DDW Editor Reece Armstrong speaks to Dr Caroline Kӧhrer, Vice President, Discovery Platform, Alltrna, about the biology behind tRNAs and how they could help produce a medicine that could treat thousands of rare diseases that share the same genetic mutation.

RA: Why are tRNA molecules so important for rare genetic diseases? 

CK: Only 5% of the 6,000 rare genetic diseases have an FDA approved drug, leaving nearly 300 million people with no treatment options. tRNA therapeutics, unlike other genetic medicines, can unify the treatment of diseases based on shared genetic mutations and accelerate rare disease drug development for millions.

To understand the potential impact of transfer RNA or tRNA molecules on rare genetic disease treatment, it is important to understand what tRNAs are and how they function within the cell.

Serving as a central component of the protein translation machinery, tRNAs are short, highly structured RNA molecules that transport amino acids to the ribosome, and the identity of the attached amino acid is determined by the tRNA’s sequence.

Within the ribosome, aminoacylated tRNAs use their anticodon sequences to read mRNA codons, seeking a complementary match. Once a tRNA molecule matches with a codon on the mRNA, the tRNA transfers its amino acid cargo to the growing polypeptide chain. This process is repeated until the translation complex reaches a stop or termination codon defining the end of the coding region. These natural termination codons are special codons for which no corresponding tRNAs exist. At the termination codon, protein translation stops and a fully formed, functional protein is released from the ribosome.

Across the 6,000-plus diseases caused by a genetic mutation, it’s been estimated that 10% of patients, representing approximately 30 million people, have a mutation somewhere in the coding region of a gene that codes for a stop codon rather than an amino acid. This is called a premature termination codon (PTC)¹.  We use the term Stop Codon Disease to represent all these diseases, regardless of the gene where this mutation occurs. 

A PTC causes protein translation to stop before a fully functional protein can be produced, resulting in truncated faulty proteins with no, diminished, or altered function which can lead to disease. Alltrna is developing engineered tRNA medicines to readthrough PTCs and restore production of affected proteins, no matter what protein is affected, and thus has the potential to unify the treatment for millions of patients with Stop Codon Disease. 

RA: Has tRNA been overlooked as a biological framework to treat disease?

CK: Part of the reason that the therapeutic potential of tRNAs has been overlooked until recently, despite dramatic advances in genomics technologies, is due to our perspectives on genetic disorders. Understanding the genome and the roles of genes in human disease led us to take a gene-centric view, which focused the industry’s attention on the DNA or mRNA sequences responsible for production of the faulty proteins. The genetic medicines generated in the last few decades show how transformative this approach has been. Within this context, few were able to see tRNAs as anything more than intermediaries in protein synthesis within cells.

Further limiting efforts to see the therapeutic potential of tRNAs was the lack of scientific tools needed to quantify and characterize these molecules in cells and – even more importantly – in tissues. Progress in this direction was greatly facilitated by advances in tools to characterize mRNAs and small-interfering or siRNAs. But not all RNAs are created equal: tRNAs offer unique challenges in terms of sequencing and chemical synthesis. Those challenges were further exacerbated by the need to identify and understand the complex modification patterns that are typical for tRNAs. Technological advances across multiple disciplines were required before the field could move forward.

Over the last six years, Alltrna has developed many innovative technologies and approaches to address these challenges, and already, has made great strides in deepening our understanding of tRNA biology and its impacts on human health and disease. That understanding comprises not just its roles in protein translation, but also in other cellular and metabolic mechanisms.

RA: How can AI and ML be used to help with drug discovery in this sector? 

CK: One of the key technological innovations facilitating this research has been the rapid expansion of artificial intelligence (AI) and machine learning (ML) tools.

Although tRNAs are relatively small – on the order of 70 to 80 nucleotides – there is incredible diversity across organisms in terms of the genes encoding tRNAs. This nucleotide-level diversity is further enhanced when one also considers the dozens of potential post-transcriptional modifications that can occur to those nucleotides.

Each of these possible variations has an impact on both tRNA function — its potential to be aminoacylated or ‘charged’ with an amino acid and its anticodon pairing properties — and its structure and stability within the cell. AI and ML tools allow researchers to evaluate the potential impacts of nucleotide sequences and chemical modifications on tRNA structure and function. Those understandings can then facilitate the design of tRNAs that have been optimised for therapeutic and pharmacological parameters such as efficacy, bioavailability, stability, toxicity, immunogenicity, manufacturability and others.

RA: How much potential do tRNA medicines have in treating a wide range of rare diseases given that many can carry the same underlying genetic mutation?

CK: As mentioned earlier, Stop Codon Disease occurs when a mutation converts what was an amino-acid-encoding codon into a premature termination codon (PTC), which stops protein translation prematurely and leads to disease. 

Alltrna is engineering tRNAs to recognise PTCs and insert the appropriate amino acid into the polypeptide chain, restoring production of full-length, functional proteins and reversing disease. 

What makes this approach different from other treatments for genetic disease, such as gene therapy, gene-editing or other RNA-based treatments is that tRNA-based therapeutics, because of their unique role in mRNA translation, can be gene- and disease-agnostic, rather than gene-specific. They have the potential to resolve the PTC error regardless of gene or disease.

