VDP-103 [Funding]: Mutation-Specific Codon Suppression for Aging and Longevity

One liner : a first-in-class intervention from an accomplished scientist entrepreneur leveraging tRNA know-how to tackle the most frequent nonsense mutation, relevant for a wide range of diseases and longevity.

Longevity Dealflow WG Team

  • Reviewers : 2 biotech entrepreneurs, 1 pharma professional, 2 geroscience professors, 1 biotech venture capitalist

  • Shepherd : Paolo Binetti

  • Other squad members : Eleanor Davies, Tuan Dinh, Eli Mohamed, Tim Peterson

  • Sourced by : proposed by principal investigator

VitaDAO Entrepreneur In Residence - Project Manager (EIR PM): Anthony Schwartz, Ph.D.

Principal Investigator (PI): Michael Torres, Ph.D.


Nonsense mutations contribute significantly to a wide range of genetic and age-related diseases by inducing premature stops of protein translation when occurring in coding regions. The most common mutation is from an arginine codon to an opal-stop codon. Dr. Torres, an RNA expert and co-founder of Recode Tx, proposes an engineered suppressor tRNA that can specifically recognize these codons and insert an arginine amino acid in its place, restoring normal protein translation. Delivery can be via be achieved using clinically validated modalities. This technology has promising preliminary results, and several clinical opportunities are possible. A stepwise research plan is outlined to confirm the feasibility and identify the most promising clinical applications.


A nonsense mutation is a point mutation in a sequence of DNA or RNA that results in a premature termination codon (PTC) or a nonsense codon, leading to a truncated, incomplete, and nonfunctional protein product.

Nonsense mutations cause disease-causing variants in about 10% of patients with genetic diseases like cystic fibrosis. They are also implicated in age-related diseases, like cancer [5, chapter 2].

Nonsense mutations at Arginine CGA codons resulting in a nonsense opal stop codon, TGA (one of the three stop codons in the genetic code), with hydrolytic deamination of 5-methylcytosine at CpG sites being the main cause, is the most frequent in tumor suppressor genes [2].

Different strategies have been attempted so far to mitigate the effects of nonsense mutations [7, 8]:

  • Nonsense-Mediated mRNA Decay (NMD) Inhibition by Drugs . NMD is a highly conserved pathway for the surveillance and degradation of abnormal mRNAs, identified based on premature termination codons. Drugs that block NMD can activate PTC readthrough, as is the case for aminoglycosides, such as G418 and NB-124, or PTC-124 (Ataluren). The drawbacks of this approach are low efficiency, the incorporation of near-cognate amino acids at PTC, and readthrough at natural termination codons (NTC), resulting in aberrant protein products and small therapeutic windows.

  • Pseudouridylation . Unlike NMD inhibition, which targets any PTC, a pseudouridylation drug can be tailored to a specific disease-causing PTC. approach. Unlike aminoglycosides, pseudouridylation raises little concern about global NTC readthrough, but like aminoglycosides, it promotes the misincorporation of near-cognate amino acids.


Arginine Suppressor tRNA

Nonsense mutations can be suppressed by a mutation in the anticodon sequence of a tRNA molecule so that it recognizes the stop codon instead. The figure below shows how such a “suppressor tRNA” works to suppress the effect of the mutation of a glutamine codon into an amber nonsense stop codon.

Figure. Mechanism of suppression of a nonsense amber codon [4]

Similarly, an opal suppressor tRNA can recognize the premature stop codon caused by a nonsense mutation in the arginine CGA codon, allowing for the incorporation of arginine at that position and the production of a full-length protein.

While suppressor tRNAs occur naturally due to mutations, they are detrimental because they imply partial loss of translation capability for a given amino acid, which is lethal in insects and mammals. In addition, naturally-occurring suppressor tRNAs bind with nonsense and normal stop codons, generating longer (and incorrect) versions of many proteins whose genes were never mutated. [4]

We propose engineered arginine suppressor tRNA capable of specifically targeting nonsense opal codons without competing with normal tRNA.

Preliminary ribosome profiling data were obtained with collaborators at Johns Hopkins that show rescue of TP53 levels in calu6, a cell line with an opal nonsense mutation in the gene (homozygous TP53 R196X), without significant NTC readthrough, performing better than known alternative interventions. See the figure below.

