In recent years, rapid advancements in gene therapy have brought about a significant transformation in modern medicine. The application of advanced molecular biology techniques and enhanced delivery systems has ushered in the era of personalized gene precision therapy. One notable breakthrough occurred in the global fight against the SARS-CoV-2 pandemic, with the remarkable success of mRNA vaccines, opening the doors to countless possibilities and inspiring exploration into innovative RNA therapies for a range of previously untreatable human diseases.
mRNA plays a crucial role in transmitting genetic information from DNA to the ribosome, the large molecular machinery responsible for cellular biosynthesis. To accurately decipher the genetic information embedded in mRNA, the ribosome relies on transfer RNA (tRNA) assistance. Traditionally regarded as interpreters of the genetic code, tRNA converts nucleotide identities into amino acids through complementary base pairing between mRNA codons and the corresponding tRNA anticodons. The four nucleotides in mRNA form 64 unique codons, comprising 61 sense codons and 3 stop codons. Certain gene mutations can convert 18 of the 61 sense codons into stop codons, leading to premature termination of protein translation and the development of dysfunctional disease phenotypes. These mutations account for approximately 11% of genetic diseases, making them a major mutation type causing diseases in the overall human population. tRNA therapy can counteract the effects of mutations by supplementing affected tRNA, thereby restoring protein synthesis and function.
However, tRNA faces numerous challenges as a therapeutic drug, including issues of inefficiency, instability, immunogenicity, and safety concerns, all of which have hindered their clinical application. Nevertheless, over the past three decades, technological breakthroughs in mRNA and small RNA therapies have begun to address many of these challenges. This includes enhancing stability and reducing immunogenicity by employing modified nucleotides and developing various materials for efficient encapsulation and delivery of RNA.
A Brief History of tRNA Therapy
Half a century ago, three pivotal studies laid the groundwork for tRNA gene therapy. Sveda et al. fused erythrocytes pre-loaded with heterologous natural sup-tRNA (from Escherichia coli or yeast) with murine cells expressing truncated hypoxanthine-guanine phosphoribosyltransferase (HGPRT), resulting in the restoration of full-length protein expression. In a separate study, Kan et al. introduced natural yeast sup-tRNASer into a solution of red blood cells obtained from a β-thalassemia patient. This patient’s β-globin gene harbored a nonsense mutation at the lysine AAG codon. The study successfully repaired up to 10% of full-length β-globin in the patient’s cells.
To broaden the application of sup-tRNA in various tissues and disease contexts, two approaches have been employed for sup-tRNA supplementation: (1) as plasmid-encoded sup-tRNA genes, expressed in the cell nucleus through exonic expression, and (2) transfection of in vitro Transcription(IVT) sup-tRNA into the cytoplasm of recipient cells. However, the activity of these sup-tRNAs in clinically relevant model systems has been inefficient. Challenges such as low activity even at higher doses in multi-gene copy supplementation and the development of delivery platforms for early-stage clinical applications have impeded clinical progress.
The past decade has witnessed novel mechanistic discoveries linking tRNA to pathologically relevant abnormalities, proposing tRNA as a new therapeutic avenue. Two recent studies indicate promising therapeutic outcomes using AAV or LNPs for tRNA delivery in preclinical experiments. In the future, the development of more RNA gene therapy delivery systems is expected to advance the application of tRNA-based therapy in treating other diseases.
Applications of tRNA Therapy
There are two types of monogenic diseases that may be addressed through tRNA gene therapy.
(1) diseases associated with nonsense mutations.
(2) diseases associated with AARS mutations related to tRNA depletion.
Correction Therapy Using Sup-tRNAs
Nonsense mutations are a common type of disease-associated mutation that introduces premature termination codons (PTCs), leading to premature termination during the protein synthesis process. To suppress PTCs associated with nonsense mutations and introduce the correct amino acids, 19 types of sup-tRNAs are required. These sup-tRNAs exhibit sufficient activity to support the production of the desired protein during the translation process, reaching the threshold required for therapeutic purposes.
One notable advantage of sup-tRNA is that a single therapeutic agent can be used to treat various diseases associated with nonsense mutations. For example, the most common nonsense mutation involves the substitution of the arginine CGA codon with the UGA premature termination codon (PTC). Therefore, a potent sup-tRNA can be employed to treat nonsense mutations in various diseases associated with the arginine codon. However, challenges to address include sequence context-driven changes in readthrough efficiency and differences in tissue-specific delivery.
Sup-tRNA selectively acts on the natural transcriptome of the affected gene, eliminating the risk of overexpression. Thus, sup-tRNA is highly suitable for treating diseases associated with nonsense mutations in genes that require precise regulation. An example is the MeCP2 gene, a well-known Goldilocks gene. Lack of MeCP2 leads to Rett syndrome, while overexpression results in MECP2 duplication syndrome.
