BioWorld International Correspondent

PARIS Two French biotechnology companies, BioProtein Technologies and Vivalis, and France’s National Institute for Agronomic Research (INRA) reported the first successful cloning of a rabbit.

The cloning was performed by INRA’s Developmental Biology and Biotechnology Unit using nuclear transfer from adult somatic cells. The head of the unit, Jean-Paul Renard, said, “The rabbits are in good health and the first two have already given birth to seven and eight kits, respectively.”

Renard pointed out that the numerous past attempts to clone rabbits always ended in disappointment “despite the rabbit’s pioneering role in defining nuclear transfer methods in mammalians.” INRA’s experiment succeeded because “we have designed a specific cloning procedure adapted to the physiological characteristics of rabbit oocytes and early embryonic development.”

In order to obtain oocytes (immature female germinal cells), females were mated with sterilized males, and the oocytes then collected and enucleated. The donor cells used to clone the rabbits originated from the pool of somatic cells surrounding the oocyte before ovulation, known as the cumulus. The nuclear transfer of a donor rabbit’s cumulus cells was performed by micro-manipulation and electrofusion in order to generate an embryo.

As the development of the cloned embryo in rabbits is slower than that of non-cloned embryos, INRA studied three different protocols, only one of which was successful. It entailed the transfer of four-cell embryos into foster mothers asynchronized with the females that gave the oocytes. The basic numbers for the procedure were as follows: a total of 371 eggs were implanted in 27 foster females, of which four gave birth to cloned rabbits; six cloned rabbits were born, of which four were still alive during weaning.

INRA waited several months before publishing the results of its successful experiment (in the April issue of Nature Biotechnology) to ensure that the clones became fertile adult animals of normal physiological appearance. It now plans to use genetically modified cells as a source of nuclei for the cloning experiments. “We consider that the rabbit is one of the most appropriate animal models to increase our knowledge of the still poorly understood process of nuclear reprogramming in mammals,” Renard said.

BioProtein Technologies and Vivalis will be the first beneficiaries of INRA’s cloning technology. INRA has granted BioProtein an option to acquire an exclusive license for using this cloning method to produce recombinant proteins in the milk of transgenic rabbits. Paris-based BioProtein Technologies already is a leader in the production of proteins in the milk of genetically modified rabbits, but CEO Marc Le Bozec told BioWorld International that using cloned rabbits would yield definite advantages.

The current procedure was wasteful and haphazard because it entailed the direct injection of the transgene directly into a cell at an embryonic stage, without any control as to whether it fell into an active or inactive zone of the genome, he said. With a cloned rabbit, “we can target the introduction of the transgene, maintain greater control over its expression and the duration of that expression, and at the end of the day use fewer rabbits.”

Le Bozec said BioProtein planned to exercise the option granted it by INRA, but only when more comprehensive results of the cloning were available, confirming that the cloned animal was industrially exploitable. The company already uses INRA technology for producing recombinant proteins and needs no convincing of the advantages of using rabbits to produce molecules of pharmaceutical interest. Its expertise spans the whole manufacturing process, from genes or cDNA expression to animal production, and it produces complex and hard-to-express proteins, including monoclonal antibodies, hormones and vaccinating antigens.

The company was founded in 1998 and completed its first funding round in May 2001, raising EUR8 million (US$7 million). Le Bozec said it had sufficient funding for three or four years, given that its burn rate was about EUR2 million a year, and that it expected to move into profit before having to raise additional external funding. As a service company, it was already generating revenues, he said.

For its part, Vivalis, which is specialized in the cultivation of embryonic stem cells and the production of therapeutic proteins in eggs, has an option for an exclusive license to apply INRA’s cloning technology in animal models for research purposes and other therapeutic applications, except for the production of therapeutic recombinant proteins in transgenic rabbit’s milk.

Based in the western city of Nantes, Vivalis was founded in 1999 and is controlled by Groupe Grimaud, a company specialized in genetic selection, while its other shareholders include INRA and two other public research establishments that developed the embryonic stem cell technology it uses. It cultivates cells and cell lines into which human genes are inserted, which code for particular proteins that express themselves naturally in the egg. It uses this technology to produce therapeutic recombinant proteins (such as antibodies, peptides and cytokines) in the eggs of transgenic chickens. It also supplies stem cells, primary cells and cell lines to the pharmaceutical industry.