In this experiment, cells were treated with G418 (a drug that significantly causes read-through), Atalruren (or PTC124) (PTC therapeutics readthrough drug), the lysine amber suppressor, and the arginine opal suppressor. The tRNAs were oligos delivered by RNAiMax. The western blot shows that G418 and Arg/Op rescued p53, but G418 had more truncated p53 than Arg/Op suggesting it is not specific. PCT124 failed to rescue p53 in this experiment.

The graph is a genome-wide ribosomal profiling study using Calu6 cells with the indicated treatments. The degree of right shifting indicates read-through beyond normal stops (into the 3’ UTR). The data demonstrate that G418 reads through normal stops, as does lysine/amber, to a certain extent, while the arg/opal tRNA does not. PTC124 either, but that’s not surprising, given the drug history and our data.

Drug delivery

We are considering both non-viral (e.g., lipid nanoparticles) and viral vectors to deliver the tRNA payloads. Amongst the viral vectors, AAV9 is a promising vector for clinical use due to its ability to efficiently transduce various tissues and organs, including the heart, liver, skeletal muscle, and central nervous system (CNS). AAV9 has been shown to have higher transduction efficiency in the CNS than other AAV serotypes, making it an attractive vector for treating neurological disorders.

In particular, AAV9-based gene therapies have shown promising results in clinical trials for treating SMA, a neuromuscular disorder caused by mutations in the SMN1 gene. AAV9-mediated gene therapy for SMA has been shown to increase the levels of SMN protein and improve motor function in patients.

Thus our lead therapeutic approach would be to use AAV9 to deliver an arginine opal suppressor. Given the validity of AAV9, this would enable us to optimize our path to first-in-human studies upon successful generation and validation of lead candidates.

We could develop an oligo-based approach as a backup.


The rate of nonsense mutations among pathologies is variable. However, the gene silencing mechanism occurring due to a nonsense mutation is shared. Consequently, common therapies can be applied to patients with various diseases, with an enormous pipeline-in-a-pill potential, as is characteristic of candidate longevity interventions.

This potential has attracted several startups that have raised significant investments in recent years, such as, in the tRNA space: Alltrna [10], hC Bioscience, ReCode Therapeutics (co-founded by this project’s PI), Shape Therapeutics, and Tevard [7].

Some of the diseases implicated with nonsense mutations are Duchenne muscular dystrophy (DMD), cystic fibrosis (CF), spinal muscular atrophy (SMA), cancer, metabolic diseases, and neurologic disorders.

We would determine lead indications with in vivo assessments in disease models. At this point, we do not intend to focus on treating a specific disease like SMA but to generate a therapy guided toward globally suppressing arginine opal mutations that occurs in multiple genes in parallel (see table below).

Relevance for longevity

An arginine opal suppressor tRNA could be a potential therapy to impact lifespan and age-related diseases. For example, cancer is an underappreciated aging-related disease. Reactivation of tumor suppressor expression due to nonsense suppression would be expected to significantly impact cancer, which would be valuable because cancer is a top cause of mortality worldwide.

Analysis of human gene transcripts reveals that CGA codons are present in aging, DNA repair, and metabolism genes, e.g., APOE, ATM, TP53, and Lamin A (REF).

Plan and IP roadmap

Phases proposed for funding

Milestone 1 – Start of the project – T0: project kickoff meeting to be organized by PM after on-chain approval of the proposal.

Phase 1 : Design & generate an arginine opal suppressor construct for use in an AAV9 vector. The rationale for AAV9 is that it is the most clinically validated AAV with the broadest tropism.

Risk mitigation: after the in-vitro data with AAV there could be a decision point to consider different delivery modalities. We can design an arginine opal suppressor as an oligo that could be conjugated or unconjugated to drive desired tropism.

Each one of these assets could be novel IP and could be selected for further development.

Milestone 2 – Go / no go check – T0 + 3 months: Completion of phase 1 and presentation of results in a dedicated meeting or at a quarterly review.

