From Senescent Cells to Restored Tissue Function
Cell cycle arrest is a defining feature of cellular senescence. SenoReverse™, the platform behind our nucleic acid medicines, reverses it. Treated cells and treated naturally aged mice had lower senescence markers than controls, and our mouse studies also showed longer telomeres, better function and longer median lifespan.
Four Ways to Target Senescent Cells
Remove
Senolytics selectively remove senescent cells.
Quiet
Senomorphics modulate the senescence-associated secretory phenotype (SASP) and inflammation.
Reprogram
Partial reprogramming uses transient expression of reprogramming factors.
Restore
SenoReverse™ reverses senescence-associated cell cycle arrest and restores function in existing cells without changing their identity.
Senescent Cells Resume Cell Cycle Activity
In senescent cells, two cell cycle inhibitors build up and hold the cell at the G1 checkpoint. SenoReverse™ lowers both, releasing the arrest and restoring normal cell cycle regulation.
We sorted senescent cells, treated them and tracked them by live-cell imaging alongside untreated controls. Treated cells resumed cell cycle activity.
Live-cell imaging of sorted senescent cells after treatment, alongside untreated controls.
Green shows a p21 reporter, a marker of senescence-associated arrest. In treated cells the signal fades, and red circles highlight cells that regained function and returned to a normal cell cycle; in controls the signal grows stronger.
Single-cell sequencing: about half as many treated cells as control cells remained in a senescent state (16.0% versus 33.2%).
Markers in Replicative Senescence
IMR90 human fibroblasts, 72-hour treatment. Direction of change only.
p16
Transcripts fell
p21
Transcripts fell
SA-β-gal
Positivity fell
Ki67
Positivity rose
- Live-cell tracking: a human senescence reporter cell line was exposed to doxorubicin for 48 hours, then returned to standard culture for 10 days. Reporter-positive cells were sorted, and treated cells were tracked alongside untreated controls.
- Replicative senescence: IMR90 cells at population doubling level (PDL) 30 served as the young reference and cells at PDL 50 as the senescent model; treatment lasted 72 hours. p16 and p21 transcripts: n=3 per group. SA-β-gal: n=6 independent biological replicates per group. Ki67: n=3 independent replicates per group.
- DNA damage and chromatin: γH2AX decreased and H3K9me3 recovered.
- With treatment, CCNG2 expression (Ccng2 in mice) fell in the senescent cells studied and in tissues from naturally aged mice.
- Binding-site reporter assays for two targets: reporter activity was lower with wild-type sites than with the vector control, and higher with mutant sites than with the corresponding wild type, supporting sequence-specific regulation of both targets.
- Single-cell RNA sequencing of sorted senescent cells after three days of treatment: 16.0% of treated cells and 33.2% of controls were in a senescent state; 34.5% and 18.4% were in a rejuvenating state.
A Younger Tissue Profile in Aged Mice
In naturally aged mice, two methods showed longer skin telomeres in treated animals than in aged controls, closer to levels seen in young mice; liver telomeres followed the same pattern. Treated mice also had lower SA-β-gal positivity in five tissues and lower levels of four serum inflammatory cytokines.
Telomere Length
Separate groups of mice. Direction of change only; not to scale.
SA-β-gal Positivity
Lower in kidney, liver, lung, spleen and skin
Serum Cytokines
Lower for IL-1β, IL-6, IFN-γ and TNF-α
- Skin telomeres were longer in treated mice than in aged controls by both qPCR and TRF/Southern blot, closer to levels seen in young mice; liver telomeres showed the same pattern. The young, aged-control and treated groups are separate cohorts, not the same animals at three time points.
- SA-β-gal positivity (kidney, liver, lung, spleen and skin): male mice dosed every two weeks from 25 months of age and assessed at 30 months; n=10 per group.
- Serum inflammatory cytokines (ELISA): IL-1β, IL-6 and IFN-γ were lower in treated mice than in controls (P<0.001), as was TNF-α (P<0.01). Male mice dosed every two weeks from 25 months of age and assessed at 30 months; n=10 per group; mean ± SEM; Student’s t test.
- Tissue transcriptomes (liver, kidney, lung and skin; n=3 per group): compared with aged controls, treated mice had higher activity in cell cycle pathways and lower activity in inflammatory pathways, the same pattern seen in young, 12-month-old mice.
