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A framework for individualized splice-switching oligonucleotide therapy – Nature

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Patients

The WGS and clinical data of 235 patients with A-T were provided by the Global A-T Family Data Platform of ATCP. Our access to the data was approved by the Data Access Committee of ATCP.

Selected patients with A-T enrolled at the Manton Center for Orphan Disease Research under the approval of the Institutional Review Board (IRB) at Boston Children’s Hospital (10-02-0053). These included the individual with c.7865C>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.

Functional studies using the cells derived from patients and their families were performed after obtaining appropriate consent under the auspices of an IRB-approved protocol maintained by the Manton Center for Orphan Disease Research Gene Discovery Core at Boston Children’s Hospital. Skin fibroblasts were derived from a 2-mm punch biopsy taken from the patient’s skin using explant culture. Fibroblasts were maintained and passaged in medium containing DMEM (Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco). Fibroblasts used in experiments were under passage 20.

Variant calling

WGS reads were aligned to GRCh38/hg38 using BWA (v.0.7.17) (ref. 46) and pre-processing and quality control were performed according to GATK Best Practice Workflows12. Multiple computational tools were used to call diverse types of variants, including GATK-HaplotypeCaller (v.3.5) (ref. 12), VarScan2 (v.2.4.4) (ref. 13) and Strelka2 (v.2.9.10) (ref. 14) for SNVs and short indels (less than 50 nt in length; Supplementary Table 2) and Delly (v.0.8.6) (ref. 15), Pindel (v.0.2.5b8) (ref. 16), MELT (v.2.2.2) (ref. 17), and xTea (v.0.1.7)18 for SVs (50 or more nt in length; Supplementary Table 3), with MELT and xTea used specifically for transposons. For large CNVs with imprecise boundaries, we manually inspected read alignments on Integrative Genome Viewer (IGV; v.2.8.9) (ref. 47) to determine rough boundaries of the variants.

Relatedness

To analyse relatedness among the 235 individuals in the ATCP cohort, we used VCFtools (v.0.1.17) with the ‘relatedness2’ option48 (Supplementary Table 1), which is based on the KING software package49. For individuals with the annotated relatedness information in the clinical record, all annotations were consistent with the inferred relatedness.

Variant effect prediction

For variant effect prediction, VEP (release 100)50 was used to annotate all SNVs and short indels. Protein-coding impact was evaluated using REVEL21 and experimental evidence of protein functionality in the literature (Supplementary Table 7); mis-splicing impact was evaluated using MaxEntScan24, SpliceAI20 and LaBranchoR25. LaBranchoR-predicted branchpoint coordinates on GRCh37/hg19 were downloaded. After converting them to the GRCh38/hg38 coordinates (using LiftOver), their potential overlap and distance to variants were examined. For variants shorter than 50 nucleotides, the allele frequencies were looked up in gnomAD (v.3.1) (ref. 51) and TOPMed (freeze 8; ref. 52). For SVs, the allele frequencies were looked up in dbVar53, DGV54 and gnomAD SVs (v.2.1) (ref. 55). The clinical significance of variants was looked up in ClinVar (as of 2 June 2020; ref. 56). All disease candidate SV events were confirmed by manually inspecting the raw sequencing data on IGV.

ACMG classification

Disease candidate variants were classified using a five-tiered system in accordance with the guidelines outlined by ACMG19,57. For predicted loss-of-function variants, we used specialized ACMG recommendations to apply the PVS1 criteria58. For CNVs, we used a specialized scoring framework proposed by ACMG and Clinical Genome Resource (ClinGen)59.

Determination of disease candidate variants

First, we defined SDVs. These include frameshift, stop-gain, start-loss and splice-site-destroying variants, and structural variants affecting one or more exons, as well as missense variants and short in-frame indels with previous experimental evidence of functional loss. Splice-site-destroying variants were defined as: (SpliceAI donor/acceptor loss score ≥ 0.1 at a canonical splice site) AND (MaxEntScan donor/acceptor score with the ALT allele < MaxEntScan donor/acceptor score with the REF allele) AND [(MaxEntScan donor/acceptor score with the ALT allele < 2) OR (MaxEntScan donor/acceptor score with the ALT allele < 0.3 × MaxEntScan donor/acceptor score with the REF allele)]. All SDVs were considered as disease candidate variants. Second, variants that were annotated as pathogenic or likely pathogenic in ClinVar were considered as disease candidate variants.

