Project PI: Jankie Bajoon M.D, MSc.
Laboratory PI: Mizukami Hiroaki M.D., Ph.D.
Disclaimer: The projects in this category are not endorsed by VitaDAO’s Longevity Dealflow Working Group, nor by the Senior Reviewers it relies upon.
Simple Summary
Evaluate the efficiency and safety of different c-MYC free partial cell reprogramming strategies (OSK and Oct3/4 alone) by viral vector gene delivery to extend healthy lifespan in vitro and in vivo. In addition, investigate possible synergistic effects with other interventions such as senolytics, intermittent fasting (IF), and TERT activation.
Problem
Ageing is a degenerative process characterized by a gradual loss of function occurring at the molecular, cellular, and organismal level, resulting in health decline, increased morbidity, and care dependency. At the chromatin level, ageing comes with a progressive accumulation of epigenetic noise that eventually leads to aberrant gene regulation, senescence, and deregulated tissue homeostasis. These changes determine “the epigenetic clock”and occur when specific DNA is methylated in the chromatin, ultimately repressing youthful gene transcription patterns in old cells. In nature, this process is reversible and occurs when a new zygote is conceived, and the epigenetic clock is reset. Artificially, cell reprogramming (CR) can reverse this clock by modifying DNA methylation patterns, which restores the youthful transcriptome, reducing the biological age of somatic cells. CR works by the expression of reprogramming transcription factors (RF); OSKM (or OSK), which are silenced during adulthood. For CR to be reversible and controllable, a trans-activator protein (TA) that regulates the temporal activation of RF, must be included along with the RF. This strategy is crucial for the technique of partial CR, which main advantage in age reversal over induced pluripotency (iPSC) is that cell identity is not abolished, hence an effective molecular rejuvenation with no carcinogenic risk. Using adeno-associated virus (AAV) mediated-partial CR, research groups have successfully ameliorated the aged phenotype in OSKM transgenic wild-type and progeroid mice (Ocampo et al., Cell, 2016), cured vision loss in mice with glaucoma (Lu et al., Nature, 2020), improved memory and delayed onset of neurodegeneration in old mice (Rodriguez-Matellan et al., Stem cell, 2020). Although this shows remarkable proof of concept, some limitations to reproducing this strategy for adult human rejuvenation include: 1. The need for a dual system that carries the TA and the RF on separate AAV vectors would negatively affect the clinical outcome due to an uneven TA: RF transfection ratio. 2. Inducible OSKM transgenic humans are still non-ethical. Interestingly, for many cell types, the ectopic expression of a single RF (Oct4 instead of OSK) is enough to rejuvenate the old age phenotype when combined with other drugs such as valproic acid or p53 inhibitors (helper drugs), that boost the initiation of the reprogramming process (Zhang et al., Aging cell, 2022).
STRATEGY
The development of a multi-tissue AAV single vector that fits both the RF (Oct4 or OSK) and TA, is presumed to be sufficient to reprogram cells and tissues in the presence of helper drugs, restoring the healthy function of major organs such as the brain and heart in animal models. This approach reduces the risk of uneven TA:RF ratio transfection, increases clinical outcome after reprogramming, and allows the objective evaluation of the therapy’s efficiency. Since biological age varies according to cell type, I will compare whole-body versus specific-organ tailored vectors to determine which is more efficient in ameliorating different aspects of the old age phenotype. In addition, I will screen the combination of gene therapy with telomerase activation, fasting, and senolytics, which have proven to extend healthy lifespan in mice (Mitchell et al., Cell metabolism, 2019) and that could act synergistically in rejuvenation since ageing is a multifactorial process.
Innovative features: the limitations of previous studies when attempting to reproduce this strategy in adult humans for reverse ageing purposes include: 1. The need for a dual AAV vector system that carries the RF and TA separately negatively impacts the clinical outcome due to an uneven TA:RF transfection ratio. 2. Inducible OSKM transgenic humans are neither feasible nor ethical. The proposed approach can overcome these issues by the use of a single vector instead of a dual AAV system,that reduces the risk of uneven transfection ratio, increases clinical outcome after reprogramming, and allows researchers to objectively evaluate its efficiency. Other innovative features include: 1.Establishment of a safe dosage: I will carefully monitor when partial CR becomes harmful(loss of cell identity) by measuring several times (before, after, and during the gene therapy activation) the biological age in vivo.2. Since ageing is a multifactorial process, I will combine partial CR with other interventions such as TERT activation, IF and senolytics, and look for synergistic effects. This combination has no scientific precedents.
