Efficacy of CRISPR-Cas9 Gene Editing for Targeting KRAS Mutations in Pancreatic Cancer: A systematic review

doi:10.21203/rs.3.rs-5001819/v1

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Efficacy of CRISPR-Cas9 Gene Editing for Targeting KRAS Mutations in Pancreatic Cancer: A systematic review

https://doi.org/10.21203/rs.3.rs-5001819/v1

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Background

Pancreatic cancer is one of the main causes of cancer-related deaths, especially pancreatic ductal adenocarcinoma and it’s characterized by a poor prognosis. The KRAS gene mutation is prevalent in about 85% of pancreatic cancer cases which is a significant factor in the pathogenesis and development of pancreatic cancer, impacting cellular tumor growth, survival, and metastasis. The targeted disruption of mutant KRAS variants through the application of various CRISPR systems has led to a marked reduction in cell viability and proliferation in vitro, accompanied by significant inhibition of tumor growth in vivo.

Method

This systematic study was adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and involved a comprehensive search of PubMed, Google Scholar, and PLOS One for original research articles published up to June 2024. Studies included those involving CRISPR-Cas9 gene editing targeting KRAS mutations in human or animal models of pancreatic cancer. Data collection and quality assessment were performed independently by two reviewers.

Results

The review identified numerous studies demonstrating the efficacy of CRISPR-Cas9 in targeting KRAS mutations. Results showed significant reductions in KRAS transcript levels, decreased tumor progression, and improved survival rates in experimental models. However, challenges such as off-target effects and efficient delivery methods were noted.

Conclusion

CRISPR-Cas9 gene editing shows significant promise as a therapeutic strategy for targeting KRAS mutations in pancreatic cancer. While the technology has demonstrated potential in preclinical studies, further research is needed to address challenges related to specificity, delivery, and long-term effects to facilitate its clinical application.

Pancreatic cancer

PDAC

KRAS mutations

CRISPR-Cas9

gene editing

systematic review

Pancreatic cancer is considered as a global health burden and is expected to become one of the leading causes of cancer-related mortality in the future (1). The average life expectancy of pancreatic cancer patients is about 1 in 56 in men and about 1 in 60 in women. But each person’s chances of getting this cancer can be affected by variable risk factors (2). Among many subtypes of pancreatic cancer pancreatic ductal adenocarcinoma (PDAC) remain the most common with 5-year survival rate of 10% (3).

The RAS gene was initially identified as a viral gene homologous to the transforming gene from the Kirsten rat sarcoma virus (4), (5). The KRAS protein, also known as p21, is a member of the Ras superfamily, found on human chromosome 12 and encoded by 189 amino acids (6). Numerous studies confirmed that the involvement of KRAS mutations in about 85% of pancreatic cancer tumors, affecting various cell processes such as enhanced migration, invasion, survival, and proliferation, while modulating cell metabolism by increasing glucose uptake, switching from aerobic glycolysis to mitochondrial oxidative phosphorylation (known as the Warburg effect), and increasing the generation of lactate and reactive oxygen species (7) (8).

The management of pancreatic cancer involves a broad-based approach, incorporating surgical, chemical (chemotherapy), and radiation therapies to maximize patient outcomes (9), (10), (11). Despite these efforts the management of pancreatic cancer might be devastating due to the late presentation of the disease and poor prognosis (12), (13).

With these barriers to treating pancreatic cancer, a new approach in molecular biology has emerged, introducing advanced technologies for treatment. Among these approaches’ gene editing has shown a promising method (14).

Genome editing technologies first introduced in 1990s. More recently, a new genome editing tool called CRISPR, invented in 2009, it has simplified the editing of DNA like never before. CRISPR is more efficient, quicker, less costly, and more accurate than older genome editing techniques. Many scientists currently utilize CRISPR for genome editing, with a significant amount of research focused on targeting genetic mutations involved in pancreatic cancer. This has opened new avenues for potential treatments and therapies for this aggressive disease (15).

The first clinical trial for treating cancer using CRISPR was done in West China hospital in 2016 (16). This non-randomized, open-label phase I study evaluates the safety of ex vivo PD-1 knockout engineered T cells using CRISPR/Cas9 in treating metastatic non-small cell lung cancer (17). While promising, the labor-intensive process of genetically modifying and infusing T-cells raises doubts about its clinical preference over PD-1 or PD-L1 antibodies. The study highlights the potential for combining PD-1 knockout with other targets and mentions ongoing and future trials, including those using CAR T-cells and in vivo genome editing approaches (18), (19).

