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Projects. Life and Matter Sciences

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

Lead Researcher:
Miguel Ángel de la Fuente García

Research Centre:
Instituto de Biología y Genética Molecular (IBGM). CSIC-Universidad de Valladolid.


Miguel Ángel de la Fuente GarcíaThe 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.

Conclusions: 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.


Scientific production
1 paper at international conferences

Researcher's web address:

Miguel Ángel de la Fuente García

He studied Medicine in Valladolid. He is a specialist in Immunology. He earned his doctorate in the University of Barcelona, and his doctoral thesis consisted of the characterisation of new leucocytic membrane receptors, above all a group of molecules in the immunoglobulin superfamily forming the family of SLAM receptors, as well as searching for their ligands.

In 1999 he moved to Boston, Massachusetts, to complete his postdoctoral training; he studied different animal models of immunodeficiency as well as molecules that are important for immune function, in particular the cause of Wiskott-Aldrich Syndrome, a rare type of primary immunodeficiency linked to chromosome X.

He returned to Valladolid in 2009 and joined the Molecular Biology and Genetics Institute (IBGM) where he is currently the Ramón y Cajal researcher. His main line of work centres on the study of gene targeting methods in the cells of patients with single gene diseases, especially primary immunodeficiencies, as well as the reprogramming by non-viral means of these patients' "corrected" cells.

*All intellectual property rights belong to the author. Reproduction of all or part of the work without permission from the author is prohibited.

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