Thus, rather than only being able to test a given treatment in a patient population that shares the same disease, tRNA therapeutic candidates can be tested in a patient population that shares the same PTC, which can occur in a diverse range of patients with different diseases. This is vital within the rare and ultra-rare disease space where individual patient populations can be so small as to make clinical trials less financially feasible.

RA: Could you discuss Alltrna’s focus on correcting premature termination codons and the prevalence of PTCs in rare diseases? 

CK: Developing and leveraging the AI/ML and technological tools mentioned earlier, Alltrna is designing novel tRNAs that can read PTCs and insert the amino acid that is originally encoded. In this way, we hope to rescue protein translation across several PTC-related diseases using a single engineered molecule. Already, in preclinical research, with a single individual tRNA, we have been able to upregulate protein expression in 25 different disease models across 14 different genes, regardless of PTC location within those genes.

This gene- and disease-agnostic approach could be transformative for patients as it has been estimated that among the 6,000-plus genetic diseases, PTCs account for about 10% of patients globally or approximately 30 million people.

As suggested above, most Stop Codon Diseases are rare or ultra-rare, and so their small patient populations have made clinical development more financially challenging. Having a single, purpose-built tRNA able to treat patients across multiple diseases, however, transforms the math and makes the once financially intractable tractable.

RA: Why do tRNAs make natural drugs?

CK: Ironically, the same molecular diversity that demanded the development and application of AI and ML tools is also a natural advantage in the development of tRNAs as drugs. Given that there are only 61 possible amino acid-coding codons (43 combinations less three stop codons), nature theoretically only requires 61 tRNA genes, and yet, many organisms have hundreds of tRNA genes. For example, humans produce more than 270 different tRNAs from 450 unique and duplicated genes, and zebrafish have more than 12,000 tRNA genes.

This diversity means that there is incredible flexibility in tRNA sequence in terms of function and structure, which allows Alltrna to leverage its computational expertise to explore the combined sequence and modification space to design therapeutic molecules optimised for innumerable characteristics important to drug design. These can range from biological activity and efficacy to tolerability, manufacturability and stability, among others.

The small size of the molecules, compared to other nucleic acid-based medicines, is also a significant advantage in designing tRNA therapeutics. Gene and mRNA-based medicines, for example, may contain thousands of nucleotides, which can complicate synthesis or formulation into delivery vehicles such as lipid nanoparticles (LNPs) and thereby limit therapeutic payload. The 70 to 80 nucleotides of a tRNA, in contrast, permit many more tRNA copies to be loaded within each LNP, offering possible advantages in dose levels and dosing frequency.

RA: What other therapeutic spaces can you target with tRNA medicines? 

CK: Although Alltrna’s initial focus is on engineering tRNAs that facilitate PTC readthrough and address Stop Codon Diseases, the same technology could potentially be extended to other genetic changes that disrupt protein production. One such change is a frameshift mutation, where nucleotides are inserted into or deleted from a gene, causing a shift in the codon triplets and large-scale changes in the resulting protein sequence. A smaller change is the missense mutation, where a single codon is altered, resulting in a protein where one amino acid has changed. In both cases, it may be possible to engineer a tRNA that overcomes the mutation, reversing or modulating the resulting disease.

There are other disease-related biological mechanisms that we might be able to influence with a tRNA-based therapy, and we expect to pursue these in time.

RA: What are the major challenges of developing tRNA therapies? 

CK: A major challenge with any drug treatment is ensuring the drug gets to the tissues and organs where it is most needed and avoiding others where it might trigger adverse events. This challenge can be exacerbated with genomic interventions, whether DNA and RNA, as these molecules can quickly be degraded by nucleases within the body and can trigger immune responses from a body primed to watch for external nucleic acids.

Alongside our efforts to engineer therapeutic tRNAs, Alltrna is also exploring and optimising the design of delivery vehicles, focusing initially on LNPs. The goal is to find vehicles that promote appropriate and optimal delivery to target tissues while protecting the tRNA from degradation and the patient from unwelcome immune responses.

Another challenge for the development of RNA-based medicines, let alone therapeutic tRNAs, has been the chemical synthesis of these molecules. The methods used to produce the much larger mRNA molecules, such as chemo-enzymatic synthesis and in vitro transcription, have not proven as effective or efficient in the production of tRNAs. Alltrna has therefore focused on developing new RNA synthesis technologies to improve production in terms of efficiency, reproducibility and scalability.

The myriad of chemical modifications mentioned earlier that significantly impact tRNA structure and function add another layer of complexity. Molecular characterisation and the application of AI and ML tools will be critical to teasing out the biological implications of each of these modifications and their locations within different tRNA species, and then applying that understanding to the design and optimisation of therapeutic tRNAs.

From DDW Volume 25 – Issue 4, Fall 2024 Read the digital issue

Reference:

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10044939/

Biography

Caroline Kӧhrer, PhD is Vice President, Discovery Platform, Alltrna. Dr Kӧhrer was Director of RNA Science at Moderna, where she developed mRNA-based technology as a new drug modality. Previous to Moderna, she spent 20 years at MIT performing research focused on protein synthesis across all kingdoms of life, with an emphasis on tRNA and RNA modifications.