Nonsense suppression rescues functional protein from mRNAs containing in-frame premature termination codons (PTCs). This approach employs small molecules that promote insertion of tRNAs at a PTC during translation (called "read-through"), enabling translation to continue past the PTC to generate a full-length, functional protein. Some aminoglycosides such as G418 promote readthrough; however, their long-term use is thwarted by toxicity and modest potency. ELX-02, a novel synthetic aminoglycoside, was developed to have both enhanced readthrough activity and reduced toxicity in mammalian cells compared to traditional aminoglycosides. Recent studies suggest that at safe doses, aminoglycosides like ELX-02 may not possess sufficient efficacy to provide clinical benefits for most diseases. "Enhancer" molecules have been identified that do not possess readthrough activity themselves but enhance the efficiency of aminoglycoside-mediated read-through. In this study, three newly identified series of enhancer molecules were tested for the ability to stimulate aminoglycoside-mediated readthrough of PTCs associated with cystic fibrosis and neurofibromatosis type 1. Although none of the enhancers alone induced readthrough, they all significantly increased readthrough via aminoglycosides at low doses (EC10). Overall, our results suggest that enhancer compounds may be a viable way to overcome potency issues associated with aminoglycosides, enabling rescue of protein function from nonsense alleles while minimizing toxicity.

Background Cystic fibrosis patients who carry a CFTR nonsense allele often express negligible CFTR protein, and thus, are unresponsive to CFTR modulators. Nonsense suppression (also called readthrough) is an emerging therapeutic approach for this patient subgroup that uses small molecules to suppress translation termination at in-frame premature termination codons (PTCs) and rescue full-length, functional CFTR protein. This study examines mechanistic aspects of readthrough at six CFTR PTCs commonly found in CF patients. Methods CFTR expression and chloride conductance were used to assess the responsiveness of six different CFTR PTCs to G418-mediated readthrough. LC-MS/MS was used to identify the CFTR variant proteins generated by readthrough of PTCs in their native, local CFTR mRNA sequence context. For each CFTR variant protein identified, the abundance, processing, activity, and responsiveness to CFTR modulators were characterized. Results CFTR expression and function varied widely among PTCs, with UGA generally being the most responsive to G418-mediated readthrough. The amino acids incorporated at PTCs during G418-induced readthrough also varied, depending on the PTC and its surrounding local mRNA context. Modulators stabilized and enhanced the abundance and activity of most CFTR variant proteins generated by PTC readthrough, with many variant proteins reaching WT CFTR activity. Conclusions Nonsense suppression therapy shows promise as a treatment for CF patients who carry a PTC, especially when combined with current CFTR modulators. Mechanistic insights of readthrough gleaned from this study can be used to develop better therapeutic strategies for treating CF patients who carry a nonsense mutation.

Rare diseases affect almost 6% of the world's population and are responsible for significant loss of health, life and economic potential. The vast majority of rare diseases are due to genetic variants that result in the loss or alteration of a protein that is needed for cells and organs to function normally. Few rare diseases have an approved treatment, making individuals with rare diseases collectively one of the most underserved communities in medicine today. The few treatments that have been available typically have focused on disease symptoms, without correcting or addressing the cause of disease, the underlying mutation. Hence, there is a drive to develop therapeutics that repair, replace, or alter DNA, RNA, or proteins responsible for rare disease. Herein, we focus on mutation targeted approaches including gene editing, gene replacement, RNA editing, and gene/protein regulation modalities including antisense oligonucleotides, nonsense suppression therapeutics, and protein modulators. We discuss how each approach works, what types of variants might be targeted, advantages, disadvantages, and successes. We also highlight other concerns such as delivery methods and the need to consider patient perspectives. Advocacy groups have been instrumental in rare disease therapeutics.

Protein synthesis terminates when a stop codon enters the ribosome’s A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3’-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient’s likelihood of improvement in response to drugs given their nonsense mutation sequence context.

specific expression of the split-ABE to understand which cell types, and how many of them, must be corrected to restore CFTR function to therapeutic levels using our BCi W1282X-CFTR model.

absence of a Cl gradient ( p < 0.0001).The variability in LUNAR-CFTRinduced Cl currents between CF hBEC donors mirrored the transfection efficiencies observed with TdT expression.The stimulated I sc measured in the absence of a Cl gradient was approximately 4 times as great in LUNAR-CFTR-treated cells as in LUNAR-TdT-treated cells and comparable with elexacaftor-tezacaftor-ivacaftor (ETI)-corrected I sc , which remained at approximately 50% of wt hBECs, although in the presence of Cl gradient, the mean I sc driven by LUNAR-CFTR was comparable with that in wt cells under similar conditions.Conclusions: LUNAR-CFTR restored CFTR expression and CFTR-dependent Cl transport in differentiated primary F508del CF hBECs in the presence or absence of a Cl gradient.Absolute current magnitude after LUNAR-CFTR treatment was comparable with that in wt controls in the presence of a Cl gradient and equivalent to ETI in its absence.Immunohistochemistry characterization is in progress to evaluate CFTR-transfected cell populations contributing to Cl transport in CF hBECs.These results support using LUNAR-CFTR for the treatment of CF.

analyzer.After normalization to cell number, basal OCR results indicated that 16hBE cells with delF508-CFTR exhibited lower adenosine triphosphate-linked respiration than wt-CFTR cells.16hBE cells with delF508-CFTR had lower maximal respiration as well, especially than that of wt.VX-770 alone, without a corrector, significantly improved oxidative phosphorylation in 16hBE cells with delF508-CFTR but not in wt-CFTR cells.CB-839, the glutaminase-1 inhibitor, exerted a corrective effect on 16hBE cells with delF508-CFTR but not on cells with wt-CFTR.Our Ussing chamber data confirmed the results from the Seahorse analysis.We found that acute inhibition of glutaminase-1 by CB-839 slightly potentiates the function of CFTR.These data suggest that inhibition of specific metabolic pathways such as glutaminolysis could be beneficial to PwCF.Conclusions: Our study has established a rationale for testing the effect of glutaminase-1 inhibitors, particularly CB-839, in primary airway epithelial cells bearing mutant CFTR.

Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.

Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin in vitro and in vivo. Nonsense suppression did not reliably increase neurofibromin in primary NF1-'- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin in vivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinically relevant tissues, including cerebral cortex, sciatic nerve, optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic of transcripts with PTCs, which could impede nonsense neurofibromas. Thus, the effectiveness of nonsense suppresof NMD inhibitors.

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene's diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC.

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.

specific expression of the split-ABE to understand which cell types, and how many of them, must be corrected to restore CFTR function to therapeutic levels using our BCi W1282X-CFTR model.

Conclusions: αCD20 treatment before lung-directed vector delivery is a promising future direction for gene therapy, because temporary abrogation of immune responses may allow for repeated vector administration in a variety of contexts.Further research is needed to determine dose optimization and duration of effects and to test this regimen with other delivery systems including adeno-associated virus vectors.