| 1. |
- Bestas, Burcu, et al.
(author)
-
Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
-
In: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
-
Journal article (peer-reviewed)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
|
|
| 2. |
- Blomberg, K Emelie M
(author)
-
Gene expression analysis of Tec family kinases in B- and T-lymphocytes
- 2009
-
Doctoral thesis (other academic/artistic)abstract
- The microarray technology is a powerful tool used in many different research areas. The technique has been around for more than a decade and has revolutionized the molecular biology field. In this thesis the Affymetrix GeneChip® arrays were used to study the transcriptomes of Tec family kinase mutant mouse models. Bruton s tyrosine kinase (Btk), IL-2 inducible T-cell kinase (Itk) and Tyrosine kinase expressed in hepatocellular carcinoma (Tec) are protein tyrosine kinases belonging to this Tec kinase family. Mutations in Btk cause a primary immunodeficiency disease called Xlinked agammaglobulinemia (XLA) in humans and X-linked immunodeficiencydisease (Xid) in mice. Gene expression profiling was performed on whole splenic Bcells as well as Transitional 1 (T1) B-cells from Xid, Btk knockout (Btk KO) and control mice. This was done in order to study differences and similarities between Btkdefective mice (Xid and Btk KO together) compared to control. Small differences were distinguished between the Btk-defective mouse strains in the whole splenic B-cell population; only seven genes differed (>2-fold) between the two. A number of potentially interesting genes were found to be differentially expressed in the Btkdefective groups compared to the controls. We also show that the Btk defect is already manifested at the T1 B-cell stage. Itk is important for the T-cell development where it has a role in regulating the conventional versus innate T-cells. Itk-deficient T-cells are of an innate, memory-like type. Tec is poorly expressed in resting T-cells, but is expressed 2-3 days after stimulation. Tec-deficient mice show no phenotypic alterations and the mice develop normally. In Papers III and IV we looked at the transcriptomes of Itk-, Tec- and Itk/Tec-deficient mice by studying unstimulated as well as stimulated CD3+ T-cells. The Itk-deficiency was also studied in CD4+ and CD8+ T-cell subpopulations. The gene expression patterns of Tec-deficient mice compared to control mice showed small differences, further supporting earlier findings. More differences were seen in Itkdeficient as well as Itk/Tec-deficient mice compared to controls. We also investigated if the Itk-deficiency could mimic calcineurin inhibition by treating Wt T-cells with cyclosporin A (CsA). CsA was shown to have a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Greater differences were seen in CD4+ and CD8+ T-cells in comparison to total CD3+ T-cells. In Paper IV we found Zbtb26, an interesting transcription factor, being up-regulated in Itk-deficient T-cells, while normalized in Itk/Tec-deficient cells compared to Wt. By the use of a combination of the microarray technology and quantitative RT-PCR analysis, we have evaluated the transcriptomes of Btk-, Itk-, Tec- and Itk/Tec-deficient mice.
|
|
| 3. |
|
|
| 4. |
- Palma, Marzia, et al.
(author)
-
Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes
- 2018
-
In: British Journal of Haematology. - : WILEY. - 0007-1048 .- 1365-2141. ; 183:2, s. 212-224
-
Journal article (peer-reviewed)abstract
- In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19(+) circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.
|
|
