title: Efficient gene transfer into primitive hematopoietic progenitors using a bone marrow microenvironment cell line engineered to produce retroviral vectors.
abstract: OBJECTIVE
  Effective gene transfer into human hematopoietic stem/progenitor cells is a compromise between achieving high transduction efficiency and maintaining the desired biological characteristics of the target cell. The aim of our work was to exploit the stromal microenvironment to increase gene transfer and maintenance of hematopoietic progenitors.
  
  
  METHODS
  The murine bone marrow stromal cell line MS-5, known to support primitive human progenitors, was modified into an amphotropic packaging cell, by the stable introduction of DNA coding for retroviral structural proteins, and a viral vector encoding a marker gene. The gene transfer efficiency of the recombinant virus was evaluated by flow cytometry, in vitro assays for committed (CFC) and primitive (LTC-CFC) progenitors, as well as a clonal assay for B and NK lymphoid progenitors.
  
  
  RESULTS
  The new packaging cell line (NEXUS) produced equivalent levels of virus as did the established GP+Am12 system, also under serum-free conditions. On average 30% of human mobilized peripheral blood CD34(+) cells were transduced by a single exposure to NEXUS supernatant, representing a three-fold increase over GP+Am12-based technology. Gene transfer into both committed and primitive progenitors increased on average two-fold using NEXUS retroviral supernatant. Furthermore, CD34(+) CD38(low) early progenitor cells purified from umbilical cord blood were efficiently transduced with NEXUS retroviral vector and gave rise to a high frequency of marked B and NK lymphocytes.
  
  
  CONCLUSIONS
  Our data show that that an established bone marrow stromal cell can be engineered to enhance the genetic modification of primitive hematopoietic and lymphoid progenitors using a clinically relevant method.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan 20132, Italy.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dando
authors.forename: Francesca
authors.initials: F
authors.lastname: Ficara
authors.forename: Sara
authors.initials: S
authors.lastname: Deola
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Roncarolo
authors.forename: Claudio
authors.initials: C
authors.lastname: Bordignon
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aiuti
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002460:Cell Line; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D017154:Stromal Cells / Q000166:cytology*; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D015678:Viral Structural Proteins