ABSTRACT
Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers.
Subject(s)
Carcinoma, Hepatocellular/genetics , Dependovirus/genetics , Genetic Vectors , Liver Neoplasms/genetics , Proto-Oncogene Proteins c-met/genetics , Transduction, Genetic , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Female , Humans , Mice , Mutagenesis, Site-Directed , Proteasome Inhibitors , Protein Structure, Tertiary , Transplantation, Heterologous , Tyrosine/genetics , Virus IntegrationABSTRACT
Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.
Subject(s)
Genetic Therapy/methods , Promoter Regions, Genetic , Retinal Cone Photoreceptor Cells/metabolism , Rod Opsins/genetics , Targeted Gene Repair , Animals , Color Vision Defects/metabolism , Color Vision Defects/therapy , Dependovirus/genetics , Dogs , Gene Expression , Genetic Vectors/administration & dosage , Green Fluorescent Proteins/genetics , Humans , Injections , Models, Animal , Transduction, Genetic/methods , TransgenesABSTRACT
The data concerning the influence of Chernobyl exclusive zone factors on cytochrome P-450 relative contents in liver microsome fractions of Balb/c, CC57W/Mv and C57 Black mice are submitted in this work Reliable diminishing of liver P-450 relative concentration in experimental animals, compared with respective isogenic control groups is shown to be varying depending on the mouse line and sex.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/radiation effects , Power Plants , Radioactive Hazard Release , Animals , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microsomes, Liver/enzymology , Species Specificity , UkraineABSTRACT
The effects of 2',5'-oligoadenilates (2,5A) on the survival of Wistar rats inoculated with Svec leukemia cells and the proliferation of tumoral cells were investigated. An agreement between the expression of c-myc protooncogene and inhibition of leukemic cell proliferation by 2,5A was found.