OBJECTIVE
To evaluate the efficiency and safety of different c-MYC free partial CR strategies, using OSK or Oct3/4 alone by AAV gene delivery, to extend healthy lifespan, reduce frailty and to reverse the aged phenotype in accelerated ageing, progeroid, and wild type mouse models. In addition, to investigate possible synergistic effects with other interventions such as senolytics, intermittent fasting (IF), and TERT activation.
EXPERIMENTAL PLAN and METHODS (Figure.1)
Phase 1: Vector development (Figure.2, 3)
Vector design and cloning simulation in snapgene > subcloning treatment cassettes (RF) and transactivator (TA) into AAV vectors by enzyme digestion, PCR and bacterial transformation > Plasmid confirmation by SANGER sequencing > Plasmid maxi prep > Vector production by Calcium Phosphate triple transfection > Vector purification by Cesium Chloride > Vector confirmation by qPCR.
Phase 2: In vitro screening.
- Human primary cells: PBMC from blood samples, liver or skin fibroblasts, lens cells, progeria skin fibroblasts.
- Murine primary cells: pyramidal neurons from hippocampus, liver or skin fibroblasts, post-mitotic skeletal muscle.
Tests: Horvath’s Epigenetic clock (gDNA methylation levels by Microarray, Illumina 850 EPIC arrays), Transduction efficiency of vectors (western blot and antibody detection), Detection and quantification of gene expression (qPCR), In vitro lifespan: (number of cell culture passages). Senescence (SA-β- Gal, p21, Phospho p53) and proliferation marker ( KI67). DNA damage (Gamma H2AX), collagen production from skin fibroblasts.
Phase 3: In vivo testing (Figure. 4)
Mouse models: C57BL/6J wild type, Progeria and SAMP8
Dose regimes
- Cyclic (positive controls)
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2 days on, 5 days off (Belmonte, 2016)
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One week off, One week on (Davidsohn, 2023)
- Continuous (positive controls)
- 13 days : optimal rejuvenation zone (Singh, 2022)
HealthTech (original Regimes and combinations)
Everyday until organic death or until biological age (eAge) and frailty significantly decrease (g.e. 18 months old mice reach 12 months of eAge)
Daily PR for 10 days followed by 24 hours food fasted + PR weekly or monthly
Daily PR for 10 days followed by 16 hours food fasted + PR weekly or monthly.
- 3.1 Whole body (AAV9 capsid). Tests for virus transduction efficiency, protein expression (RF, senescence and proliferation markers), and virus localization respectively: quantitative RT-PCR and immunostaining of brain, heart, liver, lung, kidneys, and skeletal muscle tissue. Healthspan assessment: object recognition task (memory), rotarod and tightrope test (coordination and balance), DEXA scan (body composition), multitissue epigenetic clock (biological age), body weight, walking speed, strength, endurance, physical activity (frailty). Plasma insulin and IGF-1 (metabolic markers).
- 3.2 Brain-specific (AAV.CAP-B10 capsid targeting hippocampus). Tests for virus transduction efficiency, protein expression (RF, senescence and proliferation markers), and virus localization: RT-PCR and Immunostaining of hippocampal sections. Specific tests: electrophysiology (action potential propagation), object recognition task (memory, cognition), Fear conditioning (short term memory), Barnes maze (long-term memory), rotarod test (coordination, balance), active place avoidance (spatial learning). Post mortem epigenetic clock of the hippocampus.
Phase 4: Result analysis (whole body and organ-specific)
Kaplan-Meier estimator (Survival/ Lifespan). T- test (Statistical analysis).