Phase 2 : In vitro testing of selected vectors/oligos. Using the Calu6 cell line, which is homozygous for an arginine nonsense mutation in p53 (R196X), we will evaluate activity by assessing the restoration of p53 levels. This evaluation could be performed at a CRO such as Champion’s Oncology.

Milestone 3 – End of the project – T0 + 6 months: Completion of phase 2 and presentation of results in a dedicated meeting or at a quarterly review, proposal for next round of funding. Note, a provisional patent could be filed at this stage.

Additional phases part of the plan, but not funded

Phase 3 : In vivo testing of selected vectors/oligos in relevant age-related/lifespan models. In consultation with VitaDAO, the team would identify relevant disease and lifespan models to test selected vectors/oligos to confirm the efficacy and evaluate the effects of chronic arginine opal suppression.

Phase 4 : If we see positive results in the in vivo studies, we will pursue further financing to move further into development. Business development relationships would be explored at this time as well.

Total timeline for activities until the end of phase 3 : see the figure below corresponding to 1 year (caveat: dependent on the in vivo study parameters. If lifespan studies, it could be longer)



Proposed budget for activities:

Budget item Cost
Vector design & production (CRO) $10,000
Virus production (CRO) $10,000
In vitro testing (CRO) $20,000
(*)VitaDAO EIR PM (Anthony Schwartz) $3,000/month (20hrs/month) $18,000
(*)PI consultant (Michael Torres) $2,500/month (16hrs/month) $15,000
10% contingency buffer $8,300
Total $91,300

(*) Up to 6 months or project completion. Personnel costs are only for as long as the project is ongoing. If we do not proceed at 1st no-go then there is no further personnel costs to be drawn and the remaining funds have to be returned.

Deal structure

Sponsored Development Agreement covering all the IP generated with the funding.

An IP-NFT will be minted at the beginning of the project.

IP-NFT Ownership

Proposed ownership structure upon successful completion:

  • 20% PI

  • 25% VitaDAO EIR PM

  • 5% Deal squad

  • 45% VitaDAO, in exchange for 90% of the funding (82 170$)

  • 5% Molecule, in exchange for 10% of the funding (9 130$)


Michael Torres, Ph.D., PI, is a highly accomplished biotechnology professional with extensive experience in cancer therapeutics, drug discovery, and molecular biology. As an Entrepreneur at The Accelerator for Cancer Therapeutics, Michael Torres engages with high-impact cancer therapeutic projects, develops networks, and creates resources to support product development. Michael Torres has played a pivotal role in transforming an academic project into a VC-backed company, raising $80M from prominent investors and securing a $3.2M award from the CF Foundation.

With a strong background in research and development, Michael Torres has held the position of VP of R&D at ReCode Therapeutics, Inc., where they directed cross-functional teams, managed therapeutic programs, and facilitated the preclinical development of novel tRNA/mRNA LNP drug products. Michael Torres has also contributed significantly to the field through their work as a Postdoctoral Fellow at UT-Southwestern, investigating novel therapeutics for treating Cystic Fibrosis.

With a proven track record in research and business development, Michael Torres has a keen understanding of the biotechnology landscape and is dedicated to advancing novel therapeutics for the betterment of patients worldwide.

Anthony Schwartz, Ph.D., VitaDAO EIR PM, is an entrepreneur with almost 20 years of experience in biotechnology-based startup companies. Anthony obtained his Ph.D. in Biomedical Engineering with research in regenerative stem cell therapy and using stereotactic radiotherapy to improve cancer treatment. He has founded at least 15 startups primarily focused on autoimmune diseases and cancer, which have led to large financings and an FDA-approved product. He has significant expertise in cancer immunotherapies, particularly in antisense and novel CART therapeutic modalities. More recently was part of Hibiscus BioVentures, which facilitated financings and launched several biotechnology companies. In addition, he helped in launching Hibiscus’ Mayflower BioVentures fund, which spearheaded laboratory-stage therapeutic assets from the Mayo Clinic into companies. He is a professor at Johns Hopkins, teaching finance and how to start a biotechnology company. Finally, he runs a Biotechnology based consulting agency, BioVisors, and volunteers with student-run biotechnology companies through Nucleate.


