Un quadro per la giunzione individualizzata

Natura volume 619, pagine 828–836 (2023) Citare questo articolo

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Gli oligonucleotidi antisenso di commutazione di giunzione (ASO) potrebbero essere utilizzati per trattare un sottogruppo di individui con malattie genetiche1, ma l'identificazione sistematica di tali individui rimane una sfida. Qui abbiamo eseguito analisi di sequenziamento dell'intero genoma per caratterizzare la variazione genetica in 235 individui (da 209 famiglie) con atassia-telangectasia, una malattia genetica recessiva gravemente debilitante e pericolosa per la vita2,3, ottenendo una diagnosi molecolare completa in quasi tutti gli individui. Abbiamo sviluppato una tassonomia predittiva per valutare la disponibilità di ciascun individuo all'intervento ASO di commutazione della giunzione; Il 9% e il 6% degli individui avevano varianti che erano "probabilmente" o "possibilmente" suscettibili alla modulazione di giunzione ASO, rispettivamente. Le varianti più suscettibili si trovavano in regioni introniche profonde che sono inaccessibili al sequenziamento mirato agli esoni. Abbiamo sviluppato ASO che hanno salvato con successo lo splicing errato e la segnalazione cellulare ATM nei fibroblasti dei pazienti per due varianti ricorrenti. In uno studio clinico pilota, uno di questi ASO è stato utilizzato per trattare un bambino a cui era stata diagnosticata atassia-telangectasia subito dopo la nascita e ha mostrato una buona tollerabilità senza eventi avversi gravi per tre anni. Il nostro studio fornisce un quadro per la futura identificazione di individui con malattie genetiche che potrebbero trarre beneficio da un approccio terapeutico che coinvolga ASO con commutazione di giunzione.

Si stima che 1 individuo su 10 – più di 30 milioni di persone negli Stati Uniti – abbia una diagnosi di una malattia rara4. Sebbene questo termine comprenda circa 7.000 condizioni diverse, di cui una notevole percentuale ha un'eziologia genetica nota o presunta, i trattamenti sono disponibili solo per circa il 5% di esse4,5. La rarità di queste condizioni spesso rende economicamente irrealizzabile lo sviluppo di trattamenti basati su modalità tradizionali. Il lavoro precedente ha dimostrato che, in alcuni casi, gli ASO di commutazione di giunzione possono ripristinare i livelli proteici funzionali ed essere somministrati in modo sicuro e tempestivo1. Tale terapia rappresenta un'opportunità di trattamento; tuttavia, identificare gli individui con varianti genetiche adatte a tali tentativi rimane una sfida1,6. Qui presentiamo un quadro per scoprire e sviluppare sistematicamente trattamenti di commutazione di giunzione per soggetti affetti da malattie rare, utilizzando l'atassia-telangectasia (AT) come modello.

L'AT è una malattia autosomica recessiva causata dalla perdita biallelica della funzione di ATM, un gene coinvolto nella risposta cellulare alle rotture del doppio filamento del DNA7. Clinicamente, l'AT è caratterizzata da degenerazione cerebellare progressiva, immunodeficienza e predisposizione al cancro, con manifestazioni precoci quali atassia, movimenti involontari, neuropatia, aprassia oculomotoria, disfagia, linguaggio confuso e teleangectasie oculari e cutanee3. La malattia ha una prevalenza di 1 su 40.000-100.000 nati vivi in tutto il mondo2. Nella AT “classica” (la presentazione clinica più tipica e prevalente), l'aspettativa di vita media è di 25 anni e la morte prematura è più frequentemente dovuta a malattie polmonari o cancro2. Le varianti ATM ipomorfe possono causare un fenotipo più lieve e a progressione più lenta, sebbene ancora con grave morbilità8.

L'AT è la forma più comune di atassia progressiva ereditaria infantile in molti paesi9. Tuttavia, mancano terapie efficaci, in particolare per le manifestazioni neurologiche profonde che hanno un marcato effetto sulla qualità della vita. La grande dimensione di codifica (9,2 kb) di ATM rappresenta una sfida per gli approcci di terapia genica, poiché i vettori AAV attualmente disponibili hanno una capacità di confezionamento di circa 4,7 kb (rif. 10). Il prodotto proteico di ATM è una chinasi intracellulare, che rende difficili gli approcci di sostituzione enzimatica. Al contrario, l'AT potrebbe essere un candidato interessante per la terapia ASO con commutazione di giunzione, dato che gli ASO si distribuiscono facilmente nel cervelletto quando iniettati per via intratecale11.