For the patients in whom fewer than two disease candidate events were identified in the previous two steps, we analysed the remaining variants in each patient on the basis of the population and cohort allele frequencies. We filtered out variant calls whose population or cohort allele frequencies are higher than that of c.5932G>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).

Sanger sequencing validation of a subset of disease candidate variants was performed using available patient gDNA samples. The PCR protocol comprised 10 ng template DNA, 10 µl KAPA2G Robust HotStart ReadyMix (2X; Kapa Biosystems), 1 µl site-specific primer pairs (10 µM), and PCR-grade water to a final volume of 20 µl. The cycling parameters were 94 °C for 3 min; 30 cycles of 94 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s; 72 °C for 3 min; and held at 4 °C. Validation primers are listed in Supplementary Table 4. All PCR amplicons were visualized on 2% agarose gels. Variants and corresponding genotypes were confirmed by Sanger sequencing (Supplementary Table 5).

Phasing of disease candidate variants

Trio Sanger sequencing

Trio Sanger sequencing was performed on the family of the patient (with c.7865C>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).

Homozygosity

In 32 cases (32 families), disease candidate variants were found to be homozygous. In five other cases (five families), disease candidate variants appeared homozygous owing to being in trans with a deletion at a locus overlapping the variants (Supplementary Table 1).

Read-based phasing

When the distance between two disease candidate variants is shorter than the read length, the two variants can be phased using read-based phasing methods. We used WhatsHap (v.1.0) (ref. 23), a read-based phasing tool, to analyse such cases, and found that in two cases (one family), the two disease candidate variants were in trans. These two variants were only 62 bp apart and were also confirmed by manual inspection of the raw sequencing data on IGV (Supplementary Table 1).

Variant co-occurrence

The gnomAD variant co-occurrence database can be used to predict that the two variants are likely to be in cis or in trans60. If two variants are in the same haplotype (that is, in cis), they tend to appear in the same individual. This analysis could be performed only for individuals whose two disease candidate variants are represented in the gnomAD database (v.2.1.1, in GRCh37/hg19 coordinates) at a global allele frequency of higher than 0% and less than 5%. A total of 47 individuals (38 families) in the ATCP cohort met these criteria. The analysis showed that 2 disease candidate variants are highly likely to be on different haplotypes in all of the 47 individuals (Supplementary Table 1).

ASO amenability taxonomy

General rules

(1) If a variant damages both a canonical splice site and protein-coding function at the same time, more severe damage is considered as the representative damage of the variant. (2) Solid experimental evidence on mis-splicing or coding impact of a variant, if available, can override computational predictions. For a schematic illustration of the taxonomy, see Fig. 2.

Damage to canonical splicing

(1) Severe: (i) SpliceAI donor/acceptor loss score at a canonical splice site ≥ 0.1, (ii) MaxEntScan donor/acceptor score with the ALT allele at the site < MaxEntScan donor/acceptor score with the REF allele at the site, AND (iii) [MaxEntScan donor/acceptor score with the ALT allele at the site < 2] OR [MaxEntScan donor/acceptor score with the ALT allele at the site < 0.3 × MaxEntScan donor/acceptor score with the REF allele at the site].

(2) Moderate: (i) NOT severe (as defined above), (ii) SpliceAI donor/acceptor loss score at a canonical splice site ≥ 0.1, AND (iii) [MaxEntScan donor/acceptor score with the ALT allele at the site < MaxEntScan donor/acceptor score with the REF allele at the site, MaxEntScan donor/acceptor score with the ALT allele at the site ≥ 2, AND MaxEntScan donor/acceptor score with the ALT allele at the site ≥ 0.3 × MaxEntScan donor/acceptor score with the REF allele at the site] OR [The variant is ≤3 nt away from the LaBranchoR-predicted branchpoint OR the distance between the LaBranchoR-predicted branchpoint and the site is changed by >3 nt by the variant].