  1. Mort M, Ivanov D, Cooper DN, Chuzhanova NA. A meta-analysis of nonsense mutations causing human genetic disease. Hum Mutat. 2008 Aug;29(8):1037-47. doi: 10.1002/humu.20763. PMID: 18454449.

  2. Zhang M, Yang D, Gold B. Origins of nonsense mutations in human tumor suppressor genes. Mutat Res. 2021 Jul-Dec;823:111761. doi: 10.1016/j.mrfmmm.2021.111761. Epub 2021 Aug 16. PMID: 34461460.

  3. Romanov GA, Sukhoverov VS. Arginine CGA codons as a source of nonsense mutations: a possible role in multivariant gene expression, control of mRNA quality, and aging. Mol Genet Genomics. 2017 Oct;292(5):1013-1026. doi: 10.1007/s00438-017-1328-y. Epub 2017 May 18. PMID: 28523359.

  4. Clark D, … McGehee M. Molecular Biology (Third Edition), 2019

  5. Benhabiles H, Jia J, Lejeune F, Nonsense Mutation Correction in Human Diseases - An Approach for Targeted Medicine Medicine, Elsevier 2016

  6. Dolgin E. tRNA therapeutics burst onto startup scene, Nature Biotechnology, 2022 Mar

  7. Porter JJ, Heil CS, Lueck JD. Therapeutic promise of engineered nonsense suppressor tRNAs, WIREs RNA, 2021 Feb

  8. Temaj G et.al. Recoding of Nonsense Mutation as a Pharmacological Strategy, Biomedicines, 2023 Feb

  9. Lueck JD et al. Engineered transfer RNAs for suppression of premature termination codons, Nature Communications, 2019 Feb

  10. Grinstein JD No-nonsense: Alltrna Touts tRNA Therapeutics to Treat Thousands of Diseases, GEN Edge, 2023 May


  • Therapeutic approach in the emerging tRNA space

  • Encouraging preliminary data rescuing primary tumor-suppressor gene

  • The PI is an expert in tRNA and has a successful co-founder track record

  • Pipeline in a pill: MoA shared by many diseases, giving multiple shots on goal

  • Smaller payload than gene therapies, facilitating viral delivery for age-related diseases.

  • Addresses an underappreciated aspect of aging (the high frequency of arginine CGA codon mutations)


  • No one has attempted to globally and chronically suppress arginine opal mutations.

  • Other competitors are using suppressor tRNA to treat monogenic disorders such as DMD.

  • Toxicity stemming from NTC readthrough

  • Overcoming the delivery hurdle

  • Selection of animal models relevant for tox and disease

  • tRNA biology unknowns, leading to potential off-target effects

VitaDAO’s available funds

For context, (as of May 29th, 2023) VitaDAO funded 17 projects with $4m, and has ~$6m+ in liquid funds remaining (before further fundraising), which will be used for:

  1. Funding new projects
  2. Operations, including sourcing, incubation, evaluation, & community growth
  3. Follow-on funding, including for projects VitaDAO will spin out"

Senior review digest

The reviewers have scored the proposal on different aspects including general conviction, on a scale of 1-5 (with 5 being the highest). Here are the average scores:

  • Novelty & Impact: 3.3
  • Feasibility & Data: 3.2
  • Relevance: 2.3
  • Science Team: 4.0
  • Market Advantage: 2.6
  • IP Potential: 3.4
  • Conviction score: 2.7

The full senior review digest is available here.


  • Agree

  • Revisions Requested (Detail in Comments)

  • Disagree

0 voters

Great job on fleshing this out, everyone! I am looking forward to questions from the community, especially on the science/research side.

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For budget, it looks like the annualized rate we would be paying (assuming 40 hr weeks) the PM is $312k/year and $325k/year for the PI consultant. Both of these rates surpass the NIH salary cap for academic faculty (which is $212k/year), and I think exceeds what VitaDAO has paid out for other projects. Usually for academics, the salary requested is at their current academic level, so there is some variation in the $75k-$212k range. However for DAO designed projects like this, I think a general rate should be set by the DAO. Otherwise, this will be the floor price for PIs and PMs. I think the IP-NFT ownership would justify paying closer to standard academic rates (or does industry give this amount of IP ownership to their scientists?). VitaDAO may want to hold 51%, unless the intention is to either fractionalize and/or ensure the WG, PI or PM is onboard for any IP decision.