T and c.5763-1050A>G) in this study are indicated. Deletion variants that span a splice junction were considered to be located at the junction (position 0). Short indels and SVs were considered to be located at the boundary of the variant closest to a nearby splice junction. c, Types and ASO amenability of all disease candidate variants present in the cohort. d, Cumulative fraction and box plot distribution of the age at diagnosis, based on the possession of SDVs and a known hypomorphic variant (c.5763-1050A>G). The P values (shown in the figure) were calculated through the log-rank test using the survminer R package, and were adjusted by the Bonferroni correction. Shaded areas, fraction ± 95% confidence interval. For the box plots: centre, median; lower hinge, 25th percentile; upper hinge, 75th percentile; lower whisker, smallest value greater than or equal to lower hinge − 1.5 × interquartile range (IQR); upper whisker, largest value less than or equal to upper hinge + 1.5 × IQR./p>G, prevalent in the UK) as one of the pathogenic variants (Fig. 1d)./p>T (p.Glu1978Ter), present in 16 individuals (Extended Data Fig. 3b and Supplementary Table 8)./p>

G) present in 13 of the 235 patients. Only 22% (8/36) of the ASO-amenable variants had been previously classified in ClinVar as pathogenic or likely pathogenic (Extended Data Fig. 3a); 50% (18/36) had been previously classified as likely benign, uncertain significance or conflicting interpretations of pathogenicity; and 28% (10/36) were not reported in ClinVar (as opposed to around 17% for non-ASO-amenable variants). These findings suggest that ASO-amenable variants are often misinterpreted and underrepresented in ClinVar./p>T, c.5763-1050A>G), patient fibroblasts were available for RNA-seq; for nine additional probably/possibly ASO-amenable variants, gDNA samples were available, and experimental minigene splicing assays were successfully established (for more details about minigene assays, see Methods and Supplementary Tables 10–12). All tested variants (11/11) yielded the predicted mis-splicing consequences (for RNA-seq, see the following sections; for minigene assays, see Extended Data Fig. 5). Furthermore, we conducted ASO screens for six probably ASO-amenable variants (two based on patient fibroblasts and four based on minigene assays), and successfully identified ASOs capable of correcting mis-splicing for all six (see the following sections for patient-fibroblast-based screens, Fig. 4 for minigene-based screens and Supplementary Table 9 for a summary)./p>G) and c.2839-581G>A; b, c.6348-986G>T; and c, c.3994-159A>G), small-scale ASO screening was performed, which showed that the mis-splicing events caused by all tested variants can be rescued by ASOs. NT, NT-22 (a non-targeting ASO). Blue asterisks, white (or black) arrows and white (or black) ‘x’ marks indicate bands validated by Sanger sequencing (orange asterisk indicates a band not validated by Sanger sequencing). ΔMES, change in MaxEntScan score when a given variant is introduced. For gel source data, see Supplementary Fig. 1./p>G, c.2250G>A, chr11:108243936-108243949insAlu and c.2639-22_2639-20del were found in thirteen, five, two and two individuals, respectively (Supplementary Table 9). In addition, several different ASO-amenable variants had splicing consequences that appeared addressable with a single ASO; for instance, both c.2839-579_2839-576del and c.2839-581G>A result in the inclusion of the same pseudoexon (Fig. 3). We therefore subdivided variants into ‘treatment groups’, each potentially addressable with a single ASO drug. On the basis of these patterns, around 70% (24/35) of amenable individuals were predicted to be treatable with a total of five different splice-switching ASOs, whereas developing splice-switching treatments for all 35 individuals would require 15 distinct therapeutic ASO drugs (Extended Data Fig. 4b and Supplementary Table 9)./p>G (Fig. 3). The variant, located deep in intron 38, results in the inclusion of a 137-bp pseudoexon, thereby causing a frameshift in the resulting mature product26. This variant is associated with a mild A-T phenotype owing to partial leakiness of its gain-of-splicing effect. It is a founder variant in the UK, with an estimated disease allele frequency of 18% in individuals with A-T in the UK (refs. 27,28). In the ATCP cohort, composed predominantly of