Longer Lifespan in Naturally Aged Mice
In the later of two studies using the same methods, long-term repeat dosing extended median lifespan by 38.2%. In the earlier study, mice dosed every two weeks from 20 months of age until natural death had a median lifespan of 1,029 days, versus 892 days in controls (+15.4%).
38.2%
longer median lifespan with long-term repeat dosing
An Earlier Study · Median Lifespan
- Earlier study: mice were dosed every two weeks from 20 months of age until natural death, with concurrent controls; 80 mice per group (40 female, 40 male).
- Median lifespan: 1,029 days in treated mice versus 892 days in controls (+137 days, +15.4%); hazard ratio 0.38 (95% CI, 0.27–0.53), P<0.001.
- Maximum lifespan (the longest-lived 15% of each group) was 12.13% longer in treated mice.
- Median lifespan is total lifespan from birth, not survival after the first dose.
- In the earlier study, postmortem pathology found no increase in tumor or disease burden in treated mice versus controls, and causes of death were similar.
- The 38.2% figure comes from a subsequent study that used the same methods with long-term repeat dosing; results from the two studies are not pooled.
Better Grip, Coordination and Memory in Aged Mice
With treatment, aged mice regained grip strength and stayed on an accelerating rotarod longer than controls. In a water maze, they found the hidden platform faster and spent more time in the target quadrant. They also maintained body weight better than controls.
Schematic trends of group means in naturally aged male mice, assessed monthly. Water-maze escape latency is inverted, so higher is better throughout. Not to scale.
Regained, then exceeded aged controls
Stayed longer on an accelerating rotarod
Found the water-maze platform faster
Spent more time in the target quadrant
Better maintained in old age
- Naturally aged male mice were dosed every two weeks from 25 to 30 months of age. Each readout had n=10 per group and was assessed monthly against concurrent aged controls. The function study is separate from the lifespan studies.
- Grip strength: normalized to body weight.
- Accelerating rotarod: 8 to 80 rpm over 300 seconds; latency to fall recorded.
- Water maze: escape latency (learning) and time in the target quadrant (memory).
- Body weight: recorded throughout the study.
Early Signals in Nonhuman Primates
We are studying naturally aged cynomolgus monkeys with spontaneous type 2 diabetes: four treated animals and three controls. In an interim analysis, cognition improved in treated animals and declined in controls. Fasting and premeal glucose, triglycerides and the liver enzymes ALT and AST fell in treated animals and rose in controls.
Interim analysis; direction of change only. Cognition: baseline to about 2 months. Blood measures: 6–7 weeks.
Cognition
Glucose
Lipids
Liver Enzymes
- Animals: naturally aged cynomolgus monkeys with spontaneous type 2 diabetes; 4 treated and 3 controls. The delayed matching-to-sample (DMTS) cognitive task was completed by 3 treated and 2 control animals. Observation periods differ by measure. Interim analysis; small sample.
- Both groups changed in the same direction: spontaneous activity rose (baseline to 2 weeks), and HbA1c, fasting insulin and HOMA-IR (calculated from group means) fell (6 weeks).
- Serum creatinine rose in treated animals and fell in controls (6 weeks), from low baseline values; any link to muscle would need direct measurement.
- Albumin rose slightly in treated animals and fell in controls (6 weeks).
- Total cholesterol held steady in treated animals and worsened in controls (6 weeks).
- Uric acid rose slightly in treated animals and fell in controls (6 weeks); the cause has not been determined.
- Body weight: no statistically significant change (7 weeks).
- Electrolytes (K, Na, Cl, Ca, P, Mg): only ranges over the observation period were recorded, so no baseline-to-endpoint trend is available.
Moving into Human Studies
Our two priority programs and our ALS program are each at a different stage of investigator-initiated trials (IITs).
- Sarcopenia
- IIT approved by a hospital ethics committee; working toward an FDA IND to begin Phase 1.
- Androgenetic Alopecia
- IIT read out, with hair density, follicular unit density and other endpoints improved versus control; working toward an FDA IND to begin Phase 1.
- ALS
- Early IIT completed; preliminary results showed improved function and lower levels of neurofilament light chain (NfL).