28 Development of Milk-Targeted Transgenic Animals Expressing Coagulation Factor VIII Bioengineered for High Efficiency Secretion

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The lifelong hemorrhagic diathesis of hemophilia A is treated successfully with recombinantly derived factor VIII (rFVIII). rFVIII is presently produced and purified from mammalian cell lines in liquid culture but has proven to be costly. The milk of transgenic livestock can yield an abundant source of complex therapeutic proteins such as recombinant coagulation proteins. Only the pig mammary gland has been shown to carry out all of the post-translational modifications necessary to generate the biological activity and long circulation half-life needed for complex glycoproteins. Velander and colleagues have demonstrated that human recombinant factor IX can be produced at levels up to 100 U/ml. However, yields of rFVIII have been much lower and subject to chain dissociation due to the harsh chelating environment and/or the absence of vWF. Through bioengineering strategies we have developed rFVIII variants with enhanced secretion (F309S/226aa/N6) and stability (IR8). Our goal is to express these rFVIII variants (F309S/226/N6 and IR8) in the milk of transgenic mice and pigs under the murine Whey Acidic Milk Protein (WAP) promoter and downstream 3' UTR elements. Transgenic mice provide both insight into the encoding fidelity of the transgene constructions as generally expressed by mammary epithelia but also provide useful amounts (ie. >1 ml per lactation at >20 ug/ml) of post-translationally modified, recombinant protein for evaluation. We have created 6 murine transgenic lineages that express the rFVIII variants either alone or in combination with vWF and/or alpha1-antitrypsin. We are co-expressing these constructions with vWF to help stabilize the rFVIII variants. We have expressed vWF previously in the milk of transgenic mice at greater than 100 ug/ml. We are also co-expressing alpha1-antitrypsin as this has been shown to dramatically reduce the amount of proteolytic degradation in milk. Trigenic and bigenic combinations of these constructs were mixed equal molar and microinjected into mouse zygotes to produce transgenic mice at the University of Michigan. Founder animal lineages are presently being outbred with wild-type mice. The resulting F1 lineages will undergo induced lactation and the milk analyzed for rFVIII content. Trigenic, transgenic pig fibroblast lines have also been made and lineages are awaiting nuclear transfer in August 2006.