VDP-146 [Funding] The development of oligonucleotide drugs for healthspan

One-liner: To generate a new RNA drug capable of activating a human healthspan switch mediated by the FOXO3 gene

Longevity Dealflow WG team

  • Scientific evaluation: 1 biotech executive, 1 VC, 1 biotech manager, 2 academics

  • Shepherd: Adrian Matysek

  • Other squad members: Eleanor Davies, Paolo Binetti

  • Sourced by: Adrian Matysek

  • Project PI: Lorna Harries, PhD

Simple Summary

FOXO3 is a major longevity gene, which acts as a central integration point for many aspects of cellular health and stress resilience, especially in the context of ageing and its associated diseases. Prof. Lorna Harries has identified a druggable mode of action for a longevity-associated genetic variant in the FOXO3 gene, which regulates FOXO3 isoform usage and has potential to act as a ‘switch’ for longevity and improved health span in humans. Here, she plans to exploit this finding by developing a portfolio of oligonucleotide drugs which act to influence FOXO3 isoform usage and to evaluate these in vitro and in vivo.

Problem

At present, we take a reactive, rather than a proactive, approach to ageing and its associated diseases, with clinicians treating these as unique and unlinked phenomena. This outdated approach has resulted in a system where there is an enormous healthcare and social care burden, not only in financial terms, but also in terms of multimorbidity and loss of autonomy and quality of life for our ageing population. The UK Chief Medical Officer’s Health in an Ageing society report in 2023 (which it was my privilege to contribute to) highlighted these issues; whilst we may be living longer, we are not living better. A better approach to dealing with the multimorbidity of ageing needs to be applied (1). ‘Geroscience’ approaches necessitate the identification and manipulation of crucial ‘hub’ genes and networks that control cellular ageing at the systemic level, that may be manipulated for widespread effect.

The FOXO3 gene represents one such pivotal intervention point. FOXO3 forms part of an evolutionarily conserved central hub network which integrates multiple molecular and cellular inputs that regulate cellular and organismal ageing (2). It can act either as transcriptional regulator in the nucleus, or as a stress thermostat in the cytoplasm, where under conditions of cellular stress, it has roles in regulation of autophagy (3), modulation of mitochondrial mass, clearance of defective mitochondria and protection against oxidative damage (4). FOXO3 has important roles in multiple fundamental cellular processes, such as control of metabolism, genome stability, cell division and differentiation status and response to cellular stress (5), and is now well recognised as a major geroprotective factor (2). As such, it has been implicated not only in the regulation of ageing, but also its associated diseases (6).

A genetic haplotype characterised by variants rs13217795, rs4946932 and rs9400239 located within an intron of FOXO3 has been associated with extreme long life (approx. 36% increase in lifespan by meta-analysis) and extended healthspan, offering metabolic and cardiovascular protection in multiple human populations (7). Although FOXO3 is well known as a healthspan gene and these associations have been described on multiple occasions and in multiple populations, definitive mechanistic insight into their model of action and progress in leveraging this information for potential therapeutic benefit has remained elusive.

Solution

The FOXO3 gene produces multiple RNA transcripts including 3 linear isoforms (FOXO3-long and FOXO3-short) and a circular RNA (circFOXO3). The long linear forms encode identical full-length proteins, whereas the short form encodes an N terminally truncated protein which lacks a large proportion of the DNA binding forkhead domain, and several important points of post-translational modification (figure 1). The longevity haplotype resides in the region of a bioinformatically-predicted alternative promoter and non-coding first exon located in intron 2, and my team proposed in 2021 that it may determine the relative expression of long and short FOXO3 isoforms (8).

Figure 1: Isoforms of the FOXO3 gene and the location of longevity-associated genetic variation. A. The structure of the FOXO3 gene and the location of longevity-associated variation. B. The protein structure of long and short FOXO3 isoforms.

Our published work shows this to be the case and highlights a mechanism by which FOXO3 variants impact ageing and longevity phenotypes (8). We identified that the long isoforms show ubiquitous expression, whilst the short isoform has a more limited tissue profile, being present at highest levels in skeletal muscle, but to a smaller extent also in respiratory and endocrine tissues (figure 2A). At the level of the cell, we have identified both long and short forms of FOXO3 in several important cell types such as cardiomyocytes (HCM) and fibroblasts (NHDF) by long read sequencing (figure 2B). Expression of the long isoform was also found to decrease markedly with age in human peripheral blood (figure 2C). We identified that inheritance of FOXO3 rs13217795/rs9400239 alleles associated with extreme longevity brings about an increase in long isoform usage in human peripheral blood (which expresses only the long form), but a decrease in short isoform usage in skeletal muscle, which expresses both isoforms (8) (figure 3).