In conclusion pancreatic cancer remains challenging with low survival rates, especially with coexisted KRAS mutations. Emerging CRISPR-based genome editing offers promising new promise for targeted therapies, potentially transforming treatment strategies. While early trials have focused on other cancers, ongoing research underscores CRISPR/Cas9's potential to enhance pancreatic cancer management, necessitating further investigation for clinical application.

Study Design

To evaluate the efficacy of CRISPR-Cas9 gene editing for targeting KRAS mutations in pancreatic cancer, we conducted a systematic review of original articles.

Search Strategy

This review adhered to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and is reported according to the PRISMA statement 2020. We conducted comprehensive searches in PubMed, Google Scholar, and PLOS One to identify relevant articles up to June 2024. Keywords for the search were developed with input from the contributing authors and included the following Boolean search terms: "CRISPR-Cas9" AND "KRAS" AND "Pancreatic Cancer" AND ("Efficacy" OR "Effectiveness"). Additional searches were performed manually by reviewing the reference lists of relevant articles.

Inclusion Criteria

We included studies if they met the following criteria. (1) The study had to be an original research article. (2) The publication date had to be up to and including June 2024. (3) We considered randomized controlled trials (RCTs), non-randomized controlled trials (non-RCTs), and observational studies. (4) The population needed to involve human or animal models of pancreatic cancer with KRAS mutations. (5) The intervention had to use CRISPR-Cas9 for gene editing targeting KRAS.

Exclusion Criteria

We excluded studies based on the following criteria. (1) The study design was a review, commentary, case report, or cross-sectional study. (2) The study was unpublished or non-peer-reviewed. (3) The article was not accessible in full text. (4) The article was published in a language other than English.

Data Extraction

Two reviewers independently extracted data from the included studies using a standardized form. Extracted data included study characteristics (e.g., study design, location), population details (e.g., demographics, KRAS mutation status), intervention specifics (e.g., CRISPR-Cas9 delivery method, target sequences), and outcomes (e.g., efficacy measures, off-target effects, safety). Any discrepancies between the reviewers were resolved through discussion and consensus.

Quality Assessment

We conducted a quality assessment of each included study based on predefined criteria derived from established guidelines for evaluating genetic research. Criteria included the methodological strength of the experimental design, accuracy of gene editing, and assessment of off-target effects. Each study was scored on a scale from 0 to 8 to quantify its methodological quality (Fig. 2 and Fig. 3).

Data Synthesis

We carried out a qualitative synthesis to thoroughly evaluate how effective CRISPR-Cas9 gene editing is at targeting KRAS mutations in pancreatic cancer. This process involved summarizing each study’s goals, methods, and findings, with a particular focus on the specifics of the CRISPR-Cas9 intervention and the outcomes reported. We looked for themes like the efficiency of gene editing, the impact on tumor progression, and safety concerns, using thematic analysis to identify these. By comparing the results of different studies, we highlighted both consistent findings and areas where results varied, which helped us develop a thorough understanding of how effective CRISPR-Cas9 is. We used the GRADE approach to assess the overall strength of the evidence to ensure it was comprehensive.

Study selection

We identified 98 articles from different electronic databases and additional 3 from other sources. After removing duplicates, 51 articles proceeded to the primary screening stage. 18 articles were excluded in title and abstract screening and 5 articles were excluded through full text screening. Remaining 11 studies were included in the qualitative synthesis, with 6 of these studies further included in the quantitative synthesis. Figure 1, PRISMA flow diagram summarizes the process.

Study characteristics

This systematic review included 6 clinical trials conducted between 2016 to 2023 and 3 studies were carried out in the USA, 2 in China, and 1 in Germany. The included studies were primarily preclinical trials involving in vitro and in vivo models (e.g., cell lines, mouse models). Table 1, shows the baseline characteristics of the included articles.

CRISPR-Based Tumor Induction and Metastasis Tracking

In one study a CRISPR-Cas9-based system called macsGESTALT was used as an evolving barcode to track the evolution of pancreatic ductal adenocarcinoma (PDAC) cells (Table 2 and Table 3). The study revealed that only a small fraction of cancer cell clones contributed to tumor growth and metastasis, with the M1.1 and M2.2 clones being particularly dominant in distant metastatic sites (Table 4). Clones in late-hybrid epithelial-to mesenchymal transition (EMT) states, like M1.1, exhibited higher metastatic potential. Additionally, the M2.2 clone showed elevated expression of S100 family genes, suggesting a role in metastasis (1). Table 5 presents the detailed outcomes.

