The Bubble Bursts

Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. M. Cavazzana-Calvo et al., "Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease," Science, 288:669-72, April 28, 2000. (Cited in 300 papers) Reaching positive milestones in gene therapy has been difficult since a highly publicized death in 1999,1 but some have succeeded. A major break came this year when researchers in Italy and Israel reported successfully treating two patients with ADA-SCID, a form of severe combined immunodeficiency that results from a defective gene for adenosine deaminase (ADA).2 In 2000--in the wake of gene therapy's darkest hour--a French group reported similar results for X-linked SCID, a form of the disease caused by a mutation in the common cytokine receptor gamma chain (gc).3 This Hot Paper, a culmination of years of work, may open the floodgates for therapies to come; the two patients, without further therapy, remain alive and well. "There was a time that I think everyone in the field was feeling somewhat discouraged by just the difficulties of moving things forward to clinical trials," says pediatrics professor Kenneth Weinberg, Children's Hospital of Los Angeles, who expects approval soon for a similar gene therapy trial. The French group's work invigorated a field mourning the death of Jesse Gelsinger, he says. "Ultimately [that invigoration is] going to turn out to be as important as the specific results of the transductions." BEATING THE BUBBLE X-linked SCID, the most common of a family of immune system disorders, is commonly referred to as "bubble boy" disease, named after David Vetter, a Texan who lived out his 12 years in a plastic, germ-free bubble. X-SCID, like other SCIDs, is characterized by a lack of both B-cell and T-cell functions. Additionally, patients with X-SCID have no working natural killer (NK) cells. Even the most benign infections can kill these patients, who rarely live past a very early age. Since the 1980s, bone marrow transplants (BMTs) taken from histocompatible siblings have had some success. Working hematopoietic stem cells (HSCs) from matched donors help establish a functioning im-mune system. Yet, immunoglobulins must be provided. Image: Erica P. Johnson Along with the success of BMTs, a number of factors made the disease a likely candidate for gene therapy. If a patient's own stem cells could be harvested, infected with a working copy of gc and then injected into the patient (which is exactly what the French team eventually did), the feeling was that results might be better than those for so-called half-matched transplants. Though an immunodeficient patient cannot reject a transplant, HSCs from a poorly matched donor may mature into immune cells that attack the body--a condition known as graft-versus-host disease. Another transplant worry is that even in a well-matched situation, latent infections such as Epstein Barr virus, which rest harmlessly in the blood of normal individuals, can kill a patient with SCID before immune function kicks in. Such was the cause of young Vetter's death. Hematology professor Marina Cavazzana-Calvo, University of Paris V, and the Hot Paper's lead investigator, says that the protocols for the gene therapy technique were developed over a 10-year period--a decade kicked off by new discoveries about the nature of X-SCID. One contributor, who discovered the molecular basis of X-SCID, was Warren Leonard, chief, laboratory of molecular immunology, National Heart, Lung, and Blood Institute. The finding was, in part, a matter of serendipity. "We weren't looking for the basis of the disease," says Leonard. "We were working on an important immunological system. "Leonard's studies on the interleukin-2 (IL-2) receptor mapped the locus of one component, g, to chromosome Xq13, a region where the gene defective in X-SCID had been mapped. Blood work on a number of patients, including Vetter, demonstrated that the gene for the g chain indeed was faulty.4 Obviously, questions remained. Knocking out the cytokine IL-2 in mice had less of an effect than having a defective component in its receptor. Further investigation throughout the 1990s by Leonard and others revealed that six cytokine receptors share the common g chain: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. IL-7 and IL-15, according to Leonard, explain the major T-cell and NK-cell lineage deficits. Leonard's findings gave a name to the target for X-SCID: gc. The gene's prevalence on the surface of all blood cells led researchers to believe that hematopoietic precursors could be transformed and replaced without disrupting function. "We could think that the nonregulated expression of the new gc chain couldn't have any toxic effect," says Cavazzana-Calvo. Another finding supporting the case for gene therapy came from studies on patients' mothers. X-inactivation patterns for heterozygous females--carriers of X-SCID--were not at all random. Specifically, they were skewed in favor of the correct copy of gc for T cells.5 This suggested that having the gene's correct copy conferred a selective advantage specific to immune function. That meant to researchers that even a small number of transformed cells might just win out over the established