IMPROVED TRANSFER OF THE LEUKOCYTE INTEGRIN CD18 SUBUNIT INTO HEMATOPOIETIC-CELL LINES BY USING RETROVIRAL VECTORS HAVING A GIBBON APE LEUKEMIA-VIRUS ENVELOPE

Leukocyte adherence deficiency (LAD) is an inherited immunodeficiency disease caused by defects in the CD18 leukocyte integrin subunit. Tran sduction of CD18 into hematopoietic cells from children with LAD repre sents a potential therapy for this disorder, In an attempt to maximize transfer and expression of CD18, we evaluated retroviral vectors with and without the neomycin selectable marker, with a modified tRNA prim er binding site designed to prevent inhibition of gene expression, and with two different viral envelope proteins produced by using the amph otropic retrovirus packaging cell line PA317 or the gibbon ape leukemi a virus packaging cell line PG13. The vectors were tested using transd ucing K562/CD11b cells and LAD Epstein-Barr virus (EBV) B cells and me asuring levels of cell-surface CD11/CD18 expression by fluorescence-ac tivated cell sorter analysis. The best results were obtained with vect ors made using PG13 packaging cells, for which about 25% of the K562 c ells exposed once to the vectors expressed surface CD11b/CD18 and abou t 25% of the LAD EBV B cells exposed three times over a 3-day period t o the vectors expressed surface CD11a/CD18. In contrast, transduction of cells under similar conditions with retroviral vectors produced usi ng PA317 producer cells yielded less than 2% of the K562 cells and les s than 4% of the LAD EBV B cells expressing the CD11/CD18 heterodimer on the cell surface. The presence or absence of the neomycin resistanc e gene or the modified tRNA primer had no effect on CD18 gene transfer rate or expression level. The increase in transduction with PG13 vect ors correlated with Northern blotting and reverse transcription-polyme rase chain reaction studies that indicated that both K562 cells and th e LAD EBV B cells express transcripts for the gibbon ape leukemia viru s receptor at higher levels than for the amphotropic virus receptor. T hese findings indicate that the transduction efficiency of retroviral packaging cell lines correlates with receptor gene expression in the t arget cells and that vectors made using PG13 cells may be efficacious for gene therapy for LAD and other diseases in which gene transfer to hematopoietic cells is required. (C) 1995 by The American Society of H ematology.