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Gene Therapy (2004) 11, 619–627 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt RESEARCH ARTICLE Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment L Bidou1, I Hatin1, N Perez2, V Allamand3, J-J Panthier4 and J-P Rousset1 1 CNRS UMR 8621, Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay Cedex, France; 2GENETHON, CNRS UMR 8115, Evry, France; 3INSERM U582-Institut de Myologie, IFR14, Groupe Hospitalier Pitié-Salpétrière, Paris Cedex, France; and 4 UMR955 INRA/ENVA de Génétique Moléculaire et Cellulaire, Ecole Nationale Vétérinaire d’Alfort, 7, Maisons-Alfort Cedex, France The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments. Gene Therapy (2004) 11, 619–627. doi:10.1038/sj.gt.3302211 Published online 19 February 2004 Keywords: muscular dystrophy; premature stop codons; readthrough; gentamicin; dual reporter gene; skeletal muscle Introduction While gene therapy is potentially a method of choice in the treatment of genetic diseases, alternative therapeutic methods independent of the transfer of genetic material offer an attractive option. In particular, pharmacological approaches aimed at modification of gene expression, rather than the gene itself, constitute an ideal strategy for a subset of genetic diseases in which the molecular defect is known and can be circumvented. This is the case for premature stop codon (PTC) diseases, in which the primary cause of the disease is a nonsense mutation in the gene coding sequence: about 5–10% of patients for the major genetic diseases, though the frequency may be higher for some syndromes or in specific human populations. Aminoglycosides have long been known to affect translational accuracy. In particular, they promote nonsense mutation suppression in bacteria and yeast.1 These antibiotics interact with the highly conserved decoding center of ribosomal RNA, and allow normal tRNAs to recognize incorrect codons, including stop codons.2 In 1985, Burke and Mogg3 initially suggested that some aminoglycosides presented the same properties in cultured mammalian cells. They demonstrated that paromomycin and G-418 could partially restore the synthesis of an entire protein from a mutant gene interrupted by a premature stop codon. Correspondence: J-P Rousset, CNRS UMR 8621, Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay Cedex 91405, France Received 30 June 2003; accepted 10 November 2003 Bedwell and co-workers4,5 have also shown that G-418 and gentamicin can restore the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in a bronchial cell line with a nonsense mutation in the CFTR gene. Similarly, a Hurler syndrome fibroblast cell line heterozygous for a stop mutation showed a significant increase in alpha-L-iduronidase when cultured in the presence of gentamicin.4,5 The strength of this approach was first demonstrated in vivo by the work of Barton-Davis and co-workers, who demonstrated that gentamicin could partially restore expression of fulllength dystrophin in mdx mice with the X-linked muscular dystrophy mutation (Mdmmdx), a C–T transition at position 3185, resulting in a termination codon replacing a glutamine codon in the dystrophin gene.6 Negamycin, a dipeptide antibiotic with similar stop codon-misreading activity, has also been shown to restore dystrophin synthesis in the mdx mouse skeletal muscle.7 More recently, Bedwell and co-workers demonstrated that gentamicin is effective in a transgenic mouse model of cystic fibrosis.8 Although controversial results were obtained with mdx mice,9 a very recent report showed a high level of dystrophin reexpression in a DMD patient treated with gentamicin.10 Overall, these observations suggest that antibiotic treatment is a promising alternative to gene therapy for patients with premature stop codons due to nonsense mutations. However, it is well known from experiments conducted both in cultured mammalian cells and in yeast that the stop codon context plays an important role in readthrough efficiency. First, the three stop codons are Gentamicin and muscular dystrophies L Bidou et al 620 not equally susceptible to readthrough in basal conditions: UAA is, on average, less sensitive to basal suppression than UAG and UGA.11 Secondly, several studies demonstrated the critical influence of both upstream and downstream sequences, which determine the overall efficiency of translational termination in yeast.12,13 Less is currently known on the influence of the sequence context on basal and antibiotic-induced readthrough levels in mammalian cells. Results from different laboratories have demonstrated that the antibiotic effect is dependent on the nucleotides surrounding the stop mutation; they also suggest that the nucleotide immediately following the stop codon (numbered þ 4) has a major role in readthrough.14,15 In this study, we