Vol.
171,
No.
September
2, 1990 14,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
787-795
Anthony L. Back*+, William W. Kwokb, Mohammed Adam++, Steven J. Collins+ ++, and Dennis D. Hickstein*+ *Medical
Research Division,
Seattle Veterans Administration Medical Center, Seattle, WA 98108 &Virginia Mason Research Center, Seattle, WA 98101 +Department of Medicine, University of Washington, Seattle, WA 98195
++Molecular
Medicine
Program, Fred Hutchinson Seattle, WA 98104
Cancer Research
Center,
Received August 7, 1990
Children with adherence leukocyte deficiency exhibit (LAD) heterogeneous defects in the leukocyte integrin CD18 subunit that prevent surface expression of functional CDll/CDlS leukocyte integrin adherence complexes. We used a retroviral vector, designated LCD18SN, to transfer the CD18 cDNA into KS62 human myeloid leukemia cells and into EBV B-cells from a child with LAD. Transfer of the LCD18SN retroviral construct, which expresses the CD18 cDNA from the Moloney Murine leukemia virus (MoMLV) long terminal repeat (LTR), into K562 cells resulted in relatively high levels of CD18 mRNA and intracellular protein. Retroviral-mediated gene transfer of CD18 into LAD EBV B-cells resulted in low, but readily measurable, levels of surface expression of the CDlla/CD18 complex in these previously deficient lymphocytes. The reconstitution of surface expression of the CDlla/CD18 complex by gene transfer of the CD18 cDNA into LAD EBV B-cells indicates that this syndrome represents a candidate disorder for gene therapy. 01990 Academic Press, kc. SUMMARY:
To perform their role in host defense, human leukocytes participate in a variety of adherence-related activites which are mediated in part by members of a family of surface receptors termed the leukocyte integrins (reviewed in ref. 1). The leukocyte integrins consist of one of three different, high molecular weight OLsubunits, designated CDlla (LFA-l), CDllb (Mac-l), or CDllc (p150), noncovalently associated with a common CD18 or S subunit in an dlH1 heterodimer
1This work was supported and Merit Review funds. 2Reprint VAMC,
by the Veterans Administration
Career
M.D., Medical Service request: DeMiS D. Hickstein, 1660 S. Columbian Way,Seattle, WA 98108.
Abbreviations transformed
Development
(ill),
EBV B-cells, Epstein Barr used in this paper: B-cells, FACS, fluorescence activated cell sorting..
787
Seattle Virus
-
ooO6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All righrs of reproduction in any form reserved.
Vol.
171,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(1,2). The leukocyte integrins are closely related to two other integrin families, the fibronectin receptor family and the vitronectin receptor/platelet IIb/IIIa family (reviewed in 3,4). integrin family in mediating The importance of the leukocyte adherence-related activies is underscored by a genetic disease termed leukocyte adherence deficiency (LAD) (5), or leukocyte adhesion molecule (Leu-CAM) on the deficiency (6), in which the CDll/CD18 complexes are not expressed leukocyte surface due to heterogeneous defects in the CD18 subunit (7,8). These children suffer severe, recurrent bacterial infections due primarily to the to the site of infection and adhere to and inability of neutrophils to migrate ingest C3bi-opsonized particles (5-8). Since all described cases of LAD involve defects in the CD18 subunit alone (6-8), this disorder is a candidate disease for gene therapy. In previous work the CD18 subunit, when transfected into Epstein-Barr Virus (EBV) transformed B-cells from patients with LAD, forms a heterodimer with the CDlla subunit and results in surface expression of a functional CDlla/CD18 complex (9). Retroviral vectors have been proposed to be utilized for therapy of These vectors offer an efficient means of gene specific human genetic disorders. and expression of specific genes in transfer leading to stable integration target cells (10). In the present study we describe the retroviral-mediated transduction of the CD18 leukocyte integrin subunit into both K562 cells (ll), a human myeloid cell line which does not express the leukocyte integrins, and into EBV-transformed B-lymphoblasts from a child with LAD (12).
Cell Lines - Epstein Barr virus (EBV) transformed cell lines were derived from a normal individual (P.G.B.) and from a child described previously with leukocyte adherence deficiency (LAD) (12). The leukocytes from this LAD patient are severely deficient in surface expression of the CDll/CD18 complexes (
171,
No.
