title: Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells.
abstract: Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.
authors.affiliation: Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: E
authors.initials: E
authors.lastname: Attar
authors.forename: K
authors.initials: K
authors.lastname: Cohen
authors.forename: C
authors.initials: C
authors.lastname: Crumpacker
authors.forename: D
authors.initials: D
authors.lastname: Scadden
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D005434:Flow Cytometry; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D039902:Multipotent Stem Cells / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D034741:RNA, Small Interfering / Q000008:administration & dosage; D016511:Severe Combined Immunodeficiency / Q000276:immunology / Q000628:therapy; D033581:Stem Cell Transplantation; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D018952:Antigens, CD34; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering