REDUCTION IN LEVELS OF THE CYCLIN-DEPENDENT KINASE INHIBITOR P27(KIP-1) COUPLED WITH TRANSFORMING-GROWTH-FACTOR-BETA NEUTRALIZATION INDUCESCELL-CYCLE ENTRY AND INCREASES RETROVIRAL TRANSDUCTION OF PRIMITIVE HUMAN HEMATOPOIETIC-CELLS

Successful gene therapy depends on stable transduction of hematopoieti c 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 kina se 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 ar e mandatory for cell-cycle progression, Antibody neutralization of bet a transforming growth factor (TGF beta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-me diated transduction of primary human CD34(+) cells, Although TGF beta neutralization increased colony formation from more primitive, noncycl ing hematopoietic progenitors, no increase in M-phase-dependent, retro viral-mediated transduction was observed. Transduction of the primitiv e cells was augmented by culture in the presence of antisense oligonuc leotides to p27(kip-1) coupled with TGF beta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteratio n in human hematopoietic lineage development. We conclude that neutral ization of TGF beta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent h ematopoietic progenitor populations.