Cell-penetrating peptide-conjugated, splice-switching oligonucleotides mitigate the phenotype in BTK/Tec double deficient X-linked agammaglobulinemia model

Splice-switching oligonucleotides (SSOs) have been developed as a treatment for various disorders, including Duchenne muscular dystrophy and spinal muscular atrophy. Here, the activity of several different SSOs was investigated as potential treatments for B lymphocyte disorders with a focus on X-linked agammaglobulinemia (XLA), caused by defects in the gene encoding Bruton's tyrosine kinase (BTK). In this study, the activity of locked nucleic acid (LNA), tricyclo-DNA (tcDNA), phosphoryl guanidine oligonucleotides (PGO) and phosphorodiamidate morpholino oligomers (PMO) were compared, targeting the pseudoexon region of BTK pre-mRNA. We further investigated the effect of conjugating cell-penetrating peptides, including Pip6a, to the SSOs. The effect was measured as splice-switching in vitro as well as in a further developed, bacterial artificial chromosome transgenic mouse model of XLA. Therapy in the form of intravenous infusions 2 times a week during 3 weeks of PMO oligomers conjugated to Pip6a was sufficient to partly restore the in vivo B lineage phenotype. SSOs treatment also provides a unique opportunity to get insights into a restoration process, when B lymphocytes of different maturation stages are simultaneously splice-corrected.