Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments.
Efficient transduction of reconstituting hematopoletic stem cells (HSC) is currently only possible by cocultivation of target cells directly on producer cell lines, a method not applicable to human gene therapy protocols. Our laboratory has previously shown adhesion of primitive hematopoletic stem and progenitor cells to the carboxy-terminal 30/35-kD fragment of the extracellular matrix molecule fibronectin (FN 30/35) (Nature 352:438, 1991) and increased transduction of human hematopoietic progenitor cells via retroviral vectors while adherent to this fragment (J Clin Invest 93:1451, 1994). Here we report that (1) transduction of reconstituting murine HSC assayed 12 months after infection with retrovirus supernatant on FN 30/35 is as effective as cocultivation directly on producer cells; (2) recombinant retrovirus particles directly adhere to FN 30/35 in a quantitative and dose-dependent fashion; and (3) increased transduction efficiency on FN 30/ 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 30/35 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.
Moritz
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T
Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis 46202-5225, USA.
Dutt
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P
Xiao
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X
Carstanjen
D
D
Vik
T
T
Hanenberg
H
H
Williams
D A
DA
D016475:3T3 Cells; D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002453:Cell Cycle; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism / Q000494:pharmacology*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000187:drug effects*
D005353:Fibronectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002918:Chymotrypsin; D000243:Adenosine Deaminase