individuals from the USA, it was found in a compound heterozygous state in 13 unrelated patients, representing a population frequency of 5.5% (13/235) and a disease allele frequency of 2.8% (13/469)./p>T (Fig. 3). This variant has been previously identified in the homozygous state in patients with classical (severe) A-T. A lymphoblastoid cell line with this variant had no residual protein or enzymatic activity29 (A. M. R. Taylor, personal communication); that is, this variant is a null variant. This variant is predicted to have a benign coding effect (p.Ala2622Val; predicted benign by REVEL), with a pathogenic effect that is mediated by mis-splicing: it creates a strong splice donor site within exon 53 (of 63 exons), causing truncation of the exon by 64 bp, which results in frameshift and subsequent premature translational termination. Inhibition of this splice donor site with a morpholino oligonucleotide has been previously shown to rescue the cellular phenotype of a patient-derived cell line30./p>T was encountered in one individual in the ATCP cohort (DDP_ATCP_520, currently 20 years old). Separately, we also identified a second, younger (one year old at referral; currently six years old) child with A-T with this variant as well (Extended Data Fig. 6a and Supplementary Note 4). Whereas most patients with A-T are diagnosed after the initial onset of symptoms (Supplementary Table 1), this child was diagnosed as an infant on the basis of an abnormally low T cell receptor excision circle (TREC) count. TREC assays are used in newborn screening to identify infants at risk for severe combined immune deficiency (SCID), but have also incidentally identified cases of A-T (ref. 31). Exome sequencing in this child showed compound heterozygosity for two ATM variants: c.7865C>T and c.8585-13_8598del (confirmed by trio Sanger sequencing; Supplementary Table 6). The latter is a 27-bp deletion at the intron–exon junction of exon 59, strongly predicted to cause complete loss of function (Supplementary Note 4). This combination of variants predicted a classical, early-onset A-T phenotype (Fig. 1d)./p>T. PCR with reverse transcription (RT–PCR) and RNA-seq analysis of splicing patterns in this cell line showed an abnormal truncation of exon 53 owing to premature splice donor site usage, consistent with previous studies30 (Extended Data Fig. 6b,c). Analysis using allele-specific PCR primers (designed to exclude the non-target c.8585-13_8598del allele) showed that ATM mis-splicing by c.7865C>T is complete, without detectable leakiness (Extended Data Fig. 6d)./p>T. Biologically independent experiments (independent transfections) were conducted (n = 3, initial; n = 2, fine-tuning). For some ASOs in the fine-tuning screening, the first two letters are omitted. Error bars, mean ± 95% confidence interval (shown only for conditions with n ≥ 3). A one-sample two-tailed t-test was used to assess statistical significance; means were compared to a constant value of 0 because no background normal splicing was observed in cells that were mock-transfected or transfected with non-targeting ASOs. *P < 0.05; **P < 0.01. AT010, *P = 0.0441; AT004, *P = 0.0240; AT001, *P = 0.0453; AT002, **P = 0.0010; AT005, **P = 0.0093; AT006, **P = 0.0060; AT007, **P = 0.0001; AT008, **P = 0.0082. Four top-performing ASOs (blue letters) were selected for further validation (b,c). For RT–PCR gel source data, see Supplementary Fig. 1. b, ASO-mediated restoration of irradiation-induced ATM signalling in patient fibroblasts, measured by immunoblotting. 07, 08, 22 and 26 represent AT007, AT008, AT022 and AT026, respectively; NT, NT-22 (a non-targeting ASO). pP53, phospho-P53; pKAP1, phospho-KAP1. Biologically independent experiments (independent transfections) were conducted: pP53 (n = 2, hypomorphic cases ± irradiation; n = 4, AT022, AT026; n = 5, the other conditions), pKAP1 (n = 4, AT007 and AT022; n = 5, the other conditions). Error bars, mean ± 95% confidence interval (shown only for conditions with n ≥ 3). A two-sample (comparing each condition to NT-22) two-tailed t-test was used for statistical analysis. *P < 0.05; **P < 0.01. For pP53, AT007, **P = 0.0024; AT008, **P = 0.0001; AT022, **P = 0.0471. For pKAP1, AT008, *P = 0.0201; AT022, *P = 0.0175; AT026, **P = 0.0073. Representative blot images are shown in Extended Data Fig. 8a. For blot source data, see Supplementary