(3) No to little: NEITHER severe NOR moderate (as defined above).

Damage to protein-coding function

(1) Severe: (i) frameshift, stop-gain, or start-loss variant OR (ii) missense variant predicted as pathogenic by REVEL (score > 0.5).

(2) No to little: (i) NOT severe AND (ii) synonymous variant or missense variant predicted as benign by REVEL (score ≤ 0.5).

Mis-splicing type

(1) Gain of mis-splicing (gain): (i) SpliceAI donor/acceptor gain score at a non-canonical site ≥ 0.1 AND (ii) MaxEntScan donor/acceptor score with the ALT allele at the site ≥ 2.

(2) Exon skipping or intron retention (skipping or retention): SpliceAI donor/acceptor loss score at any canonical site ≥ 0.1 without an accompanying gain of mis-splicing by SpliceAI (donor/acceptor gain score < 0.1 at any non-canonical splice site).

(3) Neither: NEITHER gain, skipping, NOR retention.

Minigene assay

Plasmid construction

To generate a minigene, we used the pSpliceExpress plasmid, which was a gift from S. Stamm (Addgene plasmid 32485; http://n2t.net/addgene:32485; RRID: Addgene_32485; ref. 61). The genomic fragment with a variant of interest was cloned into the pSpliceExpress donor vector using the BP recombination reaction. The inserted fragments for reference and alternative alleles were generated by a two-step PCR procedure. In the first round of PCR, the genomic region of interest was amplified from patient gDNA with attB tagged primers, which added 12 nucleotides of the attB1 and attB2 sites to the ends of amplicons. The second PCR reaction used the first PCR products as templates and extended them to contain complete attB sequences using universal adapter primer pairs. All PCR reactions were performed with Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) or PrimeSTAR GXL DNA polymerase (Takara Bio). Primer sequences used for minigene constructions were listed in Supplementary Table 10. Full attB PCR products were purified using the PureLink PCR Purification Kit or PureLink Quick Gel Extraction Kit (Invitrogen). Gateway BP Clonase II Enzyme Mix (Invitrogen) was used to recombine attB PCR products into pSpliceExpress. In brief, approximately 25 fmol (1 kb PCR product is 0.65 ng fmol−1) of purified attB PCR product was added to 75 ng of donor vector, TE buffer and 1 µl of BP Clonase Enzyme Mix to a final reaction volume of 5 µl. The reaction was incubated at room temperature for 1 h, after which 0.5 µl Proteinase K was added to stop the reaction. One microlitre of each BP Clonase reaction product was transformed into 25 µl OneShot TOP10 Chemically Competent Escherichia coli (Thermo Fisher Scientific). Transformed E. coli was spread on LB agar plates with ampicillin (1× LB agar with 50 µg ml−1 ampicillin) and incubated overnight at 37 °C. To screen for positive colonies containing the desired plasmids, a dozen colonies for each variant were picked up and diluted in 50 µl sterile water. Subsequently, colony PCRs were performed using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific), followed with 2% agarose gel inspection. The cycling programme was: bacteria were lysed and DNA was denatured at 98 °C for 10 min, followed by 30 cycles of 98 °C for 10 s, optimal annealing temperature for 20 s and 72 °C for 30 s, and final extension for 5 min at 72 °C. Primer sequences used for colony PCR are listed in Supplementary Table 10. Positive colonies were inoculated in liquid LB with ampicillin (1× LB and 50 µg ml−1 ampicillin) and were cultured in a shaking incubator at 275 rpm at 37 °C for 12–18 h. Plasmid DNA was extracted from overnight cultures using PureLink Quick Plasmid Miniprep Kit (Invitrogen) or ZR plasmid Miniprep Kit (Zymo Research). The genotypes and the sequences of plasmid inserts were confirmed by Sanger sequencing (Supplementary Table 11). At least one wild-type and one mutant plasmid were identified for each variant.

