (NanoBiT) system [2].
This method is based on the reversible reconstitution of NanoLuc luciferase through the association of its two fragments, Small BiT (SmBiT) and Large BiT (LgBiT), each fused to a protein of interest. When the two fusion partners interact, the SmBiT and LgBiT fragments are brought into close proximity, allowing the luciferase enzyme to refold into its active conformation. This conformational change restores the catalytic activity of NanoLuc, producing a strong luminescent signal that can be quantitatively measured in living cells.

Although interaction between progerin and its binders was previously verified through yeast two-hybrid screening and will be further evaluated by Microscale Thermophoresis (Monolith X), the NanoBiT assay provides a crucial complementary validation. Unlike those systems, which operate in in vitro or yeast contexts, NanoBiT enables the detection of protein–protein interactions within mammalian cells, reproducing the physiological environment in which the designed degradation system must ultimately function. This ensures that binding events observed in earlier assays are not artifacts of heterologous systems but are maintained in the cellular context relevant for therapeutic applications.

Although experimental validation could not yet be completed due to the limited number of MRC-5 fibroblasts available, the entire preparatory phase of the assay was successfully accomplished. This included DNA sequence optimization for NanoBiT cloning, vector assembly, and bacterial transformation. The constructs are currently awaiting sequence verification by Sanger sequencing using NanoBiT-specific primers, which will confirm successful cloning before proceeding with in-cell luminescence assays.

Through this experimental phase, we successfully established the complete workflow required to evaluate our degradation system in mammalian cells, from DNA amplification and cloning into the mammalian expression vector pcDNA3.1, to construct validation, transfection, and subsequent functional assays. The results confirmed the correct expression of the designed constructs, including HA-progerin, progerin-SpyTag, RING-SpyCatcher, progerin-mEGFP, and SpyTag-mEGFP-SpyTag.

Despite the low transfection efficiency observed in MRC-5 fibroblasts, fluorescence microscopy and Western blot analyses confirmed that the constructs were properly expressed. Cell viability tests provided preliminary but consistent indications that progerin accumulation negatively impacts cell metabolism and proliferation, while co-expression with the RING-SpyCatcher system may partially modulate these effects.

These results mark an important step toward the experimental validation of the ProgERASE strategy, in order to confirm the feasibility of using the RING-SpyCatcher platform as a programmable degradation system. Although several optimizations are still required, particularly regarding transfection efficiency, antibody validation, and assay sensitivity, the foundation for further development in mammalian models has been successfully established.

Experiment Improvements

Cell Line Optimization: HEK293T as a High-Transfection Model

To improve the consistency and interpretability of future experiments, we plan to complement the current fibroblast-based assays with HEK293T cells, which exhibit one of the highest transfection efficiencies among mammalian cell lines (often exceeding 70–90% with standard lipid-based reagents such as Lipofectamine 2000 or PEI). We chose to perform our experiments using MRC-5 fibroblasts, as their lower transfection efficiency results in progerin expression at sub-saturating levels. This condition helps preserve phenotypic variability and prevents the extensive cell loss observed under high-expression scenarios.

However, to confirm the functionality of the RING-SpyCatcher system, we plan to continue our studies using HEK293T cells. These cells are immortalized, robust, and fast-growing, providing a more homogeneous model for testing genetic constructs and fluorescent reporters. Their high metabolic activity and resilience to transfection-induced stress make them particularly suitable for optimizing plasmid ratios and analyzing the kinetics of the degradation system.
Using HEK293T cells will allow us to:

• Optimize time windows for assays (24–72 h post-transfection) to capture degradation dynamics before secondary cytotoxic effects dominate;
• Achieve clearer and more statistically reliable fluorescence and FACS readouts due to a higher number of successfully transfected cells;
• Perform various Western blot assays on progerin-transfected cell’s lysates, optimizing Diatheva ANT0046 antibody concentrations and exposure times in cells with high transfection efficiency

Refinement of the mEGFP Degradation Assay

To enhance the evaluation of mEGFP degradation mediated by the RING–SpyCatcher system, future experiments will employ a bicistronic plasmid designed to co-express RING–SpyCatcher and RFP from a single transcriptional unit. In the initial setup, MRC-5 fibroblasts were co-transfected with SpyTag–mEGFP–SpyTag and RING–SpyCatcher constructs; however, low transfection efficiency hindered the reliable detection of double-positive cells. The inclusion of an RFP reporter will make it possible to identify and gate RFP and mEGFP-positive cells, those effectively expressing both mEGFP-SpyTag and RING–SpyCatcher, and quantitatively compare mEGFP intensity between RFP-positive and RFP-negative populations. This approach will:

• Provide an internally normalized measurement of degradation efficiency;
• Reduce noise caused by uneven transfection or expression variability;
• Allow FACS-based sorting and downstream validation (e.g. Western blot or microscopy) of the RING–SpyCatcher expressing population

Optimization of the ROS Assay

To improve data reliability in oxidative stress assays, future replicates of the Total ROS assay will be performed either on Day 4 after seeding, or after a medium change at Day 3 to maintain cell viability.
Previous tests showed that by Day 6, a significant proportion of cells had detached or died, leading to inconsistent fluorescence values. Adjusting timing and refreshing the medium will help ensure that fluorescence reflects true intracellular ROS accumulation, rather than artifacts from population loss.
Normalization to cell viability (e.g., via alamarBlue, DNA content, or confluence measurements) will be integrated into future runs to provide a quantitative correction factor linking ROS signal intensity to living cell number.