analyzed the gentamicin susceptibility of individual primary stop mutations concerning Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD) patients, using a very sensitive dual reporter gene system.16 This experimental model closely corresponds to the physiological situation, in which a given mutation is embedded in a unique nucleotide context, and differs between patients. Several reasons indicated an investigation of gentamicin: its effect on readthrough is the best documented to date, and it is known to present a much lower toxicity than other suppressive molecules like G418.3 Moreover, this antibiotic is currently used in human therapy against bacterial infections, and its pharmacokinetics properties and side effects are well characterized.17 However, one cannot exclude that a long-term use of gentamicin may induce other types of side effects due to generalized readthrough of normal termination codons. DMD, the most common X-linked fatal genetic disease, is a degenerative disorder of striated muscle caused by reduction or absence of the protein dystrophin, and affects approximately 1 in 3500 newborn males. The classical form of CMD is a severe and heterogeneous neuromuscular disease caused by mutations in the LAMA2 gene encoding the a2 chain of laminin.18–20 At least 10% of DMD patients and 20% of CMD patients have a nonsense mutation resulting in premature translation termination.18,21 Our results show that a very broad variation is observed in both basal and gentamicin-induced readthrough efficiency in different mutations. They also demonstrate that the nucleotide following the stop codon is hardly the unique determinant of readthrough efficiency, and that the critical parameters might be different in the absence or presence of the antibiotic. Additionally, using electrotransfer of plasmid DNA into murine skeletal muscle, we demonstrated that both the basal readthrough level and the effect of gentamicin are comparable in cultured cells and in vivo. Our results strongly suggest that the precise quantification of readthrough levels, using reporter systems such as those described here, is a prerequisite in the identification of patients with mutations displaying a gentamicin response, justifying their inclusion in potential clinical trials. Results Quantification of readthrough levels in NIH3T3 cells A dual gene reporter system16 was used to quantify the effect of gentamicin on stop mutation readthrough in Gene Therapy culture cells (Figure 1). Sequences to be analyzed, spanning 27 nucleotides centered on the different stop mutations (Table 1), were inserted in-frame between the lacZ and luc coding sequences; every translating ribosome would thus result in b-galactosidase synthesis, but only those reading through the stop codon would yield active luciferase. Readthrough efficiency was estimated by calculating the ratio of luciferase/b-galactosidase activity. This dual gene reporter system provides an internal control permitting calibration of individual experiments for the overall expression level of each construct (vector stability, transfection efficiency, transcriptional and translational rates). Both reporter proteins are expressed from the same message, eliminating in particular potential variation in mRNA stability between the different targets analyzed. This is especially important in the comparison of constructs harboring nonsense mutations, since transcripts containing a premature stop codon have been shown to be preferentially degraded by the nonsense-mediated mRNA decay pathway (NMD).22 To establish the relative b-galactosidase and luciferase activities, expressed in equimolar amounts, we also constructed control plasmids in which each stop mutation was replaced by the sense codon found in the wildtype gene. In all, 18 DMD mutations and eight CMD mutations were analyzed (Table 1). The molecular defect in the Mdmmdx murine mutation, causing premature termination of the 427 kDa dystrophin polypeptide, was included since Mdmmdx/Y mice are used as a model for human dystrophy.23 In addition, we tested the platinum (Tyrcp) mutation, an A–T substitution, changing a lysine residue at position 489 of tyrosinase to a termination codon. This tyrosinase truncation results in misrouting of the enzyme to the melanocyte periphery; thus, Tyrcp/ Tyrcp homozygous mice have a white coat color, like albino homozygotes.24 Readthrough is expected to restore normal pigmentation in platinum mice. Finally, Figure 1 Description of the dual gene reporter system. For quantification in NIH3T3 cells, ORF1 and ORF2 correspond to b-galactosidase and firefly luciferase coding sequences, respectively, and their expression is driven by the SV40 promoter (pAC99 vectors). For in vivo quantification, ORF1 and ORF2 correspond to Renilla and Firefly luciferase, respectively, and their expression is