By David N. Leff

Shortly after World War II, so the story goes, a dog fancier in California contacted pathologist Kenneth Brinkhaus, who had just begun studying canine hemophilia at the University of North Carolina, in Chapel Hill.

The breeder had two Irish setters, which bled frequently and copiously from lack of coagulation factor VIII. With a start-up grant, Brinkhaus acquired the pair of animals in 1947, thus founding the now-famous canine hemophilia colony at Chapel Hill.

"The majority of our work is supported by the National Institutes of Health [NIH]," observed the colony's associate director, blood-coagulation specialist and cardiologist Timothy Nichols. "We also have contracts and financial support from industry, but the colony by and large, over the past 50 years, has been supported by the NIH.

"Actually," Nichols told BioWorld Today, "Brinkhaus, now 91 or 92 years old, has the longest-running NIH grant. Then, in the mid-1960s, Brinkhaus identified in some Cairn terrier-beagle crossbreeds a spontaneously occurring bleeding disorder, and they've been maintained here at Chapel Hill ever since. They were originally from a breeder's show-dog strain. Brinkhaus and his group determined in the '80s that it was due to a point-mutation deficiency of factor IX."

Cairn terriers are small, low, shaggy dogs, originally bred on farms in Scotland to exterminate rodents and other vermin. At Chapel Hill, the Cairn bleeders were carefully crossed with other breeds to obtain a larger animal model of human hemophilia B - the X-linked factor IX defect. (Hemophilia A denotes the lack of factor VIII.)

This cairn-beagle combination, Nichols pointed out, "is now a Kennel Club-recognized strain called the keagle. With their mixed-breed background, they vary in size between 20 and 40 pounds, and have a beautiful black color. But they're fragile, in that they bleed, and bleed a lot, spontaneously and severely - like human hemophilia B patients. There's absolutely no factor IX in their circulation."

Between 1987 and 1990, molecular hematologist Katherine High, then at Chapel Hill, determined the cDNA sequence for canine factor IX, by screening a library, as well as the genetic defect in the mutated gene. She is now hematology research director at the University of Pennsylvania-affiliated Children's Hospital, in Philadelphia.

High is senior author of a paper in the January issue of Nature Medicine. Its title is "Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector [AAV]."

Penn And Stanford In Tie

Back to back with her paper is a strikingly similar report - but with a difference - by the other pioneer researcher of canine factor IX deficiency, Mark Kay of Stanford University. The article of which he is senior author bears the title, "Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors."

Curiously, co-authors of both papers are Chapel Hill's Nichols; the canine colony's director, Marjorie Read; and Brinkhaus.

Both Kay and High reported their preclinical gene therapy using recombinant AAV vectors to deliver the canine factor IX cDNA to hemophilia B dogs in Chapel Hill. The difference came in as to where they deposited the gene sequence.

Kay and his co-authors chose the liver as their natural target site, because coagulation factors are synthesized in the liver. To reach this organ required entry via its portal vein feeder, which in turn involved a surgical incision, which would have provoked severe bleeding had it not been controlled in advance.

High and her associates preferred to insert their gene therapy package by fine-needle injections into various skeletal-muscle sites, which also express genomic DNA. This nearly non-invasive site, they reasoned, would also be more human-friendly in prospective clinical trials. Among High's co-authors is Gary Kurtzman of Avigen Inc., in Alameda, Calif. (See BioWorld Today, March 13, 1997, p. 1, and Sept. 22, 1997, p. 1.)

"The vector was actually produced at Avigen, at [its] own expense," High said.

In Kay's trial, infusion of some 2 trillion AAV-vector particles per canine kilogram into the portal veins of two hemophilia-B dogs produced factor IX levels that were 1 percent of normal. That partial correction remained constant for eight months, as Kay reported in Nature Medicine. A 10-percent level would provide full correction.

Human Trials High On Agenda

High's team injected intramuscular dosages of up to 8 trillion vector particles per kilogram. This had sustained partial correction for 18 months by year's end, last week.

A normal dog clots a bleeding episode in about eight minutes. A hemophiliac dog takes up to a full hour. High's five treated canines' clotting time ranged from 16 to 20 minutes. Also, their immune reactions to the AAV and factor IX antigens proved few and transient.

"I am very encouraged by these results," she said. "Keep in mind that, even between a large animal and a human, there is still a great gulf to cross. But, what is important to me about this survival is that there have been many examples of successful gene therapy strategies in mice or other small animals. They have not translated to sustained expression at levels that would be therapeutic in humans.

"But when you go to a large animal model," she continued, "the scale-up between a mouse and a dog is something between 400-[fold] and 800-fold. The difference between a dog and a human is on the order of three- to ten-fold, depending on how big the dog is. So, I think, as we demonstrated, if you can successfully cross from a mouse to a large animal, it really encourages us to predict similar outcomes in a human."

As for clinical trials, High observed that the long-term goal "of everything that I'm doing is to develop a gene therapy for hemophilia. We have actually written up a Phase I proposal and submitted it to the FDA about six weeks ago. Mark Kay has joined me on the scientific advisory board of Avigen, and we have decided to pool our resources and work together on the forthcoming clinical trials."

She concluded: "I think that if we can get levels in humans such as we're getting in the dog, we could expect at least some modest improvement in the phenotype of people with severe hemophilia." N