Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor b neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

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 p15INK4B blocks association of cyclin-dependent kinase (CDK)4ycyclin D and p27kip-1 blocks activity of CDK2ycyclin A and CDK2ycyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of b transforming growth factor (TGFb) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD341 cells. Although TGFb 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 p27kip-1 coupled with TGFb-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFb, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations. To achieve durable gene therapy for diseases affecting blood cells, corrective DNA must be introduced into pluripotent human hematopoietic stem cells (HSC). Moloney murine leukemia virus-based retroviral vectors are currently the safest and most effective vehicles for transfer of DNA into target cells with stable integration (1–3). However, transduction of HSC is thwarted by the fact that these vectors require target cell mitosis (4), and most stem cells are in the G0yG1phase of the cell cycle (5–8). Since the external factors that recruit HSC into cycle have not yet been identified, we hypothesized that a reduction in the levels of internal cell-cycle inhibitors could release HSC from quiescence to allow retroviral-mediated transduction. Cell-cycle entry depends on the sequential formation and activation of the cyclin-dependent kinase (CDK)ycyclin complexes CDK4ycyclin D, CDK2ycyclin E, and CDK2ycyclin A (9, 10). The assembly and activity of CDKycyclin complexes are regulated by the cyclin-dependent kinase inhibitors (CKI). The CKI p15INK4B is induced by TGFb (11, 12) and binds to CDK4 to prevent its association with cyclin D (12). Since CDK4ycyclin D activity is mandatory for the G1to S-phase transition, TGFbmediated induction of p15 causes cell-cycle arrest. A second CKI, p27kip-1, has been shown to be elevated in quiescent fibroblasts (13). Unlike p15, which binds CDK4 and CDK6 monomers, p27 binds to CDKycyclin complexes (14). At low levels, p27 binds to CDK4ycyclin D without altering its kinase activity (14, 15). At high levels, p27 binds to and inactivates CDK4ycyclin D and CDK2ycyclin E complexes, leaving the retinoblastoma protein in a hypophosphorylated state, preventing cell-cycle progression (16). p27 also acts through CDK2ycyclin E to negatively regulate cyclin A expression (17). Synthesis of cyclin A and activation of the CDK2ycyclin A complex are essential for S-phase progression (18). In an attempt to stimulate quiescent human hematopoietic cells to enter the cell cycle, we modulated levels of the CKI p15INK4B and p27kip-1. Reduction of TGFb levels by addition of neutralizing antibody (Ab) to the culture medium resulted in dramatic decreases in the levels of p15 in primary human CD341 hematopoietic progenitors, with a concomitant increase in the levels of colony formation and M-phasedependent retroviral transduction. However, the most primitive cells within the CD341 population [CD341y382 and 4-hydroperoxycyclophosphamide (4-HC)-resistant CD341 cells] did not enter the cell cycle when TGFb was neutralized. These data indicated that there were additional factors holding the most primitive cells in the G0yG1-phase of the cell cycle. Serum withdrawal and cell-to-cell contact elevate p27 levels and cause quiescence in fibroblasts (14–18), but the mechanisms regulating p27 expression and activity are unknown in hematopoietic cells. Cytokine addition, modulation of serum content, and loss of contact do not release HSC from quiescence. We report here that a reduction in p27 levels after treatment with antisense oligonucleotides (ONs), together with neutralization of TGFb, promoted cell-cycle progression in quiescent human hematopoietic progenitors without inducing differentiation. The entry of a portion of the cells into S phase was evidenced by induction of cyclin A and Ki67 expression, and increased levels of M-phase-dependent, retroviral-mediated gene transfer were documented in vitro and after long-term engraftment in immunedeficient mice. MATERIALS AND METHODS Transduction of Human Hematopoietic Cells. CD341 progenitors were isolated from normal human bone marrow by immuThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9513006-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: HSC, hematopoietic stem cells; CDK, cyclindependent kinase; CKI, cyclin-dependent kinase inhibitors; TGF, transforming growth factor; Ab, antibody; ON, oligonucleotide; 4-HC, 4-hydroperoxycyclophosphamide; IL, interleukin; SCF, stem cell factor; FL, Flt3 ligand; CFU, colony-forming unit; FN, fibronectin; neo, neomycin. §To whom reprint requests should be addressed at: Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, MS #62, Los Angeles, CA 90027. e-mail: [email protected].