driven by the hCMV promoter (pCRFL vectors). Oligonucleotides including the stop mutation to be analyzed, surrounded by four codons in 50 and four codons in 30 , were cloned in-frame between ORF1 and ORF2. Readthrough efficiency was determined by comparing the firefly luciferase to b-galactosidase (or Renilla luciferase) ratio obtained for each nonsense mutation, to the same ratio obtained with an in-frame control corresponding to the same sequence in which the stop codon was replaced by the amino acid naturally present in the protein. Gentamicin and muscular dystrophies L Bidou et al 621 Table 1 Basal and gentamicin-induced readthrough for DMD and CMD mutations Readthrough atb gentamicin (mg/ml) ¼ Stop targeta Wild-type denomination amino acid Nucleotidic context of stop mutation 1593d 60d 988c MDX 1240c 1143d 2726d 1437c 2125d STOP PLATI 3149d STOP DYS 651d 1417d 2522d 931d 2264d 673d 1326c 967c 3085c 744c STOP LAM 3190d 1549c 1967d 3381d 2098d 145d 319d E Q Q Q Q Q E Q Q Q W L Q E Q Q R C R R R R R R R R S GCT ACA GAT ATG TAA TTG ACA AAG AGA GGC CTG ACA GGG TAA AAA CTG CCA AAA CAA TGT TGG TGC TAA CCT GGA GTC ACA TTG AAA GAG CAA TAA AAT GGC TTC AAC AAA CTT CCA GAA TAA TTT GAA GGA AAG GAT CAC ATG TGC TAA CAG GTC TAT GCC GAA AGG CTC CTA TAA GAC TCC AAG GGA ACA TCG ATA TGC TAG AAT TGT CAA CAT ACA GAA GCT GAA TAG TTT CTC AGA AAG AAG AAG AAG AAG TAG AAG CAA CCC CAG ATG GAT ATC CTG TAG ATT ATT AAT TGT GAG GAC ACA ATG TAG GAA GTC TTT TCC TTT GCC CGG TGT TAG GAT AAT TTA GTC ATC CAA TCT GAT TAG ACA AGT CAT GAG CAG GAT TTG GAA TAG AGG CGT CCC AGT GCC AGA GAG AAA TAG CTA CAG ACA ATT TTG CCC CTG CGC TAG GGA ATT CTC AAA GTC ACC ACC ACT TAG CCA TCA CTA ACA AGA ACT GTG ACC TGA GAA GAC TTC TTG GGC TGT GTT CCC TGA AAC TGC AAT TCT AGT ATT CCG TTC TGA GGT TGC ATC AGA CCT AGG CAC AGG TGA GTT AAC GGC ACT AGG CCC TGG AAC TGA GGG GCG TTC AAC TAT GAT ACG GGA TGA ACA GGG AGG ATC TTC TGC ACG TGC TGA CCT GGA GCC ACG TTT GCT CAG TTT TGA AGA CTA AAC TTT AAA AAC AAA TTT TGA ACC AAA AGG TAT GAC CGA CAA GGG TGA TTT GAC AGA TCT CTG AGC TGG GTC TGA CAA TCA ACT CGT AGC CCA TTT CCT TGA CAG CAT TTG GAA 0 600 0.010% 0.006% 0.005% 0.006% 0.020% 0.006% 0.008% 0.010% 0.080% 0.040% 0.040% 0.030% 0.012% 0.020% 0.012% 0.370% 0.090% 0.080% 0.020% 0.010% 0.050% 0.040% 0.050% 0.110% 0.110% 0.080% 0.033% 0.730% 0.310% 0.060% 0.05% 0.05% 0.07% 0.08% 0.11% 0.15% 0.17% 0.05% 0.06% 0.10% 0.12% 0.13% 0.22% 0.30% 0.30% 0.92% 1.21% 1.90% 0.11% 0.14% 0.19% 0.20% 0.23% 0.46% 0.60% 0.70% 0.76% 1.40% 1.74% 2.65% Increase factor between doses 0 and 600 5 8 13 13 6 25 21 5 1 3 3 4 18 15 25 2 13 24 6 14 4 5 5 4 5 9 23 2 6 44 a Mutations are primary nonsense mutations, and are named by the position of the modified amino acid in the protein sequence, followed by a ‘d’ or a ‘c’ indicating, respectively, DMD or CMD mutations. The stop mutation present, of mdx mouse, is indicated as ‘MDX’. ‘STOP LAM’ and ‘STOP DYS’ correspond to the natural termination codon of laminin and dystrophin genes. ‘STOP PLATI’ indicates the platinum coat color mutation. b Readthrough efficiency has been measured as described in Figure 1. Each value of readthrough efficiency (in the absence or presence of gentamicin) corresponds to the mean of four to six independent experiments. Standard deviation errors did not exceed 20%. the natural stop codons of the dystrophin and laminin a2 chain (LAMA2) genes were also included as controls. Transfected NIH3T3 cells were cultured for 2 days in the absence or presence of 200, 300, 400, 500 and 600 mg/ml of gentamicin. Preliminary experiments with nontransfected cells demonstrated that no signs of cytotoxicity were detectable after 48 h of treatment with up to 600 mg/ml gentamicin. Indeed, following transfection, surviving cells showed similar morphology and comparable growth rate in the presence or absence of gentamicin (data not shown). Similar experiments were performed with several constructs, using C2C12 myoblasts instead of NIH3T3 cells as recipient, and identical results were obtained either in the absence or presence of gentamicin (data not shown). Figure 2 shows dose–response curves obtained with five nonsense mutations. For all of these, a linear response was obtained for doses up to 600 mg/ml. In addition, very different slopes were obtained for each mutation. For 20 other targets, readthrough level was similarly increased when higher concentrations of gentamicin were used (data not shown). For this reason, only dosages of 0 and 600 mg/ml were used in further experiments. Results obtained at doses 0 and 600 are shown in Table 1 and analyzed below. Even for targets Figure 2 Gentamicin-induced readthrough on five representative nonsense mutations 3381d (filled circle), 2522d (open circle), 1417d (triangle), 2726d (filled square) and 1143d (open square). Sequences surrounding the stop codon are presented in Table 1. These