Silencing p21waf1/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 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

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references 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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of

Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA, USA J Zhang & C Crumpacker * Center for Regenerative Medicine and Technology, Massachusetts General Hospital,

Harvard Medical School, Boston, MA, USA J Zhang, E Attar, K Cohen & D Scadden Authors * J Zhang View author publications You can also search for this author inPubMed Google Scholar * E

Attar View author publications You can also search for this author inPubMed Google Scholar * K Cohen View author publications You can also search for this author inPubMed Google Scholar * C

Crumpacker View author publications You can also search for this author inPubMed Google Scholar * D Scadden View author publications You can also search for this author inPubMed Google

Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 09 February 2005 *

Accepted: 17 March 2005 * Published: 05 May 2005 * Issue Date: 01 October 2005 * DOI: https://doi.org/10.1038/sj.gt.3302544 SHARE THIS ARTICLE Anyone you share the following link with will

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content-sharing initiative KEYWORDS * p21 * RNAi * transduction efficiency * hematopoietic stem cells