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An important percentage of disease causing mutations affects pre-mRNA splicing which is controlled by sequence elements within the exons and introns. 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 an inherited immunodeficiency resulting from mutations in the BTK gene severely impairing B cell development. BTK has a crucial role in B cell development and the absence of BTK introduces a developmental block. A prominent percentage of splicing mutations affect pre-mRNA splicing in XLA. Here, we assess the treatment-potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid (LNA) or phosphorodiamidate morpholino (PMO) backbones. In order to have access to an animal model, we engineered a novel, Bacterial Artificial Chromosome (BAC)-transgenic mouse carrying an authentic, mutated, splice-defective human BTK gene. To avoid interference of the orthologous mouse protein, mice were bred onto a Btk knockout background. This represents a unique model to study treatment strategies for diseases resulting from splice-site mutations. For the first time the capacity of SCOs to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells, is demonstrated. The corrected BTK mRNA restored expression of functional protein as shown both by enhanced lymphocyte survival and re-established BTK activation upon B cell receptor stimulation. As final proof-of-concept BTK restoration in vivo in mice and ex vivo in primary patient cells was obtained. Thus, our approach may represent a future personalized medicine for XLA. [ABSTRACT FROM AUTHOR]

Earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor CCAAT/enhancer binding protein α at very high frequencies. Using this system, we performed a systematic analysis of whether during transdifferentiation the cells transiently reactivate progenitor-restricted genes or even retrodifferentiate. A transcriptome analysis of transdifferentiating cells showed that most genes are up- or down-regulated continuously, acquiring a macrophage phenotype within 5 d. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and FMS-like tyrosine kinase 3 and a few lineage-inappropriate genes. Importantly, however, we were unable to observe the reexpression of cell-surface marker combinations that characterize hematopoietic stem and progenitor cells, including c-Kit and FMS-like tyrosine kinase 3, even when CAAT/enhancer binding protein α was activated in pre-B cells under culture conditions that favor growth of hematopoietic stem and progenitor cells or when the transcription factor was activated in a time-limited fashion. Together, our findings are consistent with the notion that the conversion from pre-B cells to macrophages is mostly direct and does not involve overt retrodifferentiation.

The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells.