6 research outputs found
The Directed Evolution process and analysis of viruses and derivative viral pools by anion-exchange chromatography.
<p><i>A,</i> Representation of the Directed Evolution process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002409#s4" target="_blank">Materials and Methods</a> for detailed description). <i>B,</i> Chromatograms of each pure Ad serotype included in the mixed serotype starting pool from which ColoAd1 was selected. <i>C,</i> Chromatograms of the passage 20 viral pools derived on the HT-29, Panc-1, MDA-231, and PC-3 tumor cell lines, respectively. The differing retention times of these pools are consistent with the predominant serotype of the pool being Ad5 or Ad40 for the Panc-1 pool, Ad11p for the HT-29 pool, Ad3 or Ad4 for the PC-3 pool, and Ad5 or Ad40 for the MDA-231 pool.</p
Potency and therapeutic indices of ColoAd1.
<p>Potency values less than 1 indicates attenuation relative to Ad5.</p><p>The potencies of ColoAd1, Ad5, ONYX-015, Ad3 and Ad11p on colon cancer (HT-29, DLD-1) and normal cells (HUVEC, HMEC) were measured by MTS assay at day 4 post-infection and is represented in the table as an IC<sub>50</sub> value. The potency (as reflected in the IC<sub>50</sub>) is the ratio of the IC<sub>50</sub> of a given virus on a given cell line relative to Ad5's IC<sub>50</sub> on that cell line. The Therapeutic Index was calculated using either HUVEC (primary endothelial) or HMEC (primary epithelial) cells and using the ratio of the IC<sub>50</sub> of a virus on these primary, normal cells and dividing it by its IC<sub>50</sub> on HT-29 tumor cells.</p
Anti-tumoral activity of ColoAd1 after systemic administration in a liver metastasis xenograft mouse model.
<p>HT-29 colon cancer cells were seeded to the liver of nude beige mice (n = 10 mice per treatment group). Plasma CEA level was used to monitor tumor establishment. <i>A</i> and <i>B</i>, Mice were treated by tail-vein (i.v.) injection with 1×10<sup>10</sup>, 5×10<sup>10</sup>, or 1×10<sup>11</sup> total viral particles of ColoAd1 per mouse. A fourth set of liver-tumor-bearing mice were i.v. injected with 1×10<sup>11</sup> total viral particles of a replication-defective [E1<sup>(−)</sup>] version of ColoAd1, ColoAd1CJ132. A fifth set of mice were injected with vehicle control (buffer). Tumor weight measurements demonstrate that ColoAd1 has anti-tumoral activity, which is dose-dependent (<i>A</i>). Blood CEA levels at the end of the study (day 12 post viral administration) corroborate the tumor weight data (<i>B</i>). <i>C and D,</i> Comparison of anti-tumoral activity of ColoAd1, Ad11p and ONYX-015 in the HT-29 liver metastasis xenograft mouse model. In a second study performed in the same model as in Panels A and B, ColoAd1 was compared to its parental virus, Ad11p, and to the clinically-approved oncolytic virus ONYX-015; each virus dosed i.v. to a total of 1×10<sup>11</sup> viral particles per mouse.</p
Genomic sequence diagram of ColoAd1.
<p>The genomic differences between ColoAd1 and Ad11p are noted in the schematic. In the E2B region there are frequent substitutions of Ad3 sequences for Ad11p sequences between base pairs 6081 and 9322. In addition, ColoAd1 has a nearly complete (2,444 bp) E3 region deletion, and a smaller (25 bp), second deletion that maps to a putative E4orf4 region of the virus.</p
Potency of ColoAd1 relative to Ad5 on a panel of colon cancer cell lines.
<p>Potency values less than 1 indicate attenuation relative to Ad5.</p><p>Potencies of ColoAd1 and Ad5 were measured by MTS on the mixed panel of tumor cell lines to derive an IC<sub>50</sub> value for each virus. These IC<sub>50</sub> values were used to derive the potency of ColoAd1 relative to Ad5 using the calculation IC<sub>50</sub> value Ad5 divided by the IC<sub>50</sub> value of ColoAd1 on the same colon tumor cell line.</p
Potency and kinetics of armed virus, ColoAd1-GFP.
<p>MTS assays were performed on ColoAd1 and ColoAd1-GFP on A) the colon tumor cell line, HT-29 and B) the primary endothelial cells, HUVEC. The reporter gene, GFP, is expressed with late kinetics (ie., is dependent upon the initiation of viral DNA replication for expression) as defined by expression C) only in the absence of AraC and D) lack of expression in the presence of AraC.</p