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Updated: Mar 3, 2026
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Published on: September 9, 2010
Selective eradication of cancer cells by delivery of adenovirus-based toxins
Shiran Shapira1, Assaf Shapira2, Diana Kazanov1
1Laboratory of Molecular Biology, The Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center, Affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.
Background And Objective:
KRAS 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.
Results:
A 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.
Design:
Efficient 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.
Conclusions:
A proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.
Insights
This study developed a novel gene therapy targeting KRAS-mutated colorectal cancer by exploiting the hyperactive RAS pathway. The engineered adenovirus effectively induced cancer cell death and reduced tumor size with minimal side effects.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- KRAS mutations are early events in colorectal cancer (CRC) development.
- Previous attempts to inhibit the hyperactive RAS pathway in CRC have largely failed.
- Exploiting aberrant signaling pathways offers a novel therapeutic strategy.
Purpose of the Study:
- To develop a gene therapy that exploits the hyperactive RAS pathway in KRAS-mutated colorectal cancer.
- To enhance therapeutic efficacy by using the bacterial MazF-MazE toxin-antitoxin system.
- To ensure tight regulation and safety of the gene delivery system.
Main Methods:
- Construction of efficient adenovirus vectors for targeted gene delivery of the MazF-MazE system.
- In vitro assessment of cell death using microscopy, colony formation assays, MTT, and FACS analysis.
- In vivo evaluation of tumor growth inhibition in a xenograft model.
Main Results:
- The MazEF-encoding adenovirus induced massive, dose-dependent cell death in KRAS-mutated cells (73% at MOI 10) compared to wild-type cells (22%).
- Increased expression of the anti-toxin MazE confirmed the system's selectivity, specificity, and safety.
- Significant tumor shrinkage (61%) was observed in vivo with no reported side effects.
Conclusions:
- A proof-of-concept for a novel, safe, and effective gene therapy for colorectal cancer is demonstrated.
- Exploiting aberrant hyperactive pathways presents a viable strategy for cancer treatment.
- The MazF-MazE toxin-antitoxin system delivered via adenovirus shows promise for targeted cancer gene therapy.
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