Modification of the bovine genome for the large-scale production of human serum albumin

There is a vast clinical need for human serum albumin (HSA) with nearly 500 metric tons used worldwide every year. As a major protein in the human body, HSA plays a vital role in many physiological processes including the maintenance of oncotic pressure and the transport of various biomolecules and pharmaceuticals. Currently, all HSA used for clinical blood expansion purposes is isolated from pooled human blood or plasma, an unpredictably fluctuating supply that can potentially fall to dangerously low levels. Furthermore, this supply is derived from thousands of different donors that could potentially result in the spread of pathogenic contaminants to recipients. One solution to the problem is the use of transgenic animals, such as cattle, as living bioreactors, enabling large-scale production of recombinant HSA (rHSA) in a cost-effective manner. Cattle are capable of producing large amounts of milk that can potentially yield large quantities of a desired recombinant protein. The production of rHSA in the milk of cattle would provide an economical resource that circumvents the current dependence on blood bank supplies. Previous bovine transgenics, which simply express ectopic rHSA in the milk, have not proved commercially viable. This is primarily due to the presence of endogenous bovine serum albumin (BSA) in the milk, a highly conserved ortholog of HSA, which necessitates a tedious and prohibitively expensive purification process. Our approach is to humanize the endogenous BSA gene, replacing it with an rHSA minigene construct, which should allow normal expression of rHSA protein in the liver as well as exogenous expression of rHSA in the milk.;As an initial step toward the large-scale and inexpensive production of rHSA in transgenic cattle, we present here the modification of the bovine genome, with the aim of humanizing the endogenous BSA gene by a nuclease-stimulated targeted integration. In Chapter 1, we demonstrate the design and development of a DNA targeting construct necessary for the simultaneous disruption and replacement of endogenous BSA expression with that of rHSA. Ultimately, the BSA targeting construct enables exclusive expression of rHSA in the blood as well as its directed expression in the milk. In Chapter 2, we demonstrate the establishment of rHSA modified bovine fibroblasts by transcription activator-like effector nuclease (TALEN)-mediated homologous recombination (HR). In Chapter 3, we demonstrate the production of transgenic bovine embryos by using the targeted fibroblasts from Chapter 2 as nuclear donors in somatic cell nuclear transfer (SCNT). In an attempt to satisfy an ever-increasing global demand, transgenic SCNT-derived bovine embryos used towards the future establishment of genetically modified (GM) cattle, provide the potential for a reliable, quality-controlled, and economically feasible source of therapeutic rHSA.