Figure 2: Expression of FOXO3 long and short isoforms across human tissues. A. Expression patterns of long and short FOXO3 isoforms across human tissues. B. Expression of the long and short isoforms of the FOXO3 gene in human cardiomyocytes and fibroblasts. C. Expression of the long FOXO3 isoforms with age in human peripheral blood.

Figure 3: The effect of genotype at r13217795/rs9400239 on FOXO3 isoform usage in human peripheral blood and skeletal muscle. The effect of longevity-associated variation on FOXO3 isoform usage in peripheral blood and skeletal muscle is given above. In blood, only FOXO3-long forms are expressed, and inheritance of at least one longevity allele is associated with elevated FOXO3-long isoform expression. Conversely in muscle, both FOXO3-long and FOXO3-short isoforms are expressed, but inheritance of two copies of the longevity allele is associated with a decrease in levels of FOXO3-short.

There have been previous efforts to modulate FOXO3 activity using agonists, and benefits have been demonstrated in cells, tissues and human participants (9, 10). The molecules concerned however are not specific to FOXO3 and have pleiotropic effects on a variety of crucial cellular signalling pathways intersecting with FOXO3 (and the other FOXOs) such as AKT, PI3K and AMPK. They will also not have selective effects on full length FOXO3 isoforms and will also promote the activity or expression of ‘adverse’ isoforms such as FOXO3-short. The significant potential for off target effects means that the safety profiles of long-term treatment with FOXO3 inhibitors remain debatable. My proposed approach is to design a precise intervention that acts only on very specific sequences within the FOXO3 gene alone, to upregulate FOXO3-long isoforms or decrease FOXO3-short isoforms, or both in combination, and thus mimic the effects of inheritance of the longevity haplotype on FOXO3 isoform expression in those that do not possess it.

As with any research, there are of course risks. The major intellectual risk at present is that we do not know whether it is the decrease in FOXO3-short which is the issue, or the increase in FOXO3-long that brings about beneficial effects. The relative importance of each isoform for cellular health will be easy to assess following moderation of isoform usage in different cell types, and systemically in the planned in vivo work. I can design oligonucleotide interventions to either increase FOXO3-long or decrease FOXO3-short. Safety profiles demonstrate that oligonucleotide drugs are well tolerated and modifications to nucleoside and backbone moieties renders them stable under physiological conditions. The largest risk for clinical application of oligonucleotide therapeutics however, is delivery. Although local delivery is now fairly routine, systemic delivery of oligonucleotides has historically been a challenge, but new technologies such for systemic delivery to specific body compartments are under rapid development (11). For this initial series of studies however delivery is not an issue since I have a proven track record in human cell oligonucleotide delivery by gymnosis in the in vitro human primary cell models to be used here. Delivering nucleic acids in simple invertebrate systems is routine.

Opportunity

This project will allow us to drug the isoform specific expression of FOXO3, a known geroprotective gene, for physiological enhancement of its activity in a precise and tuneable manner. Gene expression can be enhanced by the use of small activating RNAs (saRNAs) or by steric hindrance of negative regulatory elements. Conversely, genes can be repressed by RNAi or steric blockage of sequences necessary for expression. Based on almost 30 years’ experience in RNA biology and leveraging my knowhow in oligonucleotide design and delivery developed in my capacity as founder and CSO of SENISCA Ltd. (www.senisca.com), I will develop a panel of oligonucleotide drugs designed to allow regulated control of patterns of FOXO3 isoform expression. Such an intervention would have an extremely safe usage profile compared with a small molecule; oligo drugs act on a specific region of 20-25 nucleotides at the site of action on the target gene alone and bind nowhere else if correctly designed (12). They are the fastest growing drug modality presently with 4 new approvals in 2023 alone. Depending on the target organ, they can be delivered using lipid nanoparticles, or even naked in saline if delivered locally (13). They can be signposted to specific organs and tissues using peptides, antibody conjugates or hydrogel carriers (14) and allow researchers to drug ‘undruggable’ targets such as transcription factors, which are notoriously hard to moderate with small molecules. Outputs will include a panel of prototype oligonucleotide drugs that can then be refined for clinical development. The work described here will deliver a package of in vitro and in vivo data that will allow for immediate protection of intellectual property.

The value of a drug that is able to cause even a 1 year increase in healthspan is estimated at $38 trillion. A 10 year increase in healthspan equates to $367 trillion (15). This includes reductions in health and social care costs, and estimated increases in economic activity as well as reduction in the costs of polypharmacy. In Europe, an evaluation of the monetary savings that a geroprotective drug could provide based on reduction of polypharmacy indicated a return on investment of €2.38 per patient per year (16). This clearly provides us an enormous opportunity in terms of anticipated eventual financial return, but I would argue that real benefits will be to the resulting improvement in quality of life for our ageing population.

