Opportunity

Recent discoveries in animal cloning via nuclear transfer combined with advanced genomic manipulation have opened vistas of opportunity in livestock improvement, bioreactors for pharmaceuticals, and organ transplants, within a realistic commercial time frame.

Once techniques to manipulate genes in the test tube became generally available, developmental geneticists were eager to manipulate the genomes of whole animals for a variety of reasons. The first papers showing that DNA from an exogenous source could be stably integrated into the genome and expressed in protein appeared in the early '80s (Gordon, et al, PNAS, 77, 7380; Brinster, et al, Cell, 27, 223). Despite the technical difficulties in their construction, "transgenic mice" rapidly became part of the experimental biologists' arsenal. Totipotent cells from the mouse embryo were first established as lines in 1981 (Evans and Kaufman, Nature 292, 154 and Martin, PNAS, 78, 7634 ) and shown to contribute to the germ line in 1984 (Bradley, et al, Nature, 309, 255). In the mid '80's, geneticists (Smithies, et al, Nature, 217, 230; Thomas and Capecci, Cell, 51, 503) began publishing techniques for inactivating or "knocking out" genes in situ using these embryonic stem (ES) cells. (The previous technology was only able to add functions, not to inactivate them.) The most recent developments allow for selective gene knockouts (at different times in the animal's development or in different tissues) and actual replacement (point mutation) of genes (Sauer, Meth. Enz., 225, 890; Kuhn, et al, Science, 269, 1427; Sapolsky, et al, Nature Biotechnology, 16, 516). However, these more sophisticated procedures are still only possible in mice, utilize cells which require special handling, and require about a year for each targeted mutant.

Traditional transgenic technology (i.e. microinjection of DNA fragments to add expression of specific genes) has been very challenging in non-rodent species. While the production of transgenic livestock has been demonstrated (for review, see Cameron, Mol. Biotechnol., 7, 253), the very low efficiency of current techniques makes the use of this tool for commercial applications unfeasible. Indeed, a single transgenic founder animal with a functional transgene can cost from $25,000 (pig) to $500,000 (cow) (Wall, Nature Biotechnology, 15, 416). The embryonic stem cells that have been so useful for genetic manipulation in the mouse are not available in domestic animals, making the generation of livestock species with easily altered phenotypes beyond the reach of animal scientists.

In February 1997, the world was stunned by the appearance of Dolly the Sheep, the first animal cloned from an adult cell via nuclear transfer (Wilmut, et al, Nature 385, 753). Other investigators had previously used nuclear transfer protocols to produce clones in domestic livestock such as cattle (Sims and First, PNAS, 91, 6143; Stice, et al, Biol. Reprod. 54, 100), pigs (Prathar, et al, Biol Reprod. 41, 414), and sheep (Willadsen, Nature, 320, 63; Campbell, et al, Nature, 380, 64) using embryonic donor cells. Less that one year after Dolly's appearance, her "cousins" were made by the same research group using a genetically manipulated cell nucleus and similar techniques (Schnieke, et al, Science, 278, 2130). Cattle have also been cloned from transgenic nuclei (Cibelli, et al, Science, 280, 1256) using a slightly different strategy. Aside from the fascinating scientific questions raised by these accomplishments, the improved technology yields two dividends for an emerging industry in genetically engineered animals, namely 1) removing the species restriction on genome manipulation (previously only possible in mice), and 2) greatly increasing production efficiency by allowing the use of any quickly growing, hardy cell as the donor of the genomic material. These achievements lay the ground work for an explosion in the advanced transgenesis and animal cloning industry.

The combined strength of maturing technical skills in molecular genetics and the new techniques emerging from manipulation of mammalian embryos will pave the way for the transformation of both the agricultural and medical industries. The possible commercial payoffs from this program are very large -- improving livestock for human consumption, providing a source of tissues and organs for tissue engineering, and generating bioreactors for production of pharmaceuticals, to name a few. In addition there is great potential for a more broad-based understanding of the fundamental biological processes of cellular growth and differentiation, which could have broad implications for basic biomedical research into intractable problems like cancer and diseases of aging.

Agriculture is critically dependent on high quality plants and animals. Strain/breed improvement has historically rested upon recognition of superior phenotype and propagation of the desired trait through breeding. This is a haphazard and iterative process resulting in incremental advances. Transgenic science allows for the generation of superior animals which can pass on their improved genotype, within a fraction of the time necessary for selective breeding. Furthermore, while many animals could benefit from the addition of traits derived from other species (especially those that increase disease resistance), this is not possible through even the most creative breeding schemes. Combining transgenesis and cloning leverages the technical might of molecular biology, the enabling technology of nuclear transfer, and centuries of improvements in animal husbandry methods -- allowing for performance enhancement by orders of magnitude in a single generation. The cattle industry alone was worth over $150 billion in 1997 from 9.2 million milk cows and 33.7 million beef cows (National Agricultural Statistics Board, USDA). Healthier, more productive and efficient animals will substantially reduce maintenance cost for herds.

