695. Splice-Correction of X-Linked Agammaglobulinemia in a Human BAC-Transgenic Mouse Model Using Oligonucleotides

Abstract

The inherited immunodeficiency, X-linked agammaglobulinemia (XLA), is caused by mutations in the BTK gene, and results in a B-lineage developmental block. We have recently assessed the treatment potential of splice-correcting oligonucleotides (SCOs) targeting a mutated BTK transcript, which contains a pseudo-exon (Bestas et al., J. Clin Invest 124: 4067, 2014). In order to study the potential of SCOs, we engineered a novel, Bacterial Artificial Chromosome (BAC)-transgenic mouse carrying a mutated human BTK gene, originally found in an XLA family. In order to avoid any influence of mouse endogenous Btk protein, we bred the BAC-transgenic mice onto a Btk knockout background. In this model it was possible to correct the defect both in pro-B-cells in vitro, and also in mature B-cells, as demonstrated by the injection of SCOs in vivo. The corrected mRNA gave rise to a functional BTK protein. As a final proof-of-concept we were also able to correct the defect in primary patient cells. In this study we used different nucleotide chemistries, 2’-O-methyl, locked nucleic acid (LNA) and morpholino chemistries. We have now included also other nucleotide chemistries in order to make comparative studies. This is to our knowledge the first time that a lymphocyte defect, caused by abnormal splicing, has been corrected in vivo using a splice-correction approach.