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

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 <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 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.

One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

“…The one-step in ovo transposition might be of particular interest for genetic approaches, where two or more transgenes need to be inserted into the bovine genome. It has been shown that the expression of recombinant proteins in the udder can be enhanced by co-expression of enzymes for posttranslational modification 15 . The co-expression of recombinant proteins, which have synergistic effects 16 17 or different subunits, which assemble to functional protein complexes 18 19 would be simplified by this method.…”

Application of Genetically Engineered Pigs in Biomedical Research

“…The collected milk contained 1 g/L of this protein [48]. Other recombinant human proteins involved in the coagulation process, such as factor VIII [49], factor IX [50,51], von Willebrand factor [52], were also successfully obtained in the porcine mammary gland. Furthermore, the line of transgenic pigs producing functional recombinant human erythropoietin in their milk was demonstrated.…”

Genetically engineering milk

“…Much has been done to optimise harvest and purification of these proteins e.g. (Zhao et al 2015), with the first to successfully navigate through the appropriate regulatory process and reach the clinic in 2006 (Pollock et al 1999; Anon 2009). In parallel, a diverse range of transgene designs were evaluated providing valuable information for the entire transgenic community.…”