Fig. 1. c, ASO-mediated restoration of irradiation-induced ATM signalling in patient fibroblasts, measured by immunofluorescence staining. Scale bar, 50 μm. For a quantitative summary of the complete results, see Extended Data Fig. 8b./p>G variant, supporting the potential clinical relevance of this rescue./p>G variant. A fibroblast cell line was established from a patient with A-T from the ATCP cohort (DDP_ATCP_42) and used to confirm the mis-splicing consequences of c.5763-1050A>G (Fig. 3 and Extended Data Fig. 9). ASOs were designed to block the pseudoexon usage associated with this allele, and screening in patient fibroblasts successfully identified a lead ASO that was capable of rescuing ATM function (Extended Data Figs. 10 and 11, Supplementary Note 5 and Supplementary Figs. 9 and 11)./p>T-targeting ASO AT008 (renamed atipeksen) was selected for further clinical development. It was chosen because of the association of c.7865C>T with severe disease (classical A-T), the robustness of atipeksen-mediated RNA and cellular functional rescue and the opportunity for early therapeutic intervention before the onset of major neurological morbidity in the previously identified young child with this variant. (Note that the other identified individual with this variant, DDP_ATCP_520, was not considered to be a suitable candidate for clinical intervention owing to the advanced stage of the disease)./p>T who has been treated with AT008 (atipeksen), and individuals in the ATCP cohort, who were enrolled for WGS variant call validation by Sanger sequencing and mis-splicing validation by minigene assay and RNA-seq. gDNA samples extracted from the saliva of patients were provided by the Broad Institute. Whole-blood samples were provided by their physicians through the ATCP foundation, and RNA samples were extracted from these./p>T (p.Glu1978Ter); this variant has the highest allele frequency in this ATCP cohort among the variants annotated as pathogenic in ClinVar. It has gnomAD v.3.1 and ATCP cohort allele frequencies of 0.0000349045 and 0.034 (16/470), respectively. For the variant calls that had passed the allele frequency filter, their protein-coding and splicing impacts were examined on the basis of multiple computational tools: REVEL (for protein-coding impacts) and SpliceAI and MaxEntScan (for splicing impacts). Missense variants that were predicted as pathogenic by REVEL (score ≥ 0.5) were considered as disease candidate variants. Mis-splicing events with a SpliceAI score of 0.1 or higher were considered as likely true events. If the consequence of the mis-splicing is predicted to result in frameshift or loss of a crucial domain of the protein, the variant that caused the mis-splicing was classified as a disease candidate variant. For the patients in whom fewer than two disease candidate events were identified up to this step, we reviewed the remaining variants on a case-by-case basis (Supplementary Note 2)./p>T) who has been under treatment with atipeksen, as well as on five individuals in the ATCP cohort (four families; DDP_ATCP_42 (with c.5763-1050A>G), DDP_ATCP_218, DDP_ATCP_38/39, DDP_ATCP_96). In all six cases, we confirmed with Sanger sequencing that the two disease candidate variants in each case are in trans (Supplementary Tables 1 and 6)./p>3 nt by the variant]./p> 0.5)./p>T [in DDP_ATCP_138] and c.4801A>G [in DDP_ATCP_302]) did not pass this criterion as they showed predominant skipping of the exon of interest even in the absence of the variant of interest in the ATM gene region of the plasmids./p>T, a total of 32 ASOs were designed (12 for the initial screening and 20 for the fine-tuning screening). The ASOs were designed to be complementary to either the region encompassing the novel splice donor site in exon 53 created by c.7865C>T or predicted splice silencers surrounding the exon 53 canonical splice donor site. These silencers were predicted on the basis of a previously published hexamer-based model62. For c.5763-1050A>G, a total of 27 ASOs were designed (12 for the initial screening and 15 for the fine-tuning screening) to be complementary to the regions encompassing the novel splice donor site in intron 38 created by c.5763-1050A>G, the cryptic acceptor site of the pseudoexon in intron 38 or predicted splice silencers within the pseudoexon (also predicted on the basis of the hexamer model). For minigene-based