Dolly, Polly and other ‘ollys’: likely impact of cloning technology on biomedical uses of livestock

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Abstract

The idea of generating transgenic livestock which secrete into their milk large quantities of proteins for therapeutic use, was pioneered in the late 1980s with the disclosure of the production of a number of transgenic sheep. One particular animal, a sheep called Tracy, produced milk where over 50% of the protein consisted of human alpha 1 anti-trypsin. Sheep-derived protein has now entered clinical trials for cystic fibrosis (UK, USA) and congenital emphysema (UK). There are many other examples where this technology is making inroads into more traditional ways of making biopharmaceuticals. However, although robust, this technology has several limitations, including an inability to allow targeted insertion/modification of the animal genome, long timelines to production flocks/herds, and the rather unpredictable expression levels seen when different transgenic founders are compared. We believe that there is now a technical solution to all of these problems. Dolly is a high profile example of a new technology comprising the generation of identical animals from cultured somatic cells. This work has many implications. In the commercial context, the real benefits of this advance will be seen when genetically engineered somatic cells are shown to be suitable nuclear donors, and particularly when the manipulations are targeted to pre-determined sites in the host cell genome. The first objective has now been achieved with the birth of Polly, a cloned sheep which contains the human gene encoding Factor IX, a protein involved in preventing haemophilia.

Introduction

In 1980 it was demonstrated for the first time that micro-injection of DNA into the pronucleus of a fertilised mouse embryo could lead, in a proportion of cases, to the integration of the injected DNA into the genome of the resulting mouse; such an animal was termed transgenic, and it was quickly shown that most of these transgenic animals could transmit the the new DNA (the transgene) to the succeeding generations. Within a few years this technique had been extended to larger mammals, including pigs, sheep and goats, and improvements in the design of the DNA constructs injected, ensured that expression from the integrated transgene took place in the desired tissue and at the correct time. It was the availability of these two related technologies which led to the foundation of PPL Therapeutics in Edinburgh in 1987.

PPL produces a variety of transgenic mammals (sheep, cows, rabbits, pigs and mice) in which a human gene is expressed in the milk of the lactating females by virtue of the presence in the gene construct of a sheep milk gene promotor sequence (usually from the beta lactoglobulin gene). The human genes chosen for this purpose are those encoding proteins of therapeutic interest. Our main goal is to be able to produce and purify such proteins in high yield and at low cost from the milk of transgenic livestock for subsequent medical use combating human disease. A subsidiary goal is the use of transgenic pigs as organ donors for human recipients. In this article, I discuss our current progress in fulfilling these goals and describe how a new technique called nuclear transfer is likely to extend and improve the biomedical uses of transgenic technology.

Section snippets

Tracy: the sheep that started it all

In 1989, PPL performed a micro-injection study intended to generate transgenic sheep which expressed the human protein, alpha 1 antitrypsin (AAT) in their milk. AAT is a plasma protein whose main physiological role is to neutralise the enzyme, elastase. This enzyme, which is secreted from circulating neutrophils, has an important role in the alveolar fluid of the lung, where it serves to protect against bacterial infection. Unfortunately, if its activity is not carefully regulated by, amongst

Dolly: dawn of a new transgenic age

A great deal has already been achieved using the technology outlined above and two products, AAT (PPL Therapeutics) and antithrombin 3 (Genzyme Transgenics) are in advanced stages of clinical trial. However the micro-injection technology has several limitations. These are:

  • 1.

    Micro-injection results in gene addition but cannot effect gene removal.

  • 2.

    Only a small proportion of injected embryos results in a transgenic founder animal. The proportion of transgenic births to total births is ≈5% (sheep), 9%

Polly, Molly et al. Transgenic cloned sheep

The long term objective of the above work was to utilise nuclear transfer technology to remedy the deficiencies of the micro-injection procedure. Since all the applications involved adding genes either randomly or to specific loci, or deleting host genes, the next important step was to show that sheep could still be made if the nuclear donor cells were previously genetically manipulated. We chose primary foetal fibroblasts for this work since very large numbers of cells were available right at

Extension of nuclear transfer technology to other species

The successes reported above for the use of established cell cultures from embryonic, foetal, and adult cell cultures, all relied on the use of serum starvation to make the cells quiescent. Recently, it has been claimed that a quiescent state is not essential to the successful use of differentiated, somatic cells in nuclear transfer. Unfortunately, because of the very low efficiencies of the procedure, and current difficulties in assessing the cell cycle status of any successful nuclear donor,

Biomedical uses of nuclear transfer technology for protein production in livestock milk

The above work demonstrated for the first time that genetic manipulation of somatic cells does not compromise their totipotency. We believe this will offer the following advantages:

  • 1.

    All animals, instead of a small proportion, will be born transgenic and non mosaic.

  • 2.

    Genes can be added to, or removed from, specific regions of the chromosomes. This could ensure that high expression levels are always achieved by, for example, targeting genes to loci containing milk protein gene families like the

Other biomedical uses of nuclear transfer technology in livestock

Many of the advantages mentioned above would apply to other uses of transgenic, cloned livestock. For example, there is much discussion (and disagreement) about the use of pig organs for transplantation to desperately ill patients in circumstances where the appropriate human organs are not available. One of the major (but certainly not the only) stumbling blocks to this is caused by the presence of a particular sugar on the surface of all pig organs. The presence of this sugar, an alpha 1–3

Miscellaneous uses of the nuclear transfer technology

Many further uses of this technology have been suggested in the agricultural areas. For example, the demonstration that adult cell nuclei can be used, holds out the prospect of being able to copy and multiply up any mammal which displays a suitably attractive phenotype. For example, milk production could be increased if it were possible to clone from the very best producing cows.

The preservation of rare or dying mammalian species is another suggested use. However, it is in the area of basic

Human cloning

I do not believe that there is a good reason for human reproductive cloning in that the cost, both in financial and human terms, will be too great to justify the small number of legitimate reasons for doing it. I do believe that a case could be made for culturing pre-implantation embryos which have been reconstructed using nuclear transfer. Such embryos when cultured could give rise to different stem cell types. These stem cells could then be used to repair diseased tissue in the nuclear donor

Conclusions and discussion

We have seen above that transgenic production of therapeutic proteins in the milk of ruminants is no longer just at the concept stage and is likely to deliver useful products to the patient community in the next two to three years. The products currently in clinical trials have been made using the existing ‘micro-injection’ technology which is robust, relatively cheap, and effective. Nevertheless, I argue that this technology has major disadvantages and will be superceeded by the use of nuclear

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