sequences were cloned in the pAC99 dual reporter vector. To obtain dose–response curves, NIH3T3 cells were electroporated with the different constructs and grown for 2 days in the absence or presence of various concentrations of gentamicin. After protein extraction, luciferase and b-galactosidase activity were measured. Termination readthrough is expressed as the luciferase/b-galactosidase ratio of the test construct, normalized to the in-frame control. Gene Therapy Gentamicin and muscular dystrophies L Bidou et al 622 that exhibited a low increase effect (o4), the difference between basal and gentamicin-induced readthrough level was statistically significant (P-value ranging from 0.00197 to 0.00451). The variation between readthrough levels detected in the absence or presence of gentamicin was reflected in the level of increase observed for each mutation (Table 1). A wide variability in responsiveness to gentamicin was observed between the different mutations, but was not dependent on the identity of the stop codon. Increase factors between readthrough levels at doses 0 and 600 ranged from 1 (target 2125d) to 44 (target 319d). Figure 3 shows the readthrough levels in the absence or presence of gentamicin, for all mutations tested. For basal readthrough, values ranged from 0.005 to 0.73%, the vast majority of the mutations yielding less than 0.1% of readthrough. Previous studies reported that the UAA stop codon was a better terminator in comparison with UAG and UGA.15 We confirmed this observation; on the wide number of mutations studied here, the average basal readthrough was 0.006% for UAA codons, compared to 0.041% for UAG and 0.055% for UGA. These differences were highly significant (P ¼ 0.00089 when Figure 3 Comparison between basal and gentamicin-induced readthrough level. The distribution of readthrough levels driven by different stop codons in the absence of gentamicin (filled bars) or in the presence of 600 mg/ml of gentamicin (open bars). Experiments were performed as indicated in Figure 2. Each value corresponds to the mean of four to six independent experiments. Standard deviation errors did not exceed 20%. Gene Therapy comparing UAA versus UAG in a Mann–Whitney nonparametric test and P ¼ 0.00036 when comparing UAA versus UGA). By contrast, the readthrough level was similar for the UAG and UGA stop codons (P ¼ 0.242). Nevertheless, some of the TAG mutations in less favored contexts exhibited equivalent or even lower gentamicin-induced readthrough levels than those displayed by TAA mutations surrounded by leakier sequences (for example, compare TAA 1240c with TAG 1437c, TAG 651d, TAG 2522d and to TGA 967c) (Table 1). Like for the basal level, a very broad distribution was observed for gentamicin-induced readthrough levels, ranging from 0.05 to 2.65%. UAA stop codons also gave lower average readthrough values in the presence of the antibiotic: 0.077% compared to 0.22% for UAG (P ¼ 0.032) and 0.53% for UGA (P ¼ 0.00076). Here again, some of the TGA mutations in less favored contexts exhibited equivalent or even lower gentamicin-induced readthrough levels than those corresponding to TAA mutations surrounded by leakier sequences (for example, compare TAA 2726d with TAG 1437c, TAG 2125d and TGA 1326c, among others) (Table 1). We examined whether the mutations that gave the highest basal readthrough were particularly highly or poorly responsive to the gentamicin treatment; however, as illustrated in Table 1, no correlation was found between the readthrough efficiency and the increase factor that reflects the responsiveness to the antibiotic (correlation coefficient ¼ 0.28). The nucleotide immediately following the stop codon (position þ 4) is thought to have a major role in dictating readthrough levels. Indeed, the presence of a C residue at this position has often been reported to be correlated with maximal levels of readthrough, in the absence or presence of antibiotics.14,15 We thus analyzed the impact of the þ 4 nucleotide on readthrough level on the entire data set. A significant lower readthrough value was observed only for A þ 4 compared to C þ 4, in the presence of gentamicin (P ¼ 0.023). Figure 4 shows the percentage of readthrough level in function of the identity of the þ 4 nucleotide for different TGA mutations, in the presence of gentamicin. Obviously, there was no systematic correlation between the þ 4 nucleotide and the readthrough level. For example, a C at this position was associated either with a very high (2.65% for ‘319d’) or a moderate (0.6% for ‘1549c’) readthrough level. The same phenomenon was observed when a G was present at position þ 4 (from 1.66% for the TGAg target to 0.1% for the 1326c sequence). Similar variations are observed with A or T at this position and for the four nucleotides in the absence of gentamicin. Similar analyses have been carried