Adenoviruses mutated in the VA genes for cancer treatment
a technology of va genes and va genes, applied in the field of chemotherapy, radiation therapy, surgery, etc., can solve the problems of limited effectiveness, inability to treat cancer in clinical trials, and inability to cure most advanced cancers
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example 1
A Mutated Adenovirus in the VAI Gene Shows Ras-Dependent Replication
[0045] To demonstrate the dependence of the replication of a VAI RNA mutant (dl331) on an activated Ras pathway we have modulated the activation status of Ras in human cells.
[0046] Approximately 1.0×107 embryonic human kidney cells (line 293) are seeded on a plate 10 cm in diameter and transfected with 24 micrograms of plasmids containing either green fluorescence protein (GFP), the constitutively active form of Ras (H-Ras V12) or the negative dominant of Ras (H-Ras N17). A standard protocol of calcium phosphate was used for the transfection. Forty-eight hours after transfection the cells were transferred to new plates. To demonstrate the effect of transfection of the plasmids on the Ras pathway, we looked at the expression levels and phosphorylation of ERK (a Ras effector) on a cellular lysate by Western Blot. The dry lysate was obtained through incubation with a lysis buffer (20 mM Tris, 2 mM EDTA, 100 mM NaCl, ...
example 2
Human Tumor Cells with Active Ras Pathway Allow for Efficient Replication of an Adenovirus with Mutated VAI RNAs
[0048] Replication of a mutated adenovirus in the VAI RNA gene (dl331) was quantified in the NP9 human pancreatic cancer line that has a mutation in codon 12 of the K-Ras gene (GGT→GAT). Replication is estimated by the cytopathic effect (CPE) that the virus induces measured as a decrease in the quantity of protein in the cellular monolayer (BCA method). In short, the NP-9 cells are seeded on 96-well plates with 30,000 cells per well. On the next day the cells are infected with serial dilutions of dl331 or wild-type adenovirus from a concentration of 1000 plaque-forming units per cell. The infected cells are incubated for 5 days and the culture medium is removed to measure the quantity of protein remaining in the well. FIG. 5 shows the results obtained as the percentage of protein with respect to the uninfected wells compared to the dilution of the viral inoculum. The dilu...
example 3
A Mutated Adenovirus in the VAI RNA Gene can be used to Treat Tumors Effectively
[0049] Below we will demonstrate the antitumor effect of a VAI RNA mutant adenovirus (dl331). An in vivo experiment was performed with athymic mice of the Balb / c strain that contained tumors with an activated Ras pathway (NP9). All the experiments were performed according to FELASA guidelines (Federation of European Laboratory Animal Science Associations). A total of 1.2×107 tumor cells of the NP-9 cell line were injected subcutaneously in each posterior flank of the mouse. After 1 day the tumors formed (which reached 70-80 mm3) were distributed between different experimental groups (n=10 per group). The tumors of the control group received two intratumoral injections of saline buffer (2×10 μl). Those in the group treated with the VA mutant received two intratumoral injections (2×10 μl) of dl331 (109 viral particles per tumor). FIG. 6 shows the tumor volume compared to the initial treatment (day 0). The...
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