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Plan-les-Ouates, Switzerland

Roth J.,Yonsei University | Zuber C.,University of Zurich | Park S.,Yonsei University | Jang I.,Yonsei University | And 8 more authors.
Molecules and Cells | Year: 2010

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed. © 2010 KSMCB. Source


Kreudenstein T.S.V.,Zymeworks | Escobar-Carbrera E.,Zymeworks | Lario P.I.,Zymeworks | D'Angelo I.,Zymeworks | And 12 more authors.
mAbs | Year: 2013

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fclike biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic. © 2013 Landes Bioscience. Source


Buceta M.,University of Lausanne | Buceta M.,Selexis SA | Galbete J.L.,University of Lausanne | Kostic C.,University of Lausanne | And 2 more authors.
Gene Therapy | Year: 2011

Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors. © 2011 Macmillan Publishers Limited All rights reserved. Source


The present invention relates to purified and isolated DNA sequences having protein production increasing activity and more specifically to the use of matrix attachment regions (MARs) for increasing protein production activity in a eukaryotic cell. Also disclosed is a method for the identification of said active regions, in particular MAR nucleotide sequences, and the use of these characterized active MAR sequences in a new multiple transfection method.


Disclosed are methods and eukaryotic host cells for transgene expression. The cells may be treated and/or modified to increase homologous recombination (HR), decrease non homologous end joining (NHEJ) and/or to enhance a HR/NHEJ ratio in said cell. Such cells can be transfected with vectors comprising the transgene, which advantageously integrates into the genome of the cell to form a concatemeric structure which may comprise more than 200 transgene copies. Certain expression enhancing elements such as MARs are advantageously provided to further enhance and/or facilitate transgene expression. Disclosed is also a recombinant eukaryotic host cell, in particular a non-primate host cell, comprising a transgenic sequence encoding a protein and/or a RNA, in particular a primate protein and/or RNA, involved in translocation across the ER membrane and/or secretion across the cytoplasmic membrane.

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