In some variants, full attB PCR products could not be amplified directly from patient gDNA owing to low quality or unavailability of the patient gDNA. In these cases, a wild-type fragment was amplified from human male gDNA (Promega) and used to construct reference plasmids as described above. The Q5 site-directed mutagenesis kit was used to introduce the variants into the reference plasmids (New England Biolabs). Twenty-five-microlitre PCR reactions were set up with mutagenic primers (Supplementary Table 10) and Q5 Hot Start High Fidelity 2X Master Mix to introduce the variant into the reference plasmids and amplify the mutant plasmids. The samples were denatured at 98 °C for 30 s and subjected to 25 cycles of 98 °C for 10 s, 50–72 °C (various annealing temperatures were tested) for 10 to 30 s, 72 °C for 20–30 s per kb, followed by a final extension at 72 °C for 2 min. The linear PCR products were ligated into the plasmid through DpnI restriction digestion and ligation. The mutant plasmids were transformed into competent E. coli. Single colonies were screened and inoculated in liquid LB and ampicillin. Plasmid DNA was collected from overnight cultures.

Splicing assay

Around 1 × 105 HEK293T cells were seeded in 24-well plates. When the cells reached about 90% confluency, they were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). For each transfection, 4 µl of plasmid was added to each well along with 1.5 µl Lipofectamine, 2 µl P3000 and 50 µl Opti-MEM (Thermo Fisher Scientific). For some transfections, ASOs were also added at a final concentration of 200 µM. Twenty-four hours after transfection, total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). RNA was then reverse-transcribed into cDNA in a 4-μl total reaction consisting of 3 µl RNA and 1 µl of SuperScript IV VILO Master Mix (Thermo Fisher Scientific). The reverse transcription reactions were incubated at 25 °C for 10 min, 50 °C for 10 min and 85 °C for 5 min. To detect transcripts transcribed from the transfected plasmids, 1 µl cDNA was amplified using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific), 2× KAPA SYBR Fast qPCR Master Mix (Kapa Biosystems) or 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems). For primers, we used rat insulin primers that bind to the minigene exons flanking the inserted ATM gene region (Supplementary Table 10). The final PCR products were run and visualized on 2% agarose gel. Mis-splicing bands were extracted using the PureLink Quick Gel Extraction (Invitrogen) and confirmed by Sanger sequencing (Supplementary Table 12).

Quality control

If the amount of the canonical splicing isoform represented less than 50% of the total amount of all ATM isoforms, we disqualified and excluded the minigene assay plasmids for further analysis. We found that some of the plasmids bearing the ATM gene region did not express the normally spliced isoform even without any variant, which makes them unsuitable to assess the mis-splicing effects of variants. Therefore, we excluded them from the analysis. The minigene assay plasmids carrying the ATM gene contexts of two variants (c.3489C>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.

ASO development

ASOs

For c.7865C>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.

ASO screening

Fibroblasts were transfected with 200 nM ASOs using Lipofectamine 3000 (Thermo Fisher Scientific). Twenty-four hours after transfection, total RNA was isolated using PureLink RNA Mini (Invitrogen). cDNA synthesis using oligo-dT and random hexamers was performed using the Superscript VILO reverse transcriptase kit (Invitrogen). For allele-specific PCR, primers were designed to specifically exclude the non-target allele in each patient (Extended Data Figs. 6d and 9c and Supplementary Table 14). For c.5763-1050A>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.

