Tuning the Therapeutic Properties of tRNAs To Treat Rare Diseases

The genomics revolution has made important advancements in the prevention and treatment of human disease, as exemplified by the mRNA vaccines against COVID-19 and gene therapy for sickle cell disease. The disease-by-disease, gene-by-gene approach remains prohibitively expensive when developing treatments for the 6,000+ rare and ultra-rare diseases that exist, despite this transformative power. Small patient populations make clinical development challenging and financially unfeasible, despite the urgency that exists to address all these diseases.

In this interview, Michelle C. Werner, CEO of Alltrna, describes how the largely untapped world of transfer RNAs (tRNAs) may provide a solution. She discusses how tRNAs could transform the drug development paradigm from gene-centric to gene-agnostic and accelerate new therapies for 30 million or more patients whose genetically driven disease can be classified under Stop Codon Disease.

Anna MacDonald (AM): Why do you think that tRNA’s significance has historically been overlooked? What has spurred the increased interest in tRNA and its therapeutic potential?

Michelle C. Werner (MW): One of the factors delaying interest in tRNAs was the belief that these molecules merely served as intermediaries in protein synthesis in the cell. Our gene-centric view of disease previously focused the industry’s attention on the DNA or mRNA sequences that code for the products of those genes, an approach that has led to great advances. But despite several groups studying tRNA, few saw them as more than translational machinery.

To be fair, the ability to see tRNAs any other way was, in many ways, hampered by the lack of scientific tools needed to quantify and characterize these molecules. Advances in the technologies used to study mRNA and siRNAs helped, for sure, but tRNAs presented unique challenges in terms of sequencing and chemical synthesis, and those challenges demanded technological innovation.

Likewise, the tools to not only identify but also understand the importance of chemical modifications to those sequences – their epitranscriptomics – had to be developed as well.

Such technical developments have been a key focus at Alltrna and, more broadly, have helped the field discover that tRNA biology and its impacts on human health and disease include and extend well beyond its pivotal roles in protein synthesis.

AM: Can you tell us a little about the history and creation of Alltrna?

MW: In 2018, Alltrna’s founding scientific team questioned whether there was more to tRNAs than their role as intermediaries, amino-acid shuttles if you will, in protein translation. What if, in fact, their biology in human health and disease is much more complex?

Alltrna launched from stealth in 2021 to understand both the tRNA design rules and the breadth and power of its biology, with the mission to engineer tRNAs as a new therapeutic modality. To do so, the company first had to overcome the challenges I mentioned earlier, developing not only the technologies needed to express, synthesize, modify and quantify tRNA molecules, but also the machine learning tools to thoroughly explore the sequence and modification space to optimize their biology.

This platform has also proven critical for our efforts to tune the therapeutic properties of tRNAs, whether we’re talking efficacy and safety or more pharmacological characteristics like stability, solubility and deliverability.

In practice, Alltrna has shown that by optimizing for sequence and chemical modifications, we can substantially improve the activity of our engineered tRNAs over endogenous molecules and restore protein expression in various in vitro and animal models of what we call Stop Codon Disease.

AM: Can you explain what Stop Codon Disease is and how Alltrna is working to address the conditions it causes? What sets this approach apart?

MW: In the ribosome during normal protein translation, an mRNA copy of a gene carries a triplet code (a codon) for the amino acid sequence of a polypeptide chain. tRNAs and their bound amino acids decode these triplets via their complementary anticodons and add that amino acid to the growing protein chain. Translation then terminates when the ribosome reaches a triplet known as a stop codon for which there is no complementary tRNA, and a fully formed protein is released.

In Stop Codon Disease, however, a mutation has occurred that converts what should be an amino acid codon into a premature termination codon (PTC), stopping the protein translation process early. Such a mutation may mean no protein is produced, or the cell generates a truncated protein that may have reduced activity or may be toxic, thus triggering the disease phenotype.

It’s been estimated that across the 6,000-plus genetic diseases, somewhere around 10% of patients, representing approximately 30 million people, have a PTC that causes their disease.

Alltrna asked: what if we could engineer tRNAs that could recognize and read through these PTCs? Instead of terminating protein translation, the PTC could be recognized by the tRNA, which could insert the originally encoded amino acid to continue the growing polypeptide chain, producing full-length functional proteins and reversing patient disease.

What makes Alltrna’s approach different is that, unlike other treatments for genetic diseases, such as gene therapy, gene editing or other RNA-based treatments, which are gene-specific, tRNA-based therapies are gene- and disease-agnostic, addressing the specific PTC no matter what protein code it interrupts.

