Selective eradication of cancer cells by delivery of adenovirus-based toxins

Abstract

Background and objectiveKRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We propose to exploit the hyperactive RAS pathway, rather than to inhibit it as was previously tried and failed repeatedly. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation.ResultsA massive cell death, in a dose-dependent manner, reaching 73% at MOI 10 was seen in KRAS cells as compared to 22% in WT cells. Increase expression of MazE (the anti-toxin) protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system. Considerable tumor shrinkage (61%) was demonstrated in vivo following MazEF-encoding adenovirus treatment without any side effects.DesignEfficient vectors for cancer-directed gene delivery were constructed; “pAdEasy-Py4-SV40mP-mCherry-MazF”“pAdEasy-Py4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP“,“pAdEasy-ΔPy4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP “and “pAdEasy-mCherry”. Virus particles were produced and their potency was tested. Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT. FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptotic and dead cells, respectively. In vivo tumor formation was measured in a xenograft model.ConclusionsA proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

“…HT29‐GFP (human colorectal adenocarcinoma) cell line was imaged by both IPM and SDCM. The green fluorescent protein (GFP) gene was previously transfected into the cells using adenovirus vector [39]. HT29‐GFP clones were examined by flow cytometry and the clone with the highest GFP expression level was used in this study for imaging.…”

Label‐free discrimination and selection of cancer cells from blood during flow using holography‐induced dielectrophoresis

“…We imaged and analyzed three types of cancer cells and four types of blood cells. For cancer cells, HT29‐GFP cells were used [30]. HT29‐GFP are colon adenocarcinoma cells that have been transfected by adenovirus vector to express GFP and neomycin resistance gene.…”

Viral Vector-Based Melanoma Gene Therapy

“…Adenovirus Ad5 dsDNA Strong immunogenicity Broad host range [37,38] Brain [39] Colon [40,41] Esophaegal [42] Gastric [43] Liver [44] Lung [45] Adeno associated viruses AAV (2,3,5,6,8,9) ssDNA Slow expression onset Chromosomal integration [46][47][48][49] Brain [50] Breast [51] Liver [52,53] Lung [54] Prostate [55] Retinoblastoma [56] Alphaviruses SFV, SIN ssRNA Broad host range low immunogenicity [32] Brain [57] Colon [58] Lung [59] Melanoma [60,61] Osteosarcoma [62] Pancreas [63] Prostate [64] Herpes simplex virus HSV1, HSV dsDNA Broad host range Latent infection [65,66] Brain [67,68] Colon [69] Prostate [70] Sarcoma [71] Skin [72] Reoviruses [76,79,80] Breast [81] Gastric ...…”