339 BIOENGINEERING OF THE MAMMARY GLAND OF LIVESTOCK: INCREASED PROPEPTIDE PROCESSING OF FACTOR IX IN THE MILK OF TRANSGENIC PIGS BY CO-EXPRESSION OF FURIN

Abstract

Coagulation Factor IX is a vital protein that plays an important role in the blood coagulation cascade. Hereditary deficiency in the Factor IX gene can result in hemophilia type B, the second most common hemophilia. The milk of transgenic livestock can be an efficient vehicle for producing complex, post-translationally modified blood proteins. The advantages include decreased pathogen risk as well as 100-fold or more production efficiency over blood fractionation and cell culture biotechnology. Although the mammary gland can make many of the complex post-translational modifications necessary for biological function of blood proteins, improvements in propeptide cleavage and decreased proteolytic degradation are desirable. To explore the possibility of producing bioactive human Factor IX protein in pig milk, male and female Landrace fetal fibroblast cells were co-transfected by electroporation with 3 different transgene constructs, Factor IX (FIX), Furin, and SERPINA1. The SERPINA1 construct (containing a Neo selectable marker) was delivered into fetal fibroblasts at a 10-fold-lower molar concentration than the other 2 constructs. Following selection in Geneticin, the presence of all 3 genes was verified by PCR and then cells were used as donors for somatic cell nuclear transfer. Thirteen F₀ female piglets from 3 potential different integrations were delivered, and 6 piglets were validated by PCR to be positive for all 3 genes. Among the 6 transgenic pigs, 3 are healthy and able to reach puberty. Milk was collected by induced lactation from 2 gilts. A short murine whey acidic protein promoter-Furin gene was used to limit Furin to the lowest levels needed for pro-FIX processing. Furin was expressed to increase propeptide cleavage efficiency, with the result being complete processing of pro-FIX to FIX at ∼0.3 g L^–1 pro-FIX. Total FIX levels were ∼1 g L^–1. SERPINA1 was also co-expressed at ∼1 g L^–1 or more and this serine protease inhibitor did not seem to inhibit furin processing of the pro-FIX. Fifteen F₀ male piglets from 3 potential different integration sites were delivered and all of them were positive for all 3 genes. Four F₀ males were chosen to breed with wild-type females, and 5 litters of F₁ piglets were born. Of 63 F₁ piglets, 22 were tri-transgenic and 3 were di-transgenic (only carrying FIX and Furin). Two F₁ females were mated with wild-type males, are confirmed to be pregnant, and will be used to determine the expression level and bioactivity of the Factor IX protein in the milk.