LONDON - The commercialization of cloning has accelerated with disclosure that PPL Therapeutics plc - which produced the first cloned mammal, Dolly the sheep - has entered a research collaboration with ProBio America Inc., holder of rights to the Honolulu cloning technique.

PPL said it intends to develop this new nuclear transfer method to clone pigs for use in xenotransplantation.

The Honolulu cloning technique was developed by Ryuzo Yanagimichi and colleagues at the University of Hawaii and was successfully applied to produce multigenerational mouse clones. The approach appears to be more efficient than the technique used to create Dolly, succeeding in one in 40 cases, compared with one in 227.

The Hawaii procedure is described in the July 23, 1998, issue of Nature. ProBio America, based in Honolulu, partly funded the research and has licenses to commercialize the work.

Ron James, managing director of PPL, in Edinburgh, Scotland, told BioWorld International, “We wish to find out if it is more effective. On the face of it, it is fairly effective with mice.“

James identified the key distinction between the two processes as the microinjection of the adult cell nucleus into the enucleated egg in the Honolulu procedure vs. the use of electricity to fuse the two elements in the PPL technique.

“We really don't know what makes [Honolulu] a better technique and that is one of the things we hope to find out.“

James also pointed out that although mice are easier experimental animals to handle, mouse embryology is “less robust“ than that of sheep and other mammals, indicating the new technique may be even more efficient in other mammals.

“If the technique works in pigs, we will then apply it elsewhere,“ he said.

From a commercial standpoint, James said the publication of the University of Hawaii paper was “additive, not competitive. We have been saying for a long time that as soon as other groups of scientists show it is possible to produce clones from adult cells, it will prove that Dolly was the first clone.

“This is the next step in the development of what will ultimately become a very useful science with lots of applications.“

PPL also said it has reached the first stage of commercializing nuclear transfer with the birth three weeks ago of the first transgenic lambs produced under the procedure. The lambs carry the human gene for extracellular superoxide dismutase (EC SOD).

The East Friesian lambs, born on PPL's farm in East Lothian, Scotland, will be the founders of a commercial flock of sheep for the production of EC SOD, a natural antioxidant. PPL is evaluating its use in organ transplants and for prevention of tissue damage caused by reperfusion and ischemia following cardiac surgery.

“This is the first commercial use of nuclear transfer by PPL staff on our farm. EC SOD is a new product in large animals from PPL's own product pipeline, for which we hold the patent rights,“ James said.

Tests Substantiate Dolly Cloning

As the cloning plot thickens, PPL also disclosed last week that Dolly's provenance from an adult cell has been upheld by the inventor of DNA fingerprinting, Alec Jeffreys, of Leicester University.

Mammary tissue from the original six-year-old donor ewe was compared with a blood sample of Dolly and with cultured cells derived from the tissue that was used for nuclear transfer to produce Dolly. The DNA fingerprints of these three samples were identical in band number, position and relative intensity.

The test, also reported in Nature July 23, was carried out after it was suggested that because the ewe that produced Dolly was pregnant at the time of the experiment, Dolly could have been derived from a contaminating embryo cell or from a fetal cell present in the bloodstream of the ewe, rather than from an adult cell.

“We were confident that Dolly was a clone,“ James said, “and so this proof is perhaps more important to the academic scientists than ourselves. Nevertheless, there was an insinuation that we had got it wrong. What we didn't foresee at the time we commissioned the tests was publication of the mouse clone paper and news of cloned calves from Japan. This might have stopped people raising the question over Dolly.“

The nuclear transfer technique used to produce Dolly was developed at the Roslin Institute, in Edinburgh. PPL has a worldwide exclusive license to use the procedure to raise transgenic ruminants and rabbits, which produce therapeutic proteins in their milk. *