out for TAA and TGA mutations and lead to similar conclusions; no nucleotide can be clearly associated with a particularly low or high readthrough level (see Table 1). Finally, analyses of all other nucleotide positions in the cloned segment did not identify any major determinant involved in basal or gentamicin-induced readthrough. In vivo quantification of readthrough levels We assessed the validity of the cell culture approach by investigation of basal and gentamicin induced readthrough in vivo. For this, we used plasmid DNA electrotransfer into C57BL/6 mouse skeletal muscle.25 Using the lacZ-luc dual gene reporter system, we were Gentamicin and muscular dystrophies L Bidou et al 623 Figure 4 Impact of nucleotide in þ 4 position on gentamicin-induced readthrough. Distribution of readthrough levels driven by constructs with a TGA nonsense mutation in the presence of 600 mg/ml of gentamicin. For each construct, the nucleotide located immediately in 30 of the stop codon ( þ 4 position) is indicated above the histogram. Data are from Table 1, except for mutations indicated by an asterisk, which have been constructed to complete the schema (see Materials and methods section). unable to detect any significant b-galactosidase activity. To improve the sensitivity of our reporter vector, we replaced the SV40 promoter of the pAC vector by the strong CMV promoter, and the lacZ gene by the Renilla luc gene encoding a very sensitive luciferase marker (see Materials and methods and Figure 1), yielding the pCRFL vector. As an initial validation, we introduced the well-characterized leaky termination codon context of the Tobacco Tumour Virus in pCRFL.26 This sequence has been previously shown to promote a very high readthrough efficiency (2–3%) in cultured cells.27,28 We chose to use the same antibiotic dose (34 mg/kg) as that in previously reported in vivo studies, since it showed no toxic effects.6 Preliminary results showed that a significant readthrough level was obtained in murine skeletal muscle (4%), and that gentamicin increased this 1.5-fold. We then constructed a pCRFL reporter vector harboring the 319d mutation (pCRFL319d), which exhibited the highest gentamicin-induced readthrough efficiency and the highest induction factor (0.06–2.65%) in cultured cells assays (Table 1). Due to the changes introduced in the reporter vector, we were able to detect the very low basal readthrough level promoted by the 319d mutation. As illustrated in Figure 5, both readthrough efficiencies and the gentamicin effect were comparable in NIH3T3 cells and, in vivo, increasing from 0.12% in the absence to 4.1% in the presence of antibiotic in mouse skeletal muscle. Discussion Over the last few decades, significant progress has been accomplished in the comprehension of the physiopathological mechanisms which result in muscular dystrophies; linking this knowledge to therapeutic developments has so far proven difficult. Recently, the Figure 5 Comparison of gentamicin effect in cultured cells and in vivo. Vectors carrying the 319d mutation were either electroporated in NIH3T3 cells (pAC constructs) or in C57BL/6 mice (pCRFL constructs). Readthrough efficiencies obtained in the absence (black histogram) or in the presence of 600 mg/ml of gentamicin (gray histogram) were determined as indicated in Materials and methods. For in vivo studies, 50 mg of pCRFL plasmid DNA was used to determine readthrough levels into TA muscles of 6-week-old male C57BL/6 (n ¼ 4 per group). Plasmid DNA was injected in the left TA muscle and six transcutaneous electric pulses were applied. Groups of mice received a daily intramuscular dose of 34 mg/kg gentamicin or PBS during the next 5 days. possibly most encouraging therapeutic approach for a subset of muscular dystrophies has emerged with the report by Barton-Davis et al,6 showing restoration of dystrophin levels to 10–20% of those normally found in the skeletal muscle of mdx mice, following subcutaneous injections of gentamicin, an aminoglycoside long known for its suppression of premature termination codons. Aminoglycoside antibiotics are active in the codon– anticodon recognition of aminoacyl tRNAs during translation.3,29,30 Significantly, 10–20% of the mutations identified in DMD and DMC are nonsense mutations resulting in premature stop codons.18,21 In the analyses conducted to date, the role of the stop codon nucleotide context on readthrough efficiency has been investigated by directed mutagenesis of target sequences inserted into reporter genes.14,15,28 In consequence, the analyzed sequences are related, which may preclude the analysis of more complex effects possibly involving a larger nucleotide context. In fact, a combinatorial analysis performed in yeast showed that the context modulating readthrough extends at least six nucleotides after the stop codon.13 The gentamicin