Modulation of the extent of ritrovtral-mediated transduction of human hematopotic progenitors by antcense p271"" and amti tgfp antibody

Mo A. Oki, Jan Nolta

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Retroviral-mediated gene transfer (RMGT) into human hcmatopoietic stem cells (HSC) has been hard to achieve The pluripotent stem cells that must be transduced to allow production of corrected multilineage progeny are resistant to transducuon by Moloney-based vectors, which require target cell mitosis for integration. Using the bnx/hu xenograft system with single cell cloning and inverse PCR to track the progeny of individual human HSC, we demonstrated that gene transduction of multipotent stem cells rarely occurs (PNAS USA 93 : 2414,19% and Blood 88:428,1996). In the current studies, we have focused on finding methods to transiently bring quiescent HSC into cycle, to allow RMGT. We hypothesized that by reducing cyclin-dependent kinase inhibitor levels (P271 kcpl p21 cipl, & p15 ink4), stem cells would be released from quiescence. We first studied reduction of p27tapl levels by antiscnse oligonucleotides (ON) covering the translation initiation site. C-5 propyne modified phosphorothioate 16-mers were introduced into human CD34+ cells at a concentration of 50 uM in serum-free medium + IL-3, IL-6, and SCF. Mis-sense ON bearing the same modifications and medium alone were used as controls. The reduction in p27 levels following 12 hours incubation with anti-kip resulted in a significant increase in the number of colony-forming units obtained from 10,000 CD34+ cells treated (ave. = 2007, N=8), as compared to the same number of control cells (1270 for mis-kip (N=8, p<.05) and 1172 ave. in medium alone (N=8. p< 05)). The increase in the clonogenicity confirmed that the ON were not toxic, and suggested that cells had been recruited into cycle by anti-kip. To confirm this observation, CD34+ progenitors were purified from marrow treated with 4-HC, which kills cycling cells, to obtain a population of quiescent stem cells, and used in the same experimental schema with two changes. Cells were cultured on fibronectin, which enhances RMGT, and the LN vector was added during the 12 hr incubation. The use of the anti-kip ON alone was insufficient to bring the quiescent cells into cycle, to allow transduction. However, when coupled with antibodies to neutralize TGF, a small percentage of the cells were transduced (8%, as opposed to 0% transduced by addition of mis-kip or medium alone with anti-TGFβ (N=5)). This encouraging system will be further optimized, and the extents of transduction of multipotent stem cells will be studied following long-term engrafiment in the bnx/hu xenograft model.

Original languageEnglish (US)
Pages (from-to)881
Number of pages1
JournalExperimental Hematology
Issue number8
StatePublished - 1997
Externally publishedYes

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation


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