The new system "addresses the intractable problems of reactive oxygen species (ROS)-based SDT," Dr. Huixiong Xu of Tongii University and Drs. Yu Chen and Wei Feng of Shanghai University in China told Reuters Health in a joint email. These problems include the fact that cancer cells can activate antioxidant defense systems to counteract SDT.
SDT involves the sensitization of target tissues with a non-toxic sensitizing chemical agent, followed by exposure of the sensitized tissues to low-intensity ultrasound. Cancer cells are killed when the sonosensitizers trigger excessive production of reactive oxygen species.
The new study showed that CRISPR/Cas9 gene editing breached the cancer cells' defenses, enabling SDT to effectively shrink tumors in a mouse model of HCC.
"However, there are some obstacles to overcome before entering clinical trials," Drs. Xu, Chen and Feng said. "Firstly, due to the heterogeneity of tumors, different pathological types respond differently to the same therapeutic strategy, so we need to construct different tumor models - e.g., in situ tumors, metastatic tumors, recurrent tumors and even patient-derived xenografts models - to further evaluate the effectiveness of our therapeutic strategy."
"In addition, a more comprehensive investigation of the impact of different experimental parameters on the treatment in small (mice and rabbits) and big (pigs and primates) animals is needed," they noted. "This would include the power, duration, and frequency of ultrasound, as well as the concentration of the CRISPR/Cas9 nanosystem, among other factors."
Finally, they said, "One of the most crucial issues for the CRISPR/Cas9 system is the potential risk of being off-target. Although we have preliminarily evaluated it in mice, we also need to conduct a long-term, extensive study in big animals before the clinical trials, ensuring the biosafety of this proposed therapeutic strategy."
As reported in ACS Central Science, the team determined that cancer cells were inhibiting SDT efficacy by activating a gene called nuclear factor erythroid 2-related factor 2 (NFE2L2).
To address issue, the researchers designed a CRISPR/Cas9 system to knock down NFE2L2 and encapsulated the system and a ROS precursor molecule in lipid nanoparticles.
Subsequent experiments showed that the lipid nanoparticles were taken up by the HCC cells' lysosomes in vitro. ROS formation triggered by SDT ruptured the lysosomes, damaging certain cellular components and allowing the CRISPR/Cas9 system to enter the nucleus and knock down NFE2L2 gene expression. As a result, significantly more cancer cells died than without NFE2L2 gene editing.
The team then injected the nanoparticle treatment into mice with implanted HCC tumors. After 15 days of treatment with the new system, the tumors disappeared and did not recur. Comparative experiments showed that mice treated with SDT alone had fewer tumors than untreated mice, but the addition of the CRISPR/Cas9 system significantly improved the therapy's effectiveness. And, because the gene editing system works only in tumor tissues undergoing SDT, it will not cause mutations in healthy tissue, according to the researchers.
Summing up, the authors state, "This study provides a new paradigm for HCC management and lays the foundation for the widespread application of CRISPR/Cas9 with promising clinical translation, meanwhile developing a synergistic therapeutic modality in the combination of SDT with gene editing."
Dr. Satdarshan (Paul) Monga, Chief of Experimental Pathology Division, and Director of the Pittsburgh Liver Research Center at the University of Pittsburgh, commented on the study in an email to Reuters Health. "In its present form, the research is too preliminary to make any clinically relevant interpretations. It makes theoretical sense that knocking out antioxidant master regulator Nrf2 would have a positive impact on effectiveness of cancer cell killing effect of SDT."
HCC cells were grown subcutaneously for the tumor xenograft model, he noted. "This is a major limitation. There are many other animal models where cancer occurs in the liver, and proof-of-concept studies showing efficacy of this modality in those relevant models will be essential before making any big conclusions from the tumor xenografts."
"A major concern is that HCC occurs in the background of chronic liver injury," he added. "Antioxidant pathways like Nrf2 are also known to be activated in the peritumoral and chronically injured liver, showing inflammation and fibrosis, and are known to allow survival of remnant functioning hepatocytes."
"Hence, if the CRISPR/Cas9 targeting of NFE2L2 is more universally targeted, it could lead to enhanced liver injury," he noted. "On the other hand, gain-of-function mutations in NFE2L2 are also seen in a subset of human HCCs and in these cases, intratumoral inhibition of Nrf2 with CRISPR/Cas9-based strategies may a have notable impact independent of SDT."
SOURCE: https://bit.ly/3maKxOE ACS Central Science, online December 8, 2021
By Marilynn Larkin
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