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.

Abstract

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 (TGFbeta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-mediated transduction of primary human CD34(+) cells. Although TGFbeta 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 TGFbeta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFbeta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.