How can CRISPR Cas9 edit various mutations present in gynaecological cancers?

Supervised by: Yuhui Zhou, BA (Hons). Yuhui is a 5th year medical student at the University of Cambridge. She gained a First class degree in her intercalated year studying Pathology. She has an interest in Cancer & Immunology and has been awarded a Wellcome Trust Biomedical Vacation Scholarship to study host responses to infection.

Abstract

Since all cancers can be characterized by genetic mutations that result in an uncontrollable growth rate, scientists have experimented with CRISPR/Cas9 in in-vitro studies, by applying the technology to various cancer cell lines with the aim of restoring mutated genes. In this study, we examined the efficacy of CRISPR/Cas9 in restoring two commonly mutated genes in reproductive cancers: TP53 and BRCA1/2. Previous studies found that the restoration of wild-type TP53 mutation by repairing that 414deC mutation was reported to increase p53 mRNA expression of target genes, in concordance with an increase in apoptosis rates among PC-3 prostate cancer cell lines. Targeting HER2 which is positive mostly in BRCA2 carriers, via exons 5, 10 and 12 led to a decrease in cell proliferation and an increase in apoptosis. BRCA1 knockdown adipose stem cells were also found to result in a secretome that promoted an increase in breast cancer cell proliferation and invasion. These observations suggest that CRISPR/Cas9 has the potential to result in a phenotype that inhibits cancer cell proliferation and could therefore serve as a potential therapeutic strategy for reducing or eliminating tumors. However, CRISPR technology has not yet been tested in vivo since scientists are still experimenting with different CRISPR/Cas9 vectors that could maximise the efficiency of CRISPR/Cas9 mediated repair.

Introduction

Advances in the field of genetics have led to a deeper understanding of cancer’s pathogenesis over the years, whereby identification of genetic mutations and characterization of mechanisms involved at the genomic level can lead to better targeted therapies devoid of lethal side effects (Ahmed et al., 2021). The main focus of this literature review is to discuss how effective CRISPR Cas9 is to alter genetic mutations in reproductive cancers. CRISPR Cas9, short for clustered regularly interspaced short palindromic repeats, along with the associated protein 9, is an effective and inexpensive way to genetically modify an organism’s DNA (Medicine plus et al 2022). Since cancer is one of the leading causes of deaths in this world, this new technology with the ability to edit cancerous cells easily has caused a stir amongst doctors and scientists alike. 

The two key mutations that are discussed in this article are TP53 and BRCA1/BRCA2. TP53 mutations in certain cancers not only induce a loss of p53 tumor suppressive functions, but also confer a gain of function, promoting tumorigenesis. BRCA1 and BRCA2 were identified in the 1990s as the causative genes underlying hereditary breast and ovarian cancer syndrome. BRCA1/2 are breast cancer susceptibility genes that are involved in DNA repair, and transcriptional control. They are dysregulated in breast cancer, making them attractive therapeutic targets that can act as prognostic biomarkers in breast cancer (Yi Jin et al., 2022). 

This literature review is divided into 4 sections; an abstract, an introduction, a discussion and a conclusion. The discussion will include a detailed explanation of the TP53 mutations and the BRCA1 and BRCA2 genes. Another section of the discussion will be dedicated to CRISPR Cas9 and how it can modify mutations and genes to help specifically cure reproductive cancers.

Discussion

CRISPR Cas9

CRISPR is a naturally existing genome editing mechanism that bacteria commonly employ as a natural immunological response and is the basis of the CRISPR Cas9 genome editing system. At specific sites in the genome, the system enables the insertion, deletion, or modification of genetic material. A section known as a CRISPR array is made when bacteria take small fragments of the viral DNA and insert them into their own DNA in a certain sequence so they would be able to remember the virus in the event of a subsequent attack. The bacteria would then be able to create RNA segments from CRISPR arrays that can recognize and bind to particular sections of the viral DNA. The virus is then rendered inoperable by the bacteria’s employment of Cas9 or a related enzyme to split the DNA, so that it cannot be translated (Medicine Plus, 2022). 

