Research on a highly-efficient and tumor-specific gene therapy using autologous NK/NKT cells as gene carriers

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We examined the population of tumor infiltrating cells in the experimental primary tumor model where 3LL or B16F10 cells were injected to the lateral flank of C57BL6 mice. A number of infiltrating cells were found around the 3LL tumor. The majority of these cells were CD3+/NK1.1- Trills and neutrophils, and the remaining includes NK cells and MHC class II+ cells (macrophages and dendritic cells).Murine splenocytes were obtained and cultured for seven days in the presence ofIL-2/1, -12. The proportion of NK1.1+CD3- cells was elevated from a few % to 10-15% after the culture. These cultured splenocytes were injected around the 3LL or B16 tumors (approximately 10 mm in diameter) and the tumor infiltrating cells were immunohistochemically observed. Marked infiltration of NK1.1+CD3- cells was found in the peritumoral tissue within 48 hours after the splenocyte infection. Similar results were obtained from cultured bone marrow cells of Thalidomide-treated mice.Then the expression of p16INK4 in the tumor cell lines was examined by RTPCR and Western immunoblotting. mRNA expression of p16INK4 was observed in the both cells, but its protein expression was almost under the detectable level. Full length cDNA of p16INK4 was obtained by cDNA cloning, and inserted to a plasmid expression vector. A detectable expression was observed in the p16INK4-transfectant of 3LL and B16F10. p16INK4 overexpression resulted in a marked growth suppression of these cell lines. Now we are working for the construction of p16INK4 adenovirous vector and performing preliminary experiments using another adenovirus vector on the transport of the vectors by murine NK cells in vitro.