Study Advances Development Of Gene Therapy For Chronic Granulomatous Disease

"This is encouraging news for people born with CGD," says NIAID Director Anthony S. Fauci, M.D. "While other therapeutic advances have improved the prognosis for CGD patients in recentyears, the development of effective gene therapy would represent a big step forward."

One of more than 70 different inherited disorders known collectively as primary immune deficiencies, CGD is caused by a defect in an enzyme called phagocyte NADPH oxidase, or phox. White blood cells use this enzyme to generate hydrogen peroxide, which the cells need to kill bacteria and fungi. Mutations in one of four different genes can cause this defect, which leads to frequent and often life-threatening infections of the skin, lungs and bones with localized, swollen collections of inflamed tissue called granulomas. Approximately four to five of every million people worldwide have CGD, including about 1,000 people in the United States.

In the study reported in PNAS, researchers led by Harry L. Malech, M.D., deputy chief of NIAID's Laboratory of Host Defenses(LHD), removed stem cells, the ancestral immune cells that give riseto white blood cells, from five people with CGD. The researchersinserted the correct form of the phox gene into the stem cells and then transfused the corrected cells back into each patient. Dr. Malech and his colleagues sampled the patients' blood at regular intervals to see if the stem cells were producing white blood cells with functional phox genes.

"We detected phox activity in white blood cells from each patient for an average of three months after the gene-corrected stem cells were transfused," explains Dr. Malech. "In one patient, phox activity was still present six months after transfusion. On average, the corrected phox gene was present in one out of every 5,000 cells. While the numbers of gene-corrected cells were small, the study demonstrates unequivocally that gene therapy of stem cells can produce functionally normal blood cells in patients for a prolongedperiod."

The finding could have important clinical implications for the treatment of CGD. "Since life-threatening infections caused by CGD may require many weeks or months of therapy and relapses are frequent, use of gene therapy to provide even short- to medium-term production of phox-positive cells may be clinically beneficial," says Dr. Malech.

Studies suggest that people having 3 to 5 percent phox-positive cells in their blood might be protected from infectionsassociated with CGD, he notes. Although those levels are at least150-fold higher than levels attained in the current study, Dr. Malech predicts that they might be achieved within the next five to 10 years. "Until the tools are developed to achieve higher levels ofpermanent gene transfer to stem cells, our studies suggest that anachievable intermediate goal of gene therapy for CGD might be to augment white blood cell function in the treatment of severe infections," says Dr. Malech.

In addition to advancing the development of gene-basedtherapy for CGD, Dr. Malech notes that some of the techniques used in this study could have broad application in gene therapy protocols for other diseases. Designed to enhance the safety of gene therapy procedures involving stem cells, these techniques included the use of cell culture media containing no non-human proteins and a closed system of gas-permeable flexible plastic containers for culture andgene transfer.

Animal proteins are widely used in most cell culture media. However, animal proteins taken up by human cells during prolonged culture can stimulate an immune response when the cells are transfused back into a patient undergoing gene therapy. The closed system of flexible plastic containers, similar to those used in blood banks, reduces the contamination risk associated with procedures where cells and culture media are transferred among flasks.

"To our knowledge, this is the first human gene therapy trial targeting stem cells in which animal proteins were eliminated and stem cells were grown in sealed gas-permeable flexible plasticcontainers," says Dr. Malech. "We showed that it is possible to incorporate these safety features without compromising stem cell viability or gene transfer efficiency."

In addition to Dr. Malech, investigators involved in the study included John I. Gallin, M.D., chief of LHD and director of the National Institutes of Health (NIH) Clinical Center; other LHD and ClinicalCenter staff; as well as investigators at Baxter Healthcare, Inc., whocontributed stem cell technology, and Cell Genesys, Inc., whocontributed gene transfer technology.

NIAID, a component of the National Institutes of Health (NIH), supports research on AIDS, malaria, tuberculosis and other infectious diseases, as well as allergies and immunology. NIH is an agency of the U.S. Department of Health and Human Services.

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Reference: Malech HL, Maples PB, Whiting-Theobald N, Linton GF,Sekhsaria S, Vowells SJ, Li F, Miller JA, DeCarlo E, Holland SM, Leitman SF, Carter CS, Butz RE, Read EJ, Fleisher TA, Schneiderman RD, Van Epps DE, Spratt SK, Maack CA, RokovichJA, Cohen LK, Gallin JI. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatousdisease. PNAS 1997;94:12133-8.

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