Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs

RESCUE OF LRBA GENE EXPRESSION IN PRIMARY HUMAN FIBROBLASTS CHARACTERISED BY NONSENSE MUTATION c. 5047 (C>T).

Primary immunodeficiencies (PIDs) are rare genetic diseases characterized by susceptibility to infections, increased risk of autoimmunity, hypogammaglobulinemia, and lymphoproliferative syndromes. PIDs are associated to genetic alterations in about 400 known genes, among which, mutations of the LRBA gene. LRBA gene encodes a widely expressed multi-domain protein with highly conserved BEACH domain, involved in regulation of endosomal trafficking, particularly endocytosis of ligand-activated receptors. It was reported that stop mutations affect this gene leading to the loss of the protein expression. Recently, we identified three Translational Readthrough Inducing Drug (TRID), that showed high readthrough activity in cystic fibrosis nonsense model systems, in this study we tested one of our TRIDs in a LRBA model system. To evaluate the readthrough activity of the TRID molecule in the rescue of the LRBA expression in human primary fibroblasts characterized by an homozygous stop mutation, we treated LRBAstop cells with compound 1 after 24, 48 and 72 hours of treatment. The expression of the LRBA gene was analyzed by Real time RT-PCR and Western blot analyses. LRBA expression resulted increased after 72-hours of chronic treatment with our TRID molecule and the cells showed to growth normally. Our results confirmed that compound 1 is able to promote the readthrough of premature stop codon, besides in CF model systems, also in a different genetic context such as in LRBA expression

Translational readthrough inducing drugs: a study of toxicity in mice models and in vitro safety validation of the specific readthrough process.

Objective Nonsense mutations are responsible for 15% of Cystic Fibrosis (CF) patients due to the introduction of a premature stop codon (PTC) in the mRNA and the production of a truncated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein1. A promising therapeutic approach for stop mutations is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs) to restore the expression of the protein2,3. Recently three new TRIDS (NV848, NV914, NV930) have been proposed and validated by several assays. Our work was focused on TRIDs NV848, NV914, NV930. Important aspects of TRIDs to be evaluated are their specificity towards PTC, to demonstrate that TRIDs do not interfere with the normal translation process4 in vitro, and their in vivo toxicity in mice models, to assess their tolerability at high doses. Methods For the toxicity study, mice were treated with an acute dose as reported in standard protocols5, monitored and then sacrificed to collect organs. To study the effects of the TRIDs on natural termination codons (NTC) we have examined the molecular weight and functional activity of a panel of proteins. Results Acute toxicity studies evidenced that all three molecules used have been well tolerated in all subjects treated. Histological analyses have not shown any tissue damage. So, we can assess TRIDs are safe in vivo also at the high doses tested. About NTC preservation, experiments have not shown any impairment or dysfunction in all samples. Indeed, no aberrant protein form has been detected. Conclusions In conclusion, our experiments identified the acute dosage for each TRIDs and their tolerability also at elevated doses, encouraging to test their efficacy in in vivo CF models. Accordingly, the in vitro analyses have suggested that these TRIDs do not lead to jeopardise NTC integrity, assessing their promising use for PTC readthrough. Consequently, our results could be indicative of TRIDs safety in in vitro models, laying the basis for further investigations. Bibliography 1. Giordani B, Amato A, Majo F, Ferrari G, Quattrucci S, Minicucci L, Padoan R, Floridia G, Salvatore D, Carnovale V, Puppo Fornaro G, Taruscio D, Salvatore M; Gruppo di lavoro RIFC. Italian Cystic Fibrosis Registry (ICFR). Report 2015-2016]. Epidemiol Prev. 2019 Jul-Aug;43(4S1):1-36. 2. Pibiri I., A., Tutone, M., Lentini, L., Melfi, R., & DI LEONARDO, A. et al. Oxadiazole Derivatives For The Treatment Of Genetic Diseases Due To Nonsense Mutations, PCT Int. Appl. (2019), WO 2019/101709 A1 20190531. 3. Pibiri I, Melfi R, Tutone M, Di Leonardo A, Pace A, Lentini L. Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems. Int J Mol Sci. 2020 Sep 3;21(17):6420. doi: 10.3390/ijms21176420. PMID: 32899265; PMCID: PMC7504161 4. Grover R, Candeias MM, Fåhraeus R, Das S. p53 and little brother p53/47: linking IRES activities with protein functions. Oncogene. 2009 Jul 30;28(30):2766-72. doi: 10.1038/onc.2009.138. Epub 2009 Jun 1. PMID: 19483723. 5. OECD (1992). OECD Guidelines for the Testing of Chemicals No. 420: Acute Oral Toxicity Fixed Dose Method, 7pp. Paris, France: OECD

Inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase as possible mechanism to readthrough premature termination codons (UGAs) of the CFTR mRNA

Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10 % of the mutations affecting the CFTR gene are "stop" mutations, which generate a Premature Termination Codon (PTC), thus resulting in the synthesis of a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, that is the capacity of the ribosome to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough and for some of them the mechanism of action is still under debate. By in silico analysis as well as in vitro studies, we investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914 and NV930, could exert their readthrough activity. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase. In addition, we report that our TRIDs do not exert readthrough on natural termination codons

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with a single dose of TRIDs molecules or Ataluren, an FDA-approved TRID molecule, as control. Mice were observed continuously for the first day post-drugs administration and the behavioral changes were recorded. On the 15th day, animals were sacrificed for histological examinations. The results showed that acute administration of 2000 mg/kg of NV914 and Ataluren and 300 mg/kg of NV848 or NV930, did not induce any mortality within 14 days. Moreover, histopathological analysis of treated mice showed no differences when compared to the experimental controls. In summary, our results suggest a good tolerability for the three molecules, and include NV848 and NV930 in a category 4 and NV914 in a category 5 of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals, classifying these compounds in a low-risk scale for health