IP Roadmap
The experimental intervention aims to partially reprogram cells and tissues, extending mice’ healthy lifespan, reducing frailty, or increasing the health span with no carcinogenic effects of specific organs in mice, VitaDAO will have the shared rights to reproduce and use the gene therapy that results from Jankie Bajoon’s experiments in another lab (abroad or in Japan) for in vitro, and in vivo experiments or as an intervention for approved human clinical trials. In addition, detailed protocols for how to develop and test the gene therapy, as follows:
- Production of Dr. Jankie Bajoon’s original designs of cell partial reprogramming single and dual AAV vector systems. Selected original strategies that efficiently rejuvenate tissues, improve senescence markers, extend healthy lifespan and reduce frailty in mice and improve fitness, neurocognitive and other health span tests compared to control groups. These strategies include combination with other interventions such as dietary regimes, senolytics, and TERT activation, that act in synergy. (From western blot, qPCR, and Immunostaining to survival curves).¨
Relevance to longevity
Due to the clinical advantage of AAV-mediated therapy over other viral systems and the promising rejuvenation ability of partial CR, the main questions that this project will clarify include:
- If an original design of a single AAV vector carrying one reprogramming factor (Oct4), administered with helper drugs, is sufficient to rejuvenate cells and tissues in vivo by partial reprogramming.
- If three RF (OSK) can be compacted and fit in a single AAV vector along with TA and have a significant rejuvenation effect in vivo.
- To detect or discard carcinogenic effects.
- To compare the rejuvenating effects of partial reprogramming in organ-tailored versus whole-organism gene therapy. Specifically, I will test an AAV vector that targets the hippocampus and measure its ability to improve memory, induce hippocampal neurogenesis, and delay age-related neurodegeneration. The result will give an insight into the feasibility of organ-tailored cell reprogramming that can be accommodated to the patient’s particular condition (e.g. brain targeted CR for neurodegenerative diseases), as well as developing a whole-body therapy for prevention of the aged phenotype and healthy lifespan extension.
- Possible synergistic effects with other longevity interventions such as: senolytics, intermittent fasting (IF), and TERT activation.
- Efficiency of these strategies to ameliorate the accelerated aged phenotype and to delay early death in progeroid mice, which can serve as a complementary treatment for progeria patients.
Team
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Jankie Bajoon M.D, MSc. Ph.D student
Chief scientist
www.jichi.ac.jp/genetherapy/ -
Matilde Miranda Ph. D, UCLA
Collaborator Researcher -
Mizukami Hiroshi MD, Ph.D.
Principal Investigator
Budget
3 years program research.
Partial reprogramming rejuvenation by viral vector gene therapy + combined interventions.
In vivo models: accelerated ageing, wild type and progeria mice.
Cost breakdown:
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Jankie Bajoon M.D labour for 3 years (2023-2026), specialists consultation, and guidance: 30,000 USD
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University’s Contribution for IP. This fee corresponds to the contribution to Jichi Medical University (JMU) for the IP rights of the patent that is generated from Dr. Jankie Bajoon’s research at the division of genetic therapeutics of JMU during his doctoral program in the period of 2022-2026. It also covers the equipment and reagents used for vector development, in vitro and in vivo experiments. Licence holder (2026) of the gene therapy: Transient reprogramming rejuvenation by Adeno associated virus (AAV): 70,000 USD
Total: 100.000 USD
Strategy for indication selection, given the current regulatory landscape requiring it
- The long-term goal of this project is to produce a non-carcinogenic rejuvenating gene therapy that extends the healthy lifespan of adult humans, reduces frailty in the elderly, and compresses morbidity incidence. Selection criteria for the first clinical trials could include a biological age measured by Horvath’s multi-tissue epigenetic clock or DNAm PhenoAge of ≥ 60, a clinical frailty score of ≥ 4, and/ or a poor score on metabolic biomarkers. The objective is to avoid and delay the aged-phenotype and prevent age-related diseases (neurodegeneration, cardiovascular diseases, sarcopenia, etc)
In addition, other specific pathologies include:
- Hutchinson-Gilford progeria syndrome (Complementary treatment to farnesyltransferase inhibitors)
- Age-associated memory impairment
- Neurodegenerative disorders
- Age-related macular degeneration, glaucoma, and cataracts.
One of the most important exclusion criteria include cancer, therefore cancer screening by imaging and biomarkers is also needed.
In vitro preliminary results. 2024 Update
Background:
Collagen I and IV are downregulated with age, with collagen IV gene expression demonstrating a more dramatic reduction and a high correlation with age decline. This is because collagen IV is a major component of the basement membrane, which plays a crucial role in skin structure and function. With age, collagen IV production decreases, leading to wrinkles and other signs of skin aging. Studies have shown that collagen expression can be increased by a variety of factors, including diet, lifestyle, and certain medical interventions. However, it is important to note that the correlation between collagen expression and biological age is not perfect. Some people may have lower collagen expression than others of the same age, and vice versa, due to their different phenotypes (Varani et al., 2006; Lago and Puzzi, 2019).