This means that a single engineered tRNA may be able to correct a PTC across a number of diseases. Already, in preclinical studies, we have shown that we can use a single engineered tRNA to rescue protein expression in 25 different disease models across 14 different genes regardless of where the PTC occurs in the gene.

AM: Are there any challenges associated with tRNA that need to be addressed in order for it to meet its potential as a therapeutic?

MW: As with any genomic intervention, whether DNA or RNA, one of the major challenges is ensuring safe passage of the therapeutic molecule to the tissues and organs where it is most needed and avoiding delivery to other tissues where it might trigger adverse events.

At the same time as it is engineering therapeutic tRNAs, Alltrna’s team is also exploring and optimizing the design of tRNA delivery vehicles that help ensure these molecules are able to reach their tissue targets without being destroyed by nucleases and other defense mechanisms within the body. Given the extensive experience with the mRNA vaccines against COVID-19, Alltrna is focusing on lipid nanoparticles in the first instance.

AM: Can you explain the concept of basket trials and why Alltrna is exploring this approach?

MW: In a typical clinical trial, researchers take a disease-by-disease approach, testing a therapeutic intervention in patients with a specific disease. Our confidence in the results of that trial is heavily influenced by the number of patients included in the study. The larger that population, the more confident we can be. Unfortunately, this is a problem when studying rare and ultra-rare diseases, where global patient populations may number in the hundreds or thousands rather than millions.

To address this limitation, basket trials pool patients with different diseases caused by a mutation common to all of them and test a treatment specific to that mutation. This approach has been used widely in oncology, resulting in regulatory approvals of about a dozen cancer treatments, including KEYTRUDA (pembrolizumab), Tafinlar (dabrafenib) and Mekinist (trametinib).

The same approach is perfectly suited for potential tRNA-based interventions as the proposed therapeutic targets the PTC mutation regardless of the gene or disease. Thus, Alltrna can test a single engineered tRNA in a pool of patients with different rare and ultra-rare conditions but the same mutation. This approach would overcome the statistical and economic barriers to performing such trials in the traditional way and provide hope for the 30 million or so patients with unmet medical needs.

Right now, there are thousands of different diseases without even a single interventional clinical trial because patient numbers don’t make that kind of investment feasible.

A tRNA medicine that could universally treat patients across multiple Stop Codon Disease could accelerate and scale the development of potential disease-modifying treatments for millions of patients.

AM: Can you share Alltrna’s plans for 2024 and beyond? What do you see in store for the future of tRNA-based therapies?

MW: Initially, Alltrna is focused on the genetic liver diseases, which include upward of 400 different rare and ultra-rare diseases. In part, this is due to the already proven ability to get RNA treatments, encapsulated in lipid nanoparticles, to liver tissue. From there, we would look to prioritize diseases based on our ability to target tissues outside of the liver.

Similarly, although Alltrna’s initial focus is on Stop Codon Disease and achieving readthrough of PTC mutations, we believe that there are opportunities to extend these efforts to other types of genetic changes that disrupt protein production, such as frameshift or missense mutations.

And there is growing evidence that human health and disease is influenced by tRNA pools – the amounts of specific tRNA types – and tRNA fragments, so we may be able to address an even wider array of patients in the future.

That’s looking well down the road, however, and while we are excited about the future, there is a lot we are doing now and in the near term to make this a reality. This is a new treatment modality with a lot of open questions and a need to engage with and educate clinicians, patient groups and regulators. So, we want to make sure that we get these initial stages of development right in Stop Codon Disease.

AM: Is there anything else you’d like to highlight?

MW: I’d like to take a moment to highlight our great team who share and embody our belief that patients deserve better. Our diverse and talented team brings multidisciplinary experience and expertise tailor-made for the discovery and development of groundbreaking tRNA medicines.

Just as importantly, everyone understands and trusts that success demands both the curiosity to explore the unknown and the courage to take thoughtful risks without fear of failure. Only by working in this way can we hope to possibly improve the lives of millions of patients globally.

Michelle C. Werner was speaking to Anna MacDonald, Senior Science Writer for Technology Networks.

About the interviewee:

Michelle C. Werner is CEO of Alltrna and CEO-partner at Flagship Pioneering. She is an experienced pharmaceutical executive with more than 20 years in the industry spanning commercial and R&D. Michelle is a wife and mother to three children and is a member of the rare disease community.