Taking the p... therapeutic proteins from urine

Abstract

US researchers have developed the bladder of transgenic mice as a novel bioreactor that could prove more cost-effective than milk-based and blood-based systems for producing foreign proteins [Kerr, D.E. et al. (1998) Nat. Biotechnol. 16, 75–79]. The method should be easy to scale up to large animals such as cattle for commercial production.Bob Wall and colleagues in the US Department of Agriculture (Beltsville, MD, USA) and New York University Medical School (New York, NY, USA) used part of the mouse gene for uroplakin II (UPII), a urothelial membrane protein, to direct the production of human growth hormone (hGH) in the urothelium of transgenic mice. hGH was chosen for the experiment because ectopic (`leaky') expression is easy to detect. `We know the hormone was active in the transgenic mice, because some were very large, and all the females were sterile', says Wall.Production of pharmaceuticals by `biopharming' is cheaper and easier to scale up than cell-culture based systems, and several products derived from the milk of transgenic farm animals are now under clinical trial. A major advantage of the bladder as a bioreactor, say the authors, is the ability to harvest the product throughout the life of the animal—instead of just during lactation—and without regard for the transgenic animal's sex or reproductive status.To generate the transgenic mice, the UPII–hGH transgene (produced by ligating the 3.6 kb 5′ flanking region of the UPII mouse gene to the 5′ end of the hGH structural gene) was microinjected into fertilized eggs, which were then transferred to pseudopregnant recipients. This procedure resulted in the birth of five (sterile) female and four male founder transgenic animals. hGH was detected in the urine of three of the males, and these were bred to establish lines of mice for further study.Northern blot analysis showed `substantial' expression of hGH in the bladder—two orders of magnitude higher than in brain and kidneys; and immunofluorescent staining with an antibody to hGH revealed its expression in urothelium (Fig. 1Fig. 1). Radioimmunoassay showed hGH concentrations of up to 500 ng ml−1 in the urine of UPII–hGH transgenic mice (and none in control mice). Concentrations were relatively stable in individual animals from the first sampling at age 6 weeks to the last at 8 months. The relative levels of the expression of the founder animals were generally preserved in their offspring.Fig. 1Bladder from a transgenic mouse expressing growth hormone in the urothelial layer, stained with a fluorescently labelled antibody to human growth hormone. E denotes the epithelial layer; L denotes the lumen. Reproduced from Nat. Biotechnol. 16, 75–79. Image kindly provided by Dr Robert J. Wall, US Dept of Agriculture, Beltsville, MD 20705, USA.View Large Image | Download PowerPoint SlideMouse urine contains about 10–50 times as much total protein as bovine urine, mostly as so-called major urinary proteins produced by the liver. Thus, hGH represents only about 0.02% of urinary proteins in transgenic mice but could probably account for 0.1–1.0% of urinary protein in transgenic farm animals. This compares with a figure of 1–10% of total milk protein for pharmaceutical proteins produced by mammary-specific transgenes. However, it is unlikely that the bladder could ever come close to producing as much protein as the mammary gland, even if expression in urine could be enhanced. “Mammary glands really crank out a lot of protein—in the g l−1 range—whereas the bladder is making protein in the μg l−1 range” says Wall. But purification of specific proteins would be simpler from urine than from milk. At least one step essential—fat separation—would not be required for purification from urine. `The cost-effectiveness of purification of protein from urine will probably turn out to be the most significant benefit of a bladder bioreactor', adds Wall.Walls' team have no plans to produce other therpeutic proteins from urine. Their method is not being patented and is free to be used by others. Uroplakin genes are highly conserved in other mammalian species, including humans, cattle and sheep.Biologicals purified from urine are already used in clinical medicine—pregnant mares' urine, for instance, is a major source of natural oestrogen for postmenopausal hormone replacement. In Canada and the USA urine for this purpose is collected from 75 000 mares, each producing about 500 l of urine a year. Wall calculates that a herd of only 200 transgenic cattle could provide enough factor VIII to supply the whole world, assuming that each animal produced 20 l per day of urine containing 0.5 mg l−1 of the protein, and taking into account an estimated 50% loss of the product during purification. `And harvesting starts earlier with urine-based production', adds Wall. `It would take about 3 years from embryo microinjection to generate a herd producing a transgene in urine, compared with 7 years a milk-borne transgene'.