Dose-dependent neuroprotective effect of ciliary neurotrophic factor delivered via tetracycline-regulated lentiviral vectors in the quinolinic acid rat model of Huntington's disease.

The ability to regulate gene expression constitutes a prerequisite for the development of gene therapy strategies aimed at the treatment of neurologic disorders. In the present work, we used tetracycline (Tet)-regulated lentiviral vectors to investigate the dose-dependent neuroprotective effect of human ciliary neurotrophic factor (CNTF) in the quinolinic acid (QA) model of Huntington's disease (HD). The Tet system was split in two lentiviruses, the first one containing the CNTF or green fluorescent protein (GFP) cDNAs under the control of the Tet-response element (TRE) and a second vector encoding the transactivator (tTA). Preliminary coinfection study demonstrated that 63.8% +/- 2.0% of infected cells contain at least two viral copies. Adult rats were then injected with CNTF- and GFP-expressing viral vectors followed 3 weeks later by an intrastriatal administration of QA. A significant reduction of apomorphine-induced rotations was observed in the CNTF-on group. In contrast, GFP-treated animals or CNTF-off rats displayed an ipsilateral turning behavior in response to apomorphine. A selective sparing of DARPP-32-, choline acetyltransferase (ChAT)-, and NADPH-d-positive neurons was observed in the striatum of CNTF-on rats compared to GFP animals and CNTF-off group. Enzyme-linked immunosorbent assay (ELISA) performed on striatal samples of rats sacrificed at the same time point indicated that this neuroprotective effect was associated with the production of 15.5 +/- 4.7 ng CNTF per milligram of protein whereas the residual CNTF expression in the off state (0.54 +/- 0.02 ng/mg of protein) was not sufficient to protect against QA toxicity. These results establish the proof of principle of neurotrophic factor dosing for neurodegenerative diseases and demonstrate the feasibility of lentiviral-mediated tetracycline-regulated gene transfer in the brain.

Macrophage-derived chemokine gene transfer results in tumor regression in murine lung carcinoma model through efficient induction of antitumor immunity.

Chemokine gene transfer represents a promising approach in the treatment of malignancies. Macrophage-derived chemokine (MDC) (CCL22) belongs to the CC chemokine family and is a strong chemoattractant for dendritic cells (DC), NK cells and T cells. Using adenoviral vectors, human MDC gene was transferred in vivo to investigate its efficacy to induce an antitumor response and to determine the immunologic mechanisms involved. We observed that intratumoral injection of recombinant adenovirus encoding human MDC (AdMDC) resulted in marked tumor regression in a murine model with pre-established subcutaneous 3LL lung carcinoma and induced significant CTL activity. The antitumor response was demonstrated to be CD4+ T cell- and CD8+ T cell-dependent. Administration of AdMDC induced chemoattraction of DC to the tumor site, facilitated DC migration to draining lymph nodes or spleen, and finally activated DC to produce high levels of IL-12. Furthermore, a significant increase of IL-4 production within the tumors was observed early after the AdMDC administration and was followed by the increase of IL-12 and IL-2 production. The levels of IL-2, IL-12 and IFN-gamma in serum, lymph nodes and spleen were also found to be higher in mice treated with AdMDC as compared with that in AdLacZ- or PBS-treated mice. The antitumor response induced by AdMDC was markedly impaired in IL-4 knockout mice, suggesting an important role of IL-4 in the induction of antitumor immunity by MDC. These results suggest that MDC gene transfer might elicit significant antitumor effects through efficient induction of antitumor immunity and might be of therapeutic potentials for cancer.

[Tumor-induced immunosuppression]. / Immunitè antitumorale et tolérance immunitaire.

Tumor immunology is based on two essential concepts: immune surveillance, which implicate the host immune reactions against tumor cells, and tumor immune escape, which refers to the tumor-cell evasion process against the host immune system. The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Furthermore, expression of self-antigens on the tumor surface impose potential barriers to the development of effective immune response. Tumors are able to overcome immune surveillance by changing the polarity of effectors cells, thus down-regulating the proliferation of tumor-specific cytotoxic T cells, or altering the effector compositions of immune cells within the tumor milieu, or both. Understanding the interaction between cancer cells and host immune cells is of importance for clinical applications or immunotherapy in cancer treatment.

[Cancer genetic immunotherapy]. / Immunothérapie génique du cancer.

