Study On The Construct And Effect Of Oncolytic HSV-1s

Cancer is still a kind of malignant disease threatening people's health. The study of the therapy methods of cancer is, and will be focus of medical and biological science in the long run. A lot proof demonstrated that cancer is a kind of inherent disease, involving mutation of kinds of oncogenes and anti-cancer genes. The traditional therapy methods of cancer consists of resection, radiotherapy and chemotherapy, while the effect is not satisfactory. With the development of Molecular Biology, Oncology and Virology, using virus as a weapon fighting against cancer becomes a newly hot point. These viruses are named as oncolytic virus, which can replicate selectively within tumors, but can not replicate in normal tissues and cells. Adenoviruses, Simplex Herpes Viruses and human reoviruses are all used as oncolytic virus.Herpes simplex virus 1 is one of the viruses commonly used as oncolytic virus, due to its high infection efficiency, wide host range, clear inherent background and large volume for foreign genes. Two kinds of oncolytic HSV-1were reported. The first one consists of mutant HSV-1 lacking the enzyme involved in ribonucleotide metabolizability and can not synthesis DNA without the help of host cancer cells, which are always rich of such enzymes. The second one includes some mutant viruses with deficient icp34.5 gene, which is the most important neurotoxic gene and an apoptosis-inhibiting gene in HSV-1 genome. These mutant HSV-1 lacking ICP34.5 can replicate in cells deficient in IFN-PKR pathway, most of which are cancer cells. These normal cells with intact IFN-PKR pathway will undergo apoptosis when infected with such mutant HSV-1.2 oncolytic herpes simplex virus 1, mtHSV and HSV-EN-V1, were constructed successfully. mtHSV was constructed with traditional homologous recombination, in which 2 copies of icp34.5 gene, the apoptosis-inducing gene in HSV-1 genome, were replaced by PCMV-lacZ expression cassette. mtHSV can replicate only in those cancer cells with certain deficiency in apoptosis inducing signal path, in which IFN-PKR path may be involved. mtHSV was selected by LacZ expression in Vero cells and identified by PCR and Southern blot. Rec-A dependent recombination was used to construct HSV-EN-V1, a mutant HSV-1 cloning in bacterial artificial chromosome(BAC). UL39 gene, which encodes the large subunit of ribonucleotide reductase (rr), were replace by IRES-EGFP cassetee in HSV-EN-V1, so that the virus can only amplify in cells rich of ribonucleotide reductase , most of which are cancer cells in vivo. HSV-EN-V1 was identified by PCR and sequencing. Additionally, another mutant HSV-1 inserted with endostatin gene was also constructed with RecA-dependent recombination, but the construct is waiting for identification.mtHSV was amplified in Vero cells and also tittered on Vero cells. Four human tumor cell lines, U251, EJ, Hep3B and Hep2, shows high sensitivity to mtHSV infection and were destroyed within several days after infection. But A549, SPC-A1 and Wish cells show low sensitivity to mtHSV infection, maybe due to ras statue in these cells. Mice sarcoma cell line, s-180, also can allow replication of mtHSV. With its intact tk gene, mtHSV exhibited susceptibility to acyclovir(ACV), which provides an approach to control viral replication.In vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS. Tumor dimensions then were measured at serial time points and tumor volumes were calculated. Sarcoma growth was significantly inhibited with prolonged survival time and reduced tumor volume. The mean tumor volume of PBS-treated group is 34 days, while the early-term-therapy group is 55.8 days and mid-term-therapy group is 47.6 days. The mean tumor volume of PBS treated group is 562.6 mm3, while the early-term-therapy group is only 306.5 mm3 and mid-term-therapy group is 422.3 mm3. There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs. Immu...