response of individual PTC found in patients might thus vary considerably, depending on the position of the mutation in the coding sequence. These informations may permit a more accurate prediction of the response of a patient to antibiotic treatment, and help to establish the appropriate concentration of the drug in a potential clinical trial. Readthrough in vivo To date, the potential of gentamicin to induce readthrough of nonsense mutations has been tested by two Gene Therapy Gentamicin and muscular dystrophies L Bidou et al 624 complementary methods. First, a precise quantification of readthrough was obtained using reporter systems, either by transfection of cultured cells or through expression of synthetic RNAs in rabbit reticulocyte lysates.5,15 Secondly, the re-expression of a mutated gene after gentamicin treatment has been assessed in animal models, such as Mdxmdx or CFTR mutant mice.6,8 However, the correlation between readthrough levels obtained in vitro or ex vivo and the re-expression observed in vivo remained uncertain, since several processes might modulate the primary molecular effect of the antibiotic on readthrough, in particular mRNA stabilization and protein accumulation. In order to compare real readthrough levels obtained in cultured cells and in vivo, we used electrotransfer of a dual reporter gene in murine skeletal muscle, and compared the readthrough efficiencies obtained in vivo with those obtained in cultured NIH3T3 fibroblast cells. Remarkably, almost identical results were obtained in cultured cells and in mice. This result demonstrated, for the first time, that no potentiation of treatment was induced in muscles. This validates our approach, allowing rapid and accurate quantification of genuine readthrough levels. Effect of stop codon identity In basal conditions, targets with a UGA or UAG codon showed, on average, higher translational readthrough than those with a UAA. This was consistent with the relative termination efficiency previously described for the three stop codons (UAA4UAG4UGA).14,15 This hierarchy was also observed in the presence of 600 mg/ ml of gentamicin, UGA and UAG, with readthrough levels of 0.53 and 0.22%, respectively, and UAA only 0.076%. Nevertheless, in the presence or absence of gentamicin, this order might be completely changed due to the influence of the embedded stop condon context. Overall, although UAA was more often associated with low readthrough efficiency, in both the absence and presence of the antibiotic, the identity of the stop codon was not sufficient to predict either the basal readthrough level, or the response of a given mutation to gentamicin. Effect of the þ 4 nucleotide This position has been extensively examined and described as the major determinant of readthrough efficiency.12,13,15,26,31,32 A C residue following either a UAA or a UGA stop codon has been reported to be usually correlated with high levels of readthrough, either in the presence or absence of an aminoglycoside antibiotic. UAG gave more erratic results, with U or C being associated with high or low readthrough, possibly reflecting the difference in the surrounding context between the experimental systems used.14,15 The contribution of the þ 4 nucleotide to gentamicin-induced readthrough appeared moderate in our collection of mutations. In particular, a C at this position could be associated with some of the highest readthrough levels, but also with very moderate ones. In addition, a G, which was previously correlated with low readthrough efficiencies, appeared to be associated either with low or high readthrough levels in this study. In conclusion, the impact of the þ 4 nucleotide seemed much less critical than previously reported, and was largely dependent on the surrounding context. Apparent Gene Therapy divergences between our results and some previous observations most probably proceed from the fact that we analyzed a number of different contexts, rather than related sequences derived from a unique core by mutagenesis. In practice, this implies that the identification of the nucleotide immediately downstream of the stop codon is not sufficient to predict readthrough efficiency of a given stop mutation, either in the absence or in the presence of gentamicin. Different rules govern basal and induced readthrough Another inference drawn from our data is that no correlation could be found between the efficiency of stop codon readthrough in basal conditions and the responsiveness of the stop codon to gentamicin treatment. In other words, sequences that mediate more efficient termination at dose 0 were not systematically the least (or the most) sensitive to the aminoglycoside. Symmetrically, mutations displaying an intrinsic high readthrough level could be poorly or highly responsive to gentamicin. Thus, factors influencing gentamicininduced