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Study the therapeutic and preventive potential of targeting oncogenic mutations with CRISPR-Cas9 technology

Periodic Reporting for period 1 - Genetic Vaccine (Study the therapeutic and preventive potential of targeting oncogenic mutations with CRISPR-Cas9 technology)

Reporting period: 2019-04-01 to 2021-03-31

Lung cancer is the leading cause of death related to cancer in the world, accounting for 25.3% of all cancer deaths. Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancers, and adenocarcinoma is the most represented type among them (40%). The majority of lung cancers are caused by the accumulation of genetic alterations and the genes that are mostly mutated are EGFR (epidermal growth factor receptor) and KRAS (Kirsten rat sarcoma viral oncogene). Mutations in KRAS occur more frequently in lung adenocarcinomas (approximately 25%). When the KRAS gene is mutated, the Ras protein gets constitutively active, resulting in continuous cell proliferation. Most mutations in KRAS (approximately 80%) are located in a particular residue of the protein, therefore, we seek to prevent or disrupt such mutations in order to impede carcinogenesis and/or damage an established tumor.
We aimed first to develop a specific strategy to target KRAS mutations, and second, to evaluate its therapeutic and preventive potential.
We went through several strategies and designs to target the mutant KRAS versions while not interfering with the non-mutated protein. We were able to come up with a specific DNA-cutting tool which disrupts only the mutant versions of KRAS. This was very important because it enables a precise treatment of tumor cells.
Our DNA-editing tool showed therapeutic potential in in vitro assays, regardless the delivery mechanism used to introduce it into the tumor cells. Next, encouraged by those in vitro data, we test the in vivo tumor response using a model of xenotransplantation. Tumors engrafted in mice showed growth impairment when treated with our KRAS mutant targeting tool.
In addition, we generated a transgenic mouse carrying our validated gene-editing system to target the mutations of KRAS. The preventive value of the editing system will be interrogated using this engineered mouse with different oncogenic drives.
In conclusion, we successfully developed a gene-depletion system to specifically target oncogenic KRAS mutant alleles that led to significant tumor regression. These findings show the potential of gene-editing strategies for the treatment of tumors with driver gene mutations.
In this innovative proposal, we examined the therapeutic and preventive (work in progress) value of the DNA-editing system when destroying/avoiding predominant oncogenic mutations in lung cancer such KRAS mutations. Therapeutically, we see the regression of KRAS-dependent tumors and so, overcome the current limitations of conventional cancer therapies with their side effects caused by unselective cell killing methods. Our approach is compatible with delivering into both normal and cancer cells because cells that lack target sequences should not be edited and, therefore won’t have any adverse effect. Despite editing efficiency is not 100%, in a continuous presence context and using an efficacious delivery system, it would be expected to target the entire oncogenic DNA eventually.
Despite diagnostic and therapeutic advances, lung cancer remains highly lethal: only about 15 % of patients survive five years after diagnosis in developed countries. Currently, despite intensive effort, no effective anti-KRAS strategies have made it to the clinic. In the past 2-3 years there has been a huge effort in the pharmaceutical industry directed to develop KRAS-mutant inhibitors (G12Ci) and change the paradigm of the “undruggable” state of KRAS oncogenes. Some inhibitors are being tested in clinical trials and, even though they are showing encouraging results, they also raised some concerns such the developing of resistance. Importantly, G21Ci resistance mechanisms rely on the relentless presence of newly synthesized mutant KRAS. Conversely, our strategy induces the deletion of mutant KRAS at DNA level and the tumor cells wouldn’t be able to re-express the oncogenic protein.
The other main objective of this proposal is focused on developing a completely new angle not studied so far: its role at preventing the establishment of especially relevant mutations driving carcinogenesis in key oncogenes, thereby acting as a "genetic vaccine". Although this part of the project is being developed, the applications derived from this angle can be innumerable. For example, we can combine this technology with the advances in tissue engineering that have recently led to the development of lung tissue for in vivo implantation in order to create lung tissue resistant to mutations in KRAS or other oncogenes. In the future, we can use these “genetic vaccines” to both avoid the occurrence of especially carcinogenic mutations and to correct mutations in already developed tumors.
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