Academic literature on the topic 'Readthrough molecule'

Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their “readthrough” based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.

Abstract Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.

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.

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators.First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments.The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient.In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.

ABSTRACT IMPACT: Small molecule readthrough compounds are a promising therapeutic with the potential to overcome nonsense mutations thereby enabling the production of functional ATM protein in patients with Ataxia Telangiectasia OBJECTIVES/GOALS: To generate a novel mouse model of Ataxia-Telangiectasia for testing small molecule readthrough compounds that both expresses a clinically relevant nonsense mutation and recapitulates the major symptoms of the disease, including a progressive loss of motor coordination not previously observed in prior A-T animal models. METHODS/STUDY POPULATION: Using a double-hit strategy to increase genotoxic stress, we generated a novel A-T mouse model that expresses a clinically relevant (c.103C>T) mutation in the Atm gene and a knockout of the functionally related Aptx gene. We then characterized the mouse across multiple domains related to the various symptoms related to the disease. This includes examination of survivability, immunologic function, cancer prevalence, and motor behavior and its associated cerebellar dysfunction and atrophy. Lastly, we tested the ability of small molecule readthrough compounds to enable production of ATM from tissue explants extracted from these ATM deficient mice. RESULTS/ANTICIPATED RESULTS: The double mutant mice display reduced survivability compared to control mice (53% vs. 97%; p<0.0001), dying at a clinically relevant rate of about 30% from thymomas. At postnatal day 400 (P400), only AtmR35X/R35X; Aptx-/- mice, and none of the controls expressing at least one wildtype Atm or Aptx gene develop a motor behavioral deficits that are associate with reduced Purkinje neuron diameter (8.0 ±0.4 µm vs. 9.92 ±0.5; p<0.01) and density (4.3 ±0.2 vs. 6.0 ±0.3 per 100 µm; p<0.05) as well as cerebellar atrophy (cerebellum/forebrain area 0.26 ±0.01 vs. 0.31 ±0.01; p<0.001). ATM deficient mice also display disrupted thymocyte development and metabolic function. When exposed to small molecular readthrough compounds, greater than 50% of the ATM protein is restored. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have created a novel, clinically relevant A-T mouse model that develops a severe ataxia associated with changes in cerebellar function and atrophy as well as demonstrate the potential of SMRT compounds as an A-T therapeutic.

Abstract Frontotemporal dementia (FTD) is an early onset dementia characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5–26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its functions under physiological and pathological conditions remains to be defined. Many FTD-causing non-sense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy. Aminoglycosides are a class of antibiotics that possess a less-known function to induce eukaryotic ribosomal readthrough of PTCs to produce a full-length protein. The aminoglycoside-induced readthrough strategy has been utilized to treat multiple human diseases caused by PTCs. In this study, we tested the only clinically approved readthrough small molecule PTC124 and 11 aminoglycosides in a cell culture system on four PTCs responsible for FTD or a related neurodegenerative disease amyotrophic lateral sclerosis. We found that the aminoglycosides G418 and gentamicin rescued the expression of the progranulin R493X mutation. G418 was more effective than gentamicin (~50% rescue versus &lt;10%), and the effect was dose- and time-dependent. The progranulin readthrough protein displayed similar subcellular localization as the wild-type progranulin protein. These data provide an exciting proof-of-concept that aminoglycosides or other readthrough-promoting compounds are a therapeutic avenue for familial FTD caused by progranulin PTC mutations.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