Objectives and hypotheses
To evaluate the possible rejuvenation effects of c-Myc-free partial reprogramming (OSK) and telomerase pulse activation (TERT) in collagen IV and I gene expression of human skin fibroblasts at different chronological age. It was hypothesized that the treatment will restore the youthful levels of collagen IV gene expression and reverse the biological age in skin fibroblasts derived from seniors and elderly via epigenome remodeling and telomere elongation, respectively, without cell identity abolishion, conferring a safe and robust rejuvenating intervention.
Methods
Four in vitro assays were conducted to measure collagen I and IV gene relative expression in human dermal fibroblasts (HDF) after undergoing c-MYC-free partial reprogramming (OSK) pulse telomerase activation (TERT) or the combination of both (OSK+TERT) delivered by adeno-associated virus (AAV6) or lipofectamine. The transgenes’ expression was regulated under a tetracycline-inducible system (Tet-On); hence, expression was presumed to be active only in the presence of doxycycline. The cells were derived from elderly (62.8 years old male, one patient), middle-aged (47.5 years old male, one patient), and neonate (male, one patient). Briefly, human dermal fibroblasts from adults (HDFa) and neonate (HDFn) were cultured in T75 flasks with media containing DMEM-F12, FBS 15%, 1x NEAA, and 1x Streptomycin/Penicillin for 4 days until they reached 90% confluency. After subculture, cells were seeded in a gel-coated 12-well or 96-well plate for further lipofectamine transfection or AAV6 transduction, respectively. After 48 hours the cells were transfected with plasmids containing TeT-on.hOSK, TeT-on.hTERT, GFP, and tetracycline transactivator (rtTA) using lipofectamine3000 (In vitro1,2, and 3) or transduced with the following vectors: AAV6-TeT.on-hOSK, AAV6-TeT.on-hTERT, AAV6-hrGFP, and AAV6-rtTA (in vitro 4). After 24 hours, doxycycline was added at 2 ug/mL to activate the transgene expression and the media was changed to Essential 8 medium. The duration of the treatment was 14 days (in vitro 1, 3, and 4) and 7 days (in vitro 2). After the treatment period, total RNA was extracted from the cells, and 100 ng per sample were converted to cDNA by RT-PCR. Collagen I and collagen IV relative gene expression was measured by qPCR. ΔCt values were calculated using B-actin as the housekeeping gene, and gene fold expression was expressed from 2^-ΔΔCt values.
Results
The cells derived from the 47-year-old patient were treated for 7 days and 14 days, respectively. During the 7-day treatment, the treated cells showed a significant average gene expression increment in collagen IV, with a 5-fold increment compared to the untreated group (Figure 2). After 14 days of treatment, collagen IV relative gene expression increased by an average of 6-fold compared to untreated cells. (Figure 4). Overall, the upregulation was observed in all cells regardless of isolated treatments (OSK, TERT) or the combination of both (OSK+TERT). However, OSK+TERT showed the highest upregulation, 6.3-fold compared to the untreated groups. Collagen I relative gene expression was not significantly affected by treatment; after 14 days of transgene activation, collagen I in treated groups only increased 1.7-fold on average compared to the control group.
The cells derived from a 62-year-old patient were also subjected to 7 and 14 days of treatment in separate experiments. After 7 days, there was no significant increment in treated cells compared to the control group, both in Collagen IV and I (1,5-fold and -0.2-fold, respectively, Figure 3). Interestingly, after 14 days, the treated cells showed an average 4.2-fold increment compared to the control group in collagen IV relative gene expression, where the combination of OSK+TERT showed the highest upregulation: 4.8-fold compared to the control group (Figure 5). Once again, there was no significant difference in collagen I gene expression (0.3-fold compared to the control group).
The treated neonate dermal fibroblasts (HDFn) showed no significant upregulation compared to untreated groups, with an average of 1.1-fold and -0.26-fold gene expression in collagen IV and collagen I, respectively.
Notice that except for in vitro 3 in 62-year-old skin fibroblasts (Figure 5), all experiments showed no significant difference in gene expression for treated cells with or without doxycycline, which raised concern about the efficacy of the TeT transactivator (tetracycline-induced expression).
