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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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 2

Splice correction–induced upregulation of reporter minigene activity.

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Splice correction–induced upregulation of reporter minigene activity. Th...
The U2OS cell line stably carrying a minigene interrupted by the mutated BTK intron 4 was transfected with different SCOs, and efficacy was measured as luciferase activity or mRNA restoration. (A) The relative luciferase activity in comparison to nontreated cells (mock) 24 hours after SCO transfection. “705” indicates an unrelated control SCO, targeting position 705 of a mutated β-globin intron, not complementary to the BTK pre-mRNA. All tested SCOs are 2′OMePS based (Table 1). Data represent mean + SD of 3 independent experiments, each with 2 replicates. **P ≤ 0. 01, ***P ≤ 0.001, versus 705. (B) Increase in luciferase activity by 2′OMePS-modified SCOs versus LNA SCOs is presented as fold increase over nontreated cells (mock). Graph represents mean + SD of 3 independent experiments, each with 2 replicates. **P ≤ 0. 01, ****P ≤ 0.0001, 186.18 versus 186.15-5LNA. (C) Total RNA RT-PCR showing the 389-bp and 271-bp bands corresponding to aberrant and corrected (with BTK intron 4 excised) mRNA bands, respectively, with a lower band of ribosomal RNA (18S) serving as an RNA quality control. The lane on the right represents RNA input from nontreated (NT) cells. “Scr” represents the control LNA/2′OMePS SCO not complementary to the reporter gene sequence (Table 1). A representative gel from 2 independent experiments is shown. Statistical significance was determined using 1-way ANOVA, followed by Bonferroni’s multiple-comparison test.