Mechanisms and Delivery of tRNA Therapeutics

Abstract

Transfer ribonucleic acid (tRNA) therapeutics will provide personalized and mutation specific medicines to treat human genetic diseases for which no cures currently exist. The tRNAs are a family of adaptor molecules that interpret the nucleic acid sequences in our genes into the amino acid sequences of proteins that dictate cell function. Humans encode more than 600 tRNA genes. Interestingly, even healthy individuals contain some mutant tRNAs that make mistakes. Missense suppressor tRNAs insert the wrong amino acid in proteins, and nonsense suppressor tRNAs read through premature stop signals to generate full length proteins. Mutations that underlie many human diseases, including neurodegenerative diseases, cancers, and diverse rare genetic disorders, result from missense or nonsense mutations. Thus, specific tRNA variants can be strategically deployed as therapeutic agents to correct genetic defects. We review the mechanisms of tRNA therapeutic activity, the nature of the therapeutic window for nonsense and missense suppression as well as wild-type tRNA supplementation. We discuss the challenges and promises of delivering tRNAs as synthetic RNAs or as gene therapies. Together, tRNA medicines will provide novel treatments for common and rare genetic diseases in humans.

Figure 1

Schematic of tRNA aminoacylation and protein synthesis on the ribosome. (A) Amino acids (AA) are first activated through adenylation by their cognate aminoacyl-tRNA synthetase (aaRS) in an ATP-dependent manner. The aminoacyl-adenylate remains bound in the catalytic domain as a cognate tRNA is selectively recognized and bound to the aaRS. The aaRSs next catalyze a nucleophilic attack on the carboxyl carbon of the aminoacyl-adenylate by the 2’ or 3′-hydroxyl of the bound tRNA, resulting in an aminoacyl-tRNA product. (B) In complex with an elongation factor (not shown), aminoacyl-tRNA is then recruited to the ribosome where the tRNA recognizes complementary codons (N₁ N₂ N₃) in the mRNA via base pairing with the anticodon (green bases).

Figure 2

tRNA expression and processing in mammalian cells. RNA polymerase III is recruited to the tRNA gene by TFIIIB and TFIIIC, which are bound to a site upstream of the tRNA. Transcription of the tRNA gene (above) begins at the annotated 5′ end and continues until RNA polymerase III reaches the 3′-polyU terminator. Upstream and downstream enhancer elements vary between tRNA genes and contribute to differential expression depending on cell type. The resulting pre-tRNA undergoes several base modifications (examples of frequent modification sites are indicated by purple circles) and in some cases intron (orange) splicing before folding into its canonical 3D L-shaped structure with the help of tRNA chaperones. The 5′ leader and 3′ trailer sequence (yellow) are trimmed by RNaseP and RNaseZ respectively. After degradation of the 3′ trailer sequence, a 3′ terminal CCA sequence is covalently ligated to the tRNA and the resulting mature tRNA is exported to the cytoplasm where it becomes aminoacylated and participates in translation.

Figure 3

Consequences of aaRS disease-causing variants and cognate tRNA therapeutics. Mutated aaRSs can cause cell stress and disease through a variety of mechanisms. Pathogenic mutations in aaRSs can catalyze mis-aminoacylation of tRNAs as observed for pathogenic HisRS mutants that lead to mistranslation of the proteome. Additionally, aaRS mutations can cause reduced activity or stability known as aaRS deficiency or increased tRNA binding affinity, such as in a pathogenic GlyRS mutation that sequesters aminoacyl-tRNAs and reduces the availability of aminoacyl-tRNA for protein synthesis, leading to ribosome stalling. Ribosome stalling reduces translation efficiency and generates truncated or non-native polypeptides. Each mechanism can result in misfolded or aggregating proteins and proteotoxic stress. Supplementation with cognate tRNAs to individuals afflicted with aaRS mutations is a potential therapeutic route that can restore translation fidelity, efficiency, and protein homeostasis by increasing cognate tRNA availability for variants that are deficient or dysfunctional in aminoacylation or variants that sequester tRNAs.

Figure 4

tRNA and small molecule nonsense suppressors. Premature termination codons (PTCs) can cause disease through the production of truncated protein from premature translation termination, loss of gene expression through nonsense-mediated decay (NMD), or a combination of both processes. Both mechanisms rely on binding of the eukaryotic release factor ternary complex, as release factors are key components of the SMG-1-Upf1-eRF1-eRF3 (SURF) complex that triggers NMD. Nonsense suppressor tRNAs and small molecule translational readthrough drugs (TRIDs) function as therapeutics by competing with release factors for stop codon recognition. Nonsense suppressor tRNAs directly compete with release factors, by decoding UGA, UAG, or UAA stop codons via their mutated anticodon, while TRIDs bind to the ribosome, allowing near cognate tRNAs to better recognize PTCs. Nonsense suppressor tRNAs have a distinct advantage over TRIDs as they recognize a specific stop codon, and deliver a single amino acid, offering the potential to restore a PTC-containing protein to wild-type sequence. TRIDs enhance recognition of multiple near cognate tRNAs to decode PTCs, resulting in heterogeneity of the rescued polypeptides that may differ from wild-type protein sequence.

Figure 5

Missense suppressor tRNA. (A) Wild-type tRNA^Ala recognizes its complementary GCU codon via its anticodon. Alanine can then be incorporated into the nascent polypeptide chain, resulting in a wild-type polypeptide sequence. (B) A cytosine to guanine mutation results in the change in mRNA sequence from an alanine codon to a glycine codon. This causes recruitment of tRNA^Gly and recognition through wobble base-pairing, resulting in a mutant polypeptide sequence. (C) A missense suppressor tRNA^Ala with a G35C mutation recognizes the glycine codon through base pairing, but instead delivers alanine, resulting in a wild-type polypeptide sequence, correcting the genetic mutation during translation.

Figure 6

Schematic of dual missense and nonsense fluorescent reporters. Dual fluorescent reporters often consist of a green fluorescent protein (GFP) coupled to an mCherry protein that is sensitive to mistranslation. Mutations to key residues essential for mCherry fluorescence can be exploited to measure mistranslation or nonsense suppression levels relative to the GFP protein. Missense suppressor tRNAs that correct to wild-type sequence restore mCherry fluorescence. The ratio of red to green fluorescence can be measured to evaluate the efficiency of missense suppression. A similar approach can be applied for nonsense suppression. A PTC leads to truncation of mCherry and abolishes fluorescence, which can be rescued by a nonsense suppressor tRNA.

Figure 7

Schematic of delivery of tRNA as synthetic RNA or a gene. (A) Therapeutic tRNA is delivered as a transgene through a viral vector. The viral vector enters the cell through receptor-mediated endocytosis, escapes the endosome and is imported to the nucleus where the transgene can express endogenous levels of therapeutic tRNA. (B) Delivery of synthetic tRNA or tDNA containing tRNA genes via lipid nanoparticles. Cationic lipids allow for binding to cell membrane and triggering of endocytosis, while helper fusogenic lipids facilitate endosomal escape. (C) Cationic polymer nanoparticles package negatively charged amino acids and penetrate cell membranes by triggering endocytosis. Endosomal escape can be achieved through pH-dependent depolymerization or electrostatic interactions. Synthetic tRNA can immediately participate in translation, while plasmid DNAs containing tRNA genes are imported to the nucleus and stably express as tRNAs in cells (B and C).