Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
Burcu Bestas, … , Jesper Wengel, C.I. Edvard Smith
Burcu Bestas, … , Jesper Wengel, C.I. Edvard Smith
Published August 8, 2014
Citation Information: J Clin Invest. 2014;124(9):4067-4081. https://doi.org/10.1172/JCI76175.
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Research Article Immunology

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

Abstract

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton’s tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2′-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro–B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Authors

Burcu Bestas, Pedro M.D. Moreno, K. Emelie M. Blomberg, Dara K. Mohammad, Amer F. Saleh, Tolga Sutlu, Joel Z. Nordin, Peter Guterstam, Manuela O. Gustafsson, Shabnam Kharazi, Barbara Piątosa, Thomas C. Roberts, Mark A. Behlke, Matthew J.A. Wood, Michael J. Gait, Karin E. Lundin, Samir El Andaloussi, Robert Månsson, Anna Berglöf, Jesper Wengel, C.I. Edvard Smith

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Figure 1

Design of SCOs using bioinformatic tools to search for ESE sites in the disease-causing pseudoexon.

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Design of SCOs using bioinformatic tools to search for ESE sites in the ...
(A) Three different algorithms from the corresponding web-based servers, ESE-Finder, RESCUE-ESE, and PESX, were used, and the hits were aligned to the pseudoexon sequence to find locations with the highest correlation between algorithms. SCOs targeting the probable ESE regions were subsequently designed. SCOs are schematically presented with their binding-positions along the pseudoexon. (B) Predicted outcome of splicing, with or without SCOs, indicated schematically.