Relevance to Longevity

Successful design of an oligonucleotide therapeutic targeting a known geroprotective gene would impact multiple aspects of longevity, healthspan and lifespan. FOXO3 is one of only a handful of genes reliably and reproducibly associated with extreme longevity, which highlights its role as a systemic geroprotector. Specifically, this work will enable the development of an intervention capable of producing favourable FOXO3 isoform expression patterns for lifespan and healthspan benefit. An intervention such as the one planned here that can influence a key geroprotective nodal gene such as FOXO3, that is active in many systems and in many cellular processes stands an excellent chance of addressing the complex and systemic nature of ageing and healthspan.

IP Roadmap

​​The data packages produced from funding these early stages of this work would form the basis for a patent application to protect the IP. I envisage the end point of this initial 2 years funding would be to file an initial application. C. elegans, although a simple organism will fulfil at least part of the necessity for an in vivo model if therapeutic claims are to be made. Following initial filing, claims would be bolstered by provision of additional data from more complex in vivo systems within the following 12 months, but this would obviously be contingent on further funding. Figure 4 below illustrates the path to IND status we would take.

Figure 4: The path to clinical for a new oligonucleotide geroprotective drug. The initial phases of this work, funded by initial investment, would cover the production of prototype oligonucleotides that can be protected by patent, and evaluation of these in vivo and in vitro. Following this phase, the next steps are to optimise the oligonucleotide sequence and chemistry, and then to determine biodistribution and gross toxicology in a higher animal model system, which is usually rodents. This determines whether it is possible to deliver naked oligos in saline to the target cells and organs. In parallel, I would recommend an ex vivo track (explants) to ensure human relevance. Following on from this, a traditional preclinical package consisting of safety toxicology and PK/PD would be pursued. The nature of this and the identity of the animal models involved would depend on the outcome of our earlier work, as some oligos will be conserved and can be assayed for efficacy in rodents, whilst others may be primate specific and would require non-human primates. Milestones and estimated timing to eventual IND approval are provided.

It is likely that a future geroprotective drug would first have to be licenced in the context of an age-related disease in the current regulatory landscape, but this opens the doors as proof of concept to expanding the licence to other disorders, and eventually to healthspan more widely. This approach also permits the use of local, rather than systemic delivery mechanisms which are lower hanging fruit.

Within SENISCA, we have successfully obtained, sustained and localised lung delivery of naked oligonucleotides administered via an intratracheal route for our lung indication, without liver or kidney persistence. The route of delivery will depend on the target organ system, and in the first instance, a new oligonucleotide drug targeted to FOXO3 isoforms would be a good choice for sarcopenia or intervertebral disc degeneration, both of which are known to be associated with decline in active FOXO3 (17-20). Dealing with issues such as decline in muscle mass or integrity would bear body-wide benefits, due to the interconnected basis of systems (for example, improvements in muscle mass or function has bearing on endocrine function and insulin resistance, since muscle is an important glucose sink).

Experimental Plan

Hypothesis

Targeted moderation of FOXO3 isoform expression profiles will attenuate cellular ageing phenotypes in vitro and being healthspan and lifespan benefits in vivo.

Objectives

Milestone 1: To use oligonucleotides to influence FOXO3 isoform expression in human primary fibroblasts (dermal and lung), endothelial cells and skeletal muscle cells, and measure effect on FOXO3-FL and TR isoform levels, FOXO3 activity and subcellular localisation, autophagy and mitochondrial function, and on the phenotype and rate of accumulation of senescent cells. Outcome: This work will provide a comprehensive assessment of the activity and subcellular localisation of FOXO3 isoforms, and the impact of each on mitochondrial function, senescence kinetics, autophagy and cell survival in human primary cells.

Milestone 2: To specifically express human FOXO3-FL or FOXO3-TR isoforms or both, in a transgenic C. elegans model followed by assessment of lifespan or healthspan parameters (movement, avoidance of noxious stimuli, lipofucin accumulation, lifespan). Outcome: This aim will provide a comprehensive assessment of the relative impact of FOXO3-short and FOXO3-long isoforms on lifespan, healthspan, motor function and sensory function in an in vivo system.

The experimental plan is illustrated in figure 5 below.

Figure 5: Diagrammatic workplan of proposed project. A. Design and in vitro validation of antisense oligonucleotides capable of attenuating FOXO3 isoform usage. B. Characterisation of the effect of expression of different FOXO3 isoforms on lifespan and healthspan phenotypes in a systemic invertebrate model system C. elegans.