Abstract Genetic diseases can result from nonsense mutations that cause premature translation termination. Nonsense mutations account for ~10–15% of all cases of hemophilia A and B and this population may benefit from small molecule-induced readthrough of nonsense codons. Recent studies document the ability of an orally bioavailable small molecule, PTC124, to facilitate dose-dependent readthrough of nonsense codons in a variety of in vitro and in vivo systems, including reporter gene constructs, mdx mice (a murine model of Duchenne muscular dystrophy; Nature447:87–91, 2007), and in a mouse model of cystic fibrosis (PNAS105: 2064–2069, 2008). PTC124 was well-tolerated in healthy human volunteers (J. Clin. Pharmacol.47:430–444, 2007) and in patients with cystic fibrosis (The Lancet, online August 21, 2008). We previously constructed murine models of severe hemophilia B with nonsense mutations in a transgenic human factor IX (hF.IX) gene resulting in nonsense codons at amino acid positions 29 or 338 (R29X and R338X; Blood104:2767–2774, 2004). These mice have no circulating hF.IX detectable by ELISA. To evaluate the effect of small molecules on readthrough in the hemophilia B model, we administered PTC124 to mice carrying the R338X mutation by subcutaneous injection for three days. Measurable plasma levels of hF.IX were detected by ELISA (n=3) and high levels of hF.IX were detected in the liver by immunohistochemistry in the single mouse that was sacrificed. Following oral administration of PTC124 to R338X mice for three days, 20% of mice demonstrated detectable circulating hF.IX levels in the range of 3–5 ng/mL. In a separate experiment, we administered another small molecule, PTC-EMK, by IP injection for three days to R338X mice. With this regimen, 40% of mice showed detectable circulating hF.IX levels. To examine the effect of PTC124 or PTC-EMK on other nonsense mutations, we prepared ten of the most frequently reported nonsense mutations in the hF.IX gene (R29X, R116X, W194X, R248X, R252X, Y266X, W310X, R333X, R338X, and W407X) and transiently transfected these constructs into HEK293 cells. The cells were treated with PTC124 or PTCEMK at various concentrations for 72 hrs and the level of hF.IX protein in the medium was quantified using the ELISA. Of the nonsense containing constructs analyzed, 6/10 mutants showed positive responses with the largest response noted for cells containing the R29X mutation treated with PTC-EMK. In summary, our results demonstrate the ability of PTC124 and PTC-EMK to read through nonsense codons in the hF.IX mRNA and support the potential use of orally bioavailable small molecules as a therapeutic option for patients with hemophilia due to nonsense mutations.

La maladie de Charcot-Marie-Tooth (CMT) est la neuropathie périphérique héréditaire la plus fréquente chez l’humain. Elle touche les motoneurones (MN) et les cellules de Schwann (CS). La majorité des gènes impliqués, dont SH3TC2 et GDAP1, peuvent être affectés par des mutations non-sens. En 2021, peu de modèles cellulaires humains existaient, et aucun traitement curatif n'était disponible pour les patients. Les travaux de cette thèse se centre sur SH3TC2, responsable de la forme démyélinisante autosomique récessive la plus fréquente des CMT, nommée CMT4C ou AR-CMTde-SH3TC2 et sur GDAP1 notamment responsable d’une forme axonale AR-CMTax-GDAP1. Dans un premier temps, nous avons analysé une cohorte de 103 patients mutés sur SH3TC2 et montré que plus de 80 % des patients possédaient au moins un allèle avec une mutation non-sens, associé à une gravité clinique accrue. Nous avons également identifié 22 nouvelles mutations pathogènes sur ce gène. La seconde partie de ce travail a consisté à créer les premiers modèles cellulaires neuronaux humains pour SH3TC2. À partir de cellules souches pluripotentes induites (hiPSCs) issues d’un individu contrôle, nous avons utilisé la technologie CRISPR-Cas9 pour produire, avec plus de 90% d’efficacité, deux modèles humains in vitro contenant des mutations non-sens induisant un codon stop prématuré (PTC) : un modèle homozygote p.(Arg954*) (PTC de type UGA) et un modèle homozygote p.