readthrough might be, at least partially, distinct from factors that determine basal translational termination efficiency. Relationship between stop codon readthrough and gene re-expression As already noted, the mouse Mdxmdx mutation gave a particularly low level of readthrough upon gentamicin treatment, although re-expression of dystrophin in treated mice was reported to reach 10–20%.6 This observation strongly suggests that other levels of gentamicin action are involved in the phenotypic reversion of the mdx mutation previously reported. A good candidate is the NMD pathway, which might potentiate the antibiotic effect by stabilization of the message read by ribosomes.22 Similarly, other mechanisms like protein stability and accumulation might collectivelly participate to potentiate the primary translational effect of gentamicin. Finally, gentamicin may also affect gene expression at other levels than translation. In particular, aminoglycoside antibiotics are known to affect splicing, raising the possibility that they might be able to induce exon skipping.33 None of these potential effects is taken into account in our analyses, since we used dual reporter systems allowing accurate quantification of the translational readthrough event at the molecular level, with no interference with other levels of control. Potential use of gentamicin in treatment of genetic diseases due to nonsense mutation Overall, the readthrough levels obtained were very low: 85% of the stop codon contexts gave less than 1% readthrough after gentamicin treatment, and the highest values were lower than 3%. These results thus strongly suggest that only a subset of DMD and CMD patients carrying stop mutations would potentially benefit of a gentamicin treatment aiming at suppressing premature termination codons. They may also explain the failure or relative difficulty to obtain significant improvement of symptoms in clinical trials already reported,34–36 since the patients included in these trials were not chosen on the criteria of the response of their mutation. Similarly, Gentamicin and muscular dystrophies L Bidou et al our efforts to correct the albino phenotype associated with the platinum (Typcp) coat color mutation by local or systemic treatment with gentamicin at several stages during embryogenesis, in pups and in adults failed (data not shown). In conclusion, our opinion is that no prediction of the efficiency of readthrough – and thus of a potential benefit – can be drawn from the nucleotide context of the mutation; individual tests of each particular mutation might therefore provide useful information before submitting a patient to a potential clinical trial. These observations emphasize that more remains to be learned about the sequences and mechanisms promoting readthrough and translation termination in general, in order to allow the rational use of drugs for the treatment of nonsense mutation-associated diseases. Materials and methods Stop mutation targets Both DMD and Congenital Muscular Dystrophy (CMD) mutations were included, and are identified in the text by their code followed either by the letter c or d (for CMD and DMD, respectively). Sequences were either retrieved from databases21 or provided by several laboratories involved in a collaborative project coordinated by the ‘Association Française contre les Myopathies’. TGAg and 1952T targets used in Figure 4 have been designed specially to complete analyses on nucleotide at the þ 4 position. TGAg: 1952T: 50 TGG GGA ACA CAA TGA GAA TTA CAG CCA 30 50 TTT GCC CGG TGT TGA TAT AAT TTA GTC 30 Reporter plasmid cloning Complementary oligonucleotides corresponding to the sequences in Table 1 were annealed and ligated in the pAC99 reporter plasmid, as previously described.37 This plasmid contains a unique Msc1 cloning site between lacZ and luc genes. For a given test construct, b-galactosidase and luciferase coding sequences are separated by an in-frame stop codon corresponding to the mutation found in a patient, flanked by four codons in 50 and 30 , respectively. For each mutation, a control plasmid was constructed, in which the stop codon was replaced by the corresponding sense codon found at this position in the wild-type allele, allowing expression of 100% of the fusion protein. After transformation of Escherichia coli strain DH5a, all target DNA sequences were assessed by sequencing. Plasmids were purified using the Qiagen Midiprep Kit (Courtaboeuf, France) according to the manufacturer’s specifications. For in vivo injections, plasmids were purified using the Qiagen Megaprep endotoxin-free columns, and solubilized in NaCl (0.9%) endotoxin-free (Sigma). Construction of the dual luciferase reporter vector pCRFL The PstI/HpaI DNA fragment from the p2luci vector38 (a gift of Dr Atkins) containing the Renilla luciferase and Firefly luciferase genes was