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Silencing p21Waf1/Cip1/Sdi1 expression increases gene transduction efficiency in primitive human hematopoietic cells

Gene Therapy volume 12, pages 1444–1452 (2005)Cite this article

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 p21Waf1/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.

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Acknowledgements

We wish to thank Dr Joseph Sodroski for the generous gift of the lentivector and Dr Jason La Bonte for help in preparing the lentivector pool and titration of the recombinant virus; Drs Gregor Adams, Randolf Forkert, and Chou-Wen Lin for helpful suggestions on mouse model study, Yin Shao for performing mouse tail vein injection, LiYing Ma for laboratory technical support. This work was supported by National Institutes of Health AI055313 (JLZ) and HL044851-13 (DTS).

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Authors and Affiliations

  1. Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA, USA

    J Zhang & C Crumpacker

  2. Center for Regenerative Medicine and Technology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    J Zhang, E Attar, K Cohen & D Scadden

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Zhang, J., Attar, E., Cohen, K. et al. Silencing p21Waf1/Cip1/Sdi1 expression increases gene transduction efficiency in primitive human hematopoietic cells. Gene Ther 12, 1444–1452 (2005). https://doi.org/10.1038/sj.gt.3302544

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