Workplan

Milestone 1: In vitro

I will attenuate FOXO3 isoform usage by the use of steric hindrance translation blockers against the isoform-specific translation start site of FOXO3-short, or small activating RNAs to increase levels of the FOXO3-long isoforms. For activation of the long isoforms I will also explore the use of oligonucleotides blocking unique negative regulatory elements specific to FOXO3-long. These are standard technologies which are routinely used in both my academic team and within SENISCA (we have several oligonucleotides using this MoA). The efficiency of isoform switching will be measured by band size difference and band intensities by Western blot using an anti-FOXO3 antibody that binds an epitope present in both isoforms. By these means, it is possible to produce cells expressing primarily FOXO3-long, primarily FOXO3-short, or both. The subcellular localisation of proteins encoded by FOXO3-long and FOXO3-short isoforms will be assessed in primary human dermal fibroblasts, primary human skeletal muscle cells and cardiomyocytes by immunofluorescence microscopy. Changes in the relative ability of FOXO3 isoforms to transactivate a commercially-available luciferase reporter construct containing FOXO binding sites will then be assessed as a measure of FOXO3 activity. Effects on mitochondrial function will be assessed using live cell measurement of mitochondrial respiration and glycolysis on the Seahorse XF24 platform (21, 22) following oligonucleotide manipulation. I will also measure the impact of isoform usage on rate of senescence by monitoring senescence-associated beta galactosidase (SA-b-Gal) activity, EdU incorporation (for effects on proliferation), gH2AX (for the accumulation of senescence-associated DNA damage foci) and autophagy using fluorescence microscopy on the Operetta high content imager where we have built a unique machine learning algorithm for the classification and quantification of senescent cells. We will also assess effects on the senescence-associated secretory phenotype (SASP) and molecular markers of senescence (CDKN2A [p16], CDKN1A [p21], TP53 [p53] and LAMINB1) by quantitative PCR and ELISA where appropriate.

Milestone 2: in vivo

Although C. elegans contains a single FOXO gene, daf-16, it is expressed as several isoforms including daf-16a1 and daf-16a2, which encode full length versions, and daf-16b, which is an N-terminal deletion structurally similar to FOXO3-short (23). Daf-16 shares a binding site with its mammalian orthologs. These observations indicate that engineered human FOXO3 isoforms are likely to be able to target the same functional endpoints in C. elegans. We will create precise sequence knock-ins encoding human FOXO3-long or FOXO3-short isoforms in the daf-16(mu86) null and wild type N2 backgrounds to account for the effect of endogenously expressed FOXO. I will obtain C. elegans overexpression constructs from commercial suppliers (Genscript). Human FOXO3 isoform expression will be measured in viable, sequence verified animals using RTqPCR. Genetically modified animals will then undergo assessment of healthspan/lifespan phenotypes in 3 biological replicates (n=~80 per replicate) of manipulated worms. Lifespan and healthspan experiments will be performed at 20 °C, performed over 6 timepoints in triplicate (total n = ~240 per condition and time point). Censored lifespan data will be plotted as Kaplan-Meier curves and P values calculated using the log-rank (Mantel–Cox) analysis method. Healthspan will be measured by movement rate as one of the most relevant indices (24) as well as the accumulation of ageing pigments such as lipofucin (25). Sensory perception will also be determined using chemical nociception (acetylcholine; ACh) and recording of escape behaviours by avoidance index. C. elegans work will use the NemaLife microfluidic system as we have in our previous work (26).

Budget

Financing and VitaDAO Funding Terms

VitaDAO, the Researcher and the University of Exeter will enter into a Sponsored Research Agreement. Payments will be unlocked per milestone accomplished. Such operations will fall under the supervision of the VitaDAO Builder Squad.

If milestones 1 and 2 are met, the project will be spun out as a VitaDAO-supported company. The team will proceed to raise via Intellectual Property Token (IPT) mechanism. Details will be determined in a future VDP proposal. Reference spin out frameworks include Fission Pharma and Matrix Bio.

Team

Professor Lorna Harries

Professor Lorna Harries gained her PhD from University College London in 1994. Lorna established the RNA-mediated disease mechanisms group at Exeter in 2006 and holds a personal chair in Molecular Genetics at the University of Exeter Medical School and a position as co-founder, co-director and Chief Scientific Officer at SENISCA Ltd, a spin out company founded on the Harries lab’s research. Professor Harries also heads the Exeter Animal Free Research Centre of Excellence (ARC 2.0) funded by Animal Free Research UK. Lorna was awarded the Diabetes UK RD Lawrence rising star award in 2011, was a te4asm member of the Queen’s Anniversary Prize for research excellence in 2006 for our work on monogenic diabetes, and a direct recipient in 2019 for her work on the human health implications of microplastics in 2019. She was awarded the Proteomass Lifetime Career Award in 2021 for her work on senescence and RNA processing. The Harries lab have interests in -omics approaches to the study of ageing and age-related disease processes in humans, and her work takes a genes to systems approach, ranging from ‘big data’ analyses to detailed individual molecular analysis of particular genes. She has published over 140 peer reviewed publications in her career which have accrued over 19000 citations, and she has an H index of 53. Lorna has a proven track record of translation of basic research; she is inventor on two patents to date, one of which is under preliminary filing and the other is currently under examination in 7 global territories. Her team were the first to report dysregulation of alternative splicing as a new, and druggable, hallmark of ageing; a finding which is now being developed as the basis for a new generation of interventions to treat the underpinning causes, not just the symptoms, of age-related chronic disease. Lorna is overall project manager and responsible for successful completion of the project and delivery of any IP that arises.

