title: Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells.
abstract: A major challenge in gene therapy is to achieve efficient transduction of hematopoietic stem cells (HSC). It has previously been shown that lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). Here we studied the effect of cytokines during the short ex vivo incubation with vector. Although SRC transduction was efficient without stimulation, the presence of cytokines significantly improved it. The treatment did not affect the engraftment level or the SRC frequency, but seemed to enhance SRC susceptibility to LV. SRC transduced in both conditions repopulated primary and secondary recipients, maintaining stable multi-lineage transgene expression. Using linear amplification-mediated PCR, we then analyzed vector integration in the bone marrow and CFC of the engrafted mice to monitor the clonal activity of the transduced SRC in vivo. We showed polyclonal engraftment, multi-lineage differentiation, and propagation to secondary recipients of individual SRC. We observed multiple integrations in most clones. These results provide the first formal demonstration that primitive human HSC with self-renewal and multi-lineage repopulation capacities were transduced by LV. Our findings are relevant for the design of clinical protocols that exploit this system to reach significant engraftment by genetically modified HSC in the absence of in vivo selection or strong conditioning regimens.
authors.affiliation: Laboratory for Gene Transfer and Therapy, University of Torino Medical School, Candiolo, Italy.
authors.forename: Laurie
authors.initials: L
authors.lastname: Ailles
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Francesca Romana
authors.initials: FR
authors.lastname: Santoni de Sio
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Simona
authors.initials: S
authors.lastname: Cavalieri
authors.forename: Stefania
authors.initials: S
authors.lastname: Bruno
authors.forename: Wanda
authors.initials: W
authors.lastname: Piacibello
authors.forename: Christof
authors.initials: C
authors.lastname: Von Kalle
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D016026:Bone Marrow Transplantation; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D016133:Polymerase Chain Reaction; D019076:Transgenes
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins