Genetic Modification of Glycosylation Pathway in Epithelial Cells of Mammary Gland".
Currently there are several systems for therapeutic recombinant proteins expression, and their choice will depend on the type of protein that needs to be expressed, since the system must provide correct post-translational processing, such as tertiary and quaternary folding structure, glycosylation,...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2021 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/253226 |
| Acceso en línea: | https://hdl.handle.net/10533/253226 |
| Access Level: | acceso abierto |
| Palabra clave: | Ciencias Naturales Otras Ciencias Naturales |
| Sumario: | Currently there are several systems for therapeutic recombinant proteins expression, and their choice will depend on the type of protein that needs to be expressed, since the system must provide correct post-translational processing, such as tertiary and quaternary folding structure, glycosylation, among others. These modifications will guarantee their biological functionality, with similar characteristics to native protein. The in situ transformation of goat mammary gland epithelial cells using viral vectors for the genetic modification of adult females is presented as a feasible alternative for recombinant proteins expression with complex tertiary and quaternary structure. Moreover, in this system, the recombinant protein is secreted into the milk, which is an easily collected daily fluid. However, the proteins expressed in this system present a characteristic and defined N-glycosylation pattern, which consists majority in presence of bi-antennary structures and with a single terminal sialic acid bond. Although this pattern is functional for the biological activity of some therapeutic proteins, it is not sufficient for proteins such as human erythropoietin, which presents a complex, tetra-antennary and tetra-sialylated glycosylation pattern. Erythropoietin expressed in goat mammary gland epithelial cells does not show biological activity due to the glycan pattern added using this system. The aim of this thesis was to modify in vivo the glycosylation pathway of goat mammary gland epithelial cells, using human erythropoietin as a model protein. XIV For this purpose, adenoviral vectors containing the sequence of this hormone fused to the human immunoglobulin G Fc fragment (EPOFc) were used. In addition, these vectors contain the sequences of key glycoenzymes for obtaining branched glycans terminated in sialic acid, thus assuring a modification in the glycosylation pattern of EPOFc expressed in goat milk. The glycosyltransferases used were N-acetylglucosaminyltransferases IV and V (GNT-IV, GNT-V), in addition to sialyltransferase 1 (ST6N). Goat mammary glands were transduced with EPOFc vectors as control and with the combination EPOFc+GNT-IV, EPOFc+ST6N, EPOFc+GNT-V+ST6N. Analysis of glycosylation patterns of different variants showed relevant differences compared to the control. In all the variants obtained, structures with a higher degree of branching were observed. In addition, the variants that were expressed together with ST6N showed an increase in sialic acid-containing structures compared to EPOFc, however, none of them showed a significant increase in hematopoietic activity measured as the percentage of hematocrit. These preliminary results allow us to demonstrate that it is possible to modify the glycosylation pathway of epithelial cells in the goat mammary gland. |
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