(Gln71*) (PTC de type UAG). Ces hiPSCs contrôle et mutées ont ensuite été différenciées en CS. Nous avons mis en évidence une expression précoce de SH3TC2 dans les CS contrôle. Dans les modèles CS AR-CMTde-SH3TC2, une expression réduite de SH3TC2, un retard de maturation, une capacité réduite à soutenir les MN en coculture, et des anomalies dans le recyclage des récepteurs à la transferrine ont été observées. Enfin, nous avons testé plusieurs molécules thérapeutiques ciblant les mutations non-sens, des agents de translecture et des inhibiteurs du mécanisme de surveillance des ARN non-sens (NMDi). Sur un modèle de progéniteurs neuronaux dérivés d’hiPSCs portant la mutation homozygote non-sens p.(Ser194*) (UGA) sur GDAP1, nous avons testé une de ces molécules et montré qu’elle stabilisait l'ARNm muté GDAP1, restaurait son expression protéique et corrigeait la morphologie mitochondriale. Dans les modèles CS créés dans cette thèse pour SH3TC2, nos premiers résultats suggèrent l’effet positif de deux de ces molécules sur la réexpression de la protéine pour les deux types de codons UGA et UAG. Dans la quatrième partie de ce travail, nous avons développé un modèle 3D de coculture CS/MN permettant d’induire la myélinisation, étape ultime pour étudier les maladies démyélinisantes comme l’AR-CMTde-SH3TC2. Les molécules thérapeutiques identifiées pourront être testées sur ces modèles cellulaires de coculture et potentiellement in vivo pour évaluer leur capacité à induire une remyélinisation. Ce travail de thèse souligne l'importance des modèles cellulaires adaptés pour comprendre les mécanismes physiopathologiques de la CMT et ouvre des perspectives prometteuses pour de nouvelles approches thérapeutiques
Charcot-Marie-Tooth disease (CMT) is the most common hereditary peripheral neuropathy in humans. It affects motor neurons (MNs) and Schwann cells (SCs). Most of the genes involved, such as SH3TC2 and GDAP1, can be affected by nonsense mutations. As of 2021, few human cellular models existed, and no curative treatment was available for patients. This thesis primarily focuses on SH3TC2, responsible for the most common autosomal recessive demyelinating form of CMT, known as CMT4C or AR-CMTde-SH3TC2, and on GDAP1, notably responsible for an axonal form, AR-CMTax-GDAP1. In the first part of this work, we analyzed a cohort of 103 patients with SH3TC2 mutations and demonstrated that more than 80% of the patients carried at least one allele with a nonsense mutation, associated with increased clinical severity. We also identified 22 new pathogenic mutations in this gene. The second part of my work involved creating the first human neuronal cell models for SH3TC2. Using induced pluripotent stem cells (hiPSCs) derived from a control individual, we employed CRISPR-Cas9 technology to generate, with over 90% efficiency, two in vitro human models containing nonsense mutations inducing a premature stop codon (PTC): a homozygous p.(Arg954*) model (UGA-type PTC) and a homozygous p.(Gln71*) model (UAG-type PTC). These controls and mutated hiPSCs were then differentiated into Schwann cells (SCs). We observed early SH3TC2 expression in control SCs. In AR-CMTde-SH3TC2 SC models, reduced SH3TC2 expression, delayed maturation, impaired ability to support MNs in co-culture, and abnormalities in transferrin receptor recycling were noted. Finally, we tested several therapeutic molecules targeting nonsense mutations, including readthrough agents and inhibitors of nonsense-mediated mRNA decay (NMDi). In a model of neuronal progenitors derived from hiPSCs carrying the homozygous nonsense mutation p.(Ser194*) (UGA) on GDAP1, we tested one of these molecules and demonstrated that it stabilizes the mutated GDAP1 mRNA, restores its protein expression, and corrects mitochondrial morphology. In the SC models created in this thesis for SH3TC2, our early results suggest a positive effect of two of these molecules on protein re-expression for both UGA and UAG codons. In the fourth part of this work, we developed a 3D co-culture model of SCs/MNs that enables myelination, the ultimate step to studying demyelinating diseases such as AR-CMTde-SH3TC2. The identified therapeutic molecules can be tested on these co-culture cellular models and potentially in vivo to evaluate their capacity to induce remyelination. This thesis highlights the importance of appropriate cellular models to understand the pathophysiological mechanisms of CMT and opens promising perspectives for new therapeutic approaches

