Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
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
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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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 5

Enhanced survival and anti-IgM–induced BTK tyrosine phosphorylation in SCO-treated BAC transgenic B cells.

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Enhanced survival and anti-IgM–induced BTK tyrosine phosphorylation in S...
(A) Spleen B cells from BAC transgenic mice were treated with 3.2 μM SCO (186.15-3LNA or scrambled control) and stimulated with 20 μg/ml anti-IgM 48 hours after electroporation (time 0). Cells were counted, and viability was determined by trypan blue exclusion 24 and 48 hours after stimulation. The initial number of cells from both WT and BAC transgenic mice was 2 × 106. Data represent 2 independent experiments with duplicates. Note that nontreated and scrambled ON–treated cell numbers overlap. Statistical significance was analyzed by using 1-way ANOVA followed by Bonferroni’s multiple-comparison test. ****P ≤ 0.0001 for BAC SCO treated versus BAC Scr and NT at 24 hours. (B) Analysis of BTK tyrosine phosphorylation after 3.2 μM SCO (186.15-3LNA) treatment and anti-IgM stimulation (20 μg/ml). Blots show pY551 phosphorylation of BTK and total BTK. Whole cell lysate (WCL) analysis shows BTK protein and actin. Each duplicate (–/+) corresponds to starved or anti-IgM–activated conditions. A representative blot is shown from 2 independent experiments. Note that the higher molecular weight band, located immediately above the pY551-BTK band in activated samples (IP pY551 gel), is of unknown origin and unrelated to BTK, since it also exists in B cells obtained from Btk KO mice following stimulation. The bar graph shows the quantitative analysis of Y551 phosphorylation as a percentage of relative intensity signal from the blots according to ImageJ Software.