Lastly, the results were reproduced using a second gene delivery system, AAV6. This experiment was only conducted on a 47-year-old HDFa. Two different doses were administered. The low dosage of AAV6-TeT.on-OSK + AAV6-Transactivaotor (total of 2x1010vg/ cell) showed the highest upregulation in Collagen IV compared to control groups (5.6-fold), while the high dosage (total of 4x1010vg/ cell) resulted in a 3.2-fold upregulation. Collagen I showed no significant upregulation, with only a 1.5-fold increase on average in the treated groups vs. untreated groups (Figure 6).
Conclusions:
-Collagen IV relative gene expression was significantly upregulated in HDFa treated with either c-Myc partial reprogramming (OSK), telomerase pulse activation (TERT), or the combination of both. As collagen I gene expression is not affected during aging, it showed little or no significant changes between treated and untreated groups. The greater upregulation of Collagen IV compared to Collagen I is consistent with similar studies using maturation phase transient reprogramming (MPTR) to rejuvenate adult human dermal fibroblasts (Diljeet Gill et al., 2022).
-The results were reproduced throughout all trials using two different gene delivery methods: lipofectamine and AAV6.
-The duration of treatment may be an important factor in determining the magnitude of the rejuvenation effect of the tested interventions. A one-week exposure to partial reprogramming seemed enough to restore youthful levels of collagen IV in skin fibroblasts of a middle-aged subject; however, a longer period of 14 days was necessary to achieve a similar effect in the cells derived from the elder patient, suggesting that longer treatment duration may be necessary to achieve the maximal benefits of interventions involving epigenetic remodeling and telomere elongation by gene-based therapy to ameliorate the downregulation of collagen genes associated with age decline.
-The results of this study suggest that c-Myc partial reprogramming, telomerase activation, or the combination of both may be a promising approach to rejuvenating skin in adults.
Limitations
-In order to develop a safe gene-based intervention, dosage regulation of the treatments is essential. The Tetracycline (TeT) inducible expression system was chosen to control the transgene dosage in this project, which activates the transgene expression in the presence of doxyxicline (Tet-on). We notice that in most of the experiments, the treated groups showed similar gene fold expression regardless of the presence or absence of doxycycline. Although the protein expression of all plasmids was evaluated previously by western blot (March–June 2023), further evaluation is necessary to assess the efficacy of the tetracycline-inducible expression system before advancing to in vivo steps.
-The rejuvenating effects of the tested interventions need to be verified with other methods relevant in the fields of aging and longevity, such as epigenetic clocks and cell migration assay. Both assays are being conducted in 2024.
-This study was conducted with a small sample size. A larger sample will be ideal to validate the findings.
I think reprogramming is an interesting idea, but there are a few things that could kill it no matter how the project progresses. Turning on transcription factors smacks of cancer, and it doesn’t look like there’s a good plan to see if cancer risk will kill the project early. A spontaneous cancer model should be the first in vivo test instead of B6.
Indeed, in many types of cancer such as hepatocellular carcinoma, prostate, melanoma, bladder, ovarian, breast, and lung cancer, Oct4 is universally expressed by mechanisms that include epigenetic modifications. SOX2 is also overexpressed in some types of human cancer and it’s correlated with poor survival of patients (Li, 2020. Sun, 2020). However, recent work has shown that the risk of cancer is not increased in mice after in vivo partial cell reprogramming (PR) compared to untreated control groups. This includes teratoma, which is a side effect of in vivo full reprogramming (Davidsohn, 2023. Sinclair, 2023). A possible explanation is the removal of c-MYC from the reprogramming cocktail in their strategy, and mine too. As you might know, MYC activation is a hallmark of cancer Initiation and maintenance and it has been implicated in the pathogenesis of most types of human tumors (Felsher, 2014). Furthermore, deregulation of these transcription factors during carcinogenesis is continuous and uncontrolled while regulatory systems such as Tetracycline-inducible expression (integrated in my strategy), allow the expression of the Yamanaka factors to be dosed. I wrote that any diagnosed cancer is an exclusion criterion, nevertheless, since expression of these factors can increase the survival of cancer stem cells (CSC), and it is unpractical to take biopsies of all the targeted tissues, I agree that spontaneous tumor animal models have to be included.