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An important percentage of disease causing mutations affects pre-mRNA splicing. The mechanism of pre-mRNA splicing is controlled by sequence elements within the exons and introns. These sequence elements recruit the spliceosome for directing the splicing process. Mutations disrupting the sequence elements commonly result in exon exclusion or cryptic exon inclusion named as pseudoexon. The result is typically a frame-shift in the pre-mRNA causing production of partially functional or a defective protein. A common way to correct splicing mutations is to use antisense oligonucleotides (AONs) that are short complementary sequences that bind to the pre-mRNA and re-direct the splicing.X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease that is caused by the mutations in the gene named Bruton's Tyrosine Kinase (BTK). BTK has a crucial role in B cell development and the absence of BTK introduces a developmental block at the stage where the transition between pro-B and pre-B cells takes place. A prominent percentage of splicing mutations affect pre-mRNA splicing in XLA [1]. Previously, we have published a proof-of-concept study and shown that AONs can correct a cryptic exon mutation and restore a functional BTK in a humanized transgenic mouse model both in vitro and in vivo [2]. AONs in the previous study were designed to sterically block an intronic mutation and prevent the recruitment of the splicing factors around the cryptic exon.We are here addressing a different scenario, where defective BTK is produced due to exon exclusion. This is typically due to mutations that weaken the splice sites or splicing enhancer sequences in their vicinity, and constitutes the most common type of splicing defect. Here we have designed bifunctional AONs that have a complementary binding site and a free tail region that is able to recruit splicing factors to the site in order to enhance exon inclusion. Initially, we constructed a library of BTK reporters with mutations known to cause XLA, and the bifunctional AONs were then tested in cultures of cells stably transfected with various BTK reporters for intronic mutations affecting the inclusion of BTK exons 16 and 17. These exons were selected owing to the rather small size of the corresponding introns simplifying the generation of relevant reporter cell lines. Screening of different tail sequences show that AONs designed to recruit TIA-1 or TDP-43 proteins can correct the splicing defects and give rise to wild-type BTK mRNA. Optimization of the antisense and tail parts of the AONs has also revealed the importance of chemical modifications (2’-O-methyl, phosphorothioate, and/or locked nuclei acid) for enhancing the efficacy of the AONs. We have also studied the influence of combining bifunctional AONs and found that this can profoundly enhance exon inclusion. Thus, depending on the exact type of mutation single or combinatorial approaches provide different outcomes. To the best of our knowledge, this is the first time that exon inclusion has been achieved in a hematopoietic setting and experiments are currently underway to further optimize and explore the design of bifunctional AONs for potential in vivo use.1. Bestas, B., et al., Splice-correction strategies for treatment of x-linked agammaglobulinemia. Current Allergy and Asthma Reports, 2015. 15(3): p. 510.2. Bestas, B., et al., Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model. The Journal of Clinical Investigation, 2014. 124(9): p. 4067-81.