For Phase 1, it’s not clear what data would be needed for a go/no-go. I’m also not clear how Phase 1 would fail, since Phase 2 is the in vitro testing and design. Is this a check to see if the CRO rugs?

While I get the preliminary data are with p53 and Calu6 cells, and first validation should be to repeat earlier results, I would like to see the project branch out closer to longevity from there. Also important to check other cell types to ensure that it’s not a quirk of Calu6 cells. p53 is a cancer target, not a longevity target. Since transduction isn’t 100%, one concern with the overall approach of “stuff things in cancer cells to fix them” is that you will select for cancer cells that either lack AAV9 tropism or resist transduction and fail to clear the cancer.

Also, I missed this the first time around-- did the p53 expression halt growth of the cells? From the gel, it looks like there’s less actin in the blot, which could be cell death (or a bunch of other things). But a proper BrdU uptake/CFSE dilution experiment would give a cleaner answer on growth, or AnnV/PI + cell counts for apoptosis. Also, for the longevity angle, does p53 push senescence in these cells instead of apoptosis or other outcomes?


Could you maybe elaborate what the envisioned competitive advantage would be compared to the well funded competitors e.g. alltrna? If it’s not the vector/delivery system and not the indication, then what is it?


I will let the VitaDAO address the economics while I will address the project-related

The timeline graphic shows an internal check. This is a status update and not a true go/no-go. That would come after in vitro testing, which is captured here:

@Paolo, perhaps you can revise to make it clearer that phase 2 is a true go/no-go.

I won’t wade into whether a tumor suppressor and “guardian of the genome” is an longetivity target or not, but I agree that it would be good to test in other cell lines. The Calu6 is simply a confirmation that the vector does what we expect it to do. It is the cheapest way to do so. It would be good to test in another model, if there are funds available to do so. Ideally, we will move to more “longevity” focused models once we’ve confirmed the construct is active. This is where the fun begins in my view.

Again, p53 is just the model protein to establish proof of concept. I would not advocate trying to address longevity by specifically correcting mutant p53. The concept of restoring tumor suppressors for anti-cancer is what others are working on. This approach addresses an underappreciated aspect of biology…hypermutation of CGA codons across many genes at the mRNA and DNA level. This is why I am excited about this project as no one has tried to tackle this. We simply don’t know what will happen. I like AAV9 (again, an oligo could be a backup) because that can be easily translated into a drug as there are approved drugs using AAV9. Confirmation of activity means we may have a strong hammer that could be valuable to pharma and other drug developers.

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Hi, thanks for replying. If you have additional insight into competitors (Alltrna is a Flagship Co, so ultra hush-hush) please share.

I’d answer this way (borrowing from a comment I made above):

The concept of restoring tumor suppressors for anti-cancer is what others, like Alltrna, are working on. This approach addresses an underappreciated aspect of biology…hypermutation of CGA codons across many genes at the mRNA and DNA levels. This is why I am excited about this project as no one has tried to tackle this.

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Thank you for your comments. Concerning compensation, while it is useful to check comparables, academic salaries for-full time positions might not be the ideal reference, because: both the PI and the EIR in this proposal are experienced biotech operators/entrepreneurs; their compensation is not a salary but a fee, which does not come with the overheads of a salary; these are not full time positions. In addition, because both the PI and the EIR are not affiliated with a university, there is no overhead on top the direct costs. Considering closer comparables, such as biotech consultants or EIRs standards, the proposed fees are relatively low.

This comes back to one of the ongoing discussions about mission/purpose for VitaDAO. Is the focus working with academics to bridge the Valley of Death and get initial IP into commercial tracks, or is it to work as a venture fund? If the former, academic salaries are the best reference. If the latter, biotech consultants makes more sense.

I would suggest targeting earlier career people who will be cheaper and who can’t as easily afford an L as people who have a reputation for wins. One way to do that would be to set compensation near the associate prof level. Those under it will be incentivized to participate, and those above it will be disincentivized.

Regardless of what compensation is set, I would prefer it remain equal across the VitaDAO initiated VDPs, and not be as dependent on time in field or previous startups. Should be a ‘qualified’/‘not-qualified’ decision for PIs, and if qualified, give the same rate.

