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A framework for individualized splice-switching oligonucleotide therapy

July 12, 2023

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were ‘probably’ or ‘possibly’ amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

It is estimated that 1 in 10 individuals—more than 30 million people in the USA—have a diagnosis of a rare disease4. Although this term encompasses an estimated 7,000 different conditions, with a notable proportion having a known or presumed genetic aetiology, treatments are available for only around 5% of them4,5. The rarity of these conditions often makes it economically infeasible to develop treatments based on traditional modalities. Previous work has shown that, in some cases, splice-switching ASOs may restore functional protein levels and be administered in a safe and timely manner1. Such therapy presents a treatment opportunity; however, identifying individuals with genetic variants suitable for such attempts remains a challenge1,6. Here, we present a framework to systematically discover and develop splice-switching treatments for individuals with rare diseases, using ataxia-telangiectasia (A-T) as a model.

A-T is an autosomal recessive disease caused by biallelic loss of function of ATM, a gene involved in the cellular response to DNA double-strand breaks7. Clinically, A-T is characterized by progressive cerebellar degeneration, immunodeficiency and predisposition to cancer, with early manifestations including ataxia, involuntary movements, neuropathy, oculomotor apraxia, dysphagia, slurred speech and ocular and skin telangiectasias3. The disease has a prevalence of 1 in 40,000 to 100,000 live births worldwide2. In ‘classical’ A-T (the most typical and prevalent clinical presentation), the average life expectancy is 25 years, and early death is most frequently due to lung disease or cancer2. Hypomorphic ATM variants can cause a milder, slower-progressing phenotype, albeit still with severe morbidity8.

A-T is the most common form of inherited childhood progressive ataxia in many countries9. Nevertheless, effective therapies are lacking—particularly for the profound neurological manifestations that have a marked effect on quality of life. The large coding size (9.2 kb) of ATM poses a challenge for gene therapy approaches, as AAV vectors that are at present available have a packaging capacity of around 4.7 kb (ref. 10). The protein product of ATM is an intracellular kinase, which makes enzyme replacement approaches difficult. By contrast, A-T could be an appealing candidate for splice-switching ASO therapy, given that ASOs readily distribute to the cerebellum when injected intrathecally11.

The Global A-T Family Data Platform is an international initiative led by family advocates and primarily funded by A-T Children’s Project (ATCP), a patient-advocacy foundation for A-T. ATCP has systematically collected clinical information and genomic DNA (gDNA) from individuals who have been clinically or genetically diagnosed with A-T. Through this effort, whole-genome sequencing (WGS) was performed, yielding a WGS dataset of 235 individuals with A-T. We analysed this ‘ATCP cohort’ to establish molecular diagnoses, identify pathogenic variants that could be amenable to splice-switching ASO therapy, develop proof-of-concept individualized ASOs and initiate a pilot investigational trial.

The ATCP cohort comprised de-identified phenotypic and genomic data from 235 individuals who were clinically or genetically diagnosed with A-T, from 209 families. Of these individuals, 46% were female (108/235) and the median age of clinical A-T diagnosis was 3.6 years (range 0–32; Extended Data Table 1 and Supplementary Table 1).

The results of molecular genetic testing were available for only a small subset of individuals within the cohort (23%, 55/235), mostly from sequencing of ATM coding regions (Fig. 1a and Extended Data Table 1). We therefore performed an extensive set of WGS analyses on all individuals to generate a comprehensive picture of genetic variation in A-T. We identified single-nucleotide variants (SNVs) and short indels using GATK12 (Supplementary Table 2), VarScan213 and Strelka214; and copy number variants (CNVs), structural variants (SVs) and mobile DNA element insertions using Delly15, Pindel16, MELT17 and xTea18 (Supplementary Table 3). For completeness, we manually inspected sequencing reads across the ATM locus using the Integrative Genomics Viewer to identify variants that might have been missed by the aforementioned methods. Variants were named with respect to the GRCh38/hg38 reference genome and NM_000051.3, the canonical RefSeq transcript for ATM.

Kim, J., Woo, S., de Gusmao, C. M., Zhao, B., Chin, D. H., DiDonato, R. L., Nguyen, M. A., Nakayama, T., Hu, C. A., Soucy, A., Kuniholm, A., Thornton, J. K., Riccardi, O., Friedman, D. A., El Achkar, C. M., Dash, Z., Cornelissen, L., Donado, C., Faour, K. N. W., … Yu, T. W. (2023, July 12). A framework for individualized splice-switching oligonucleotide therapy. Nature News.

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