Pig islets ready to Rocky and roll

Rocky the pig may not be too aware of what's going on, but his keepers at Living Cell Technologies' (LCT) breeding facilities in New Zealand are eagerly awaiting the signature of one very important man.

That man is the country's health minister, Pete Hodgson, who is expected to give the go-ahead for an encapsulated porcine islet cell transplantation trial at Auckland's Middlemore Hospital.

LCT has received all of the regulatory and ethical approvals required and the trial just awaits Hodgson's signature. It will be the second such trial for LCT, which is conducting another at Moscow's Sklifosovsky Institute, and it will be small, involving eight patients.

However, the trial's effect on the looming debate about the xenotransplantation moratorium in Australia, due to be re-considered in 2009, might be another matter.

Professor Bob Elliott, LCT's co-founder and medical director, puts his views on the potential of ending the moratorium in 2009 bluntly.

"It will end a great deal quicker than that if we get people off insulin long term," he says. "If it is working, then clearly the benefits would outweigh any possible, highly unlikely, risks."

And it does seem to be working. LCT's case on the public relations front has been boosted recently by two notable examples: one is Michael Helyer, the Auckland man who took part in an early trial and was happy to make international headlines when it was found that he still has functioning porcine islet cells making small amounts of insulin 10 years after the transplant.

The other was the release of very early results in the Moscow trial, in which one patient has come off insulin injections altogether.

Although it was an unusual step, LCT was obliged to release early results about the Russian patients because it is listed on the Australian stock exchange, which requires companies to release information that could affect the share price.

And the whispers from the Moscow trial were becoming a roar by September: the first patient, a 26-year-old man, has had his insulin requirements reduced by almost 40 per cent after the first of two transplants. The second, a 40-year-old woman, no longer required insulin after a month.

"We knew it was starting to leak out from Russia because we were getting people we didn't know popping up who had heard about the results," Elliott says.

"We'd made that decision [to announce early results] ahead of the second patient coming off insulin, and that's the Holy Grail of course. That news was getting around too. There is no obligation on the Russians to retain secrecy - there were hundreds of people in Moscow who knew what was going on."

Elliott is extremely confidant about the efficacy of his technology but knows there will still be many doubters who will require a lot of delicate persuasion.

There are those who point to the zoonotic disease risk, in particular the risk of transmission to humans of porcine endogenous retroviruses (PERV), the driving force behind the halting of trials in 1997 and the subsequent worldwide moratorium.

This is where Rocky and his colleagues come in. Rocky is a very special pig indeed, bred from an isolated and disease-free herd resident on Auckland Island, south of New Zealand.

"The animals we use do not produce PERVs," Elliott says. "They've got the sequence in their genomes but they do not produce infective particles and they cannot produce them because they don't have the right genetic code. It's defective, so they've been designated null pigs.

"That's the basis we work on but some people worry they might mutate again, so we are following all of the guidelines the FDA has laid out. Thousands of exposures to full-length, infective PERVs have been carried out by mistake, when people were given factor VIII from pigs, repeatedly given it intravenously, but nobody came to any harm by actually receiving the virus. It was a genuine scare and we stopped just like everyone else but it has turned out to be a red herring."

Then there are those who argue that microencapsulation of islet cells has been tried for 25 years and doesn't work, or just doesn't work well enough, allowing the problem of immune rejection to rear its ugly head.

A big issue for any xenotransplantation is the alpha 1-3 galactose epitope, or alpha-GAL, which all animals bar humans and Old world monkeys such as baboons and macaques carry on their cells.

"Man is very peculiar in this respect and it does mean that man has acquired antibodies against all animal cells," Elliott says.

"This is a particular problem but it is antibody-dependent, complement-mediated, so by doing the encapsulation that is not a problem. It eliminates the immediate rejection that is seen with pig cells put into humans."

Elliott says it is also essential to perfect the technology so that macrophages aren't attracted to the islets, which means weeding out all the dead ones.

"If you put dead cells in then the macrophages will come. So we had to make sure that we had completely viable cells. Again it was a matter of technology and treating the cells as gently as we could.

