Readthrough Of Premature Termination Codons Research Articles

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough.

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors’ clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.

2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Translational readthrough at F8 nonsense variants in the factor VIII B domain contributes to residual expression and lowers inhibitor association.

In hemophilia A, F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favor inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTC) may contribute to immune tolerance by producing full-length proteins through the insertion of amino acid subset(s). To quantitatively evaluate the readthrough output in vitro, we developed a very sensitive luciferase-based system to detect very low full-length FVIII synthesis from a wide panel (n=45; ~60% patients with PTC) of F8 nonsense variants. PTC not associated with inhibitors displayed higher readthrough-driven expression levels than inhibitor-associated PTC, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six hemophilia A patients with PTC, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTC not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (4 out of 57). These original insights into the molecular genetics of hemophilia A, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favor PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.

Ataluren suppresses a premature termination codon in an MPS I-H mouse

AbstarctSuppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs.Key messagesAtaluren promotes readthrough of PTCs in a wide variety of contexts.Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models.Ataluren has a bell-shaped dose–response curve and a narrow effective range.

FRI248 - In vitro rescue of nonsense variations by drug-induced readthrough of premature termination codons in progressive familial intrahepatic cholestasis type 2

FRI248 - In vitro rescue of nonsense variations by drug-induced readthrough of premature termination codons in progressive familial intrahepatic cholestasis type 2

Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903

Nonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4–C1, and TRPC5–C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8–18 μM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.

Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis.

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

Reducing the Toxicity of Designer Aminoglycosides as Nonsense Mutation Readthrough Agents for Therapeutic Targets.

A significant proportion of genetic disease cases arise from truncation of proteins caused by premature termination codons. In eukaryotic cells some aminoglycosides cause readthrough of premature termination codons during protein translation. Inducing readthrough of these codons can potentially be of therapeutic value in the treatment of numerous genetic diseases. A significant drawback to the repeated use of aminoglycosides as treatments is the lack of balance between their readthrough efficacy and toxicity. The synthesis and biological testing of designer aminoglycoside compounds is documented herein. We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds by modification of the overall cationic charge of the aminoglycoside scaffold through ring I modifications.

Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.

Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough.

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.

Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X, and p.E1302X) identified in patients with PFIC2. The ability of G418, gentamicin, and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in human embryonic kidney 293 (HEK293), HepG2, and Can 10 cells using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [3 H]-taurocholate transcellular transport in stable Madin-Darby canine kidney clones co-expressing Na+-taurocholate co-transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4-phenylbutyrate [4-PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and ursodeoxycholic acid significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In Madin-Darby canine kidney clones, gentamicin induced a 40% increase of the BsepR1090X [3 H]-taurocholate transport, which was further increased with additional 4-PB treatment. This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.

Effect of small molecule eRF3 degraders on premature termination codon readthrough.

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.

MMADHC premature termination codons in the pathogenesis of cobalamin D disorder: Potential of translational readthrough reconstitution

Mutations in the MMADHC gene cause cobalamin D disorder (cblD), an autosomal recessive inborn disease with defects in intracellular cobalamin (cbl, vitamin B12) metabolism. CblD patients present methylmalonic aciduria (MMA), homocystinuria (HC), or combined MMA/HC, and usually suffer developmental delay and cognitive deficits. The most frequent MMADHC genetic alterations associated with disease generate MMADHC truncated proteins, in many cases due to mutations that create premature termination codons (PTC). In this study, we have performed a comprehensive and global characterization of MMADHC protein variants generated by all annotated MMADHC PTC mutations in cblD patients, and analyzed the potential of inducible translational PTC readthrough to reconstitute MMADHC biosynthesis. MMADHC protein truncation caused by disease-associated PTC differentially affected the alternative usage of translation initiation sites, protein abundance, and subcellular localization of MMADHC. Aminoglycoside compounds induced translational PTC readthrough of MMADHC truncated variants, allowing the biosynthesis of full-length MMADHC in a PTC-specific manner. Our results suggest that translational PTC readthrough-based interventions could complement current therapies for cblD patients carrying specific MMADHC PTC mutations.

Translational Read-Through Therapy of RPGR Nonsense Mutations.

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

The position of nonsense mutations can predict the phenotype severity: A survey on the DMD gene.

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used the DMD gene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, the DMD gene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, because DMD mutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5’ nonsense mutations, if reinitiation can occur, or due to late 3’ nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool “REPAIRv2” (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively.

261 Assessment of readthrough of premature termination codons in xeroderma pigmentosum group C patients by real-time PCR and immunohistochemistry

Xeroderma pigmentosum (XP) is a rare autosomal recessive DNA repair disorder caused by mutations in 8 genes, XP-A to XP-G, and Variant with severe sun sensitivity and10,000-fold increased risk of skin cancer. XP-C is the most common type in the U.S. About 12% of XP-C patients have premature termination codon (PTC) mutations that can lead to XPC protein elongation arrest and XPC mRNA template degradation by nonsense mediated decay. Our previous studies have shown treatment with aminoglycosides promote readthrough of PTC in XP-C cells. Amlexanox, an anti-inflammatory, antiallergic and immunomodulator, was reported to increase readthrough in human cells with PTC associated diseases. We treated parallel pairs of fibroblasts and lymphoblasts derived from two unrelated XP-C patients along with 2 normal control cells with amlexanox and/or previously tested aminoglycosides. Levels of XPC mRNA increased in XPC fibroblasts exposed to both G418 and amlexanox to a different extent in different cells. We are also developing a more direct assessment of level of XPC protein in human skin by use of immunohistochemistry with XPC antibodies. Two mm skin punch biopsies are stained with IHC and assessed microscopically. The slides are then scanned and the intensity of the staining is assessed by computer algorithm. In skin from normal donors XPC staining was prominent in the nuclei of epidermal cells and sweat glands in the dermis. In contrast, only background XPC staining was observed in cells from XPC patients. These preclinical tests are a step towards precision medicine that should assist patient selection and determining which drug combination would be optimal to increase XPC function for each patient.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.