Optimization of Enzyme Replacement Therapy Using Vesicles as Delivery Vehicles for alpha galactosidase (GLA) for the treatment of Fabry disease
Coello Quihuiri, Edwin Patricio
Abasolo, Ibane, dir. (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Corchero Nieto, José Luis (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Universitat Autònoma de Barcelona. Facultat de Biociències

Date:	2025
Description:	32 pag.
Abstract:	In the first part of the project, the production of α-galactosidase A (GLA) was carried out using two strategies: transient transfection in HEK293 cells and stable cloning in CHO cells, and three variants were obtained: HEK GLA HIS, HEK GLA cMYC, and CHO GLA CMYC, with the highest expression observed in CHO GLA CMYC, in line with previous reports highlighting CHO cells for their high efficiency and stability in recombinant protein production; the HEK GLA CMYC variant outperformed HEK GLA HIS, potentially due to the stabilizing effect of the CMYC tag, while in contrast, the HIS tag may negatively affect the solubility and functionality of GLA, and for purification, soluble GLA was isolated using affinity chromatography with His SpinTrap columns, although exposure to imidazole reduced enzymatic activity, necessitating a subsequent dialysis step; the second phase of the project focused on the purification of extracellular vesicles (EVs) loaded with GLA, using two methods: bottom-through filtration (BTF) and tangential flow filtration (TFF), where TFF outperformed BTF in terms of yield and preservation of enzymatic activity owing to its gentler flow conditions, and CHO GLA CMYC cells not only produced a higher quantity of EVs but also generated vesicles with enhanced enzymatic activity, which is attributed to the superior folding capacity and post-translational modification machinery of CHO cells, so the CHO GLA CMYC system combined with TFF purification emerges as an efficient and scalable platform for the development of an extracellular vesicle-based enzyme replacement therapy (ERT) for Fabry disease.
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons en que s'entrega l'obra al domini públic. Podeu copiar, modificar, distribuir l'obra i fer-ne comunicació pública, fins i tot amb finalitat comercial, sense demanar cap mena de permís.
Language:	Anglès
Studies:	Bioquímica, Biologia Molecular i Biomedicina [4313794]
Study plan:	Màster Universitari en Bioquímica, Biologia Molecular i Biomedicina [1599]
Document:	Treball de fi de postgrau
Subject:	4-Methyllumbelliferone ; Alpha galactosidase (GLA) ; HEK 293 cells ; CHO cells ; Fabry disease ; Vesicles ; Enzymatic replacement therapy ERT

33 p, 1.2 MB

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Research literature > Dissertations > Biosciences. MT