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Evaluation of a polypeptide that can potentially improve the pharmacokinetic properties of therapeutic proteins
KTH, School of Biotechnology (BIO).
2013 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Utvärdering av en polypeptid med förutsättning att kunna förbättra de farmakokinetiska egenskaperna hos terapeutiska proteiner (Swedish)
Abstract [en]

Sanfilippo syndrome is a lycosomal storage disease and a third type of mucopolysaccharidosis (MPS III type) caused by the deficiency of three lysosomal enzymes. Sulfamidase (SGSH) is involved in the degradation of glycosaminoglycan heparan sulphate. The defect of the enzyme leads to accumulation of heparan sulphate and MPS IIIA; a disease where the central nervous system (CNS) is damaged. The involvement of CNS has obstructed the development of an effective treatment. An approach of treatment is enzyme replacement therapy (ERT); already established for efficient treatment of many other lysosomal storage diseases. The challenge is the cross-over of protein across the blood-brain barrier (BBB) and avoidance of rapid clearance of the protein by renal and hepatic elimination and also the decrease of enzyme endocytosis through receptors involved in the uptake of the protein. A potential way to come across these difficulties is by extending the serum jalf-life of SGSH Such effect would potentially reduce clearance rates in peripheral tissues while still mediate a desired receptor-mediated uptake in cells on the other side of the BBB.

In this study four recombinant variants of SGSH have been constructed, where the protein is attached to four lengths of XTEN; a hydrophilic and unstructured ciopolymer developed by Amunix to extend the serum half-lives of drugs. The constructs have been expressed in eukaryotic cells and purified. Characterization of SGSH-XTEN variant's propensity for cellularuptake and potency of the proteins have been investigated using already established in vitro assays. Experimental procedures have been performed in order to analyze the uptake of the recombinant proteins in mouse fibroblasts (MEF-1 cells) and protein distribution in MPS IIIA patient cells.

The attachment of longer XTEN polymers to the C-terminal of SGSH appear to enhance the protein expression in HEK-293 Freestyle cells. All four constructs had a low uptake in MEF-1 cells, indicating that XTEN greatly affects and modulates the cellular uptake. However only the variants of SGSH with shorter length of XTEN appeared to have a significant uptake compared to the background/control. The uptake of these variants differed in MPS IIIA fibroblasts, where their uptake were above of what was expected. This is probably due to higher expression of mannose-6-phosphate receptor. Based on the results, the SGSH constructs with longer XTEN should be further assessed. The ultimate evaluation of the usefulness of the polypeptide fused to the C-terminal of SGSH is to perform a pharmacokinetic study in vivo.

Place, publisher, year, edition, pages
Keyword [en]
Protein engineering, pharmacokinetics, clearance, receptor, half-life, blood brain barrier, peptide
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-150701OAI: diva2:744738
Available from: 2015-04-17 Created: 2014-09-08 Last updated: 2015-09-18Bibliographically approved

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