Scientists at the ChristianaCare Gene Editing Institute have tried to improve the safety and efficacy of CRISPR gene editing technology in patient treatments by demonstrating how to identify and evaluate the effects of gene regulation and gene knockout on target tissues. The work, recently published in Gene Therapy, is the result of the use of CRISPR systems to regulate a major regulatory gene, preventing it from expressing a protein that reduces the effectiveness of chemotherapy in patients.
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"We found that when USING CRISPR for gene editing, sometimes it changes rather than completely silence the expression of the gene of interest, so we developed a program to more fully understand the effects of this mechanism on patients," explains Dr. Eric Kmiec, executive director and chief scientific officer of the ChristianaCare Gene Editing Institute and lead author of the study.
Referring to the lung cancer study his team, Dr. Kmiec said, "Our study found that even though CRISPR genetic manipulation did not completely disable the target gene, it changed the expression of the target gene, making lung cancer tissue more sensitive to chemotherapy."
Use CRISPR to validate lung cancer studies
"We were fortunate to find that our strategy for using CRISPR to improve lung cancer treatment was once again validated." He added. "However, we still want to make an objective assessment of our study, also to highlight the importance of using CRISPR to knock out specific genes for all the potential consequences." Specifically, anyone developing CRISPR therapy needs to pay attention to edits that don't completely knock out the entire piece of coded DNA and assess their potential impact on patients. These results may be as positive as our situation, or they can be negative or neutral, but they need to be fully understood. ”
Many exciting medical applications for CRISPR involve using this tool to knock out a specific DNA sequence or harmful gene, causing it to lose its original function. But more evidence suggests that CRISPR-edited cells may simply alter the expression form of the target DNA sequence, and that these target genes can still continue to produce biologically active proteins.
Gene editing | clbiomed.com
Scientists at the Gene Editing Institute are investigating the possibility of using CRISPR to silence a gene called NRF2, which can express a protein that can block squamous cell carcinoma tissue from chemotherapy or radiation therapy. In tumor cell experiments and animal studies, it has been demonstrated that these effects do not interfere with healthy genes in normal cells and are targeted and selective for the NRF2 gene.
In order to build on the current research. Scientists want to fully understand how CRISPR gene editing allows the NRF2 gene to retain enough DNA code and continue to produce deteriorated versions of the protein. The research team is laying the groundwork for a clinical trial that will use CRISPR technology to improve the efficacy of conventional chemotherapy and radiation therapy. Dr. Kmiec said that before the trial moves forward, he wants his team to develop a clear process for identifying and evaluating all the results of CRISPR edits.
Identifying and understanding the diversity of product outcomes in the gene editing process has been a core basic research project since the establishment of the gene editing institute.
Use CRISPR safely
Dr Kmiec said: "We conduct our experiments in an objective and unbiased manner, with patient safety and treatment effectiveness as the guiding direction of our scientific research efforts. "Whatever we find or elucidate, they will help the ChristianaCare Institute and the field as a whole to use CRISPR in a safer and more efficient way." ”
The researchers found in multiple cells that the strands of the target DNA code in the NRF2 gene were not completely destroyed. Instead, after CRISPR editing, some cells that retained enough of the original code and could continue to produce another form of protein emerged. Trials have shown that cancer tumor cells that produce these mutated proteins may be more vulnerable to chemotherapy drugs.
Altered NRF2 transcripts are detected by smFISH imaging | References[1]
Dr Kelly Banas, lead author of the paper, said: "For our work around NRF2, the abridged version of the protein produced by CRISPR editing seems to make tumor cells more sensitive to treatment. But the point is that these proteins are clearly biologically active, which means we need to determine their potential impact on the safety and efficacy of using CRISPR to treat lung cancer patients. ”
Dr. Banas notes that the study points to the limitations of the detection criteria that would otherwise have been considered successful in CRISPR editing simply by confirming that the original protein form of interest was missing. If this standard is followed, she says, their gene editing is successful. The edited NRF2 gene no longer produces the original form of the protein. But she adds that if that's what the ChristianaCare team is after, they'll ignore the mutated proteins produced by the NRF2 gene alterations, and thus ignore an important outcome. In this case, this result refines the original hypothesis and experimental approach of using CRISPR-targeted editing of the NRF2 gene to improve treatment outcomes in lung cancer patients.
The importance of due diligence
Dr Kmiec said: "The process we described in this study should serve as a template that should be followed in any effort to treat CRISPR as a drug. As practitioners in healthcare facilities, ensuring patient safety is our top priority. We are also playing a pioneering role in the exciting field of cutting-edge medicine, where if rigorous due diligence is not performed, it has the potential to set the field back decades. Through this research, we validated a process that could help the field move forward quickly and safely. ”
CRISPR stands for "regularly spaced clusters of short palindrome repeats," a defense mechanism found in bacteria that recognizes and excises viral DNA sequences that invade viruses. Scientists have learned how to modify this mechanism so that it can be directed to "edit" specific DNA coding sequences.
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[2] https://www.eurekalert.org/news-releases/946824
Compiler: Yin Chen
Edit: Crispy fish
Typography: Yin Ningliu
Title image source: Stand Cool Helo
Research team
Corresponding author Eric B. Kmiec: Known for his pioneering work in molecular medicine and gene editing. Since 2014, he has been the director of the Institute of Gene Editing. Throughout his career, Dr. Kmiec has led research teams that study the reaction mechanisms, biochemistry, and molecular genetics of gene editing in human cells. His early work on sickle cell disease led to the research and development of a new generation of gene-editing tools, including CRISPRs and more promising variants such as single-stranded DNA oligonucleotides (ssODNs) for the treatment of genetic diseases.
Lead author Kelly Banas: Researcher whose research interests are in developing and evaluating novel gene-editing strategies for non-small cell lung cancer. Her current focus is on targeting cancer-specific NRF2 mutations with CRISPR gene editing tools and evaluating the short- and long-term effects of these specific targeted strategies on affected and neighboring cells. Kelly's discovery of a unique CRISPR target within tumor cells that does not exist in normal cells surrounding tumors establishes an innovative foundational approach to treating solid tumors through CRISPR-guided gene editing. She recently presented her major findings to the U.S. Food and Drug Administration, and her findings led to the development of a clinical protocol. She graduated from the University of Delaware in 2021 with a Doctor of Molecular Medicine degree.
Research group website
https://christianacare.org/people/eric-kmiec-phd/
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