Organ availability for transplantation is an increasingly serious problem in the U. S. Surgical skills and immunological intervention are advancing rapidly, but the tissue sourcing has remained static, and the supply of organs cannot even approach the need. In 1996, there were 20,300 lifesaving transplants to the 50,000 patients on waiting lists, while over 4000 people died for lack of a suitable donation (United Network for Organ Sharing: http://www.unos.org/Data/anrpt_main.htm). The total direct costs for organ transplantation in the United States in 1996 were $3 billion. Should the goals of the program be realized, there would be an essentially unlimited availability of organs engineered to avoid immune rejection. Some physicians estimate that such an unlimited supply would increase the use of transplantation as a disease treatment by a hundred-fold (Nature, 391, 325). The market impacts of this radical change in supply are not clear, but there would be substantial effects in at least two costly areas. The procurement costs for human organs total one-half a billion dollars annually. In addition, using current immunosuppressive therapies and other long-term follow-up costs, each organ recipient adds $7000 to $29,000 to annual health care costs.

Pharmaceuticals produced from transgenic livestock would have at least two advantages over state-of-the-art vat fermentation technology: (1) a bioreactor that eats hay and produced over 1 gm/kg of milk is much less costly to operate than a large scale culture facility, and (2) many complex proteins, such as coagulation factors are difficult if not impossible to manufacture in their biologically active forms in the available cells lines. Since dairy animals can give high rates of heterologous protein secretion in the milk, and typically lactate for 10 months out of the year, production levels can easily be 100-fold higher that than currently achieved in cell culture. Thus, the cost of producing a protein pharmaceutical, even while maintaining a dairy herd under Good manufacturing Practices, is $1 per gram compared to $1000 per gram in tissue culture (Meade and Ziomek, Nature Biotechnology, 16, 21). The Biotechnology drug market is currently $10.6 billion annually

Program Goals and Technical Scope

The goal of this focused program is to provide a "toolbox" of techniques in molecular genetics and mammalian development for the efficient and economical production of animals with phenotypes altered to suit specific needs. Such changes as increased disease resistance, enhanced characteristics (e.g. more muscle, less fat), production of novel proteins (e.g. pharmaceuticals) in easily harvested form (e.g secreted into the milk), or reduced tissue immunogenecity are envisioned. Protocols for a variety of commercially useful animals such as chickens, cows, fish, goats, horses, pigs, or sheep are needed, as well as technologies dealing with non-human primates and endangered species.

Many technical hurdles remain before the economic potential of this nascent industry can be realized. Some of the more pressing barriers include:

• understanding the cell cycle coordination of the cytoplasm and nucleus in different species (nuclear transfer protocols);
• delineating factors and cell-cell interactions controlling proliferation and differentiation of stem cells;
• generating "generic" stem cell lines from different species;
• designing efficient vectors for genetic exchange in embryonic stem cells;
• improving gene replacement, point mutation, and regulatable expression techniques.

Any proposal addressing these issues, or other techniques for improving genetic manipulation of whole mammals would be in scope. Competitive proposals would aim to produce a genetically altered animal with therapeutic potential or agricultural superiority. Exclusions from the technical scope include production of transgenic mice using established techniques, and generation of disease models rather than therapies.

Industry Commitment

There are only a handful of small businesses publicly engaged in animal cloning and transgenesis for commercial purposes: Advanced Cell Technologies (Worcester, MA), Genyzme Transgenics (Framingham, MA), Infigen (De Forest, WI), Pharming (The Netherlands and Rockville, MD) and PPL Therapeutics (Scotland and Blacksburg, VA). Informal discussions with some ATP awardees reveal great interest in this topic, and indeed some preliminary work. There is much activity in the academic sector. A recent workshop on "Embryonic Stem Cells" co-sponsored by the NIH and the Wisconsin Regional Primate Center drew almost 200 participants. There were at least three meetings about agricultural, commercial, and medical implications of cloning in the summer of 1998. Most agree that the major barrier to an explosion of economic development is a lack of investment due to the controversial nature of related research, and the need for patient capital due to the long term nature of the investment.

An ATP-sponsored electronic workshop is planned for the summer of 1998. Invited participants will post papers outlining the current status of research and development in the arena, and suggestions for how an ATP focused program could make a real contribution toward advancing commercialization of these emerging technologies. After a two-week on line discussion among the invited participants, the public will be invited to comment. It is expected that many small companies who have not published papers or otherwise declared themselfes in the arena will come to the table.

While the possible benefits of this technology are clear, private investment has been lagging. There are at least three possible explanations for the reticence: (1) while proof-of-principle has been shown, the work is still very high risk, (2) there is a long lag between investment and return since the time between experimental manipulations and results are dependant on the long gestation and maturation periods of the animals in question, and (3) critics fear that animal cloning technology will make human cloning an inevitability. The ATP focused program can address all of these concerns. ATP funding can bridge the gap between basic science and routine production of engineered animals for the numerous species involved. Discovery of the necessary technology will empower the venture capital community to make the patient investment necessary for product development. In addition, a federal investment in this blossoming research area would reassure the private sector that there is a very clear distinction between research using human tissue which is restricted by Executive Order, and the potentially beneficial and profitable genetic improvement of animals.

This work certainly will be done, but more slowly without the U. S. government's involvement. ATP could make a major difference in not only the pace of technological developments, but also in securing the U. S. position in an industry where we have had little impact.

An electronic workshop exloring this technology area was held in the fall of 1998.