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

Sci Rep. 2015 Sep 21:5:14176. doi: 10.1038/srep14176.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Bioreactors
  • Factor IX / genetics*
  • Factor IX / metabolism
  • Factor IX / therapeutic use
  • Female
  • Furin / genetics*
  • Furin / metabolism
  • Hemophilia B / therapy*
  • Humans
  • Lactation / metabolism
  • Male
  • Mammary Glands, Animal / metabolism*
  • Milk / metabolism*
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / therapeutic use
  • Swine

Substances

  • Recombinant Proteins
  • Factor IX
  • Furin