IIRC, fringe benefits for academics usually add 20-30%, and health insurance another $1k/month or so. But I don’t recall seeing fringe broken out in prior budgets, which would mean the prior salaries have all included the fringe costs, too.

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This is why you need to confirm that p53 is functional in those cells. If these readthrough proteins are messed up, the amounts may not matter. And p53 is notorious for being non-functional in cancers.

In the world where the AAV9 construct recapitulates the prior results with p53, restores function, and holds up in multiple cell lines, what next for longevity? It seems like there is no plan for that. Is it just ‘patent the approach and sell to alltrna or the highest bidder’?

I would prefer to see the planning for disease target/longevity connection up front.


As I understand e.g. Tevard and Shape are also working on genetic diseases and not cancer. According to this the Nature paper ‘tRNA therapeutics burst onto startup scene’ (can’t link it here but you are also mentioned in the piece Michael!) these companies are also working on suppressor tRNAs so I’m just trying to understand the difference of the proposed approach here. It could be my lack of understanding on tRNA biology but any clarification would be much appreciated!


No problem. At a high level, everyone (alltrna, tevard, hc biosciences, et al.) is working on developing suppressor tRNA therapeutics. As you dig deeper, each has its approach to designing and delivering tRNAs. This proposal encompasses one of many approaches for a specific area of biology. This is an opportunity for VitaDAO to generate one focused on longevity.

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“An IP-NFT could be minted at the end of the project.” It would make sense to mint an IP-NFT at the beginning of the project rather, fractionalized per the distribution mentioned above. This also could create a longer-term fundraising mechanism for the project.


“An IP-NFT could be minted at the end of the project.” It would make sense to mint an IP-NFT at the beginning of the project rather, fractionalized per the distribution mentioned above. This also could create a longer-term fundraising mechanism for the project.

Totally agree

following about the conversation since beginning. I agree with @bowtiedshrike that it is important to clarify the focus of VitaDAO; and the conecpt of taking the compensation ref level of “disincentivized”.

Whom we are taking the ref represents the position of VitaDAO is.

I would like to ask if there is the preliminary ribosome data about TP53 R213X as well. According to the related paper, R213X and R196X are the two most frequent nonsense TP53 mutations in human tumors.

Or any reason that not choosing R213X but using R196X as the target type?


“Around 10% of TP53 mutations are nonsense mutations (6–8) that give rise to premature termination codons (PTCs), resulting in the expression of unstable truncated p53 or complete lack of p53 expression due to nonsense-mediated mRNA decay (NMD) (11). R213X and R196X are the two most frequent nonsense TP53 mutations in human tumors, and R213X is present in about 1% of all human tumors (7, 8), corresponding to roughly 141,000 new cancer cases worldwide 2012 and estimated 236,000 cases in 2030 (12, 13). R213X is the 6th most common TP53 mutation in 12 common cancer types and the 2nd most common TP53 mutation in lung squamous cell carcinoma after R158L (6).”

Ref: https://www.frontiersin.org/articles/10.3389/fonc.2017.00323/full

Would it be possible for Molecule to co-invest for 10% of the funding amount requested? We would be interested in supporting if this will be an IP-NFT. @Paolo @mykalt45

Also, reupping the comment that the IP-NFT would need to be minted at the beginning of the project for VitaDAO to secure its ownership and rights. Can this be edited, @Paolo? Otherwise, a company would need to be set up and support done via VHF and not VitaDAO, which I do not believe is the intent.

Hi @tylergolato, yes I will update the proposal accordingly

This is acceptable to me. @tylergolato I will follow up on Discord as I have some clarifying questions. @Paolo

I like the project. However:

  • I also think the IP-NFT fractions of 20% for PI and 25% for VitaDAO EIR PM are quite steep given that they already receive USD compensation. Maybe 5-10% for each sounds more reasonable.

  • Also have to include Molecule fraction of the IP-NFT I’d assume.

  • Agreeing that VitaDAO should have 51% of it.

  • Moreover not seeing the Senior review yet, would be good to see the ratings of the senior reviewers (assuming this is needed before it can even go on chain).