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Updated: Sep 10, 2025

Using the E1A Minigene Tool to Study mRNA Splicing Changes
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Context Matters: The Importance of a Comprehensive Genomic Region When Assessing the Therapeutic Potential of

Dyah W Karjosukarso1, Julia F Kiefmann1, Femke Bukkems1

  • 1Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Nucleic Acid Therapeutics
|August 25, 2025
PubMed

Insights

Mini/midigene splicing assays using antisense oligonucleotides (AONs) showed surprising efficacy for ABCA4 variants. A maxigene with full genomic context improved AON candidate selection for splicing modulation therapy.

Area of Science:

  • Genetics
  • Molecular Biology
  • Ophthalmology

Background:

  • Splicing modulation therapy, often using antisense oligonucleotides (AONs), is a promising approach for genetic disorders.
  • Assessing AON efficacy typically involves mini/midigene splicing assays.
  • A recurrent variant in the ABCA4 gene (c.768G>T) causes a splicing defect relevant to retinal diseases.

Purpose of the Study:

  • To evaluate the efficacy of 25 AONs targeting the ABCA4 splicing defect.
  • To investigate the impact of genomic context on AON efficacy in splicing assays.
  • To determine the necessity of including proper genetic context in splicing assays for therapeutic assessment.

Main Methods:

  • Testing 25 AONs in a midigene assay containing parts of ABCA4 intron 5, exon 6, and intron 6.
  • Comparing AON efficacy in the midigene assay versus a maxigene assay with extended genomic context (introns 5-11).
  • Corroborating findings with patient-derived retinal cells.

Main Results:

  • Nearly all tested AONs demonstrated high efficacy in the midigene assay, hindering candidate selection.
  • The maxigene assay, incorporating a larger genomic region, successfully differentiated between efficacious and less efficacious AONs.
  • Results from the maxigene assay aligned with observations in patient-derived retinal cells.

Conclusions:

  • The lack of extensive genomic context in mini/midigene assays may overestimate AON efficacy.
  • Including a comprehensive genomic context in splicing assays is crucial for accurately assessing AONs for splicing modulation therapy.
  • Maxigene assays provide a more reliable platform for selecting effective AON candidates for ABCA4-related disorders and potentially other genetic conditions.
Keywords:
antisense oligonucleotidesgenomic contextmidigenessplicing assay

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