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Type: Artigo de periódico
Title: Transgene-host Cell Interactions Mediate Significant Influences On The Production, Stability, And Function Of Recombinant Canine Fviii.
Author: Crawford, Bredon
Ozelo, Margareth C
Ogiwara, Kenichi
Ahlin, James
Albanez, Silvia
Hegadorn, Carol
Harpell, Lori
Hough, Christine
Lillicrap, David
Abstract: Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII.
Rights: aberto
Identifier DOI: 10.1038/mtm.2015.33
Date Issue: 2015
Appears in Collections:Unicamp - Artigos e Outros Documentos

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