A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.

Inhibiting the expression of the HIV-1 coreceptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report stable knockdown of human CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/liver/thymus (BLT) mouse model. We delivered a potent shRNA against CCR5 into human fetal liver-derived CD34(+) hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced HPSCs solidified with Matrigel and a thymus segment under the mouse kidney capsule. Vector-transduced autologous CD34(+) cells were subsequently injected in the irradiated mouse, intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues, including gut-associated lymphoid tissue, the major site of HIV-1 replication. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T-cell development as assessed by polyclonal T-cell receptor Vbeta family development and naive/memory T-cell differentiation. CCR5 knockdown in the secondary transplanted mice suggested the potential of long-term hematopoietic reconstitution by the shRNA-transduced HPSCs. CCR5 tropic HIV-1 infection was effectively inhibited in mouse-derived human splenocytes ex vivo. These results demonstrate that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 in systemic lymphoid organs in vivo.

Generation of T lineage cells from human embryonic stem cells in a feeder free system.

Human embryonic stem cells (hESC) have the potential to revolutionize certain medical treatments, including T-cell-based therapies. However, optimal approaches to develop T cells from hESC are lacking. In this report, we show that T-cell progenitors can be derived from hESC cultured as embryoid bodies (EBs). These EB-derived T-cell progenitors give rise to phenotypically and functionally normal cells of the T lineage when transferred into human thymic tissue implanted in immunocompromised mice, suggesting that introduction of these progenitors into patients may also yield functional T cells. Moreover, hematopoietic progenitors demonstrating T-cell potential appeared to be CD45+/CD34+, resembling those found in normal bone marrow. In contrast to T cells developed from hESC cocultured on murine stromal cells, the EB-derived T cells also expressed normal levels of CD45. Importantly, the EB system eliminates the previous need for murine cocultures, a key impediment to developing a protocol for T-cell progenitor derivation suitable for clinical use. Furthermore, following lentiviral-mediated introduction of a vector expressing enhanced green fluorescent protein into hESC, stable transgene expression was maintained throughout differentiation, suggesting a potential for gene therapy approaches aimed at the augmentation of T-cell function or treatment of T-cell disorders.

Molecular characterization, reactivation, and depletion of latent HIV.

Antiretroviral therapy is unable to eliminate HIV infection in a small, long-lived population of latently infected T cells, providing a source for renewed viral replication following cessation of therapy. Analysis of individual latently infected cells generated in the SCID-hu (Thy/Liv) mouse demonstrated no functional viral RNA produced in the latent state. Following reactivation viral expression was dramatically increased, rendering the infected cells susceptible to an anti-HIV immunotoxin. Treatment with the immunotoxin in conjunction with agents that activate virus expression without inducing cell division (IL-7 or the non-tumor-promoting phorbol ester prostratin) depleted the bulk of the latent reservoir and left uninfected cells able to respond to subsequent costimulation. We demonstrate that activation of latent virus is required for targeting by antiviral agents and provide the basis for future therapeutic strategies to eradicate the latent reservoir.

Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1.

Human bone marrow expresses a pseudogene that encodes an antimicrobial peptide homologous to rhesus monkey circular minidefensins (delta-defensins). We prepared the putative ancestral human peptide by solid-phase synthesis and named it "retrocyclin." Retrocyclin did not cause direct inactivation of HIV-1, and its modest antibacterial properties resembled those of its rhesus homologs. Nevertheless, retrocyclin had a remarkable ability to inhibit proviral DNA formation and to protect immortalized and primary human CD4(+) lymphocytes from in vitro infection by both T-tropic and M-tropic strains of HIV-1. Confocal fluorescent microscopy studies performed with BODIPY-FL-labeled RC-101, a close analog of retrocyclin, showed that the peptide formed patch-like aggregates on the surface of CD4(+) cells. These findings suggest that retrocyclin interferes with an early stage of HIV-1 infection and that retrocyclin-like agents might be useful topical agents to prevent sexually acquired HIV-1 infections.