The concept of cancer immunotherapy and the resulting technical advances have evolved considerably during the last decade. However, cancer treatment by recombinant IL-2 or IFN-alpha still represents today the best therapeutic way for the treatment of renal carcinoma, melanoma and in some cases lymphoma. The immunotherapy approaches such as vaccination, gene and cellular therapy, have not yet demonstrated a sufficient clinical efficacy for the treatment of solid tumors. The goal of this review is to summarize the different approaches to cancer immunotherapy developed today. Specific approaches such as antigenic vaccination will be first described, then non-specific approaches such as gene transfer on the tumor site of immuno-stimulating genes will be discussed.

CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection.

Increases in circulating CD14+/CD16+ monocytes have been associated with HIV dementia; trafficking of these cells into the CNS has been proposed to play an important role in the pathogenesis of HIV-induced neurological disorders. This model suggests that events outside the CNS leading to monocyte activation initiate the process leading to HIV dementia. To investigate the role of this activated monocyte subset in the pathogenesis of HIV dementia, we examined brain specimens from patients with HIV encephalopathy (HIVE), HIV without encephalopathy, and seronegative controls. An accumulation of perivascular macrophages was observed in HIVE. The majority of these cells identified in microglial nodules and in the perivascular infiltrate were CD14+/CD16+. P24 antigen colocalized with both CD14 and CD16 suggesting that the CD14+/CD16+ macrophage is a major reservoir of HIV-1 infection in CNS. Using CD45/LCA staining, the perivascular macrophage was distinguished from resident microglia. In addition to perivascular and nodular localizations, CD16 also stained ramified cells throughout the white matter. These cells were more ramified and abundant than cells positive for CD14 in white matter. Double staining for p24 and CD16 suggests that these cells were often infected with HIV-1. The prominent distribution of CD14+ cells in HIVE prompted our analysis of soluble CD14 levels in cerebrospinal fluid. Higher levels of soluble CD14 (sCD14) were observed in patients with moderate-to-severe HIV dementia, suggesting the utility of sCD14 as a surrogate marker. CD14+/CD16+ monocytes may play a role in other neurological disorders and sCD14 may be useful for evaluating these conditions.

[Molecular basis of oncogenesis]. / Bases moléculaires de l'oncogenèse.

After a quarter century of rapid advances, cancer research has generated a rich and complex body of knowledge, revealing cancer to be a disease involving dynamic changes in the genome. Several lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives. The barriers to development of cancer are embodied in a teleology: cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis. This review concern the description of novel capabilities acquired during tumor development.

Autoantibodies to TNFalpha in HIV-1 infection: prospects for anti-cytokine vaccine therapy.

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine principally involved in the activation of lymphocytes in response to viral infection. TNFalpha also stimulates the production of other cytokines, activates NK cells and potentiates cell death and/or lysis in certain models of viral infection. Although TNFalpha might be expected to be a protective component of an antiviral immune response, several lines of evidence suggest that TNFalpha and other virally-induced cytokines actually may contribute to the pathogenesis of HIV infection. Based on the activation of HIV replication in response to TNFalpha, HIV appears to have evolved to take advantage of host cytokine activation pathways. Antibodies to TNFalpha are present in the serum of normal individuals as well as in certain autoimmune disorders, and may modulate disease progression in the setting of HIV infection. We examined TNFalpha-specific antibodies in HIV-infected non-progressors and healthy seronegatives; anti-TNFalpha antibody levels are significantly higher in GRIV seropositive slow/non-progressors (N = 120, mean = 0.24), compared to seronegative controls (N= 12, mean = 0.11). TNFalpha antibodies correlated positively with viral load, (P = 0.013, r = 0.282), and CD8+ cell count (P = 0.03, r = 0.258), and inversely with CD4+ cell count (P = 0.003, r = - 0.246), percent CD4+ cells (P = 0.008, r = -0.306), and CD4 :CD8 ratio (P = 0.033, r = - 0.251). TNFalpha antibodies also correlated positively with antibodies to peptides corresponding to the CD4 binding site of gp160 (P = 0.001, r = 0.384), the CD4 identity region (P = 0.016, r = 0.29), the V3 loop (P = 0.005, r = 0.34), and the amino terminus of Tat (P = 0.001, r = 0.395); TNFalpha antibodies also correlated positively with antibodies to Nef protein (P = 0.008, r = 0.302). The production of anti-TNFalpha antibodies appears to be an adaptive response to HIV infection and suggests the potential utility of modified cytokine vaccines in the treatment of HIV infections as well as AIDS-related and unrelated autoimmune and CNS disorders.

Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.

Improvement of mouse beta-thalassemia upon erythropoietin delivery by encapsulated myoblasts.

The goal of the present study was to analyze if sustained delivery of elevated doses of recombinant erythropoietin (Epo), by genetically modified and immunoprotected allogenic cells, was able to correct the chronic anemia, characteristic of a spontaneous mouse model of beta-thalassemia (Hbb thal 1). Mouse C2C12 myoblast cells were transfected with a plasmid containing the mouse Epo cDNA and a mutated dihydrofolate reductase (DHFR) gene for gene amplification upon administration of increasing doses of methotrexate. In order to immunoprotect the transplanted cells, the stably modified cells were loaded into polyethersulfone microporus hollow fibers which were implanted subcutaneously into Hbb thal 1 mice. An increase in hematocrit starting 2 weeks after implantation was associated with elevated blood levels of Epo and an improved red blood cell phenotype. The latter indicated an improvement of cell morphology and membrane defects, in particular a reduced amount of free alpha hemoglobin chain, the hallmark of globin chain imbalance in beta-thalassemia. A reduction of reticulocyte count contrasting with the increase in hematocrit was also observed suggesting an improved erythrocyte survival. We conclude that the phenotype can be durably improved in some beta-thalassemic mice upon in vivo delivery of recombinant Epo by polymer encapsulated cells. Sustained elevated delivery of recombinant Epo holds promise for the treatment of beta-thalassemia-associated chronic anemia.

Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts.

The transplantation of polymer encapsulated myoblasts genetically engineered to secrete erythropoietin (Epo) may obviate the need for repeated parenteral administration of recombinant Epo as a treatment for chronic renal failure, cancer or AIDS-associated anemia. To explore this possibility, the human and mouse Epo cDNAs under the control of the housekeeping mouse PGK-1 promoter were transfected into mouse C2C12 myoblasts, which can be terminally differentiated upon exposure to low serum-containing media. Pools releasing 150 IU human Epo per 10(6) cells per day and 390 IU mouse Epo per 10(6) cells per day were selected. Polyether-sulfone (PES) capsules loaded with approximately 200,000 transfected myoblasts from these pools were implanted on the dorsal flank of DBA/2J, C3H and C57BL/6 mice. With human Epo secreting capsules, only a transient increase in the hematocrit occurred in DBA/2J mice, whereas no significant response was detected in C3H or C57BL/6 mice. On the contrary, all mice implanted with capsules releasing mouse Epo increased their hematocrit over 85% as early as 7 days after implantation and sustained these levels for at least 80 days. All retrieved implants released Epo and contained well preserved myoblasts. Moreover most capsules were surrounded by a neovascularization. Mice transplanted with nonencapsulated C2C12 cells releasing mouse Epo showed only a transitory elevation of their hematocrit reflecting the poor engraftment of injected myoblasts. These results indicate that polymer encapsulation of genetically engineered myoblasts is a promising approach for the long-term delivery of bioactive molecules, allowing the resolution of the shortcomings of free myoblast transfer.

A gene therapy approach to regulated delivery of erythropoietin as a function of oxygen tension.

Current therapy for several forms of anemia involves a weekly regime of multiple subcutaneous injections of recombinant human erythropoietin (hEpo). In an effort to provide a physiologically regulated administration of erythropoietin, we are developing cell lines genetically engineered to release hEpo as a function of oxygen tension. C2C12 cells were transfected using a vector containing the hEpo cDNA driven by the hypoxia-responsive promoter to the murine phosphoglycerate kinase gene. In vitro, these cells showed a threefold increase in hEpo secretion as oxygen levels were shifted from 21% to 1.3% oxygen. To test in vivo response, C2C12-hEpo cells were encapsulated in a microporous membrane and implanted subcutaneously on the dorsal flank of DBA/2J mice. On average, serum hEpo levels in animals exposed to 7% oxygen were two-fold higher than values seen in their control counterparts kept at 21% oxygen. Similar studies employing rats confirmed that hEpo delivery is regulated as a function of oxygen tension. These results suggest the feasibility of developing an oxygen-regulated, encapsulated cell-based system for hEpo delivery.