By David N. Leff

A baby boy born without a functioning gene for the blood-clotting cascade's Factor VIII is thrice doomed in life.

Lacking the crucial coagulation factor, he must receive frequent replacement doses of it, either from donor blood extracts or recombinant Factor VIII. The annual tab for such maintenance treatment can reach the neighborhood of $100,000.

But this child enters the world with an immune system that has never seen the Factor VIII protein. So antibodies promptly mark that clotting factor for elimination as a dangerous outsider molecule. This immune rejection renders the substitute Factor VIII substance — one of the costliest medicaments in the medical armamentarium — ineffective.

In an attempt to outgun the circulating antibodies trashing his only therapeutic recourse against life-threatening hemorrhages, a hemophiliac must greatly step up his normal dosage of injected human factor VIII.

"To date," Richard Morgan pointed out, "about 75 percent of patients making high responses to antibody inhibition of Factor VIII have been 'cured' of inhibitors long term. But that approach," he said, "requires very frequent injections of Factor VIII concentrate. In some cases, this treatment exceeds one million dollars."

When third-party carriers balked at such a price-tag, he recalled, "patients have been obliged to declare personal bankruptcy, sell off their assets, and go on Medicaid."

Morgan directs gene transfer at the National Human Genome Research Institute's Clinical Gene Therapy Branch, in Bethesda, Md. He is trying to lift the triple curse on Factor VIII hemophiliacs by a gene therapy strategy.

His story thus far appears in the current Proceedings of the National Academy of Sciences (PNAS), dated May 12, 1998, under the title: "Genetic induction of immune tolerance to human clotting factor VIII in a mouse model for hemophilia A."

Hemophilia A denotes the form of the disease that affects victims devoid of Factor VIII. It afflicts one in 10,000 U.S. males, who represent 70 to 80 percent of all hemophiliacs; Factor IX deficiency accounts for most of the rest. (See BioWorld Today, April 21, 1998, p. 1.)

"More and more clinical trials of gene therapy for more and more inherited diseases seem probable," Morgan told BioWorld Today. "Their success will be increasingly handicapped by frequent presentation to their patients' naive immune systems of proteins they have never seen before."

Hemophilia Mouse Model Offers Proof-Of-Principle

In a proof-of-principle attempt to blunt this threat, Morgan chose hemophilia A, he recalled, "because a very good mouse model of this disease was available. It had had its murine Factor VIII gene knocked out." He also noted, "Of all hemophilia A patients, 20 percent will get an immune response to their replacement Factor VIII.

"Our idea," he recalled, "was to produce a powerful life-alteration in the genome of the mouse surrogate. This would model re-education of the blood-forming stem cells in adult human patients. Their immune cells — antibodies and T lymphocytes — would learn to recognize the Factor VIII protein as 'self,' during their development from a single stem cell to hematopoiesis of red blood cells, white cells and T lymphocytes."

Morgan and co-author Gregory Evans transformed that hemophilia mouse model's bone-marrow stem cells with the gene encoding human Factor VIII, delivered by a retroviral vector. Then they collected this factor-positive bone marrow and transplanted it by injection into a second factor-negative mouse. But first they wiped out this recipient rodent's Factor-VIII-lacking bone marrow with whole body irradiation.

"Half of these reconstituted mice," Morgan recounted, "did not develop anti-Factor VIII immune responses. But every one of the controls did produce lots of antibodies. And one-half represents a good result for this experimental gene therapy."

To test whether this bone-marrow transplant and gene transfer could bring about immune tolerance to human Factor VIII, Morgan and his co-author immunized 18 or 19 animals in each vector group, transformed and control, with recombinant Factor VIII. "Four weeks later," he recounted, "our test animals' sera had four-fold lower levels of antibody inhibition than the controls."

Making The Vector Shoe Fit

What accounts for the 50 percent that did generate factor-inhibiting antibodies, despite their gene therapy, is still under investigation. Part of the explanation, Morgan suggested, "may be that the Factor VIII protein has at least half a dozen immune-signaling epitopes on its surface, and is unprecedentally huge for a gene-therapy molecule. Its size is 180 kiloDaltons. Most gene-therapy products are under 50, and none is this big."

To shoe-horn his outsize Factor VIII gene into its tight-fitting retroviral vector, Morgan deleted about one-third of its redundant sequence.

In fact, one of his conceptual explanations for achieving immune tolerance is "giving back such an enormous protein that it exhausts the antibody-producing B cells."

Unlike many gene therapy preclinicals, which look forward to human trials as soon as possible, Morgan's prototype protocol, he observed, needs a lot more preclinical work.

"A clinical approach based on whole-body bone marrow irradiation," he said, "is not safe." In fact, 25 percent of his fragile recipient mice died following the procedure.

He and his team are now "using various tricks to make irradiation unneccessary." They will subject cohorts of their mice — and eventually hemophiliac dogs — to prophylactic and therapeutic treatment — before and after the onset of Factor VIII-degrading immune inhibition.

"The issues for the biotech community," Morgan suggested, "are that as one starts to engineer cells to express, or administer to patients, proteins to treat genetic disease, if their bodies haven't seen this protein before, their immune systems could bedevil your efforts to treat them.

"If this type of approach works for hemophilia," he concluded, "it really should work for most any protein that one can get expressed in the hematopoietic system, in order to tolerize the immune system against the gene you want to transfer or express." *