ABSTRACT
The CELO recombinant avian adenovirus carrying the gene coding the human angiogenine (ANG) synthesis was obtained. Expression of the angiogenine gene was shown in the LMH cell culture after infection with the CELO-ANG virus. The ability of CELO recombinant adenoviruses to carry out the delivery and expression of alien genes in muscle cells was demonstrated in experiments with laboratory animals (Wistar line rats). The induced neovascularization in rat muscles after the animals were administered the CELO-ANG viruses was shown.
Subject(s)
Angiogenesis Inducing Agents , Aviadenovirus/genetics , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Ribonuclease, Pancreatic , Animals , Aviadenovirus/metabolism , Cell Line , Gene Expression , Humans , Male , Muscle, Skeletal/metabolism , Rats , Rats, Wistar , Recombinant Proteins/biosynthesis , Ribonuclease, Pancreatic/biosynthesis , Ribonuclease, Pancreatic/genetics , Tibia , TransfectionABSTRACT
In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.
Subject(s)
Fowl adenovirus A/genetics , Interleukin-2/genetics , Melanoma, Experimental/immunology , Animals , Chick Embryo/virology , Cloning, Molecular/methods , Female , Humans , Interleukin-2/therapeutic use , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , Plasmids , Survival AnalysisABSTRACT
Human adenovirus (Ad) vectors are extensively used as gene transfer vehicles. However, a serious obstacle for the use of these vectors in clinical applications is due to pre-existing immunity to human Ads affecting the efficacy of gene transfer. One of the approaches to circumvent host immune response could be the development of vectors based on non-human Ads that are able to transduce genes into human cells. In this study, we explored the possibility of using avian Ad CELO vectors as gene-transfer vehicles. For this purpose, we constructed a set of recombinant CELO viruses and demonstrated that they are able to deliver transgenes into various organs on the background of pre-existing immunity to human Ad5. The created CELO-p53 vector restored the function of the p53 tumor suppressor both in cultured human tumor cells in vitro and in their xenografts in nude mice in vivo. The latter effect was accompanied by inhibition of tumor growth. Noteworthily, the delivery of CELO-p53 led to activation of p53 target genes in cells showing inactivation of endogenous p53 by three different mechanisms, that is, in the human epidermoid carcinoma A431, lung adenocarcinoma H1299, and cervical carcinoma HeLa.
Subject(s)
Aviadenovirus/genetics , Genes, p53 , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Neoplasms/therapy , Animals , Cell Line, Tumor/metabolism , Gene Expression , Humans , Injections , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms/metabolism , Transgenes , Tumor Suppressor Protein p53/analysisABSTRACT
Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.
Subject(s)
Aviadenovirus/genetics , Gene Transfer Techniques , Genetic Vectors , Interleukin-2/genetics , Luminescent Proteins/genetics , Allantois/metabolism , Allantois/virology , Animals , Cells, Cultured/virology , Chick Embryo , Green Fluorescent Proteins , Interleukin-2/metabolism , Melanoma, Experimental/genetics , Melanoma, Experimental/virology , Mice , Mice, Inbred C57BL , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.