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Gene transfer into T lymphocytes is currently being tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor (TCR) activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LVs) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as interleukin 7 (IL-7), IL-2, and IL-15. Late-generation LVs transduced efficiently nonproliferating T cells that had progressed from G0 to the G1 phase of the cell cycle on cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15, stimulation preserved physiologic CD4/CD8 and naive-memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than for IL-15. Thus, LV transduction of IL-2 or IL-7 prestimulated cells overcomes the limitations of retroviral vectors and may significantly improve the efficacy of T-cell–based gene therapy.

We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence. (C) 2003 by The American Society of Hematology. We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence. (C) 2003 by The American Society of Hematology.

Lentiviral vectors transduce at high efficiency human hematopoietic stem/progenitor cells (HSCs) without compromising their self-renewing and repopulation capacity. Last-generation helper constructs and self-inactivating (SIN) vectors have minimized the chances of generating recombinant, replication-competent HIV derivatives during packaging, and drastically reduced the potential risks of insertional activation of oncogenes. To allow the clinical use of lentiviral vectors, development of packaging cell lines and standardized procedures for vector manufacturing, downstream processing and quality/safety testing appears necessary. However, generation of stable packaging cell lines has been hampered so far by the high toxicity of VSV-G and of some of the HIV helper proteins used in the packaging steps. More recently, lentiviral vectors have been pseudotyped with alternative, non-toxic envelope proteins such as derivatives of the RD114 feline leukemia virus envelope glycoprotein, allowing the generation of a prototype, stable packaging cell line (Ikeda et al., Nature Biotech 21:569, 2003). The receptor for RD114 is abundant on human hematopoietic cells, allowing efficient transduction by RD114-pseudotyped lentiviral vectors. We report a comparison between lentiviral vector preparations pseudotyped with VSV-G and the RD114-TR fusion glycoprotein in transducing human cord blood-derived CD34+ HSCs and peripheral blood lymphocytes (PBLs). RD114-TR-pseudotyped vectors transduce both cell types at much lower m.o.i than VSV-G-pseudotyped vectors (5–10 vs. 50–200), resulting in lower toxicity, higher persistence and less vector copy number per genome. In addition, RD114-TR-pseudotyped vectors transduce at higher efficiency human clonogenic progenitors (BFU-E, CFU-GM, CFU-GEMM), as assayed in semi-solid cultures in vitro. Transduction efficiency and average vector copy number in human repopulating HSCs and their progeny was determined after xeno-transplantation in NOD-SCID mice. Our results indicate that pseudotyping with RD114-TR is a promising alternative to VSV-G for developing clinical-grade lentiviral vector preparations aimed at genetic modification of human HSCs.