David A. Williams

Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells

Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long–term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.

Large-scale mobilization and isolation of CD34+ cells from normal donors

We describe collection and purification of peripheral blood CD34+ cells from volunteer, normal donors and allogeneic stem cell donors. A total of 98 aphereses were performed on 68 volunteer donors using peripheral venous access. The mean number of nucleated cells collected was 4.6 × 1010 which included 1.9 × 108CD34+ cells corresponding to 2.7 × 106 CD34+ cells/kg. The number of CD34+ cells collected did not differ between males and females but did correlate with the donors weight and the total number of nucleated cells collected. The Nexell Isolex 300i cell separator was used to isolate CD34+ cells from 30 of the collections. A mean of 0.36% of the total cells was recovered and included 43 ± 18% of the CD34+ cells. CD34+ cells represented 85 ± 11% of the recovered cells. The total number of CD34+ cells recovered was not influenced by the number of nucleated cells placed on the Isolex 300i. The percentage of CD34+ cells recovered was not related to the number of CD34+ cells placed on the Isolex 300i. The purity of the final product was influenced by the number of CD34+ cells but not the total number of nucleated cells. An additional 38 CD34+ cell isolations were performed on normal allogeneic stem cell donors with similar results. These observations further support the safety and feasibility of peripheral blood CD34+ cell collection and purification. Bone Marrow Transplantation (2000) 26, 1271–1279.

Protection of hematopoietic cells against combined O6-benzylguanine and chloroethylnitrosourea treatment by mutant forms of O6-methylguanine DNA methyltransferase.

This report demonstrates that expression of the P140A O6-methylguanine DNA methyl transferase (MGMT) mutant via retrovirus-mediated gene transfer leads to significant, but modest, resistance of cells to both 6-benzylguanine (6-BG) depletion and treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Expression of the P140A/G156A double mutant appeared to be associated with reduced or unstable protein in hematopoietic cells. Bone Marrow Transplantation (2000) 25, Suppl. 2, S105–S109.