Dr Sam Gould

Dr Gould is a cell and molecular biologist with a background in cellular ageing. Sam is currently 0.5FTE PDRF in the Harries academic team and 0.5FTE in SENISCA, which provides her an insight into translation pipelines. Sam brings a background in tissue engineering and cellular ageing biology and will contribute to the in vitro workplan alongside Dr Appleby.

Dr Sarah Appleby

Dr Appleby is currently a 0.5FTE technician within the Harries team. She has valuable skills in gene editing, primary cell culture and xenotransplantation. We are proposing to use half the staffing funds if awarded to bring Sarah up to full time, to contribute to the in vitro alongside Dr Sam Gould, whilst we recruit the remaining 0.5FTE to undertake the worm work.

Collaborators

Professor Tim Etheridge

Professor Tim Etheridge is an established expert in the use of C. elegans as a model for understanding human ageing. His work established a new cell-adhesion mechanism regulating C. elegans muscle health, and since directly translated this into human clinical trials investigating ageing muscle regeneration. He brings a wealth of experience in the use of invertebrate models for ageing research and access to all facilities necessary for this work. His approaches involve the high-throughput assessment of phenotype changes in genetically manipulated or drug-treated worms using microfluidics. Tim will provide oversight and advice on the worm in vivo work.

References

1.CMO. Chief Medical Officer’s Annual Report 2023 Health in an Ageing Society. 2023.
2. Morris BJ, Willcox DC, Donlon TA, Willcox BJ. FOXO3: A Major Gene for Human Longevity–A Mini-Review. Gerontology. 2015;61(6):515-25.
3. Zhao Y, Yang J, Liao W, Liu X, Zhang H, Wang S, et al. Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nat Cell Biol. 2010;12(7):665-75.
4. Tseng AH, Shieh SS, Wang DL. SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative damage. Free Radic Biol Med. 2013;63:222-34.
5. Huang H, Tindall DJ. Dynamic FoxO transcription factors. J Cell Sci. 2007;120(Pt 15):2479-87.
6. Chang ZS, He ZM, Xia JB. FoxO3 Regulates the Progress and Development of Aging and Aging-Related Diseases. Curr Mol Med. 2023;23(10):991-1006.
7. Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, et al. FOXO3A genotype is strongly associated with human longevity. Proc Natl Acad Sci U S A. 2008;105(37):13987-92.
8. Frankum R, Jameson TSO, Knight BA, Stephens FB, Wall BT, Donlon TA, et al. Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels. Geroscience. 2021.
9. Chen X, Li M, Li L, Xu S, Huang D, Ju M, et al. Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway. Sci Rep. 2016;6:28423.
10. Kaplon RE, Hill SD, Bispham NZ, Santos-Parker JR, Nowlan MJ, Snyder LL, et al. Oral trehalose supplementation improves resistance artery endothelial function in healthy middle-aged and older adults. Aging (Albany NY). 2016;8(6):1167-83.
11. Kakimoto T, Ogasawara A, Ishikawa K, Kurita T, Yoshida K, Harada S, et al. A Systemically Administered Unconjugated Antisense Oligonucleotide Targeting DUX4 Improves Muscular Injury and Motor Function in FSHD Model Mice. Biomedicines. 2023;11(9).
12. Bennett CF. Therapeutic Antisense Oligonucleotides Are Coming of Age. Annu Rev Med. 2019;70:307-21.
13. Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, Hog A, et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 2010;38(1):e3.
14. Cohen SA, Bar-Am O, Fuoco C, Saar G, Gargioli C, Seliktar D. In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides. Cell Death Dis. 2022;13(9):779.
15. Scott AJ, Ellison M, Sinclair DA. The economic value of targeting aging. Nature Aging. 2021;1(7):616-23.
16. Campins L, Serra-Prat M, Palomera E, Bolibar I, Martinez MA, Gallo P. Reduction of pharmaceutical expenditure by a drug appropriateness intervention in polymedicated elderly subjects in Catalonia (Spain). Gac Sanit. 2019;33(2):106-11.
17. Gellhaus B, Boker KO, Gsaenger M, Rodenwaldt E, Huser MA, Schilling AF, et al. Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes. Cells. 2023;12(17).
18. Jing Y, Zuo Y, Yu Y, Sun L, Yu Z, Ma S, et al. Single-nucleus profiling unveils a geroprotective role of the FOXO3 in primate skeletal muscle aging. Protein Cell. 2023;14(7):497-512.
19. Alvarez-Garcia O, Matsuzaki T, Olmer M, Miyata K, Mokuda S, Sakai D, et al. FOXO are required for intervertebral disk homeostasis during aging and their deficiency promotes disk degeneration. Aging Cell. 2018;17(5):e12800.
20. Hao Y, Ren Z, Yu L, Zhu G, Zhang P, Zhu J, et al. p300 arrests intervertebral disc degeneration by regulating the FOXO3/Sirt1/Wnt/beta-catenin axis. Aging Cell. 2022;21(8):e13677.
21. Gero D, Torregrossa R, Perry A, Waters A, Le-Trionnaire S, Whatmore JL, et al. The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro. Pharmacol Res. 2016;113(Pt A):186-98.
22. Szczesny B, Modis K, Yanagi K, Coletta C, Le Trionnaire S, Perry A, et al. AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro. Nitric Oxide. 2014;41:120-30.
23. Lee RY, Hench J, Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol. 2001;11(24):1950-7.
24. Bansal A, Zhu LJ, Yen K, Tissenbaum HA. Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants. Proc Natl Acad Sci U S A. 2015;112(3):E277-86.
25. Clokey GV, Jacobson LA. The autofluorescent “lipofuscin granules” in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev. 1986;35(1):79-94.
26. Manni E, Jeffery N, Chambers D, Slade L, Etheridge T, Harries LW. An evaluation of the role of miR-361-5p in senescence and systemic ageing. Exp Gerontol. 2023;174:112127.