validation of ASO amenability, a total of 24 ASOs were designed for 4 ASO-amenable variants (c.2839-579_2839-576del, c.2839-581G>A, c.6348-986G>T and c.3994-159A>G). The ASOs were designed to block either the splice donor/acceptor site or predicted exonic splicing silencers within a pseudoexon of interest. NT-20 and NT-22 (non-targeting oligonucleotides with the same chemistry) were used as negative controls1. For in vitro toxicity testing, ASO-tox, a gapmer with known toxicity, was used. All ASO sequences and detailed chemical modifications of ASOs are provided in Supplementary Table 13. All ASOs were manufactured by Microsynth. The ASO drug substance used in the atipeksen N-of-1 clinical trial was manufactured by ChemGenes in accordance with GMP guidelines./p>G, the distance between the two ATM variants was too far (around 2 kb) to distinguish the two bands representing normally and abnormally spliced products (which differ by 137 bp) on a agarose gel; therefore, a nested PCR was performed. PCR was performed using 1 µl of cDNA and a standard condition (35 cycles; 98 °C for 5 s, 60 °C for 15 s, 72 °C for 45 s). Relative quantities of the normally and abnormally spliced transcripts were measured by 1.5% agarose gel electrophoresis and densitometry analysis using ImageJ./p>

G (chr11:108236504-108236504T>G) variant, identified in DDP_ATCP_368, is located in a distal enhancer-like element, as catalogued by ENCODE. The absence of this variant in both gnomAD v3.1 and TOPMed freeze 8, with an allele frequency of 0.0, is consistent with a potential pathogenic role. Declaring this variant as disease-causing could complete the genetic diagnosis for DDP_ATCP_368. However, owing to a lack of substantial evidence confirming that the enhancer-like element is functional, and that the variant disrupts its function, we have refrained from declaring this variant as a disease candidate. For more details, see Supplementary Note 2./p>T, n = 1; c.2639-22_2639-20del, n = 2) have strongly predicted mis-splicing potential and have not been found in gnomAD v3.1 and TOPMed freeze 8 (Supplementary Table 1); because no other variants can explain the clinical diagnosis in the respective patients, we considered them as disease candidate variants. b, Recurrence of disease candidate variants. The recurrence distribution of disease candidate variants in 235 patients in the ATCP cohort is depicted. Homozygous variants are double-counted. The inset shows a magnified view of the dotted box region in the main graph./p>T (probably), c.2839-579_2839-576del (probably), and c.2839-581G>A (probably). b, Validation of complex mis-splicing effects (pseudoexon inclusion and exon extension) of an ASO-amenable variant, c.3994-159A>G (probably). c, Validation of exon extension effects of an ASO-amenable variant, c.6573-15T>G (possibly). d, Validation of complex mis-splicing effects (exon truncation and exon skipping) of ASO-amenable variants, c.2639-21A>G (possibly) and c.2639-22_2639-20del (possibly). e, Validation of exon skipping effects of ASO-amenable variants, c.496+5G>A (possibly) and c.2250G>A (possibly). For a–e, black asterisks and black arrows indicate PCR products confirmed by gel extraction and Sanger sequencing; grey arrows indicate bands not validated by Sanger sequencing (Supplementary Table 12). REF, reference allele; ALT, alternative allele; NTC, no-template control. For gel source data, see Supplementary Fig. 1./p>T. Note: this case is not in the ATCP cohort and is not the same case as DDP_ATCP_520, who is a currently 20-year-old patient with c.7865C>T. The paternally inherited c.8585-13_8598del variant disrupts the canonical splice acceptor site of exon 59, leading to the skipping of exon 59 (87 nucleotides), and removes critical residues from the highly conserved PI3K/PI4K kinase domain of the ATM protein. Although it is in-frame, because the skipped region contains a critical kinase domain, it results in complete loss of function. The maternally inherited c.7865C>T variant creates a novel splice site in exon 54, leading to mis-splicing that results in an out-of-frame truncation of 64 nucleotides from the 3′ end of exon 53. b, RNA-seq analysis of the trio’s fibroblast cell lines shows the gain-of-splicing effect in exon 53 by c.7865C>T in the patient and the mother. c, RNA-seq analysis of the trio’s fibroblast cell lines shows the gain-of-splicing