September
2, 1990 14,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
787-795
Anthony L. Back*+, William W. Kwokb, Mohammed Adam++, Steven J. Collins+ ++, and Dennis D. Hickstein*+ *Medical
Research Division,
Seattle Veterans Administration Medical Center, Seattle, WA 98108 &Virginia Mason Research Center, Seattle, WA 98101 +Department of Medicine, University of Washington, Seattle, WA 98195
++Molecular
Medicine
Program, Fred Hutchinson Seattle, WA 98104
Cancer Research
Center,
Received August 7, 1990
Children with adherence leukocyte deficiency exhibit (LAD) heterogeneous defects in the leukocyte integrin CD18 subunit that prevent surface expression of functional CDll/CDlS leukocyte integrin adherence complexes. We used a retroviral vector, designated LCD18SN, to transfer the CD18 cDNA into KS62 human myeloid leukemia cells and into EBV B-cells from a child with LAD. Transfer of the LCD18SN retroviral construct, which expresses the CD18 cDNA from the Moloney Murine leukemia virus (MoMLV) long terminal repeat (LTR), into K562 cells resulted in relatively high levels of CD18 mRNA and intracellular protein. Retroviral-mediated gene transfer of CD18 into LAD EBV B-cells resulted in low, but readily measurable, levels of surface expression of the CDlla/CD18 complex in these previously deficient lymphocytes. The reconstitution of surface expression of the CDlla/CD18 complex by gene transfer of the CD18 cDNA into LAD EBV B-cells indicates that this syndrome represents a candidate disorder for gene therapy. 01990 Academic Press, kc. SUMMARY:
To perform their role in host defense, human leukocytes participate in a variety of adherence-related activites which are mediated in part by members of a family of surface receptors termed the leukocyte integrins (reviewed in ref. 1). The leukocyte integrins consist of one of three different, high molecular weight OLsubunits, designated CDlla (LFA-l), CDllb (Mac-l), or CDllc (p150), noncovalently associated with a common CD18 or S subunit in an dlH1 heterodimer
1This work was supported and Merit Review funds. 2Reprint VAMC,
by the Veterans Administration
Career
M.D., Medical Service request: DeMiS D. Hickstein, 1660 S. Columbian Way,Seattle, WA 98108.
Abbreviations transformed
Development
(ill),
EBV B-cells, Epstein Barr used in this paper: B-cells, FACS, fluorescence activated cell sorting..
787
Seattle Virus
-
ooO6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All righrs of reproduction in any form reserved.
Vol.
171,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(1,2). The leukocyte integrins are closely related to two other integrin families, the fibronectin receptor family and the vitronectin receptor/platelet IIb/IIIa family (reviewed in 3,4). integrin family in mediating The importance of the leukocyte adherence-related activies is underscored by a genetic disease termed leukocyte adherence deficiency (LAD) (5), or leukocyte adhesion molecule (Leu-CAM) on the deficiency (6), in which the CDll/CD18 complexes are not expressed leukocyte surface due to heterogeneous defects in the CD18 subunit (7,8). These children suffer severe, recurrent bacterial infections due primarily to the to the site of infection and adhere to and inability of neutrophils to migrate ingest C3bi-opsonized particles (5-8). Since all described cases of LAD involve defects in the CD18 subunit alone (6-8), this disorder is a candidate disease for gene therapy. In previous work the CD18 subunit, when transfected into Epstein-Barr Virus (EBV) transformed B-cells from patients with LAD, forms a heterodimer with the CDlla subunit and results in surface expression of a functional CDlla/CD18 complex (9). Retroviral vectors have been proposed to be utilized for therapy of These vectors offer an efficient means of gene specific human genetic disorders. and expression of specific genes in transfer leading to stable integration target cells (10). In the present study we describe the retroviral-mediated transduction of the CD18 leukocyte integrin subunit into both K562 cells (ll), a human myeloid cell line which does not express the leukocyte integrins, and into EBV-transformed B-lymphoblasts from a child with LAD (12).
Cell Lines - Epstein Barr virus (EBV) transformed cell lines were derived from a normal individual (P.G.B.) and from a child described previously with leukocyte adherence deficiency (LAD) (12). The leukocytes from this LAD patient are severely deficient in surface expression of the CDll/CD18 complexes (