Another study demonstrates that how efficiently CRISPR/Cas9 induces pancreatic cancer through multiplexed gene targeting and electroporation, leading to rapid tumorigenesis (Table 6). (3) Additionally, it enabled precise tracking of metastases, highlighting its effectiveness in generating and analyzing complex cancer models, highlighting its utility in creating and studying intricate cancer models (Table 7).

Efficacy of CRISPR system in PDAC suppression

Different methods were used to deliver CRISPR-Cas9 into target cell including exosome, electroporation and viral vectors in the included articles (Table 8).

Exosome-mediated delivery of CRISPR/Cas9 plasmid DNA effectively targeted KrasG12D in pancreatic cancer cells, leading to significant suppression of KrasG12D mRNA and reduced cell proliferation in vitro. Similarly, MSC exosomes loaded with Cas9/KrasG12D sgRNA1 resulted in a reduction in tumor volume, weight, and burden in the mouse model (2).

The AAV8-CasRx-gRNA system effectively targeted and silenced mutant KrasG12D in PDAC tumors, reducing Kras mRNA levels by 50% and significantly suppressing tumor growth in mouse models. Mice treated with AAV8-CasRx-gRNA had a 33% increase in survival and showed decreased tumor metabolic activity (Table 9 and Table 10). The system also enhanced sensitivity to Gemcitabine. In patient-derived xenografts harboring KRAS G12D mutations, this approach significantly slowed tumor growth (4). Table 11, summarizes the study’s findings (Table 9).

The CRISPR-Cas13a system, using crRNA19-14, specifically targeted KRAS-G12D mRNA, achieving up to 94% reduction in mRNA levels and 70% suppression of tumor growth in pancreatic cancer cell lines in vitro. Similarly, in mice, it significantly inhibits tumor growth, reduces proliferation, and increases apoptosis (5).

In another study, CRISPR/Cas9 was used to identify genes that affect sensitivity to CDK4/6 inhibitors in KRAS-mutant PDAC cell lines. The screen revealed key genes involved in PI3K–AKT–mTOR signaling, cell-cycle regulation, and DNA damage repair that modulate response to these inhibitors. Validation showed that targeting genes like CDK2 and BAP1 increased inhibitor efficacy (Table 12). Additionally, CRISPR findings guided the use of synergistic drug combinations, such as SRC and HDAC inhibitors, to enhance CDK4/6 inhibitor effectiveness, providing new insights for improving treatment outcomes (6). Table 4 summarizes the finding of this study.

Figure 1: The flow chart of studies selection

Figure 2. Risk of bias graph

Figure 3. Risk of bias summary

This systemic review, emphasizes how pancreatic cancer, a deadly illness with few available treatments, may benefit from CRISPR-Cas9 gene editing to target KRAS mutations. Promising outcomes from the included studies include effective editing of KRAS mutations, inhibition of tumor growth and proliferation, and higher rates of animal model survival.

Considering the difficulties with conventional treatments, the effectiveness of CRISPR-Cas9 in addressing KRAS mutations is noteworthy. KRAS mutations are frequently found in pancreatic cancer, and their existence is frequently linked to a worse prognosis and treatment resistance (1, 2).

The included researches used a range of delivery techniques, such as exosomes, electroporation, and viral vectors. Exosome administration has been shown to significantly decrease tumor development and boost survival rates, indicating that this strategy could be a practical and effective way to target KRAS mutations.

Nevertheless, the existing body of evidence has flaws and limits. To close these knowledge gaps and maximize the application of CRISPR-Cas9 in the therapy of pancreatic cancer, more research is required. To be more precise, additional in vivo research is needed to completely comprehend the safety and effectiveness of CRISPR-Cas9 in animal models (4, 5).

Additionally, in order to use these discoveries in the clinic, human clinical trials are required. It's also important to consider the possibility of CRISPR-Cas9 combination therapy. The potential for combination therapeutic methods is shown by the synergistic effect of CRISPR-Cas9 and CDK4/6 inhibitors in decreasing tumor development and enhancing survival rates (7).

