title: Tightly regulated gene expression in human hematopoietic stem cells after transduction with helper-dependent Ad5/35 vectors.
abstract: OBJECTIVE
  Inducible and transient expression of transcription factors, growth factors, or mitogenic factors can be used to influence proliferation or differentiation of hematopoietic progenitor/stem cells (HSC). Furthermore, transient expression of proteins with site-specific endonuclease activity, potentially, can support targeted integration of exogenous transgenes into specific sites in the genome, a task that is currently a focus in development of gene therapy vectors.
  
  
  METHODS
  We constructed a set of helper-dependent adenovirus (Ad) vectors with serotype 35 fiber knob domains (HD-Ad5/35), which can efficiently transduce human CD34(+) cells, particularly subsets with potential stem cell capacity. These vectors contain Tet-inducible expression systems that were shielded by insulators and transcription stop signals to minimize unspecific interference by transcriptional elements present in viral and stuffer DNA. We compared two vectors, containing a fusion between the Krüppel-associated box (KRAB) domain and the tetracycline repressor (HD-Ad5/35.Tet-1) or an autoregulated rtTA (HD-Ad5/35.Tet-2) for regulated transgene expression in Mo7e cells, a model for HSC, and primary human CD34(+) cells.
  
  
  RESULTS
  HD-Ad5/35.Tet-1 conferred lower background expression than HD-Ad5/35.Tet-2, although levels of induced gene expression were higher for HD-Ad5/35.Tet-2. In CD34(+) cells, while HD-Ad5/35.Tet-1 allowed for activated gene expression in all transduced cells, induced gene expression from HD-Ad5/35.Tet-2 was restricted to a small subset of CD34(+) cells. Importantly, clonogeneic assays and repopulation studies in nonobese diabetic/severe combined immunodeficient mice showed that both HD-Ad5/35.Tet-1 and -2 vectors mediated induced gene expression in primitive hematopoietic cells. These studies also confirmed that transduction of CD34(+) cells with HD-Ad5/35 vectors is not associated with cytotoxicity, a problem observed with first-generation Ad5/35 vectors.
  
  
  CONCLUSIONS
  Both HD-Ad5/35 vector expression systems confer tightly regulated, transient transgene expression in human HSC.
authors.affiliation: Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Hongje
authors.initials: H
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Cao
authors.forename: Martin
authors.initials: M
authors.lastname: Wohlfahrt
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
authors.forename: André
authors.initials: A
authors.lastname: Lieber
mesh_terms: D000256:Adenoviridae; D018952:Antigens, CD34 / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology
chemical_list: D018952:Antigens, CD34