By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
The world learned Friday of gene therapy's first therapeutic triumph. The journal Science, in its issue dated April 28, 2000, headlined the story: "Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease." The report was authored by a team of 13 gene therapists at the Necker Children's Hospital in Paris, with INSERM - France's National Institute of Health and Medical Research, and the Pasteur Institute in Paris. Its senior author is Necker pediatric immunologist Alain Fischer.
SCID-X1, an X-linked genetic disorder, leaves its infant victims bereft of a functioning immune system - no T or B lymphocytes, no natural killer cells - to fight off invading pathogens. Death usually intervenes before the first birthday. The disease first gained notoriety in the U.S. a generation ago. In 1971, at Texas Children's Hospital in Houston, a newborn boy identified only as "David V." was delivered by ultrasterile Caesarean section directly into a germ-proof plastic "bubble," where he spent the next half-decade before deadly infection, following failed therapy, finally ended his life.
In Paris, the first two victorious SCID babies, aged 11 and 8, received infusions of retroviral vectors that delivered healthy gamma-chain (gc) transgenes into their blood. (The 1-month-old third patient came later.) This gamma chain, when mutated, can't activate the receptor of interleukin-2, a key cytokine that mobilizes the body's immune defenses.
In Patient One (P1), T-cell counts increased from the 30th day following infusion, whereas gc-expressing T cells appeared in the blood of P2 after 60 days. By 120 to 150 days post-therapy, these cell counts had climbed to 1,700 per microliter, and reached 2,800 by eight months. In both recipients, this T-cell proliferation equaled that of age-matched normal, healthy control children. When the replicating new T cells were immunized in vitro with tetanus and polio vaccines, their generation of antibodies was on a par with controls.
The transgene-expressing natural killer (NK) cells were detected in the blood of P2 by day 30; in P1 only on day 150.
Parallel to these immunological parameters, clinical conditions also responded. P1 had Pneumocystis carinii pneumonia. P2 suffered from thrush (oral candidiasis), protracted diarrhea, skin lesions and failure to thrive. In both babies, these symptoms disappeared.
As the Science paper reported, "Both patients left protective isolation at days 90 and 95, and are now at home 11 and 120 months, respectively, after gene transfer without any treatment. Both enjoy normal growth and psychomotor development. No side effects have been noted."
It added in a footnote, "A similar result has since been achieved in a third patient four months after gene transfer. This infant was treated at one month of age, and within three months, T and NK lymphocyte counts reached age-matched control values. The gc-expression at T and NK cell surfaces was fully restored. The child is at home without any therapy, four months after treatment."
The article pointed out, "These overall positive results contrast with the failure of previous attempts to perform ex vivo gene therapy in adenosine deaminase (ADA)-deficient patients." That maiden effort to perform gene therapy took place just a decade ago, at the National Institutes of Health, under the direction of pioneer pediatric gene therapist W. French Anderson. (See BioWorld Today, Oct. 3, 1995, p. 1.)
Anderson, now at the University of Southern California, Los Angeles, wrote the news release accompanying the French announcement in Science. Its title: "Gene therapy frees two children from sterile 'bubbles,' Science authors report." He said that with the gc gene's direction, "SCID X1 patients are left fatally vulnerable to even slight infectious insults to the body such as a cold sore or common childhood diseases like chickenpox."
Inner-Ear Hair Cells From 30 Bullfrogs Track Hearing Pathway From Sound To Brain
The bullfrog (Rana catesbeiana) doesn't place very high in the popularity contest for animal models of human disease. Yet neuroscientists at The Rockefeller University in New York enlisted 30 of the bulky amphibians to help them trace the circuit of neurons that convey audible sound from the inner ear to the auditory centers of the brain.
In this endeavor, R. catesbeiana has it all over the ears of fruit flies, roundworms, mice and people. In the human inner ear, some 160,000 sound-sensitive hair cells align themselves with the precision of church organ pipes to detect sounds. These epithelial receptors translate mechanical stimuli into electrical signals. An adult bullfrog's inner ear contains a receptor organ called the sacculus, attended by 2,000 to 3,000 hair cells, each surrounded by half a dozen supporting cells.
Saccular hair cells have been extensively studied for their biophysical properties, but molecular analysis of their proteins is frustrated by the lack of starting material accessible from the inner ear. The researchers sought to turn from biochemistry to immunology, by raising antibodies to the hair-cell proteins, but were thwarted by the limited quantity of these antigenic targets. The way they found around that obstacle was recombinant-antibody technology - specifically, building libraries of antibody fragments expressed by filamentous bacteriophage. So they chose to produce such a phage display expression library of antibody fragments directed against inner-ear proteins from the bullfrog's sacculus.
The title of their research report, in the Feb. 29, 2000, issue of the Proceedings of the National Academy of Sciences (PNAS) says as much: "A library of bacteriophage-displayed antibody fragments directed against proteins of the inner ear."
The co-authors homogenized the auditory epithelial cells from the 30 bullfrog sacculi, and injected the material near the ankle of a laboratory mouse. They then boosted this immunization by five additional injections over subsequent days. From the murine immune response to this antigenic challenge, they used single-chain, variable-region antibody fragments to generate the phage-display library.
"Using this approach," their PNAS paper observed, "we have created a source for immunological reagents that can be repeatedly used for the isolation of reagents against proteins in the inner ear." The team concluded: "With these immunological tools in hand, it should now be possible to more thoroughly characterize inner-ear proteins and to ascertain their roles in our senses of hearing and balance."