Il trattamento delle malattie genetiche, pur avendo notevolmente migliorato la qualità di vita dei pazienti, presenta spesso limiti dovuti, ad esempio, all'inaccessibilità del tessuto da trattare, alla breve emivita del farmaco, o alla difficoltà nel veicolare transgeni di grandi dimensioni. Scopo di questa tesi è stato di indagare tre approcci terapeutici alternativi per altrettante patologie genetiche, scelte come modelli paradigmatici. Nella prima parte della tesi è stata indagata l’induzione del readthrough nel contesto della malattia di Fabry, un disordine da accumulo lisosomiale dovuto a carenza dell’enzima α-galattosidasi A (AGAL). Questo approccio correttivo si basa sulla capacità di alcune molecole (e.g. aminoglicosidi) di stimolare la soppressione di mutazioni nonsenso che causerebbero la produzione di una proteina tronca, ed ha il vantaggio di sfruttare sostanze in grado di raggiungere il sistema nervoso centrale al momento non accessibile alla terapia sostitutiva disponibile sul mercato. In questo studio, l’analisi di un pannello di varianti nonsenso causanti la malattia di Fabry ha permesso di identificare tre mutazioni suscettibili alla correzione mediata dall’aminoglicoside G418, fornendo una prima dimostrazione dell’efficacia di questo approccio. I risultati ottenuti dalla co-espressione di varianti wild-type e missenso (predette derivare da readthrough) sembrano inoltre indicare un potenziale effetto negativo di alcune missenso sulla dimerizzazione e quindi sulla funzione di AGAL, un’ipotesi che verrà approfondita in studi futuri. Nella seconda parte della tesi è stata prodotta e caratterizzata una proteina di fusione tra il fattore IX della coagulazione (FIX) e l’albumina, con lo scopo di ottimizzare la terapia sostitutiva dell’emofilia B, una malattia emorragica dovuta a carenza di FIX. In particolare, sono state fuse geneticamente una variante naturale ed iperattiva del FIX e una variante dell’albumina ingegnerizzata per avere maggiore affinità per il recettore neonatale del frammento Fc e quindi emivita prolungata. Studi in vitro ed in vivo, in modelli murini specifici, hanno mostrato che questa nuova proteina di fusione è caratterizzata da maggiore attività coagulante ed emivita prolungata rispetto alla proteina di fusione attualmente in commercio. Le proprietà migliorate di questa molecola, se traslate al trattamento di pazienti affetti da emofilia B, permetterebbero di ridurre la frequenza di somministrazione e migliorare l’aderenza alla terapia e la qualità di vita dei pazienti. Nella terza parte della tesi, è stato indagato un approccio di correzione dello splicing nel contesto dell’emofilia A, una malattia emorragica causata da carenza del fattore VIII della coagulazione. L’analisi tramite minigeni di tutte le varianti riportate a carico dell’esone 19 del gene F8 ha identificato tredici varianti che causano splicing aberrante. Per correggere il processamento del messaggero è stato utilizzato un piccolo RNA nucleare modificato e specifico (ExSpeU1), in grado di reclutare il macchinario di splicing anche in presenza di mutazioni. Una prima analisi ha dimostrato l’effettiva capacità correttiva di questa molecola su cinque diverse mutazioni, sia esoniche che introniche. Studi futuri valuteranno l’efficacia dell’ExSpeU1 nel ripristinare la sintesi di una proteina funzionale (studi in vitro) e nel correggere il fenotipo patologico (in un modello murino di emofilia A). Le piccole dimensioni della cassetta di espressione dell’ExSpeU1 ne permetterebbero la veicolazione tramite vettori virali adeno-associati, attualmente considerati il sistema d’elezione per approcci di terapia genica. Nel complesso, questa tesi fornisce una dimostrazione preliminare delle potenzialità di tre diversi approcci applicati a tre patologie specifiche. Se traslate ai pazienti, queste strategie potrebbero ampliare le opzioni terapeutiche attualmente disponibili.
Currently-available treatments for genetic diseases are still hampered by limitations such as inaccessibility of specific tissues to the treatment, short half-life of infused drugs and difficulty in delivering large therapeutic transgenes. In this context, alternative approaches targeting a specific subset of patients or exploiting precise protein engineering may offer substantial improvements. The aim of this work was to explore three different targeted molecular strategies in paradigmatic genetic disease models. First, we investigated the induction of ribosome readthrough in the context of Fabry disease, a lysosomal storage disorder caused by deficiency of the lysosomal hydrolase α-galactosidase A (AGAL). We identified three nonsense mutations that, due to favourable nucleotide and protein features, could be rescued by G418-mediated readthrough induction, supporting the feasibility of this approach. Moreover, we suggested that readthrough-induction to rescue a dimeric enzyme such as AGAL may result in potentially dominant-negative effects, caused by the interaction of wild-type and missense variants producing dysfunctional heterodimers. The nonsense suppression strategy could provide remarkable advantages for the relevant subset of Fabry disease patients harbouring nonsense mutations, since readthrough-inducing compounds can reach the central nervous system, currently inaccessible to enzyme replacement therapy, and since even low levels of functional AGAL seem sufficient to ameliorate the disease phenotype. The second part of the thesis focused on the rational engineering of a novel factor IX (FIX)-albumin fusion protein to improve replacement therapy for Haemophilia B (HB), an X-linked bleeding disorder caused by deficiency of coagulation FIX. In particular, we exploited a gain-of-function FIX endowed of 8-to-15-fold improved pro-coagulant activity, as well as a rationally engineered albumin variant characterised by enhanced binding to the neonatal Fc receptor (FcRn) and thus endowed with extended half-life. Studies in a panel of mouse models with different FcRn/albumin settings showed a 2.5-fold half-life improvement of the engineered chimaera compared with the commercial fusion protein, thus supporting further studies in animal models. If translated to HB treatment, the improved features of the novel fusion protein would have the potential to address many of the current limits of replacement therapy by widening the therapeutic window and reducing injections frequency, thus ameliorating patients’ quality of life. Finally, in the third part of the thesis, a splicing correction approach was explored for Haemophilia A (HA), an X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). We first characterised all reported point mutations in exon 19 of F8 gene, identifying thirteen variants associated to aberrant splicing, including three exonic variants with no detrimental effect on FVIII secretion and cofactor activity. Subsequently, we identified a unique ExSpeU1 able to completely rescue three exonic and two intronic variants, thus widening the therapeutic potential of this molecule and providing the first proof-of-principle of this approach for HA. The short length of the ExSpeU1 cassette would represent a considerable advantage in the context of HA, since the large dimensions of F8 gene still hamper gene therapy attempts. Future studies will address this splicing correction strategy at the protein level through an in vitro expression system, and at the phenotypic level through the adeno-associated virus-mediated delivery of ExSpeU1 in a HA mouse model. Overall, this thesis provided a preliminary proof-of-concept of three different molecular strategies applied to specific disease models. If translated to patients, these alternative strategies would display relevant improvements compared to available treatments, thus supporting further investigation in this direction.

