Rapid adenoviral transduction of freshly resected tumour explants with therapeutically useful genes provides a rationale for genetic immunotherapy for colorectal cancer

Abstract

To develop protocols for the molecular immunotherapy of can reproducibly be explanted and established in shortcolorectal cancer, we compared the efficacy of three separterm culture. Finally, a rapid transduction protocol has ate classes of therapeutic genes to induce antitumour been developed by which, using adenoviral vectors, as responses in a murine colorectal cell model. Thus, the many as 90% of the cells in these fresh tumour explants effects of two cytokines (IL-2 and GM-CSF) were comcan be engineered to express high levels of the clinically pared with those of a costimulatory gene (B7.1) and a suicrelevant genes (GM-CSF or IL-2) within 1-2 weeks of suride gene (HSVtk). The rank order of efficacy against prigery. Adenovirus-mediated gene delivery was reproducibly mary tumour growth was HSVtk[GCV], B7.1 Ͼ puro, IL-2 and significantly more efficient than retroviral transduction ϾGM-CSF, neo whereas the order of efficacy in inducing using the MFG-␤-Gal retroviral vector over the time-frame antitumour immunity was GM-CSF, IL-2, ϾB7.1, of importance for vaccination. Hence, combination of the HSVtk[GCV]Ͼ puro, neo in a prophylactic vaccination animal model data with the ex vivo modification protocol model. To exploit these data in a clinically relevant and suggests that vaccination of colorectal patients of the realistic way, we also demonstrated that colorectal tumours appropriate stage will be possible and effective.

Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells

“…2,3 Several genes such as the tumor suppressor p53 4,[37][38][39] or the prodrug-activating gene HSVtk may be useful for vector-mediated cancer gene therapy 5,21,40 and have been tested in vitro and in animal models in vivo. Clinical phase I and II studies using a p53-expressing AdV have been started which investigate its potential to disrupt colorectal carcinoma metastases.…”

“…1 Because of the high prevalence and mortality of cancer in humans 2 and the limitations of conventional cancer therapy against metastatic cancer, experimental gene therapies such as suicide gene therapy, tumour suppressor gene therapy, and molecular immunotherapy have been investigated. 3,4 Although suicide gene therapy…”

Potent Selection of Antigen Loss Variants of B16 Melanoma following Inflammatory Killing of MelanocytesIn vivo

“…1D). Of the nine tumors that were explanted, seven (B16ova-VAR1-7) were successfully reestablished in culture (30). Of these seven B16ova explants, all were noticeably depigmented C and D, mice bearing B16ova tumors seeded 9 days previously instead of the 3 days of A were treated with just two rounds (six total injections) of plasmid injections (days 10, 11, 12 and 17, 18, 19) and given ganciclovir or PBS at days 10-14 and 17-21.…”

“…C, IFN-g enzyme-linked immunospot assays (PharMingen, San Diego, CA) were done using splenocytes harvested from C57BL/6 mice that had been cured of established B16ova tumors by treatment with Tyr-HSVtk /CMV-hsp70 /ganciclovir. Splenocytes were stimulated in the presence of the synthetic, H-2D b -restricted peptides hgp100 [25][26][27][28][29][30][31][32][33] , KVPRNQDWL, TRP-2 180-188 SVYDFFVWL, and H-2K b restricted Ova SIINFEKL as described (11) in triplicate cultures at a density of 250,000 splenocytes per well. Spot numbers were determined 72 hours later by computer-assisted image analyzer.…”

“…Tumors were explanted and dissociated rapidly on ice (30), and RNA was prepared with the Qiagen RNA extraction kit (Qiagen, Chatsworth, CA). One-microgram total cellular RNA was reverse transcribed in a 20 AL volume using oligodeoxythimidylate as a primer.…”