Oncolytic viruses (OVs) are natural or recombinant viruses that prefer to infect cancer cells. This feature has made them one of the main current research topics on cancer therapeutics and the significant element in the future of cancer treatment. Over the past two decades, there has been growing evidence that OVs are effective in treating cancer in both preclinical models and clinical trials. The most tested OVs in preclinical and clinical trials include the Herpes simplex virus (HSV), adenovirus (AdV) and Vaccinia virus (VV). In one study, an oncolytic HSV (T-VEC) coding for granulocyte/macrophage-colony stimulating factor (GM-CSF) was administered by direct intratumoral injection to patients with metastatic malignant melanoma and this resulted in complete regressions of injected and uninjected lesions in eight of 50 patients. In addition to the single therapy, T-VEC has also been used in combination with radiotherapy and cisplatin in clinical trials to treat stage III/IV head and neck cancer. An oncolytic vaccinia virus (JX-594) armed with GM-CSF, showed hopeful results in preclinical and clinical trials treating liver cancers.
Methods for Editing the Oncolytic Viruses Genome
Viruses have become crucial potential vectors for cancer therapy. The conventional method for generating tumor-targeted OVs with large genomes (such as HSV, adenovirus, and vaccinia virus) is based on homologous recombination by a shuttle vector, either in bacteria or in mammalian cells. Currently, three major established methods are used to edit the adenovirus genome: bacteria-based homologous recombination system, hybrid yeast–bacteria cloning system and bacterial artificial chromosome (BAC) system. These systems require laborious multi-step methods with low efficiency. Thus, it would be beneficial to develop a more efficient and straightforward method for editing large viral genomes to construct mutant or recombinant DNA viruses.
The discovery of the CRISPR system as the adaptive immune system of bacterial cells and the advanced idea of using Cas9-gRNA molecular complex for specific targeting of any genomic area have made genome editing much more feasible than before. Since its initial application in human cells in 2013, CRISPR-Cas9 has been adapted for genomic editing in mammalian cells and this has been utilized in many ways. For example, CRISPR-Cas9 has been used to generate genetically engineered mice for the study of human diseases and for the induction of genomic alterations in plants, Drosophila, zebrafish, Caenorhabditis elegans, and yeast. Recently, it has been successfully employed in manipulating the genomes of various viruses, including HSV, adenovirus, and vaccinia virus.
CRISPR-Cas9 System Induces High Efficiency of Homologous Recombination
Generation of mutant vaccinia viruses mainly depends on homologous recombination to delete a particular target gene, or to arm the virus with a gene in the target region. DNA double-stranded breaks can effectively induce homologous recombination in mammalian cells and this mechanism can be harnessed to improve efficiency in the generation of VV mutants. The CRISPR-Cas9 system has been successfully applied to improve homologous recombination in eukaryotic organisms. Besides, the CRISPR-Cas9 system has been used to generate mutant viruses with greater efficiency including HSV1, AdV and VV mutants. This system can induce homologous recombination with efficiencies up to 2%-3% in introducing the DsRed gene into the AdV genome, where whole-genome sequencing of mutant AdV showed the incorporation of the DsRed gene into the target region and also that no off-target mutations were induced. Moreover, the results demonstrated that a nickase guided by a single gRNA combined with a repair donor DNA can generate more precisely targeted viral mutants.
Create Mutant Oncolytic VV by CRISPR-Cas9 System
Oncolytic viruses can be developed by attenuating and modifying viruses or arming the virus with a therapeutic gene. The deletion of the thymidine kinase (TK) gene in VV increases the selectivity so that the virus replicates preferentially in cancer cells. Thus, using an oncolytic virus or a virus as a vector for vaccines has better security. The TK gene was replaced with the RFP gene by the CRISPR-Cas9 system with a greater than 90% rate in the RFP positive plaques, thus making the CRISPR-Cas9 system an attractive method for modification of VV. Indeed, the A46R deletion and N1L deletion viruses were created with high efficiency by the CRISPR-Cas9 system as well. A mutant vaccinia virus with a deletion in specific region can be easily created with the incorporation of a reporter gene in the presence of a combination of repair donor DNA, gRNA and Cas9. It is undoubtedly that the CRISPR-Cas9 system will play a large part in the development of future generation oncolytic viruses, as well as basic research in viral biology.
The CRISPR-Cas9 system has been used successfully in creating mutations with high efficiency in the target region of HSV-1 and AdV, which indicates the CRISPR-Cas9 can be an attractive approach to generate oncolytic viruses. QVirusTM Platform, a division of Creative Biogene, can offer various oncolytic virus production services using the CRISPR-Cas9 system to accelerate your cancer immunotherapy projects. If you have any special requirements, please feel free to contact us.
References
- Yuan M, et al. CRISPR-Cas9 as a powerful tool for efficient creation of oncolytic viruses. Viruses, 2016, 8(3): 72.
- Rezaei R. A Cas9-based Oncolytic Virus (OnCas).
- Biagioni A, et al. Delivery systems of CRISPR/Cas9-based cancer gene therapy. Journal of biological engineering, 2018, 12(1): 1-9.
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