effect in exon 59 by c.8585-13_8598del in the patient and the father. d, Determination of the gain-of-splicing effect of c.7865C>T in the trio’s fibroblast cell lines by allele-specific PCR that is specifically designed to exclude the non-target (parental) allele. This result of the allele-specific PCR on the naive patient fibroblasts was replicated in 11 additional biologically independent experiments (n = 3, initial; n = 2, fine-tuning; n = 3, AT008 dose–response; n = 3, AT026 dose–response; Fig. 5a and Extended Data Fig. 8c). For gel source data, see Supplementary Fig. 1./p>T in patient fibroblasts. It is depicted as in Fig. 5a, except the twelfth ASO of the initial screening round, AT012, is shown. AT012, designed to inhibit an intronic splicing silencer downstream of exon 53, showed no efficacy. b, Combined analysis of initial and fine-tuning rounds of screening for c.7865C>T. As AT008 was used in both the initial and fine-tuning rounds of screening (Fig. 5a), the ASO efficacy measurements of the two rounds were combined into a single figure by normalizing the measurements to the matched mean efficacy of AT008 of each round. All statistical information (sample size, error bar, statistical test, P value) is as in Fig. 5a, because this is a reanalysis of the data from that figure. For RT–PCR gel source data, see Supplementary Fig. 1./p>T. Phosphorylation of P53 and KAP1 in response to irradiation-induced DNA damage. Five independent replicate experiments were performed and band intensities were quantified to generate Fig. 5b. For AT007, AT008, AT022, and AT026, “AT0” was omitted. NT represents NT-22, a non-targeting ASO. For blot source data, see Supplementary Fig. 1. b, Quantification of immunocytochemistry analysis of the rescue of ATM signalling by the ASOs targeting c.7865C>T, demonstrating recovery of P53 and KAP1 phosphorylation following treatment with AT008 and AT026. For representative micrographs, see Fig. 5c. For quantification, 10 microscope field images were taken from each of three biologically independent wells for each condition. The % positive cells of the 10 microscope field images were averaged to yield the % positive cells value for each well. Error bars, mean of the three biological replicates ± 95% confidence interval. Two-sample (comparing each condition to NT-22) two-tailed t-test was performed. *, P < 0.05; **, P < 0.01. For pP53, AT008, **P = 9.5E-7; AT026, **P = 2.5E-7. For pKAP1, AT008, **P = 5.0E-6; AT026, **P = 8.5E-8. c, Dose–response analysis of rescue of ATM mis-splicing by AT008 and AT026. NT-20 and NT-22 are non-targeting ASOs. The fraction of the intensity of the normally spliced transcript of the total intensity of the normally and abnormally spliced transcripts was calculated for each condition. Three biologically independent experiments (independent electroporations) were conducted. Error bars, mean ± 95% confidence interval. The regression curves and EC50/IC50 values were calculated by using “log(agonist) vs. response - Variable slope” model in GraphPad Prism 8. 1,000 nM concentrations of AT008 and AT026 were excluded from analysis because they showed cell-level toxicity and anomalous RT–PCR results. Representative agarose gel images are shown at the bottom of the dose–response curves. For gel source data, see Supplementary Fig. 1. d, RNA-seq analysis of the rescue of mis-splicing in patient fibroblasts by AT008 and AT026. Numbers marked in red and blue represent the number of reads that can be unambiguously determined to be originated from either paternal or maternal allele. The percentage of functional splicing was calculated by scoring the fraction of transcripts that were without exon 52/53/52-53 skipping, intron 51/52/53 retention, and exon 53 truncation. pP53, phospho-P53; pKAP1, phospho-KAP1./p>G in one allele of DDP_ATCP_42 creates a novel splice site that causes the out-of-frame (137 bp) inclusion of a pseudoexon and premature translation termination of ATM. c.3993+1G>A in the other allele of the patient destroys a splice donor site, which leads to the use of a cryptic splice donor site in exon 26. The resulting mis-splicing truncates 120 nt from the 3’ end of exon 26. b, RNA-seq analysis of whole-blood samples from the patient and three other patients without c.5763-1050A>G shows the pseudoexon inclusion effect between exon 38 and exon 39 by c.5763-1050A>G. c, Determination