Funding proposal for a project to reverse ageing by gene-therapy-based transient cell reprogramming

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)

Phase 3: In vivo testing (Figure. 4)

Mouse models: C57BL/6J wild type, Progeria and SAMP8

Dose regimes

  1. Cyclic (positive controls)
  • 2 days on, 5 days off (Belmonte, 2016)

  • One week off, One week on (Davidsohn, 2023)

  1. 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), 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 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:

  1. 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:

  1. 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.
  2. 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.
  3. To detect or discard carcinogenic effects.
  4. 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.
  5. Possible synergistic effects with other longevity interventions such as: senolytics, intermittent fasting (IF), and TERT activation.
  6. 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

  • Jankie Bajoon M.D, MSc. Ph.D student
    Chief scientist
    www.jichi.ac.jp/genetherapy/

  • Luca Perrino
    Chief Executive Officer

  • Matilde Miranda Ph. D, UCLA
    Collaborator Researcher

  • Mizukami Hiroshi MD, Ph.D.
    Principal Investigator

December 2022 Progress Report

Budget

4 years program research.

Transient reprogramming rejuvenation by viral vector gene therapy.

In vivo models: accelerated ageing, wild type and progeria mice.

Cost breakdown:

  • Doctoral program tuition and admission fees, includes Jankie Bajoon M.D labour for 3 years (2023-2026), specialists consultation, and guidance: 22,680 USD
  • 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): 30,000 USD

HealthTech Lifestyle (HT) Tokyo Longevity and Wellness Center (Partnership with VitaDAO)

Centre specialised in Reversing biological age, decreasing frailty and maximising healthy longevity and to collect data from natural and artificial interventions.

*All products generated from HT can be commercialised by VitaDao and will carry the logo of VitaDAO as main sponsor and right holder.

Cost breakdown:

  • HealthTech Epiclock mobile app development (First stage): 2,000 USD
  • HealthTech Epiclock kits production (First lot): 10,000 USD
  • Exercise equipment: Bioelectrical impedance Body composition analyzer (InBody 770) x 1: 17,000USD
  • Exercise equipment (weights, cardio machines, benches, blood pressure monitors, chest heart sensors): 52,000 USD
  • Salaries: 1 year for Jankie Bajoon (Chief scientist, Nutrition and Training expert), Luca Perrino (Business chief executive), and Staff (Medical specialists consultants, trainers and neuroscientist consultant): 91,000 USD
  • Supplements production (β -Nicotinamide Mononucleotide -NMN- 250mg capsules - First lot): 7,000 USD
  • Supplements production (plant based protein powder HT original - different flavours - First lot): 5,000 USD
  • Supplements production (plant based protein bar HT original - First lot): 5,000 USD
  • Supplements production (plant based pre workout/ fat loss boosting formula HT original - First lot): 5,000 USD
  • Fitness related food production (premium muesli HT original - First lot): 3,000 USD
  • Overhead costs (Wellness centre rent - 1 year): 54,000 USD

Total: 304,680 USD

HT Tokyo Longevity and Wellness Center description: centre specialised in Reversing biological age, decrease frailty and maximise healthy longevity by natural interventions (exercise, diet, meditation, and supplements), artificial interventions (blood dilution protocols and gene-therapy based rejuvenation) and human clinical trials consented data collection. Some of the first clinical trials that can be carried out from 2023-2026 while the gene therapy is available:

Fasting-mimicking diet (FMD) and intermittent fasting (IF) protocols to reduce biological age, improve metabolic markers and body composition. Effectiveness will be measured by HealthTech biological age test (Horvath epigenetic clock), body composition, exercise performance, and metabolic markers. Subjects will rank from 25 to 70 years old. FMD protocols have proven to extend health span, improve metabolic markers and cognition, increase fat loss, while keeping muscle lean mass, hence, appropriate for elderly people at risk of sarcopenia or healthy individuals who want to keep muscle mass and maximise longevity. Reference:

  1. A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan - PubMed

  2. Fasting-mimicking diet and markers/risk factors for aging, diabetes, cancer, and cardiovascular disease - PubMed

In addition, we can offer a holistic reverse ageing program which fuses HealthTech Wellness and Anti-aging programs. In summary, this program encompasses the following interventions: guided meditation, exercise routines (both 1on1 or online), customised nutrition (longevity diets), and supplements for maximising longevity (NMN). We will measure the effectiveness of the program by epigenetic clock (Horvath clock), body composition, exercise performance and frailty improvement. Prospect clients range from 30 to 50 years old but anyone from 20 (maintenance) to 85 years old (elderly support) can benefit from this program. Scientifically proven: reversal or biological age by natural interventions has already been proven in a small randomised clinical trial. Reference:

  1. Potential reversal of epigenetic age using a diet and lifestyle intervention: a pilot randomized clinical trial | Aging

HealthTech’s program content:

  1. https://healthtechlifestyle.com/en/anti-aging-support/

  2. https://healthtechlifestyle.com/en/disease-prevention-wellness/

Innovative features and commercial interests of HT clinical trials with natural interventions:

  • Can be replicated worldwide: the programs can be reproduced regardless of the location. A team of nutritionists, trainers, and physicians would be needed to implement and monitor the program correctly.

  • Expandable: the intention is to grow in effectiveness and efficiency towards maximising longevity and extending healthy lifespan. Eventually, according to a business schedule, HT and VitaDAO can share a partnership to establish health centres worldwide which can even serve as data collection to foster and compare other natural interventions as well as consented experimental interventions (senolytics, blood dilution and cell reprogramming).

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.

2 Likes

Thanks for posting @jankiebajoon ! There seems to be two separate proposals here. I’m particularly excited about the reprogramming project. The budget seems small for it though. Can you comment please?

