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The correction of fibroblasts with mutations causing Wiskott Aldrich syndrome by means of adeno-associated viruses (AAV). The effects of corrective methods on cellular reprogramming by means of episomal vectors

16th national competition for scientific and technical research

Rare diseases

Senior Researcher : Miguel Ángel de la Fuente García

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Research Centre or Institution : Instituto de Biología y Genética Molecular (IBGM).Valladolid.


The objective of this work is gene targeting (GT) in human fibroblasts obtained from patients with Wiskott-Aldrich's syndrome (WAS) under several experimental conditions that favour the process of individual or combined homologous recombination, using recombinant adeno-associated virus (rAAV) vectors. Once the most efficient correction protocols are known, a second phase will produce induced pluripotent stem cells (iPSs) by means of reprogramming with episomal vectors or recombinant proteins to prevent the integration of foreign DNA. Gene correction treatments will be evaluated to see whether they affect the success of the reprogramming.

In an initial phase, human cell line HCT116 was used as the model. Two donor adeno-associated viruses were produced, AAV-WASP1/2 and AAV-WASP 3/4/5/6 to correct mutations in exons 1‑2 and 3‑6 of WASp, respectively. These viruses were trialled in HCT116 cells, having obtained a GT frequency for both of around 2%.

Pre-treatment with fusion protein ScRAD52 increased the GT frequency by a factor of 3 over that of the control. Bacterial expression plasmids were generated for the reprogramming factors as fusion proteins including the tag 6xHis-Tat-NLS in their amino-terminal end. Their purification in soluble form was prepared using inclusion bodies.


optimum assay conditions have been created for several of the treatments increasing GT frequency. The production of reprogramming factors as fusion proteins in bacteria has been found to be efficient.

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