defective-cell population in the patient. Gaining this benefit over established cells is an important hurdle to cross. In the Italian trial on ADA-SCID released this year, researchers used two techniques to give transduced cells an advantage. They stopped administering Peg-ADA, a modified form of bovine ADA used to treat patients with ADA-SCID. This presumably gives a leg up to cells that can produce the detoxifying protein. And, the group used chemotherapy to kill some of the established HSCs and allow the small numbers of transduced cells to compete. Cavazzana-Calvo says that ADA-SCID is a much more difficult disease to model for gene therapy because the enzymatic defect can affect other organs. The Italian group had good success with two patients, but Cavazzana-Calvo laments, "Because they used the two modifications at the same time--condition regimen plus the stop of Peg ADA administration--we don't know which of these two changes have permitted the success for treatment of the patient." "Probably both contributed, but we cannot define at what extent in this study alone," writes Claudio Bordignon, lead investigator for the Italian study, in an E-mail. He adds, "When you have good reasons to believe that the patient may benefit from a procedure, you must use it even if the science of it will not be immediately clarified." Cavazzana-Calvo says that 10 patients have since had the treatment under the protocol reported in 2000; eight have responded positively without further treatment. The two initial recipients from the paper are doing well and living a normal life, she says. They have overcome infections, and they have been inoculated against other diseases. One patient did not respond with reconstituted T-cell function even though gc-positive cells were detected. Another patient with an atypical form of X-SCID was treated at age 15. "Probably it is a very difficult case," Cavazzana-Calvo says, "and probably gene therapy is not working." Photo: Courtesy of Duke Medical Center News Office THE BUBBLE BOY: David Vetter, the boy with SCID whose fate was dramatized in the 1970s by a documentary film, shares a moment with his mother, Carol Ann Vetter. AFTER THE BURST, AND BEYOND Research continues. Rebecca Buckley, professor of pediatrics and immunology, Duke University Medical Center, says, "The expression of the good gene in the B-cell lineage was very minimal compared to the T-cell and natural killer cell expression. That's why it's been surprising and reassuring that even with minimal expression in the B-cell lineage, these patients are able to produce antibodies." Buckley has worked with patients with SCID for nearly four decades. She developed the bone marrow transplant procedures that are the most common form of treatment. Weinberg says that low gc expression, along with NK cell levels that decrease over time, lead him to question whether a sufficient number of HSCs were transduced. "The message is that for immunologic diseases it is better to get transduction of hematopoietic stem cells than of cells that are further along in lymphoid differentiation. Permanent reconstitution of patients with these diseases probably depends on [that]." In the gene therapy trial Weinberg is initiating, a different retroviral vector will be used that he says should increase the number of transduced HSCs. Many say the trial in 2000 has opened the door for other gene therapies on blood-related diseases. Sickle cell anemia and thalassemia rise to the surface both because of their blood association, and because they pose a greater public health problem. Cavazzana-Calvo says she's forming collaborations in the United States to work on sickle cell anemia; Weinberg says that such diseases will pose a formidable challenge. "If you start looking at diseases like hemoglobinopathies, it gets much more complicated ... Can you in fact get lineage-specific expression?" While researchers are working on such specialized vector types, most agree that SCID was and is the correct starting point. Leonard says, "It really was as ideal a model as one could choose for an initial gene therapy protocol, because the selective growth advantage was known to be conferrable to cells that take up the proper genes. Now the challenge is to see will it work forever, and what are the best ways of doing it most efficiently?" Brendan A. Maher can be contacted at bmaher@the-scientist.com. References 1. J.P. Roberts, "Psst! Gene therapy research lives," The Scientist, 16[13]:12-14, June 24, 2002. 2. A. Aiuti et al., "Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning," Science, 296:2410-13, June 28, 2002. 3. M. Cavazzana-Calvo et al., "Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease," Science, 288: 669-672, 2000. (Cited in 300 papers) 4. M. Noguchi et al., "Interleukin-2 receptor g chain mutation results in X-linked severe combined immunodeficiency in humans," Cell, 73:147-57, 1993. 5. J.M. Puck et al, "Maximum-likelihood analysis of human T-cell X-chromosome inactivation patterns--Normal women versus carriers of X-linked severe combined immunodeficiency," American Journal of Human Genetics, 50:742-8, 1992.

The Bubble Bursts

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