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.
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 .
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.
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. 
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.
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 , hC Bioscience, ReCode Therapeutics (co-founded by this project’s PI), Shape Therapeutics, and Tevard .
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).
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).
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.
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:
|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||$8,300|
(*) Up to 6 months or project completion. Personnel costs are only for as long as the project is ongoing. If we do not proceed at 1st no-go then there is no further personnel costs to be drawn and the remaining funds have to be returned.
Sponsored Development Agreement covering all the IP generated with the funding.
An IP-NFT will be minted at the beginning of the project.
Proposed ownership structure upon successful completion:
25% VitaDAO EIR PM
5% Deal squad
45% VitaDAO, in exchange for 90% of the funding (82 170$)
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.
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.
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.
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.
Clark D, … McGehee M. Molecular Biology (Third Edition), 2019
Benhabiles H, Jia J, Lejeune F, Nonsense Mutation Correction in Human Diseases - An Approach for Targeted Medicine Medicine, Elsevier 2016
Dolgin E. tRNA therapeutics burst onto startup scene, Nature Biotechnology, 2022 Mar
Porter JJ, Heil CS, Lueck JD. Therapeutic promise of engineered nonsense suppressor tRNAs, WIREs RNA, 2021 Feb
Temaj G et.al. Recoding of Nonsense Mutation as a Pharmacological Strategy, Biomedicines, 2023 Feb
Lueck JD et al. Engineered transfer RNAs for suppression of premature termination codons, Nature Communications, 2019 Feb
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
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:
- Funding new projects
- Operations, including sourcing, incubation, evaluation, & community growth
- Follow-on funding, including for projects VitaDAO will spin out"
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
Revisions Requested (Detail in Comments)