Fibronectin Improves Transduction of Reconstituting Hematopoietic Stem Cells by Retroviral Vectors: Evidence of Direct Viral Binding to Chymotryptic Carboxy-Terminal Fragments

EMATOPOIETIC cell adhesion in the complex hemaH topoietic microenvironment contained in blood-forming organs is mediated both by integrin- and proteoglycanmediated mechanisms.’.’ Primitive hematopoietic stem cells (HSC) that are capable of multilineage differentiation and long-term reconstitution in vivo adhere to the extracellular matrix molecule fibronectin (FN)’ and endothelial cell vascular-cell adhesion molecule (V-CAM)’ via the integrin u4/p1 and cell-surface glyc~saminoglycans~*~*~ which interact with heparin-binding sequences located in several extracellular matrix molecules, including FN.7 More differentiated hematopoietic progenitor and precursor cells, especially of the erythroid lineage, interact with the integrin (Y~/PI .8-11 FN is a complex extracellular matrix molecule with multiple cell adhesion sites (Fig 1). Cell adhesion to the central cell binding domain (Cell) is mediated through the tetrapeptide Arg-Gly-Asp-Ser (RGDS) located in the middle of the FN molecule.” Cell adhesion to the carboxy-tenninus of FN is mediated by sequences contained within peptides termed CS-1 and CS-5 located within the non-type 111 connecting segment (IIICS) in some, but not all, fibronectin pep tide^.'^,'^ Additionally, RGDS-independent cell adhesion occurs in this region of FN to a heparin-binding domain (Heparin, Fig 1) and is mediated by at least two sequences represented in synthetic peptides FN-C/HI and FN-C/HII.’’s’6 Genetic transduction of more primitive long-term hematopoietic reconstituting cells has been one problematic goal for the use of retroviral-mediated gene transfer technology in human gene therapy protocols aimed at lifelong cure of genetic hematopoietic diseases. Previous work by Moore et all7 and recent work by Nolta et all8 have shown increased gene transfer into hematopoietic cells adherent to stroma using retroviral vectors, although the mechanism involved in this increased transduction was unclear, Our laboratory has shown that retroviral-mediated gene transfer into human hematopoietic progenitor cells is substantially increased using cell-free virus to infect these cells while adherent to the carboxy-terminal chymotryptic fragment of fibronectin, FN 30/35 (Fig l), which contains both the CS-1 site and the heparin-binding domain. l9 Transduction of more primitive producer cells; (21 recombinant retrovirus particles directly adhere to FN 30135 in a quantitative and dose-dependent fashion; and (3) increased transduction efficiency on FN 301 35 does not appear to be associated with increased cell proliferation or activation of protein phosphorylation typically induced by integrin-fibronectin interactions. Therefore, we speculate that supernatant infection of HSC on FN 30135 leads to colocalization of retrovirus particles and target cells on FN 30/35 molecule with a large increase in local virus titer presented to the cell. These findings have direct and important implications for the modification of current human gene therapy protocols. 0 1996 by The American Society of Hematology. long-term culture-initiating cell (LTC-IC) was significantly better on FN 30/35 versus intact FNI9 and this result parallels differences in adhesion of primitive hematopoietic cells versus progenitor cells to FN 30/35.* Ward et alZo have recently confirmed increased transduction of human progenitor cells using fibronectin-coated plates. In this report we show that transduction of murine longterm reconstituting HSC is substantially increased when these targets are infected on the FN 30135 fragment. In addition, we demonstrate that recombinant retroviral vector particles bind quantitatively and in a dose-dependent fashion to this same FN 30/35 fragment. These data suggest that the mechanism of increased genetic transduction of primitive HSC may be related, in part, to colocalization of stem cells adherent to CS-1 and virus adherent to FN 30/35.