Senior Review Digest - Quantitative

Below is the average scores out of 5 per category from 5 reviewers, who all recommended that the project should be advanced for token-holders vote.

Average Scores

  • Team Expertise: 4.4
  • Feasibility & Data: 4.4
  • Commercial Potential & Impact: 4.0
  • Novelty & Market Advantage: 3.8
  • IP Defensibility: 4.0
  • Relevance to Longevity: 5.0
  • Deal Terms: 3.8
  • General Conviction Score 4.4 (for reference, the average score of past funded projects is 3.7)

Majority of the reviewers think that this project is a moonshot.

Senior Review Digest - Qualitative

Each reviewer was asked whether they would endorse the project, below are their answers.

Reviewer 1
I am enthusiastic about the idea and approach to modulate FOXO3 isoforms to promote healthspan. A tantalising idea that needs more research in order to be feasible for translation. Thus, recommend funding to enrich our biological understanding.

Reviewer 2
Yes, and will refer the project to an investment firm which I am actively involved in.

Reviewer 3
Yes. If the tech is indeed unique and the IP can be protected, this project has high scientific merit and would allow VitaDAO to engage with high impact tech at an early phase, potentially increasing future ROI.

Reviewer 4
I fully endorse funding this project. Lorna and her team have identified a druggable mechanism for a longevity-associated genetic variant in the FOXO3 gene. This mechanism regulates FOXO3 isoform usage and has the potential to act as a “switch” for longevity. This discovery is very promising and offers potential for treating various age-related conditions. Shortcomings of prior attempts in targeting FOXO3 have been thoroughly considered and are addressed by the proposed approach.

Reviewer 5
Yes. The final decision making criteria was answered satisfactorily from the team, I endorse the project.

  • Agree
  • Revisions Requested [Details in Comments]
  • Disagree
0 voters
7 Likes

Huge kudos to @Adrian for sourcing this deal and for bringing this VDP over the line! Looking forward to hearing the VitaDAO community’s feedback.

5 Likes

I appreciate that this was written more from an academic standpoint getting right to the meat of the issue than flashy business pitch deck.

It sounds like the desired oligos are not yet in hand. That’s a huge risk, even if the team works with RNA. I think milestone 0 should be ~$10k or so to generate oligos that alter FOXO isoform expression in C2C12 cells, primary skeletal muscle, or some other muscle cell type. Release the next set of funding once functional oligos are in hand. Then the focus for milestone 1 would be testing the longevity hypotheses in vitro.

How will senescence be induced?

I don’t like the worm part of the project. It seems like a tangential project to the main goal, which is developing new oligos that work. If the worm part fails, is that due to the xenogeneic expression or because the longevity hypothesis is falsified? If it’s falsified, who cares because worms are not humans. If it works, while encouraging, worms are not humans. The “ex vivo human explant” studies would be a better next step in my opinion. Or test in the mouse disease model if possible, which is closer.

A few other questions that come up with the worms:
What is the phenotype of the daf16 ko worms? Why not express long/short daf16 isoforms first? How similar is the intronic region? Can the human-specific oligos cross-react with worm (or mouse, rat, etc)? Would Rapamycin be used as a control for longevity increase?

3 Likes

Great job on this write-up and sourcing Lorna @Adrian .
I agree with @bowtiedshrike on milestones.

