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

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.

Recombinant human fibrinogen that produces thick fibrin fibers with increased wound adhesion and clot density.

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominantly contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ'-chain transcriptional variant that has been reported to bind thrombin and Factor XIII. In spite of these differences, rFI and pdFI were kinetically similar with respect to the thrombin-catalyzed formation of protofibrils and Factor XIIIa-mediated formation of cross-linked fibrin polymer. However, electron microscopy showed rFI produced fibrin with much thicker fibers with less branching than pdFI. In vivo studies in a swine liver transection model showed that, relative to pdFI, rFI made a denser, more strongly wound-adherent fibrin clot that more rapidly established hemostasis.

Secretion of recombinant human fibrinogen by the murine mammary gland.

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.