The review also emphasizes how crucial it is to optimize the design and delivery of CRISPR-Cas9 guide RNA (gRNA) in order to target KRAS mutations in an effective and targeted manner. Reducing off-target effects and improving gRNA design can be achieved through the use of in vitro validation assays and computational techniques (1, 5).

The review also emphasizes the necessity of more investigation into the effectiveness and safety of CRISPR-Cas9 in human subjects. Although the included studies show encouraging outcomes, issues with immunogenicity, mosaicism, and off-target effects remain.

For CRISPR-Cas9 to be successfully translated into the clinic, these issues must be resolved. Subsequent research endeavors have to concentrate on refining CRISPR-Cas9 delivery strategies, investigating combination treatments, and resolving safety issues (4).

The review also highlights how crucial it is to take the tumor microenvironment into account and how it affects the effectiveness of CRISPR-Cas9. Treatment resistance and the advancement of cancer are significantly influenced by the tumor microenvironment.

It is crucial to comprehend the interactions between CRISPR-Cas9 and the tumor microenvironment in order to design successful treatment plans. The review also emphasizes the need for more investigation into the possibility that CRISPR-Cas9 could be used to target additional carcinogenic mutations in pancreatic cancer (3).

One major advantage of CRISPR-Cas9 over conventional treatments is its capacity to address many mutations at once. Improved patient survival and more successful treatment results could result from this strategy.

To fully grasp the therapeutic potential of CRISPR-Cas9 in the treatment of pancreatic cancer, additional study is necessary. CRISPR-Cas9 may provide pancreatic cancer patients new hope with more research and development.

This systemic review concludes that CRISPR-Cas9 gene editing has potential as a targeted treatment for pancreatic cancer. Notwithstanding certain restrictions and gaps in the available data, the results imply that CRISPR-Cas9 might be a useful therapeutic option for this incurable illness (2).

To fully grasp CRISPR-Cas9's therapeutic potential in the treatment of pancreatic cancer, more study is required. Treatment for pancreatic cancer appears to have a bright future, and CRISPR-Cas9 may be essential to bettering patient outcomes.

In conclusion, while the results of this review highlight the promising role of CRISPR-Cas9 in the treatment of KRAS-mutant pancreatic cancer, significant challenges remain before this technology can be safely and effectively implemented in clinical practice. Continued research, particularly in optimizing delivery methods and minimizing off-target effects, will be essential in translating these preclinical successes into tangible clinical benefits.

collectively support the potential of CRISPR-Cas9 as a viable therapeutic strategy for targeting KRAS mutations in pancreatic cancer. The consistent efficacy in tumor suppression, combined with the generally low incidence of off-target effects, positions CRISPR-Cas9 as a promising candidate for clinical development. However, the variability in delivery systems and the need for long-term safety assessments are critical factors that must be addressed in future studies to ensure the successful translation of this technology into clinical practice.

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and materials: All data generated or analysed during this study are included in this published article

Competing interests: The authors declare that they have no competing interests

Funding: No funding was received.

Authors' contributions:

Hashim Talib Hashim: Conceptualized the study, designed the systematic review and meta-analysis protocol, and supervised the research process.

AQMA: Conducted the literature search, data extraction, and initial data analysis.

TF: Performed statistical analyses and contributed to the interpretation of the results.

TH: Reviewed and selected studies for inclusion, and ensured the accuracy of data extraction.

FAS: Assisted in the literature search, data extraction, and provided critical revisions of the manuscript.

MGM: Contributed to the design of the data extraction forms and assisted in the critical appraisal of studies.

MAA: Participated in data extraction, manuscript drafting, and formatting.

RAK: Reviewed the manuscript for important intellectual content and contributed to the writing process.

ADA: Assisted in statistical analysis, data interpretation, and manuscript preparation.

AA: Provided expertise in ophthalmology, reviewed the clinical aspects of the included studies, and contributed to manuscript revisions.

All authors read and approved the final manuscript.

Acknowledgements: None

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Tables 1 to 12 are available in the Supplementary Files section.

No competing interests reported.

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Efficacy of CRISPR-Cas9 Gene Editing for Targeting KRAS Mutations in Pancreatic Cancer: A systematic review

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Abstract

Background

Method

Results

Conclusion

Figures

Introduction

Methodology

Study Design

Search Strategy

Inclusion Criteria

Exclusion Criteria

Data Extraction

Quality Assessment

Data Synthesis

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1