of the pseudoexon inclusion effect of c.5763-1050A>G in the patient’s fibroblast cell line by allele-specific RT–PCR that is specifically designed to exclude the non-target allele (c.3993+1G>A). Because the distance between the two ATM variants were too far (~2 kb) to distinguish the two bands representing normally and abnormally spliced products (which differ by 137 bp) on an agarose gel, a nested PCR was performed. This result of the allele-specific PCR on the naive patient fibroblasts was replicated in 7 additional biologically independent experiments (n = 3, initial; n = 2, fine-tuning; n = 4; Extended Data Fig. 10b). For gel source data, see Supplementary Fig. 1./p>G. These ASOs were tested in the fibroblasts from DDP_ATCP_42 by using the allele-specific RT–PCR assay (Extended Data Fig. 9c). The positions and efficacy of the ASOs are illustrated (a, mean efficacy is represented using a grey scale; b, mean efficacy, along with a 95% confidence interval, is illustrated using bar graphs). Biologically independent experiments (independent transfections) were conducted for initial screening (n = 6, naive; n = 2, AT049, one of the three experiments was excluded from statistical analysis as it showed a highly anomalous result, Supplementary Fig. 1; n = 3, the other conditions) and fine-tuning screening (n = 3, AT046, AT059, AT060, AT063, AT064; n = 4, the other conditions). Error bars, mean ± 95% confidence interval (shown only for conditions with n ≥ 3). Two-sample (comparing each condition to NT-22) two-tailed t-test was performed for statistical analysis. *, P < 0.05; **, P < 0.01. For initial screening, AT051, **P = 0.0046; AT054, **P = 0.0058; AT050, **P = 0.0002; AT042, **P = 0.0048; AT053, **P = 0.0002; AT048, **P = 0.0004; AT045, **P = 3.3E-5; AT044, **P = 0.0003; AT047, **P = 0.0006; AT043, **P = 0.0002; AT046, **P = 0.0011. For fine-tuning screening, AT068, **P = 0.0030; AT066, **P = 0.0006; AT064, **P = 0.0006; AT065, **P = 0.0004; AT067, **P = 0.0042; AT063, **P = 0.0004; AT061, **P = 4.1E-5; AT060, **P = 3.2E-5; AT059, **P = 0.0002; AT046, **P = 2.9E-5; AT062, **P = 3.7E-5; AT043, **P = 3.6E-5; AT055, **P = 8.8E-7; AT057, **P = 2.8E-5; AT058, **P = 6.8E-5; AT056, **P = 1.6E-6. c, Combined analysis of initial and fine-tuning rounds of screening for c.5763-1050A > G. As AT043 was used in both the initial and fine-tuning screening, the ASO efficacy measurements of the two rounds were combined into a single figure by normalizing the measurements to the matched mean efficacy of AT043 of each round. All statistical information (sample size, error bar, statistical test, P value) is as in b because this is a reanalysis of the data from that panel. For a–c: six top-performing ASOs (blue letters) were selected from each length group (picking one ASO from each of the 17-, 18-, 19-, 20-, 21-, and 22-mers) for further validation (Extended Data Fig. 11a,b); three ASOs (underlined in red) were subjected to additional RNA-seq validation (Extended Data Fig. 11c); for RT–PCR gel source data, see Supplementary Fig. 1./p>G. pP53, phospho-P53; pKAP1, phospho-KAP1. “AT0” was omitted in AT043, AT056, AT058, AT062, AT065, and AT067. NT represents NT-22, a non-targeting ASO. b, Quantification of the immunoblots using GAPDH as the loading control. Irradiated, control fibroblasts (ATM +/−) were used for normalization. All six tested ASOs showed comparable efficacy in restoring the phosphorylation of the two downstream effectors. Three biologically independent experiments (independent transfections) were conducted for pP53 and pKAP. Two-sample (comparing each condition to NT-22) two-tailed t-test was performed to assess the statistical significance of the means normalized to carrier case with irradiation. Error bars, mean ± 95% confidence interval. *, P < 0.05. For pKAP1, AT043, *P = 0.0386; AT056, *P = 0.0395; AT058, *P = 0.0232; AT062, *P = 0.0145; AT065, *P = 0.0447; AT067, *P = 0.0287. For a,b, for blot source data, see Supplementary Fig. 1. c, RNA-seq validation of ASOs targeting c.5763-1050A>G. RNA-seq analysis of the fibroblast cell line established from DDP_ATCP_42 shows the pseudoexon inclusion effect of c.5763-1050A>G can be mitigated by AT043, AT056, and AT057. Among the tested ASOs, AT056 showed the highest efficacy in reducing the pseudoexon inclusion, making it the lead candidate. NT-22, non-targeting ASO./p>