The CRISPR Cas9 genome editing system was then developed and by Jenniffer Doudna and Emmanuelle Charpentier, earning them the Nobel prize in Chemistry in 2020 (Zhang et al., 2021). 

CRISPR can be used to treat cancer by changing the activity of oncogenes, which are mutated genes with the ability to cause cancer. For example, CRISPR Cas9 mediated knockdown of CD133 reduced vimentin expression in colon cancer cells, cell proliferation and colony formation. It also significantly inhibited cell migration and invasion. Other studies also prove that the CRISPR/Cas9 system is a powerful tool to identify and target oncogenes. (Zhang et al., 2021). 

Using this same technique, other mutated genes such as TP53 and BRCA1/2 can be modified. For example, TP53, a common mutated tumor suppressor gene that can lead to human cancer, usually gains oncogenic features that stimulate tumorigenesis. In prostate cancer cells that express TP53 mutants the wild type TP53 genotype and phenotype can be restored by replacing the TP53 414delC frameshift mutation locus with a functional copy (Zhu et al., 2020). BRCA1 is a common tumor suppressor gene expressed in breast and ovarian tissues. When the cysteine is mutated, this results in changed functions, including a decrease in ubiquitin ligase activity, which can increase cancer risks (Ahmed et al., 2021). 

The type II (2) Cas system is most commonly used for genome editing. The mature crRNA joins with a tracerRNA, which is complementary to the CRISPR sequence, (both part of a gRNA). crRNA base pairs at the target site and a double strand break is induced in the complementary DNA by Cas9’s nucleus domain while an RuvC-like nucleus domain breaks the non-complementary strand. Researchers can then edit the DNA by replacing a part of the DNA with a customized sequence (Zhang et al., 2021).

TP53 and Gynaecological Cancers

P53 protein, which is a tumor suppressor protein, plays a vital role in preventing cancer. Wild-type p53 protein can normally be found in cells in an inactive form. When there is damage in the cell, the p53 protein induces cell-cycle arrest and initiates apoptosis. If the damage to the cell is repairable, the cell can undergo repair instead of apoptosis at the end of the cell cycle. Therefore production of the cells that are irreparably damaged in the body can be stopped with the help of p53 protein. Other functions of the p53 protein also include promoting genomic stability, exerting anti-angiogenic effects, controlling tumor inflammation and immune response, repressing metastases, etc. If the p53 protein goes through mutation and loses its function, the cells that are damaged continue to divide uncontrollably and therefore cancer occurs. Normally, p53 is regulated through its interaction with MDM2 which is its negative regulatory partner, which exports p53 to the cytoplasm to be degraded by E3-ubiquitin ligases (Duffy et al., 2022). The activated p53 has many effects on gene expression like the transcriptional activation of p21, which is an inhibitor of different cyclin/cyclin-dependent kinase complexes such as BCL2 survival proteins (Psyrri et al., 2007). p53 also binds to apoptotic initiators such as PUMA and NOXA (Duffy et al., 2022). 

A study made by Malkin shows that people with Li-Fraumeni syndrome that carries TP53 mutations have a high risk of developing early onset cancers (Malkin et al., 1990). TP53 gene is inactivated in half of the sporadic cancer (Hollstein et al., 1991). Mutations in the TP53 gene are associated with many types of cancers, especially gynaecological cancers. TP53 mutations are the most frequent genetic alterations in breast cancer and are observed in 30% of breast carcinomas (Bertheau et al., 2013). Also mutations in TP53 gene occur in 70-80% of cases of triple-negative breast cancer. The mutation in the p53 protein includes 96% of high-grade serous ovarian cancer. Mutant p53 promotes epithelial ovarian cancer by regulating tumor differentiation, metastasis, and responsiveness to steroid hormones (Ren et al., 2016). P53 protein is mutated and altered in 50% of advanced ovarian cancer cases. 

