Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Authors

• Jianguo Zhao, University of Missouri, Columbia
• Weijie Xu, University of Nebraska-LincolnFollow
• Jason W. Ross, University of Missouri, Columbia
• Eric M. Walters, University of Missouri, Columbia
• Stephen P. Butler, ProGenetics, LLC
• Jeff J. Whyte, University of Missouri, Columbia
• Lindsey Kelso, University of Missouri, Columbia
• Mostafa Fatemi, University of Nebraska-Lincoln
• Nicholas C. Vanderslice, University of Nebraska-LincolnFollow
• Keith Giroux, University of Missouri, Columbia
• Lee D. Spate, University of Missouri, Columbia
• Melissa S. Samuel, University of Missouri, Columbia
• Cliff N. Murphy, University of Missouri, Columbia
• Kevin D. Wells, University of Missouri, Columbia
• Nick C. Masiello, LFB USA, Inc.
• Randall S. Prather, University of Missouri, Columbia
• William H. Velander, University of Nebraska-LincolnFollow

Date of this Version

2015

Document Type

Article

Citation

Scientific Reports, 5:14176

Comments

This work is licensed under a Creative Commons Attribution 4.0 International License.

Abstract

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.