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

Scientific reports

PubMedID: 26387706

Zhao J, Xu W, Ross JW, Walters EM, Butler SP, Whyte JJ, Kelso L, Fatemi M, Vanderslice NC, Giroux K, Spate LD, Samuel MS, Murphy CN, Wells KD, Masiello NC, Prather RS, Velander WH. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk. Sci Rep. 2015;514176.
Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete ?-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 ?-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 ?-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.