Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Baseline metabolic disturbances and the twenty-five years risk of incident cancer in a Mediterranean population.

BACKGROUND AND AIMS: Obesity is predictive of metabolic syndrome (metS), type 2 diabetes, cardiovascular (CV) disease and cancer. The aim of the study is to assess the risk of incident cancer connected to obesity and metS in a Mediterranean population characterized by a high prevalence of obesity. METHODS AND RESULTS: As many as 1133 subjects were enrolled in two phases and followed for 25 years (859 subjects) or 11 years (274 subjects) and incident cancer was registered in the follow-up period. Anthropometric measures and biochemical parameters were filed at baseline and evaluated as predictors of incident cancer by measuring hazards ratios (HR) using multivariate Cox parametric hazards models. Best predictive threshold for metabolic parameters and metS criteria were recalculated by ROC analysis. Fasting Blood Glucose >5.19 mmol/L [HR = 1.58 (1.0-2.4)] and the TG/HDL ratio (log10) (Males > 0.225, Females > 0.272) [HR = 2.44 (1.3-4.4)] resulted independent predictors of survival free of cancer with a clear additive effect together with age classes [45-65 years, HR = 2.47 (1.3-4.4), 65-75 years HR = 3.80 (2.0-7.1)] and male gender [HR = 2.07 (2.3-3.1)]. CONCLUSIONS: Metabolic disturbances are predictive of cancer in a 25 years follow-up of a Mediterranean population following a traditional Mediterranean diet. The high prevalence of obesity and metS and the observed underlying condition of insulin resistance expose this population to an increased risk of cardiovascular disease and cancer despite the healthy nutritional habits.

Cytotoxic T lymphocyte and antibody responses generated in rhesus monkeys immunized with retroviral vector-transduced fibroblasts expressing human immunodeficiency virus type-1 IIIB ENV/REV proteins.

The immune response against human immunodeficiency virus type-1 (HIV-1) is believed to play a role in controlling the early stages of disease progression. The cellular immune response, in particular cytotoxic T lymphocyte (CTL) activity, may be important for eliminating virally infected cells in HIV-1-infected individuals. Genetic immunization using retroviral vectors provides an effective means of introducing antigens into the antigen presentation pathways for T cell stimulation. A nonreplicating, amphotropic murine retroviral vector containing the HIV-1 IIIB env gene has been used to transduce primary rhesus monkey fibroblasts for the expression of HIV-1 antigenic determinants. Rhesus monkeys were immunized with four doses of either vector-transduced autologous fibroblasts (VTAF) expressing the HIV-1 IIIB ENV/REV proteins or nontransduced autologous fibroblasts (NTAF) administered at 2-week intervals. The animals were evaluated for both the induction of HIV-1-specific immune responses and potential toxicity associated with this ex vivo treatment. The VTAF-immunized monkeys generated CTL responses specific for HIV-1 ENV/REV expressing autologous target cells, whereas, NTAF-immunized monkeys showed negligible CTL activity. The cytotoxic activity was mediated by CD8+, major histocompatibility complex (MHC)-restricted CTL. In addition, antibody responses directed against the HIV-1 gp120 protein were also detected in the sera of VTAF-immunized monkeys. Clinical and histopathological evaluation of immunized monkeys showed no evidence of significant adverse events. Several animals that received either VTAF or NTAF had detectable anti-cytoplasmic antibodies, but were not positive for anti-nuclear antibodies or rheumatoid factor. Subsequent evaluation of renal, synovial, and hepatic tissue samples from these monkeys revealed no autoimmune disease-associated lesions. This study demonstrates the safety and ability of autologous retroviral vector-transduced cells expressing HIV-1 IIIB ENV/REV proteins to stimulate immune responses in a non-human primate model, and provides a basis for this form of genetic immunization in HIV-infected humans.

Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer.

A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.

Transduction of human melanoma cells with interleukin-2 gene reduces tumorigenicity and enhances host antitumor immunity: a nude mouse model.

Human melanoma tumor cells were genetically modified in vitro by transferring the interleukin-2 (IL-2) gene via a retroviral vector into established or fresh tumor cells. In addition, human melanoma cells were transduced in vivo by the direct injection of the IL-2/retroviral vector into melanoma xenografts in nude mice. The gene-modified melanoma cells expressed the IL-2 cytokine gene and secreted biologically active IL-2. Transduction of melanoma cells with the IL-2 gene did not affect the antigenic profile of the cells, but caused a strong abrogation of their tumorigenicity. One million parental cells formed subcutaneous tumors in nude mice. In contrast, various doses of up to 20 x 10(6) IL-2-transduced cells failed to form tumor in the mice. Coinjection of IL-2-producing cells with parental cells inhibited tumor formation even when highly tumorigenic doses of parental cells were used. Histochemical analysis of the injection sites of IL-2-modified cells showed an influx of host immune cells, predominantly macrophages, as early as the third day after inoculation. Neutrophils, mast cells, and eosinophils were also seen in the inflammatory exudate. Eventually, transduced cells showed signs of degeneration and necrosis and ultimately died in 4 weeks. Macrophages were seen in parental tumor sites only during the first few days after injection, and then parental tumors exhibited fast, progressive growth. The study suggests that melanoma cells transduced with the IL-2 cytokine gene may provide an effective vaccine for melanoma patients, whereas the in vivo transduction of tumors with cytokine genes is feasible and may represent a novel approach for the immunotherapy of cancer patients.