Protein synthesis terminates at the first stop codon encountered by the ribosome. In stop codon readthrough, translation termination is suppressed, enabling the ribosomes to continue translation beyond the canonical stop codon upto a downstream in-frame stop codon resulting in a C-terminally extended polypeptide. Several cis- or trans-acting elements contribute to a programmed stop codon readthrough event. Evidence of stop codon readthrough and the functional significance of the readthrough isoforms have been reported for VEGFA, AGO1, AMD1, AQP4, LDH, MDH, MTCH2 and VDR. The first evidence of functionally relevant, programmed stop codon readthrough was reported in VEGFA. In the same study, genome-wide bioinformatic based analysis was done to identify other potential readthrough candidates. NNAT (encodes Neuronatin), the focus of this study, was one of the potential readthrough candidates identified. Neuronatin (NNAT) is a small proteolipid expressed from chromosome 2 in mice and chromosome 20 in humans. The gene encoding NNAT, resides in the opposite strand within the intronic region of a larger gene, BLCAP (Bladder Cancer Associated Protein). Also known as Peg5 (Paternally Expressed Gene 5), NNAT is maternally imprinted and is thus expressed only from the paternal allele. Conservation of the proximal 3ꞌUTR of NNAT upto a second in-frame stop codon in different mammals led us to the hypothesis that NNAT mRNA undergoes stop codon readthrough. In this study, we have demonstrated readthrough in NNAT using luminescence- and fluorescence-based reporters as well as by Western blot. Analysis of previously reported ribosome profiling data coupled with the detection of endogenous NNATx (readthrough isoform) by Western blot using an antibody specific for the C-terminal extension of NNATx, confirmed stop codon readthrough in NNAT. We have also identified several cis-acting factors and an RNA-binding trans-acting factor, NONO/p54nrb, that drive stop codon readthrough in NNAT. To understand the functional role of the readthrough isoform, NNATx, we have overexpressed the canonical (NNAT) and the readthrough (NNATx) isoforms in a mouse neuroblastoma cell line, Neuro-2a. NNAT increases cytoplasmic Ca2+ levels by inhibiting SERCA2 (Sarco/endoplasmic reticulum Ca2+-ATPase isoform 2). SERCA2, a P-type ATPase, is localized on the membrane of the endoplasmic reticulum and pumps Ca2+ from the cytoplasm to the ER. By inhibiting SERCA2, NNAT increases Ca2+ levels in the cytoplasm and promotes neuronal differentiation. Unlike NNAT, the readthrough isoform, NNATx, fails to interact with SERCA2 and does not increase cytoplasmic Ca2+ levels. As a result, NNATx does not promote neuronal differentiation. To further understand the specific role of NNATx, we decreased the endogenous expression of NNATx using an antisense oligonucleotide (named as +43 ASO) that targets a region between 43 to 66 nucleotides in the inter stop codon region (the 3ꞌ untranslated region between the 1st and 2nd stop codons) of NNAT. With reduced expression of endogenous NNATx, +43 ASO treated cells showed increased cytoplasmic Ca2+ compared to a non-targeting oligonucleotide treated cells. Thus, by modulating stop codon readthrough of NNAT, we show that NNATx fails to promote neuronal differentiation as it fails to increase cytoplasmic Ca2+ levels. Overall, these results demonstrate regulation of neuronal differentiation by SCR of NNAT. Importantly, this process can be exogenously regulated using a synthetic antisense oligonucleotide.
MHRD