ASO validation

Immunoblotting

Fibroblasts were transfected with 400 nM ASO as described above. Forty-eight hours after transfection, cells were irradiated with 10 Gy using a caesium-137 source, and then incubated for 30 min at 37 °C. Cell lysates were then collected using RIPA buffer (Boston Bioproducts) supplemented with Roche PhosSTOP (Sigma-Aldrich). Lysates were incubated with 4× Laemmli buffer (BioRad) and loaded onto 4–15% precast gradient protein gels (BioRad) and separated by electrophoresis. Protein samples were then transferred to PVDF membranes, which were subsequently incubated overnight with primary antibodies for phospho-P53 (Cell Signaling Tech, diluted 1:500) and phospho-KAP1 (Bethyl Lab, diluted 1:1,000). GAPDH was used as a loading control and primary antibody for GAPDH (Proteintech) was diluted to 1:250. Following incubation with secondary antibodies that were diluted to 1:5,000 for phospho-P53, phospho-KAP1 and GAPDH (Li-Cor), targets were visualized with the Li-Cor Odyssey system and quantified with densitometry analysis (ImageJ).

Immunocytochemistry

Fibroblasts were transfected with 200 nM of ASOs as described above. Forty-eight hours after transfection, cells were irradiated with 1.5 Gy using a caesium-137 source, and then incubated for 60 min at 37 °C. Cells were washed in PBS, fixed in 4% (w/v) paraformaldehyde and permeabilized with 0.1% (w/v) Triton X-100 in PBS at room temperature. Cells were then incubated overnight in PBS with 3% BSA and antibodies to phospho-P53 (Cell Signaling Tech) and phospho-KAP1 (Bethyl Lab) and were visualized with immunoglobulin G Alexa Fluor conjugates (Life Technologies). DNA was counterstained with Hoechst 33342. Images were collected with the ImageXpress Micro microscope (Molecular Devices) and processed with MetaXpress (Molecular Devices). The abundance of targets expressed in nuclei was quantified.

Dose–response

Fibroblasts were electroporated using the Neon Transfection System (Thermo Fisher Scientific) with varying amounts of ASOs: 0–1,000 nM (0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000 nM; final concentrations). Twenty-four hours after electroporation, total RNA was isolated as described above. cDNA synthesis, RT–PCR, gel electrophoresis and densitometry were performed as described above.

RNA-seq

Fibroblasts were transfected with 200 nM of ASOs as described above. Forty-eight hours after transfection, total RNA was isolated as described above. RNA-seq libraries were prepared using the KAPA Hyper Prep kit (KAPA Biosystems). Sequencing was performed on an Illumina HiSeq 2500 (for sequencing; 2 × 100 bp). For alignment, STAR (v.2.7.5c) (ref. 63) was used to map reads on GRCh38/hg38 in the paired-end, two-pass mode to yield BAM files that were sorted by chromosomal coordinates. Gene annotation was not provided to the alignment program to avoid any biased alignment favouring annotated splice junctions. The sorted BAM files were indexed using SAMtools (v.1.10) (ref. 64). IGV was used to draw sashimi plots, which showed the number of reads supporting splice junctions.

Off-target analysis

The following derivative sequences were computationally generated from the sequences of AT008 (atipeksen), AT026, AT056, nusinersen and milasen: (1) sequences with progressively trimmed ends, starting from the full-length ASO sequences down to 16 nt in length; (2) sequences with up to 2 nt mismatches; and (3) sequences with a 1-nt internal insertion or deletion (Supplementary Figs. 58 and 11). BWA (v.0.7.17) (ref. 46) was used to align the generated sequences on GRCh38/hg38 and the RefSeq transcriptome sequences, downloaded from the UCSC Genome Browser.

In vitro ASO toxicity assay

An FITC Annexin V Apoptosis Detection Kit I (BD 556547, BD Biosciences) was used to quantitatively measure the percentage of cells undergoing apoptosis after transfection with ASOs at different concentrations as described above. Cells were collected, washed with PBS and resuspended in 1× binding buffer four days after transfection. Five hundred microlitres of the resuspended cells was stained with 5 µl of Annexin V-FITC and 5 µl propidium iodide (PI) in the dark at room temperature for 15 min. The cells were analysed using a flow cytometer (BD FACSAria III system) and were quantified by FlowJo software. The Annexin-V-positive and PI-negative fraction was ‘early apoptotic’, and the Annexin-V-positive and PI-positive fraction was ‘late apoptotic or necrotic’.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.



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