Use of Antisense Morpholino Oligonuckeotides in correction of a novel splicing defect the BTK gene causing Bruton agammaglobulinemia

Abstract

X-linked agammaglobulinemia (XLA) is an inherited primary humoral immunodeficiency characterized by a profound deficiency of all immunoglobulins, mature B cells and plasma cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene. We studied six patients with clinically diagnosed XLA. Five BTK mutations were identified; three of which were novel and two were known mutations. One of them was an intronic point mutation, a C>A mutation in intron 3 of the BTK gene (c.240+109 C>A). Interestingly, its pathogenicity was evidenced by the presence of aberrant splicing of BTK mRNA, in which there was a 106-bp insertion between exons 3 and 4 resulting in a frameshift. Treatment of cultured peripheral blood mononuclear cells (PBMCs) from the patient with this splicing defect with Antisense Morpholino Oligonucleotides (AMOs) complementary to the mutation splice site sequence was performed. The AMOs were able to restore correctly spliced mRNA in the patient’s PBMCs. Using various concentrations of AMOs, the minimal level with maximal effect in correcting aberrant splicing of the BTK gene in the patient’s PBMCs was about 10 μM. The products of correctly spliced mRNA persisted for up to 30 days after AMOs administration. This approach in which AMOs are used to restore correct splicing of the target gene may be applicable to other splicing mutations and is of potential clinical interest.