Do you know of other groups or start ups aiming to target Foxo directly (eg Rofoxy Pharma)?

3 Likes

Hello and thank you for your useful comments!

The oligo design is not as risky as one might think - there are many tools available for this that we have in regular use at SENISCA (and in my other academic work). I have designed and validated hundreds of oligos with different molecular mechanisms over the years, and although you do sometimes get tricky sequences, there are always workarounds using slight movement of sequence placement or chemistry. I have never yet been unable to design something that does what it needs to!

Senescence is done by natural ageing of cells by continuous culture - we don’t use chemical or damage induced senescence as its likely that this induces different subsets of senescent cells. We already have banks of near senescent cultures that can be expanded for most of the cell lines in this work (Cardiomyocytes, fibroblasts). We have the option to induce senescence by other means if necessary, we have previously used metabolic and oxidative stress stimuli to achieve this.

I appreciate your concerns about the worm work, Although it’s not directly linked to the development of the oligos, it is necessary for proof of principle for the action of different FOXO3 sequences in a systemic setting, and will add to the scene setting for the importance of these specific isoforms in the resulting patent (we will need some form of in vivo work supporting the action of the specific isoforms if we want to be able to claim therapeutic action, especially in the US). The explant work (and definitely the mouse work) would not be possible within the financial constraints of this initial investment, it would cost significantly more. The mouse work needs mouse model validation, biodistribution (which is complex and expensive for oligos needing bespoke assays for each oligo) and at least one pilot with prototype oligos before selection of the eventual candidate using oligo walks. For reference, this work for our lead asset in SENISCA for one output has so far cost about £150K compared with around 20K to do the worm work in its entirety. To do lifespan work in mice will take at least a year more, even with a progeroid model. I think its prudent to test the system in something less expensive before we take that leap. The worm work actually gives a lot of bang for its buck; its relatively cheap but will give valuable data on the importance of the targeted isoform in a systemic setting. There is enough evidence in the literature for the importance of FOXO3 in human longevity, and this together with the FOXO work in model species for me to be able to be very confident of the longevity hypothesis. The knockout is necessary to provide an endogenous null background for us to be able to say definitively that activity we see is because of the introduced isoforms. There are well established positive controls for worm longevity (age1 knockout) but we can also include a rapamycin arm in both in vitro and in vivo if desired.

In terms of cross reactivity, these oligos will be primate specific - even mouse sequences are variant, so when the time comes for the mammalian in vivo we will use a mouse sequence surrogate oligo. This is absolutely convention in the oligo field when sequences show little conservation and well accepted by regulators. The worm work is not a test of the oligos per se, but it’s an important step in demonstrating the candidacy of the different isoforms that will augment and support the in vitro work and the development of the eventual asset.

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There are a few folks looking at targeting FOXO genes - ones like Refoxy Pharma don’t specify between the different FOXOs. There are entities around FOXO4 peptides as senolytics (Cleara Therapeutics). There’s quite a lot of academic activity in this space, but mostly using a small molecule approach that does not differentiate between isoforms.

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Appreciate your feedback.

In theory, I agree that oligo design should be straightforward, and that you are likely able to do it. But in practice, I’ve seen lots of “straightforward” things go sideways even when experts try them. Since the whole project depends on this key reagent, I’d like to see it in hand and working as advertised first.

You addressed a lot of my concerns about the worm work. I’m still not sure why you need to introduce the human alleles instead of targeting the endogenous daf16. Does FOXO rescue the ko phenotype?

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Thanks, I appreciate your comments. It would be perfectly possible to divide the first milestone into 2; the development and validation of the oligos, and the assessment of the full phenotype (and I agree this is a senisible move). In terms of mitigating risk to your investment however, I am not sure that it would save much however, since many of the activities that would fall under the second half of milestone 1 are actually necessary for proper validation of the oligos, and the staffing to actually do the work would have to be committed to up front.

RE the work KO, worms have only one FOXO gene (daf 16) whereas humans have 4. Targeting the endogenous only would replicate targeting the activity of all four human genes (FOXO1, FOXO3, FOXO4 and FOXO6). Introduction of the human forms is necessary for us to be able to visualise the differential effects of the isoforms.

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Thank you all for this great proposal @gweisha and @Adrian

I agree with bowtied shrike that if we can find a way to split the first milestone into two or three it would be great.

One for designing oligonucleotides
One for an initial set of experiments in vitro to provide proof of concept
The rest and or continuation of the in vitro experiments after successful completion of the second milestone

The reason is that among our eligibility criteria there is the need to provide TRL3 data supporting the intervention - these criteria are quite flexible and given the novelty of the approach and the background of Lorna, it makes perfect sense to be flexible.

However creating smaller milestones might help us de-risk somehow the investment. I let the squad and Lorna decide the best way to do so.

Another question , more for the squad than for Lorna, is why we are relying on a post-doc to carry out this work rather than on our network of CROs.

