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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 ?-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.

SUBMITTER: Zhao J

PROVIDER: S-EPMC4585688 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Zhao Jianguo J Xu Weijie W Ross Jason W JW Walters Eric M EM Butler Stephen P SP Whyte Jeff J JJ Kelso Lindsey L Fatemi Mostafa M Vanderslice Nicholas C NC Giroux Keith K Spate Lee D LD Samuel Melissa S MS Murphy Cliff N CN Wells Kevin D KD Masiello Nick C NC Prather Randall S RS Velander William H WH

Scientific reports 20150921

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 hum ...[more]

PMID: 26387706

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Project description:BackgroundUnlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation.Methodology/principal findingsDistal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization.Conclusions/significanceA progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland.

Dataset Information

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

Publications

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

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