In certain cases, due to the evolutionary age and selectivity of tRNA, engineered modifications may not render it resistant to decoding PTC, or the inhibitory potency may be insufficient to achieve therapeutic effects. In such instances, powerful sup-tRNA carrying different amino acids can be used to restore translation of PTC. This strategy mimics missense mutations and may be applicable to specific proteins. For example, the myotubularin gene associated with Duchenne muscular dystrophy can tolerate missense mutations, while disease-associated proteins in Dravet syndrome or cystic fibrosis are sensitive to missense mutations. Combining sup-tRNA with approved therapeutic approaches can mitigate the misfolding effects of erroneously inserted amino acids caused by sup-tRNA.
tRNA Supplementation Therapy
Recent research indicates that certain diseases associated with different pathologies share characteristics of transient or permanent depletion of tRNA isoacceptor families. This depletion leads to abnormal translation of the affected codons. For instance, Charcot-Marie-Tooth (CMT) pathology associated with heterozygous mutations in several AARS genes is a typical example. The hallmark of this pathology is length-dependent axonal atrophy and degeneration.
In most cases, mutations in AARS genes do not alter the enzyme’s aminoacylation activity, suggesting that the loss of enzymatic activity is not a prerequisite for the disease. Recent studies have found that mutations in glycyl-tRNA synthetase associated with CMT alter the dynamics of tRNAsGly release. This transient tRNAsGly fixation significantly reduces translation speed for all four glycine codons, alters the expression of glycine-containing transcripts, and activates an integrated stress response.
Increasing the copy number of the most abundant tRNAGly(GCC) in CMT mice alleviated atrophy and degeneration, suggesting that supplementing the corresponding tRNA may be a suitable therapeutic strategy. Elevating the levels of major tRNA isoacceptors is sufficient to overcome tRNA fixation caused by mutations in glycyl-tRNA synthetase. However, a more favorable approach is to use a mixture containing all isoacceptors corresponding to the mutated AARS, with proportions mimicking their natural concentrations. This ensures overall balance in restoring translation function across all glycine codons, leading to enhanced therapeutic efficacy.
tRNA Delivery for Therapeutic Applications
To fully harness the potential of tRNA in precision treatment for monogenic diseases, specific and tailored strategies related to tRNA loading need to be developed. Due to its negative charge and susceptibility to degradation, delivering tRNA into cells poses a challenge. Therefore, efficient delivery to specific tissues or cell types is crucial to ensure the clinical effectiveness of tRNA gene therapy.
Significant progress has been made in both viral and non-viral delivery systems, offering feasible options for delivering various RNA payloads and targeting different tissues. While the potential of tRNA as a therapeutic agent has only recently been recognized, and no clinical studies have been conducted yet, recent research indicates that using delivery platforms developed for other RNAs can successfully administer sup-tRNA, supporting the potential applicability of tRNA therapeutic drugs.
To design clinically relevant drug delivery vehicles for transporting tRNA, considering the mechanism of action and the tissue starting points corresponding to the pathology is crucial. Virus-derived carriers or exosomes exhibit natural tropism for crossing the blood-brain barrier (BBB), making them attractive for diseases with the central or peripheral nervous system as the primary affected tissue. Synthetic carriers such as liposomes and biocompatible polymers may be more suitable for repeated systemic administration and offer a supplementary option for tissues inaccessible to AAV carriers, such as the kidneys and lungs.
Biocompatible Polyethylene Oxide–Polypropylene Oxide Copolymers are emerging as potential substitutes for LNPs or AAV as carriers for tRNA. Leveraging the low immunogenicity and excellent safety profile of polyethylene oxide, a self-assembling particle has been developed for the delivery of plasmid DNA to the lungs. This system comprises three synthetic peptides: an anchoring peptide with a hydrophobic block from polyethylene oxide, a cationic segment containing basic amino acids for nucleic acid encapsulation and endosomal escape promotion, and a targeting block for directing particles to specific tissues. These particles have been tested in vitro, cell models, and mouse models for mRNA replacement therapy in cystic fibrosis.
The Clinical Trial Journey of tRNA
tRNA therapy represents an emerging treatment approach with high expectations for its potential and prospects. Currently, all tRNA therapy platforms are still in the discovery or preclinical development stages, but significant progress has been made. As research on tRNA therapy continues to advance, clinical trials for tRNA therapy are becoming an inevitable trend.
Clinical trials using tRNA therapy require consideration of safety, efficacy, and regulatory compliance. Preclinical studies are crucial for establishing the pharmacological and toxicological characteristics of tRNA therapy. These studies need to comprehensively assess the biological distribution, metabolism, and potential adverse reactions of tRNA molecules to provide support for the safety and efficacy of clinical trials.
The potential immunogenicity and risk of immune reactions to exogenous tRNA molecules must be carefully assessed. Quantifiable disease biomarkers and reliable methods for quantifying and characterizing tRNA molecules in biological samples should be established to ensure accurate analysis and comparison during clinical trials. It is essential to closely collaborate with regulatory agencies throughout the trial process and adhere to ethical guidelines to ensure the safety and reliability of tRNA therapy.
Prospects for tRNA Therapy
tRNA therapy is a novel gene therapy approach with the potential to treat various diseases. Currently, most research is still in the preclinical stages, but several companies are exploring the therapeutic possibilities of tRNA. While it may take a few more years for the first tRNA therapy to receive clinical trial approval, breakthroughs in tRNA biology and design, nucleotide chemistry, delivery systems, and bioinformatics are paving the way. This concept of tRNA therapy, which has a history of over half a century, may soon enter a new era as an innovative treatment modality for monogenic diseases.
References
1. A, k, Irem.; et al. Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology. Sec. RNA Networks and Biology. 2020, 7.
2. J, D, Lueck.; et al. Engineered Transfer RNAs for Suppression of Premature termination Codons. Nature Communications. 2019, 10: 882.