Optimization of the alamarBlue^TM Viability Assay

To refine the assessment of cellular viability and better capture the dynamic effects of progerin expression and RING–SpyCatcher co-transfection, we plan to optimize the alamarBlue^TM Cell Viability assay protocol. In the initial experiments, viability was measured at Day 4 and Day 6 post-transfection. While the Day 4 assay revealed clear and significant differences in metabolic activity between the experimental conditions, by Day 6 most cells had already lost viability, preventing further quantitative interpretation.

Future experiments will therefore include daily viability monitoring over an extended time course to generate a detailed growth profile. Specifically, fluorescence will be first recorded at Day 0 (immediately after seeding) to establish a common baseline for all wells, and then measured daily for approximately ten days, with medium replacement performed as needed to maintain optimal culture conditions.

This approach will enable us to:

• Monitor the progressive effects of progerin expression and RING–SpyCatcher co-expression on cell proliferation and metabolic recovery;
• Identify the exact time window in which viability differences are most pronounced;
• Distinguish between acute cytotoxicity and long-term adaptive responses;
• Ensure that the assay reflects true metabolic variations rather than cumulative cell death due to culture stress

Tracking viability across multiple timepoints will thus provide a more accurate and continuous representation of cell health and growth dynamics in response to progerin expression and its targeted degradation.

Future Perspectives

NanoBiT Assay

After confirming successful cloning through vector sequencing, we will proceed with testing the NanoBiT control vectors (N203, N204) to verify:

• The functionality of the assay reagents;
• The compatibility of MRC-5 fibroblasts with the NanoBiT detection system

Following control validation, we will assess the interaction between LgBiT–laminA and SmBiT–progerin, as well as between SmBiT–progerin and LgBiT–progerin. Detection of a luminescent signal in these conditions will confirm that progerin is correctly folded and functionally expressed within the NanoBiT assay environment. Moreover, the observation of progerin–progerin interaction would provide further insight into its ability to self-associate, supporting the possibility of RING dimerization within the degradation system.
Once these preliminary validations are completed, we will perform assays between SmBiT–Progerin and LgBiT–Interactor constructs to evaluate specific binding, while SmBiT–lamin A will be used in parallel as a specificity control. This comparison is essential to ensure that the designed interactors selectively recognize progerin without binding to wild-type lamin A.

If the NanoBiT system performs as expected, we will extend the screening to a broader panel of interactors, enabling a systematic evaluation of binding strength and specificity directly within mammalian cells.

Toward Viral-Mediate Delivery

In the next stages of development, we aim to translate our approach into a disease-relevant context by testing the system in HGPS primary fibroblasts. This step will be undertaken only after completing the characterization of our degradation mechanism in laboratory-engineered fibroblasts, ensuring full validation under controlled conditions.

To enable targeted delivery in patient-derived cells, we plan to use viral delivery systems, such as Adeno-Associated Virus (AAV) or Lentiviral Vectors, to deliver our therapeutic construct.

AAVs represent a well-established platform for gene delivery, offering stable, long-term expression without genomic integration, and the possibility of tailoring tissue tropism and minimizing immunogenicity through serotype and capsid selection. On the other hand, Lentiviral Vectors are effective gene delivery tools that enable stable, long-term expression by integrating into the host genome. They can transduce both dividing and non-dividing cells, making them versatile for therapeutic and research applications. By modifying their envelope proteins, lentiviruses can be tailored for specific tissue targeting and minimized immune responses, making them ideal for gene therapy and functional gene studies.

This preclinical phase will bridge our current in vitro validation to future translational applications, providing a robust framework for assessing safety, efficiency, and specificity in a physiopathological model of HGPS.

AAV-Immune Considerations and Pediatric Window

Although AAV can elicit innate and adaptive immune responses to both capsid and transgene, studies indicate that anti-AAV antibody seroprevalence is significantly lower in children (often <15% in ages 3–12, increasing to >50% in adults). [3]
This suggests a therapeutic window in pediatric populations, including HGPS patients, in which viral transduction could be achieved with minimal immune interference. Nonetheless, standard NAb screening will remain essential prior to any application.
Given that HGPS cells exhibit a pro-inflammatory phenotype—characterized by elevated NF-κB activity and cytokine secretion—careful immunological monitoring (IL-6, TNF-α, interferon-stimulated genes) will be implemented in translational stages.