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 4

Full-length human mutated BTK pre-mRNA corrected by SCOs yielding normal-sized transcripts and detectable BTK protein.

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Full-length human mutated BTK pre-mRNA corrected by SCOs yielding normal...
(A) B cells from human BAC transgenic mice were treated with 1.6 μM or 3.2 μM 186.15-3LNA SCO and analyzed by RT-PCR. To identify human RNA in cells from BAC transgenic and KO mice, a primer pair only detecting human BTK was used. To measure endogenous mouse Btk mRNA levels in splenocytes derived from WT mice, a different primer set selective for mouse Btk was used. A representative gel from 3 independent experiments is shown. Ribosomal RNA (18S) was used as an RNA quality control. (B) FACS analysis of cells 48 hours after treatment with 186.15-3LNA. B cells were stained with antibodies directed against CD19 or BTK protein (recognizing both mouse and human protein). Histograms for BTK expression in each sample from a representative experiment as well as the percentage of corrected cells from 3 independent experiments are shown (mean + SD). A concentration of 3.2 μM was used for the scrambled control SCO (Table 1). Mean fluorescence intensity, WT NT: 2,314; BAC SCO treated: 1,210. Percentage of WT BTK RNA was calculated as WT RNA fraction × 100/(misspliced + WT RNA fractions).