By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
When researchers clipped 1-centimeter tips from the tails of six mice with hemophilia, those unfortunate animal models bled to death. But 46 percent of their littermates, treated with a novel gene-therapy vector, survived.
"After years of disappointments and setbacks in the field of gene therapy," wrote molecular hematologist Katherine High, "the stage appears set for success, and many observers agree that the first convincing demonstration of efficacy for gene therapy will likely be for hemophilia." High, at the University of Pennsylvania in Philadelphia, is supervising early clinical trials of gene therapy in human hemophilia patients. (See BioWorld Today, June 15, 1999, p. 1.)
She made her optimistic forecast in a commentary on a preclinical experiment reported by Belgian scientists in the current Proceedings of the National Academy of Sciences (PNAS), dated Aug. 31, 1999. Its title: "Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice."
Those neonatal animals acquired their hemophilia by having their inborn genes for murine blood coagulation factor VIII knocked out. Then they received intravenous injections of a retroviral vector aiming human factor VIII sequences at their livers, the main organ that manufactures the clotting protein.
In humans, that gene resides on the sex-linked X chromosome, which means that boys, but not girls, inherit the recessive hemophilia A (factor VIII deficiency) or hemophilia B (lack of factor IX). Clinically, the two disorders can't be told apart.
In both versions, bleeding doesn't happen only in reaction to a bump, blow or fall - let alone external cuts and bruises. It occurs spontaneously and frequently inside the body, too, with blood pouring into joints and other internal tissues, resulting in chronic severe arthritis. Such extravasation in closed spaces, notably cranium or abdomen, can result in death.
Yet lack of factor VIII is not an all-or-nothing proposition. "Mild" hemophilia A defines 5 percent to 30 percent of normal protein levels; "moderate" as 1 percent to 5 percent; and "severe" as less than 1 percent. In other words, a small input of exogenous factor VIII can make a large difference in disease severity. That treatment consists of injected factor VIII concentrate, purified from donor blood, or more recently from recombinant synthesis.
But such replacement therapy, aside from its astronomical cost, packs a grim Catch-22 factor: When a patient's immune system meets an incoming bolus of foreign factor VIII, it reacts to reject the alien protein. This weakens its clotting effect, and requires escalating infusions of the protein. The Belgian trials, at the University of Louvain, sought to overcome these drawbacks by gene therapy."
Thirteen hemophilic 2-to-3-day-old baby mice received injections of the gene-transfer construct. Six of them (46 percent) expressed "physiologic or higher levels" of the human factor. Five of the six survived the lethal tail-clipping test, and two of them have retained their human clotting-factor expression - ranging from 20 percent to 1,250 percent of normal human levels - for more than 14 months. But six of the seven animals that failed to express the gene developed immune inhibitors of the protein.
In Hemophilic Patients, French Discover Immune Mechanism That Disarms Factor VIII
Elsewhere on the hemophilia front, a French team of immunologists, at the Pierre and Marie Curie Hospital in Paris, unraveled the inhibitory action of human immune defenses against factor VIII therapy. Their report, in the September 1999 issue of Nature Medicine, bears the title: "Catalytic activity of antibodies against factor VIII in patients with hemophilia A."
They pointed out that 25 percent of all hemophiliacs with the severe form of the disease develop inhibiting antibodies to the infused clotting factor. In two of three patients studied, they found that these proteolytic antibodies actually cleave and neutralize the factor VIII molecules by hydrolysis, "demonstrating a previously unknown mechanism by which factor VIII inhibitors may prevent the pro-coagulant function of factor VIII."
Their paper makes the point, "This is the first report, to our knowledge, of the induction of catalytic antibodies in humans in response to exogenous administration of a protein antigen." It concludes: "The characterization of factor VIII inhibitors as site-specific proteases may provide new approaches for the treatment of inhibitors."
Correlating Mosquito Migration, Insecticide Resistance, For Better Spraying Management
Another French group, on a totally different kick, has tracked the evolution of insecticide- resistance genes in the common house mosquito, Culex pipiens. Reporting in Nature dated Aug. 26, 1999, the molecular geneticists, at the University of Montpellier, in the south of France, observed, "The evolution of pesticide resistance provides some of the most striking examples of Darwinian evolution occurring over a human life span."
Between mid-1995 and late 1997, the co-authors charted the migration of 8,594 mosquitoes over a narrow land belt bordering the Mediterranean Sea, which was sprayed with organophosphate, and north into the insecticide-free hinterland. They sampled diapausing (hibernating) females in four overwintering caves, and larvae in breeding-site pools of water.
The team correlated this field data with laboratory and statistical studies of the annual frequencies of two major gene loci conferring organophosphate resistance. One resistance allele (parental gene variant) coded for a modified acetylcholinesterase neurotransmitter - the insecticide's target. The other encoded overproduced detoxifying esterases.
"Allelic frequencies," their paper noted, "were computed separately for each sex during overwintering as only females overwinter as adults, whereas males survive as spermatozoa in fertilized females." It made the point, "A precise understanding of the conditions under which insecticide resistance occurs is necessary for the development of successful management. . . . Unrelated insecticides such as Bacillus sphaericus could then be used on the remaining [area of treatment] to achieve the same treated area size but without the evolution of organophosphate resistance."