TP53 mutations can happen in different ways. One of the most common somatic aberrations is point mutations, followed by small deletions and insertions. Most of the missense mutations in breast and ovarian cancers are found in exons 5 through 8, with mutations, especially at particular codons such as codon 75, 245, 273, 285, 220, 306. TP53 mutations can exert two different effects on cells. One of them is the loss-of-function (LOF) effect. With this effect, the cell loses its ability to protect itself from cancer. This is also called the dominant-negative effect as the mutant allele masks the function of the wild type allele. The other one is called the gain-of-function (GOF) effect. This effect gives the ability of novel tumor-promoting. Loss of function (LOF) is the most significant functional outcome of TP53 mutations both in breast and ovarian cancer (Silwal-Pandit et al., 2017). 

TP53 mutations in breast and ovarian cancers are very aggressive. These mutations are characterized by poor differentiation, increased invasiveness, and high metastatic potential (Silwal-Pandit et al., 2017). Mutations in TP53 are not very common in low-grade serous ovarian carcinomas and serous borderlines, whereas TP53 mutations are found in 100% of the high-grade serous ovarian cancer cases (Ahmed et al., 2010). Breast tumors that are very aggressive and have poor clinical outcomes such as large tumor size, axillary lymph node metastasis, high histological grade, and estrogen receptor (ER) negativity mostly have mutations in TP53 gene (Langerod et al., 2007). 

Since the majority of TP53 mutations are missense, CRISPR/Cas9 shows promise for restoring wild type TP53 because it has previously been reported to successfully correct single nucleotides in models, both in-vitro, and in-vivo (Batir et al., 2019). CRISPR Cas9 is an RNA guided endonuclease with a complementary sequence to the target gene so that it may bind and induce double-strand breaks in the DNA. This encourages use of DNA-repairing mechanisms such as non-homologous end-joining or homology directed repair. When cells use HDR, the donor DNA (intended sequence) changes to be incorporated into the edited genome, causing nucleotide exchange (Batir et al. 2019). In wild-type-p53-positive human pluripotent stem cells, CRISPR Cas9 was found to induce a genotoxic response since recognition of the genetic alteration by the p53 protein activated apoptotic pathways. Hence the efficacy of CRISPR Cas9 was tested in prostate cancer PC-3 cell lines which present a loss of TP53 function, mainly due to the TP53 414del-C mutation in the study described below (Batir et al., 2019). 

The RNA guided endonuclease was introduced in the form of single-guide RNA (sgRNA) which introduced a double-strand-break in the sequence following the PAM. The donor DNA was then delivered as a single-stranded-oligodeoxynucleotide at the target sequence marked by sgRNA, to minimize toxicity and maximize targeted integration into the genome, improving the accuracy of homology directed repair up to tenfold (Batir et al., 2019). To prevent recutting of the intended repaired sequence, a second guide blocking mutation was introduced opposite the 414delC mutation (on the other side of the PAM to no longer enable rebinding of the RNA guided endonuclease (Quart D et al., 2017; Paquet D et al., 2016) 

Two different sgRNAs (sgRNA1 and sgRNA2) and 3 different ssODNs (antisense ssODN1, sense strand ssODN2) to sgRNA2 and neutral ssODN3 (as a control) were designed, where sgRNA1 induced a double-strand break (DSB) seven nucleotides away from the 414delC mutation, while sgRNA2 induced the DSB 3 nucleotides away. Gene modification mediated by sgRNA2 was calculated to be much more efficient (30%) than sgRNA1 (4%), so sgRNA2 was then coupled with either ssODN1 or ssODN2 to determine overall efficacy in repairing the TP53 414delC mutation, where ssODN2 combined with sgRNA2 was found to be doubly as effective (5%) as sgRNA2 + ssODN1 (2.5%). This was in concordance with the findings that sgRNA2 + ssODN2 exhibited around 4 times more p53 mRNA expression compared to sgRNA2 + ssODN1 as determined by a PCR analysis and with the fact that wild-type p53 protein expression was highest in the sgRNA + ssODN2 group, according to an immunofluorescence analysis. Furthermore the ratio of percentage of apoptotic PC-3 cells transfected with sgRNA2 was of interest – PC-3 cells treated with sgRNA2+ssODN1 was higher after 72 hours was higher compared to 48 hours while percentage apoptotic cells treated with sgRNA2+ssODN3 displayed no significant change, suggesting that PC-3 cells transfected with ssODN2 sustain a greater apoptosis rate. 

