This project aims to develop leukocyte adherence deficiency (LAD) as a model for applying advances in understanding of stem cell biology to transduction of the hematopoietic stem cell. LAD is characterized clinically by recurrent, life-threatening, bacterial infections due to the inability of leukocytes to adhere to the vessel wall and migrate to the site of infection. These adherence defects stem from the inability of leukocytes from affected children to express the CD11/CD18 leukocyte integrin heterodimers on the leukocyte surface due to primary genetic defects in the CD18 subunit. Leukocyte adherence deficiency is an attractive model for the proposed studies for several reasons: 1) the disease is life-threatening in the severe deficiency form, and except for bone marrow transplantation there is no treatment other than supportive therapy; 2) the disease is due to a defect in a single gene, the CD18 subunit, and the gene has been cloned; 3) transduction of a normal CD 18 gene into LAD EBV B cells has been shown to correct the biochemical and functional defect; 4) results from bone marrow transplantation indicate that the defect resides in the hematopoietic stem cell; 5) based upon gene expression by leukocytes in the moderate deficiency phenotype of LAD, low levels of CD11/CD18 expression appear to be sufficient to correct the severe clinical manifestations of the disease; and 6) since the defect involves a membrane receptor, a quantitative assessment of gene transfer and expression can be accomplished using flow cytometry of peripheral blood leukocytes. In the current studies we will build on our preliminary data identifying retroviral vectors, packaging cell lines, and culture conditions facilitating transduction of long term culture initiating cells.
The specific aims of this project are: 1) to determine the conditions and vectors required for effective transduction of long term culture initiating cells; 2) to determine the ability of peripheral blood stem cells from children with LAD to be transduced using ex vivo retroviral- mediated gene transduction of the leukocyte integrin CD 18 subunit followed by reinfusion of the transduced cells; ,and 3) to determine if prior administration of a conditioning regimen enhances expression of the leukocyte integrin CD18 subunit in leukocytes following ex vivo transduction of CD 18 into peripheral blood stem cells from children with LAD. We anticipate that these studies will incorporate advances in understanding of the hematopoietic stem cell from the other projects in the SCOR, including those identifying ways of eliciting or selecting stem cells that are most susceptible to transduction, and those developing vectors and culture conditions supporting transduction of hematopoietic stem cells. The results of these studies should be applicable to a variety of diseases involving the hematopoietic stem cell.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center (P50)
Project #
5P50HL054881-04
Application #
6110536
Study Section
Project Start
1998-09-01
Project End
1999-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
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