2 Likes

Hello Tim. Thanks for the interest and comment.

We integrated the research project with the longevity center since, in the future, we want to use it as a chanel where we offer gene therapy based interventions, hence the price includes both proposals. As for now, the clinic can only offer natural interventions and serve as a consented data collection center, more importantly, it will be the platform that generates income, since research is just taking resources.

Nevertheless, those interested in investing, only in the research project, are welcome to do so with different arrangements that must be agreed with VitadDAO first. If you have interest I can enquire about this matter.

Let me know if you need further information.

Jankie M.D

2 Likes

I agree this looks like two distinct projects.

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. You can measure both health span and carcinogenesis in that. As an aside, you might want to consider multiple behavior tests beyond novel object recognition. Both in case one test doesn’t work, and to provide more confidence in the effect.

It’s not clear what the timing/dosage on the doxycycline treatment would need to be. Or if the plan is to take doxy once per month or something to control the levels of your transcription factor.

AAV also runs into some immunogenic issues. The strength of doing a one shot rejuvenation is that you should avoid most of that risk. But if you have to do 2 or more (which I would expect in a human living decades), then you start running into potential immunogenicity issues. I didn’t see a plan to control for those risks. You may need a different delivery system, which would avoid the need for dual dleivery systems. Lipid nanoparticles are hot right now.

Proposed IP roadmap needs more clarity. It’s not clear what you have protected, what is not protected/obvious, and/or when the plan to file additional patents would be. There might be people here that can better comment on your prospects if none of this is protected.

The budget looks inadequate. $22k covers tuition and stipend for 3 years? $22k is below recommended NIH stipend levels without tuition for 1 year. Is the $30k the patent filing fees, buying a license or option on the IP rights, or buying out the university’s IP rights? There is no budget for supplies, cell culture, or mice.

This also looks earlier stage than some of the other projects VitaDAO has funded. It looks like this is still an idea without preliminary data. It may be that the longevity fellow mechanism VitaDAO has used in the past might be a better mechanism to support a PhD student interested in longevity research.

I would want to see promising in vitro data before agreeing to fund.

6 Likes

@bowtiedshrike

Thanks for the insightful comment. Let me reply one section at a time:

:large_blue_circle: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.

:arrow_forward: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.

:large_blue_circle:As an aside, you might want to consider multiple behavior tests beyond novel object recognition. Both in case one test doesn’t work, and to provide more confidence in the effect.

:arrow_forward:Thanks for the suggestion, I am working with a neuroscientist on an experimental design to include not only a variety of behaviour tests but also imaging when evaluation cognitive decline, age related memory loss, and glaucoma.

:large_blue_circle:It’s not clear what the timing/dosage on the doxycycline treatment would need to be. Or if the plan is to take doxy once per month or something to control the levels of your transcription factor.

:arrow_forward: Regimes will be described in more detail in the proposal but briefly:

  1. Cyclic
  • 2 days on, 5 days off (Belmonte, 2016)

  • One week off, One week on (Davidsohn, 2023)

  1. Continuous:
  • 13 days : optimal rejuvenation zone (Singh, 2022)

HealthTech:

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 fasted (protein and carbs) + PR weekly or monthly

Daily PR for 10 days followed by 16 hours fasted (protein and carbs) + PR weekly or monthly.

:large_blue_circle:AAV also runs into some immunogenic issues. The strength of doing a one shot rejuvenation is that you should avoid most of that risk. But if you have to do 2 or more (which I would expect in a human living decades), then you start running into potential immunogenicity issues. I didn’t see a plan to control for those risks. You may need a different delivery system, which would avoid the need for dual dleivery systems. Lipid nanoparticles are hot right now.

:arrow_forward: Liposomal vectors have many advantages including: they are released into the cytosol (do not integrate into the genome), have low immunogenicity, and surface ligands can be modified to target specific cell type. However, it seems that the expression of factors in this delivery system can’t be regulated, so transduction of the polycistron will be constant

Regarding the AAV dual system (in case the one single Factor + drugs fails), if viral titers decrease over the years or if a high dosage is required to reach clinical significance, some groups have been able to mitigate the immune response with prednisolone or methylprednisolone protocols for 3-6 months, pre and post gene therapy administration (Flotte 2023). This will require strict monitoring and a team of specialists though.

:large_blue_circle:Proposed IP roadmap needs more clarity. It’s not clear what you have protected, what is not protected/obvious, and/or when the plan to file additional patents would be. There might be people here that can better comment on your prospects if none of this is protected.

:arrow_forward: Is there someone you can suggest? We are still working on the IP rights and I’m not an expert.

The budget looks inadequate. $22k covers tuition and stipend for 3 years? $22k is below recommended NIH stipend levels without tuition for 1 year. Is the $30k the patent filing fees, buying a license or option on the IP rights, or buying out the university’s IP rights? There is no budget for supplies, cell culture, or mice.

:arrow_forward:As I linked both proposals into one, I included my stipends as salary alone with the rest of the team (check budget) but I’m open to suggestions if the proposals are split.

On the other hand, 30k is what the university first agreed for IP rights, + first year of usage of the lab and reagents. This was a long lasting burocratic step and in the last meeting we had with vitaDAO, the PI stated that, as I advance in the experiments, the university might ask for more in the next 2 years. I will update accordingly.

This also looks earlier stage than some of the other projects VitaDAO has funded. It looks like this is still an idea without preliminary data. It may be that the longevity fellow mechanism VitaDAO has used in the past might be a better mechanism to support a PhD student interested in longevity research.

:large_blue_circle:I would want to see promising in vitro data before agreeing to fund.

:arrow_forward: Absolutely, I will keep this proposal updated once we have some in vitro results. As for now we are testing protein expression by western blot.

Thanks again for the interest.

Jankie

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