ABSTRACT. X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of host defense that results from mutations in the gene encoding gp91phox, the large subunit of the phagocyte NADPH oxidase flavocytochrome b. In this study, we constructed a recombinant adeno-associated virus-2 (AAV) vector in which the constitutively active promoter from the human elongation factor- 1alpha (EF-1alpha) gene drives expression of the murine gp91phoxcDNA, and tested its ability to integrate and express in a human X-CGD myeloid cell line. The nitroblue tetrazolium (NBT) test of NADPH oxidase activity was used to screen transduced cells for vector-mediated expression of recombinant gp91phox. Between 2 - 14 % of cells were NBT-positive in the first several weeks after transduction. Clones with NBT-positive cells persisting several months after transduction had integrated vector by Southern blot analyses, with high level reconstitution of NADPH oxidase activity. In some clones, oxidase activity persisted for at least 8 to 14 months. In the majority, however, vector-derived RNA transcripts declined, although integrated rAAV genomes persisted. Decreased transgene expression was not directly correlated with methylation of the provirus. This study indicates that rAAV vectors can be successfully used for stable gene transfer, integration, and expression of recombinant gp91phoxin a human myeloid cell line for at least 8 - 14 months in the absence of any selection. The EF-1alpha promotor, however, was subject to silencing in a high percentage of clones with integrated rAAV, suggesting that alternative promotors may be desirable for achieving long-term expression in myeloid cells.

Keywords: Adeno-associated virus 2, neutrophils, chronic granulomatous disease, elongation factor-1alpha, gene therapy.

Reprint requests to: Mary C. Dinauer, M.D.,Ph.D., Herman B. Wells Center for Pediatric Research, Cancer Research Institute, Indiana University School of Medicine, 1044 West Walnut Street, Room 466, Indianapolis, IN 46202, phone: (317) 274-8645, fax: (317) 274-8679, email: mdinauer@iupui.edu.

No Abstract

Keywords: Hereditary spherocytosis, mutation, spectrin, ankyrin, erythrocyte.

Reprint requests to: Patrick G. Gallagher, M.D., Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208064, New Haven, CT 06520-8064, phone: (203) 688-2896, fax: (203) 785-6974, e-mail: patrick.gallagher@yale.edu.

ABSTRACT. The myeloproliferative disorders (MPD) are clonal diseases that originate from a transformed stem cell and involve all myeloid lineage. The affected cells have both proliferative and functional impairment. Therefore, we evaluated and compared neutrophil function in 31 patients with polycythemia vera (PV), idiopathic myelofibrosis (MF), chronic myeloid leukemia (CML), and essential thrombocytosis (ET). Neutrophil chemotaxis, random migration, bactericidal activity and superoxide anion release in these patients were simultaneously compared to those of 31 healthy controls. In this study, chemotactic activity was significantly impaired in patients with PV and CML as compared to controls (M+/-SE: 42 +/- 6 vs. 69+/- 5 cells/field; p<0.005 and 47+/-7 vs. 68+/- 5; p<0.05, respectively). The assessment of the bactericidal activity of neutrophils showed no impairment in most of the patients. In the CML group, the serum had a very strong "lytic" effect on bacteria, possibly due to the high levels of serum lysozyme (22 +/- 2 ug/ml). The superoxide anion release was found to be normal in most of the patients. Nevertheless, in 25% of PV patients the superoxide production was impaired (less than 60% of the simultaneous controls). In ET most patients had normal neutrophil function. Regarding the effect of treatment, neutrophil chemotactic activity was found to be significantly reduced in the hydrea-treated patients, as compared to the non- treated patients (p<0.001) or healthy controls (<0.0001). We conclude that disturbances in neutrophil function are present in patients with various MPDs, except ET. This probably reflects abnormal maturation of ancessors of the damaged stem cells. Nevertheless, we should keep in mind that therapy itself could affect neutrophil functions. This matter should be studied more extensively. Although infections are not common in MPD disorders, they occasionally occur. It is possible that impairment in the phagocytic function contribute to the development of infections in patients with myeloproliferative disorders.

Keywords: Myeloproliferative disorders, neutrophil chemotaxis, random migration, bactericidal activity, superoxide production, serum lysozyme.

Reprint requests to: Baruch Wolach, M.D., Department of Pediatrics, Meir General Hospital, 44281 Kfar Saba, Israel, phone: 972-9-7472555, fax: 972-9-7425967.