cloned into the pBC SK( þ ) vector (Stratagene) at the PstI/HincII restriction site. At the BglII site of this first construct, a BglII, MscI, HpaI, and ClaI polylinker (GCCATGGAGGCCCCGGGG ATC) was added. To remove the AUG translational start codon of the Firefly luciferase gene, the sequence delineated by HpaI/ClaI restriction sites was replaced by the Firefly luciferase sequence contained within the MscI/ClaI sites from the pAC99 vector.37 Finally, the NotI fragment containing the 2 luciferase fusion genes was cloned at the NotI restriction site on the pCDNA3 vector from Invitrogen Company. In this construct, the expression of the protein fusion Renilla luciferase/Firefly luciferase was under the control of the CMV promoter. This dual reporter vector was named pCRFL. 625 Cell culture and transfection NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 7% fetal calf serum (Gibco BRL), and incubated at 371C in humiditysaturated 6.5% CO2. Cells were electroporated with 18 mg of each pAC (lacZ-luc) plasmid construct, as described,39 and plated immediately in culture medium. Culture medium was replaced 7 and 30 h later with fresh medium at a concentration of gentamicin (GIBCO BRL) varying from 0 to 600 mg/ml. At 2 days after transfection, cells were harvested, spun 5 min at 41C and lysed by repeated pipetting in 150 ml of cold luciferase assay buffer.39 For each construct, at least four independent transfection experiments were performed. Electric pulsed delivery in murine skeletal muscle C57BL/6 males, 6-weeks old (IFFA Credo, France), were anesthetized during the entire procedure by intraperitoneal injection of ketamine (30 mg/kg) and xylazine (10 mg/kg). One or two legs were shaved, and 50 mg of DNA from the pCRFL319d (eight mice) or the in-frame control plasmid (eight mice) in 0.9% endotoxin-free NaCl (25–35 ml) was injected in the left Tibialis anterior (TA) muscle, percutaneously and longitudinally, using a Hamilton syringe with a 30-gauge needle. Immediately after injection, transcutaneous electric pulses were applied using two stainless steel plate electrodes placed on either side of the hind limb as previously described,40 with some modifications. Briefly, six square-wave electric pulses were generated by an electropulsator (ECM-830 BTX, San Diego, CA, USA) with an output voltage of 300 V/cm, a pulse length of 15 ms, and a frequency of pulse delivery of 2 Hz. Electric field strengths (in V/cm) are reported as the ratio of applied voltage to the distance between the electrodes. In vivo gentamicin treatment At 15 min after electrotransfer, four mice having received the pCRFL319d and four mice having received the inframe control vector were injected with 17 mg of gentamicin in each muscle. Gentamicin (17 mg) was then injected each day for the next 3 days. The gentamicin was from the same batch as that used for the cell culture experiments. The remaining mice were injected with phosphate-buffered saline. Gene Therapy Gentamicin and muscular dystrophies L Bidou et al 626 Muscle extracts and determination of luciferase gene expression At the indicated times after DNA injection, mice were euthanized, the Tibialis anterior was dissected and rapidly frozen in liquid nitrogen for further analysis. Muscle extracts were prepared as described.25 Briefly, muscle extracts were prepared by homogenizing the muscle tissue with an Ultra-Turrax homogenizer (Bioblock Scientific, Illkirch, France) in 500 ml of Promega lysis buffer containing protease inhibitors (Sigma). Extracts were frozen-thawed twice and immediately cleared by centrifugation (5000 g for 15 min at 41C). Enzyme assays Luciferase and b-galactosidase activities were measured from the same crude extract as described.39 For in vivo tests, dual luciferase detection was achieved using the dual detection kit from PROMEGA, following the manufacturer’s instructions. Stop codon readthrough was calculated by dividing the ratio of luciferase to b-galactosidase activity obtained with a given test construct by the ratio obtained from the corresponding control construct. Statistical analysis was performed using the Mann– Whitney nonparametric test, which investigates the difference between two independent samples carrying different values of an individual subject. This test is an alternative to the independent group t-test, when the assumption of normality or equality of variance is not met. This, like many nonparametric tests, uses the ranks of the data rather than their raw values to calculate the statistic (Richard lowry, 2000, Inferential statisticshttp://faculty.vassar.edu/lowry/ch11a.html). Acknowledgements We are especially indebted to Maryse Godon for help with plasmid preparations. We are grateful to Laetitia Baudry for animal experiences and Jerome Poupiot for technical assistance. 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