Reduction in levels of the cyclin-dependent kinase inhibitor p27(kip-1) coupled with transforming growth factor beta neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Dao, M. A.; Taylor, N.; Nolta, J. A.

Proceedings of the National Academy of Sciences of the United States of America

1998-10-27 / vol 95 / pages 13006-13011


Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15(INK4B) blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27(kip-1) blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression, Antibody neutralization of beta transforming growth factor (TGF beta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-mediated transduction of primary human CD34(+) cells, Although TGF beta neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27(kip-1) coupled with TGF beta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGF beta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.



expression; gene-transfer; vectors; bone-marrow transplantation; 4-hydroperoxycyclophosphamide; cdk inhibitor; immune-deficient mice; mediated transduction; progenitors; stem-cells

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