title: Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor.
abstract: BACKGROUND
  One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application.
  
  
  METHODS
  In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials.
  
  
  RESULTS
  A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC).
  
  
  CONCLUSIONS
  MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.
authors.affiliation: Gene Therapy Program, Department of Pediatrics, and Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA. Dao.Pan@cchmc.org
authors.forename: Dao
authors.initials: D
authors.lastname: Pan
authors.forename: David F
authors.initials: DF
authors.lastname: Stroncek
authors.forename: Chester B
authors.initials: CB
authors.lastname: Whitley
mesh_terms: D018952:Antigens, CD34; D019149:Bioreactors; D018929:Cell Culture Techniques; D005374:Filtration; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D006801:Humans; D008059:Mucopolysaccharidosis I / Q000235:genetics*; D012190:Retroviridae; D013234:Stem Cells; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D018952:Antigens, CD34