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Hi all, following community feedback this VDP has been updated to include @Lorna_Harries revised budget plan for the project.

In addition, the soft vote is now open as a precursor to conducting the Senior Review. Thank you for reviewing!

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Hi Michelle, I can shed some light on why it would be best for a PDRA to do this rather than a CRO. The cell types involved are primaries, and are very tricky to work with, particularly when senescent. For reference, within SENISCA, we have just brought our oligo optimisation back in house because our CRO (Selvita) was unable to culture these cells without selective loss of cell subtypes, despite being probably the best in class for senescent cell senolytic screening. CROs are great at what they do but they don’t go beyond preliminary troubleshooting and many of our approaches are bespoke. By and large, we have also found using a CRO to be more expensive.

I favor supporting a postdoc in the lab over a CRO, too.

Training new academics in longevity research advances VitaDAO’s mission.

Another advantage is that if this endeavor is successful, you have someone with ownership/investment in the project who can get hired into the spin-out company and keep the momentum going.

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That makes perfect sense @bowtiedshrike . I was trying to figure out ways to commit as little as money as possible upfront. If we hire a post-doc, we need to pay upfront $90K, and then if the experiments fail, that would be a sunk cost.
Given the project’s stage, $120K is still a lot of money. I would rather do as you suggest: spend $10K to design the oligonucleotides. Then i would give $50K or some to do some POC experiments, and if these go well, commit the rest of the money, no questions asked.
But it looks like the community is happy with the project, so not a hill to die on for me

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Impressive conviction score! - can the team please expand the view of senior reviewers - as it was done for Fission Pharma? The community relies on these reviews to form their opinion - personally would expand the view of reviewers 1, 2, 3 and 5

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Hey @michele.gallia! I am more than happy to expand the answers from Senior Reviewers. Please see below additional comments from the Reviewers:

Reviewer 1

I am enthusiastic about the idea and approach to modulate FOXO3 isoforms to promote healthspan. A tantalising idea that needs more research in order to be feasible for translation. Thus, recommend funding to enrich our biological understanding.

They have the unique technical knowledge to design such an oligo tx for foxo3. The idea is exciting and should be worked on!

Reviewer 2

Yes and will refer the project to an investment firm which i am actively involved.

  • strong domain expertise in FOXO3 and design of oligonucleotides,
  • novel approach and market entry,
  • clear and well-thought planning.

Reviewer 3

Yes. If the tech is indeed unique and the IP can be protected, this project has high scientific merit and would allow VitaDAO to engage with high impact tech at an early phase, potentially increasing future ROI.

  1. High Scientific merit,
  2. strong research team,
  3. potential for high impact in the health span space.

Reviewer 4

I fully endorse funding this project. Lorna and her team have identified a druggable mechanism for a longevity-associated genetic variant in the FOXO3 gene. This mechanism regulates FOXO3 isoform usage and has the potential to act as a “switch” for longevity. This discovery is very promising and offers potential for treating various age-related conditions. Shortcomings of prior attempts in targeting FOXO3 have been thoroughly considered and are addressed by the proposed approach.

Reviewer 5

Yes. The final decision making criteria was answered satisfactorily from the team, I endorse the project.

  1. Expert scientists team with experience in translational projects and spinning out a company,
  2. Known in the longevity community and investors network,
  3. Novel approach to develop longevity therapeutics and for age related diseases.
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Could you please provide information on the current amount of funds available in the VitaDAO treasury for funding purposes?

Additionally, could you specify what percentage of the total available funds this project would represent?

Perhaps we should consider initially funding it with $10K or through Catalyst.

We can raise additional funds as the project meets key milestones through tokenization as an IPT.

Hey @SimiKol , I have no access to information about the percentage of this project in the context of whole fund, but at this point, I can tell you that the project passed to the final voting on Snapshot this morning. The final voting, with the terms outlined above, will start in the next few days.

  1. How can VitaDAO move to voting without knowing the current amount of funds available in the treasury? It’s essential to ensure we have enough funds before committing to any project. Are we just funding anything without this critical information?
  2. The suggestion to initially allocate $10K for designing the oligonucleotides seems prudent, yet it hasn’t been addressed or incorporated into the proposal. Why is this important point being overlooked?
  3. What is the process for improving this proposal and ensuring that community feedback is considered? Who is responsible for making these decisions and ensuring that suggestions, like the initial $10K funding and milestone-based funding, are integrated into the plan?
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Once enough people vote ‘agree’ here, it will move to Snapshot. If you do not like the current terms, vote ‘no’ on Snapshot with your $Vita. That is the final vote for the DAO.

This forum is where proposals are considered and improved. If enough people vote ‘agree’, on the proposal as is, why would it be revised?

Prior to reaching this stage, the proposal is considered by one of the Working Groups.

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