"The other thing was to make sure that as far as possible our encapsulation was complete and that we didn't have some unprotected cells - they would have attracted macrophages and they'd start excreting cytotoxic cytokines and could kill off even the innocent bystanders. We had to make sure there were no duds in there.

"A lot of people say this can't work. Well, it does work, but not because of any great leap of science. It has been a slow grind with a very old idea. It was just a matter of perfecting it so that it did what it was supposed to do."

---PB---

Elliott first became interested in the potential of xenotransplantation for diabetes during his paediatric medicine training, over 40 years ago. He was involved in some experimental research even then, looking at transplanting rat islets to the anterior chamber of the eye - an immune-privileged site - of diabetic rabbits.

He kept an eye on ongoing research in human foetal islets while he did further training in Colorado, and after he moved to the University of Auckland in 1970, but it wasn't until the mid-80s that practical interest resurfaced.

This was when he met a businessman called David Collinson, who had a son with diabetes and had come looking for answers. Together, they set up LCT and have been working on perfecting porcine islet transplantation ever since.

"There are never going to be enough human islets so it would have to be animal islets, and if it is animal islets somehow they have got to be done without immune suppressants," he says.

"With those two things in mind, we initially tried to get islets from unborn pigs, just before term, and transplanting those into a variety of animals. We were using a non-immuno-suppressant called nicotinamide, which arrests the immediate immune response, with a small degree of success."

While on study leave in Germany in 1991 he met a researcher who recommended alginate encapsulation as a way to avoid immune rejection, so he began looking around for people doing work in that area.

LCT became involved with a Californian research group and used their encapsulation technology, along with LCT's cells, in early trials, including the one Michael Helyer was involved in. Then to Mexico, where a group was experimenting with Sertoli cells from the testis, which produce a local immuno-suppression effect.

It was a group from the University of Perugia in Italy, however, led by Professor Riccardo Calafiore, that turned out the most promising. This group has found a way to purify alginate to make smooth, long surviving capsules without surface pitting, meaning the cells inside are not exposed.

LCT and the Perugia team work closely together and have recently published details of their methods in the Journal of Biomedical Materials Research Part A.

"They didn't have patents, as they were old-fashioned academics who worked their butts off getting the stuff made," Elliott says.

"We have subsequently taken out a patent that is an improvement on the original method. The standardisation of materials and the way you go about doing it - we put a lot of work into making sure it would be the same each time we did it. We needed to get it dead right."

So have they got it dead right? The proof of the pudding is in the eating and LCT is getting good results, Elliott says.

"There is room for improvement and that's going on consistently. There are all sorts of issues turning up, like how long do you keep the cells in culture before you put them in.

"Initially we thought the quicker the better because they don't go too well when you culture for a long period of time. But after encapsulation they last quite well and they seem to improve in the first couple of weeks."

No one quite knows how long the capsules and their precious contents remain productive in the body, but Michael Helyer is proof that a decade is not out of the question.

Elliott says those surviving islets were probably the hardiest survivors, but considering that the technology used at the time was quite poor, it looks promising. "How long that will go on I don't know, but the prospects are that with the technology we have now they will last as long as they would in a pig. A pig lives for about 20 years."

So, onwards to clinical trials. Six patients will be involved in Russia and eight in New Zealand, with the Russian patients receiving two half-doses six months apart. The New Zealand patients will be split into two groups of four, one of which will receive double the dose of the Russian patients and the other triple.

The primary endpoints of the trials are safety and efficacy, and to ensure the procedure is worthwhile. The cells must remain productive for more than six months, but preferably more than 12 months, to make the procedure economically viable.

In LCT's favour for moving the trials along quickly, however, is the fact they involve transplants, not pharmaceuticals. "We are following the methods used for drug trials, but this is not a drug: it's a transplant procedure," Elliott says.

"Transplant trials are not carried out that way - they don't go through Phase I, II and III. My suspicion is that this will never go through those phases - it will be a number of pilot studies, some larger pivotal studies and it will be free for all after that."