Cell-penetrating peptide-conjugated, splice-switching oligonucleotides mitigate the phenotype in BTK/Tec double deficient X-linked agammaglobulinemia model

Burcu Bestas; H. Yesid Estupiñán; Qing Wang; Shabnam Kharazi; Chenfei He; Dara K. Mohammad; Dhanu Gupta; Oscar P. B. Wiklander; Taavi Lehto; Karin E. Lundin; Anna Berglöf; Mikael C. I. Karlsson; Frank Abendroth; Samir El Andaloussi; Michael J. Gait; Matthew J. A. Wood; Christian J. Leumann; Dmitry A. Stetsenko; Robert Månsson; Jesper Wengel; Rula Zain; C. I. Edvard Smith

doi:10.1039/D4CB00312H

Cell-penetrating peptide-conjugated, splice-switching oligonucleotides mitigate the phenotype in BTK/Tec double deficient X-linked agammaglobulinemia model†

Burcu Bestas,

^a H. Yesid Estupiñán,^am Qing Wang,^a Shabnam Kharazi,^a Chenfei He,^b Dara K. Mohammad,^a Dhanu Gupta,^af Oscar P. B. Wiklander,^an Taavi Lehto,^acp Karin E. Lundin,^a Anna Berglöf,^a Mikael C. I. Karlsson,^b Frank Abendroth,^do Samir El Andaloussi,^ae Michael J. Gait,^d Matthew J. A. Wood,^f Christian J. Leumann,^g Dmitry A. Stetsenko,^ij Robert Månsson,^ah Jesper Wengel,

^k Rula Zain^ael and C. I. Edvard Smith

*^ae

Author affiliations

* Corresponding authors

^a Department of Laboratory Medicine, Karolinska Institutet, ANA Futura, Alfred Nobels Allé 8 Floor 8, SE-141 52 Huddinge, Sweden
E-mail: edvard.smith@ki.se

^b Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

^c Institute of Techology, University of Tartu, Tartu, Estonia

^d Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK

^e Karolinska ATMP Center, Karolinska Institutet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden

^f Department of Paediatrics, University of Oxford, Oxford OX3 7TY, UK

^g Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland

^h Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden

ⁱ Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentiev Ave., Novosibirsk 630090, Russia

^j Department of Physics, Novosibirsk State University, 2 Pirogov Str., Novosibirsk 630090, Russia

^k Department of Physics, Chemistry and Pharmacy, Biomolecular Nanoscale Engineering Center, University of Southern Denmark, Odense, Denmark

^l Centre for Rare Diseases, Department of Clinical Genetics and Genomics, Karolinska University Hospital, SE-171 76 Stockholm, Sweden

^m Departamento de Ciencias Básicas, Universidad Industrial de Santander, Bucaramanga, Colombia

ⁿ Breast Center, Karolinska Comprehensive Cancer Center, Karolinska University Hospital, Stockholm, Sweden

^o Department of Chemistry, University of Marburg, Marburg, Germany

^p Institute of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, 50411 Tartu, Estonia

Abstract

Splice-switching oligonucleotides (SSOs) have been developed as a treatment for various disorders, including Duchenne muscular dystrophy and spinal muscular atrophy. Here, the activity of several different SSOs was investigated as potential treatments for B lymphocyte disorders with a focus on X-linked agammaglobulinemia (XLA), caused by defects in the gene encoding Bruton's tyrosine kinase (BTK). In this study, the activity of locked nucleic acid (LNA), tricyclo-DNA (tcDNA), phosphoryl guanidine oligonucleotides (PGO) and phosphorodiamidate morpholino oligomers (PMO) were compared, targeting the pseudoexon region of BTK pre-mRNA. We further investigated the effect of conjugating cell-penetrating peptides, including Pip6a, to the SSOs. The effect was measured as splice-switching in vitro as well as in a further developed, bacterial artificial chromosome transgenic mouse model of XLA. Therapy in the form of intravenous infusions 2 times a week during 3 weeks of PMO oligomers conjugated to Pip6a was sufficient to partly restore the in vivo B lineage phenotype. SSOs treatment also provides a unique opportunity to get insights into a restoration process, when B lymphocytes of different maturation stages are simultaneously splice-corrected.

This article is part of the themed collection: Nucleic Acid Chemistry: celebrating Christian Leumann’s retirement

RSC Chemical Biology

Cell-penetrating peptide-conjugated, splice-switching oligonucleotides mitigate the phenotype in BTK/Tec double deficient X-linked agammaglobulinemia model†

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Cell-penetrating peptide-conjugated, splice-switching oligonucleotides mitigate the phenotype in BTK/Tec double deficient X-linked agammaglobulinemia model

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