The final crucial step is to increase the efficiency of gene transmission. Adenoviral-associated-vector shows promise in improving HDR efficiency, hence potentially enabling trials with CRISPR to restore TP53 in vivo. In addition, other proposed strategies aimed at targeting the tumor without targeting similar sequences in healthy cells includes encapsulating the survivin-promoter-gene in the vector, which is only expressed in tumor cells, or utilising other RNA endonuclease enzymes to minimize off-target effects. 

However, drug therapies using COTI2 that have also been tested in vitro appear effective in restoring mutant p53 protein in triple-negative breast cancer (TNBC). An immunoblot analysis on a study carried out on the results demonstrated a decrease in staining of the PAb240 (mutant-protein specific antibody) and an increase in staining of the PAb1620 (wild type p53) suggesting refolding of mutant p53 into its wild-type conformation (Q. Zhang et al., 2018). Still, the study compared reduction in cell proliferation in TNBC cells treated with COTI2 to TNBC treated with traditional chemotherapy drugs rather than the CRISPR Cas9 therapy, which showed COTI2 to reduce cell proliferation by 70% more than the chemotherapy drugs carboplatin and cetuximab, with no data available on rates of apoptosis (Duffy et al., 2022). In addition, COTI2 displays p53-independent anticancer effects such as activation of AMPK and inhibition of mTOR pathways, leading to replication stress (Duffy et al., 2022). 

APR-246 is another drug that is broken down to MQ, which then binds covalently to the thiol groups Cys277 and Cys124, converting mutant p53 back to its wild-type conformation (Duffy et al., 2022). Like COTI2, APR-246 also appears to exert p53-independent anticancer effects since its metabolite, MQ, is known to inhibit the redox enzymes thioredoxin reductase and glutaredoxin reductase, depleting antioxidant glutathione which leads to oxidative-stress-mediated apoptosis (X. Peng et al., 2018) (L. Haffo et al., 2013). In one study where APR-246 was administered to patients with T-prolymphocytic leukaemia, expression of target genes as measured by the apoptosis-initiating proteins PUMA and NOXA increased by 40-80% (S Lehman et al., 2012). However, no measurements regarding tumor size were taken and results have yet to be published about the efficacy of APR-246 on reproductive cancers. 

Hence, it is difficult to determine whether the efficacy of the drugs APR-246 and COTI2 is a relevant meter of comparison to CRISPR/Cas9 to compare their effectiveness as anticancer treatments, based on their p53 restorative properties. 

In conclusion, both the CRISPR Cas9 as well as drug therapies using COTI2 and APR246 were successful in restoring wild type p53 function through repair of the 414delC mutation (CRISPR Cas9), and through mediating refolding of the mutant p53 into its wild-type conformation in addition to p53’s independent anticancer effects. To maximize the efficacy of a future experiment focused on in-vivo cancer treatment, further in-vitro research could be carried out to determine the efficacy of encapsulating the survivin-promoter-gene within lentiviral and adeno-associated-viral vectors (respectively). In addition, more in-vitro research needs to be carried out to assess the efficacy of HDR mediated by lentiviral or adeno-associated viral vectors in cancer cell lines. In future, designing a further in-vitro study using identical quantities of various reproductive cancer-cell-lines treated with various doses of CRISPR Cas9, COTI2 and APR246 would be ideal. This would help determine the optimal treatment for each reproductive cancer based on which treatment displays highest apoptosis rates, and the highest sustenance over varying time-frames. This can then be tested in-vivo, followed by clinical trials.

BRCA1/BRCA2

BRCA1/BRCA2 are genes that help in the process of repairing damaged DNA, and are also known as tumor suppressor genes that have the job of controlling cell death and cell growth. BRCA1 was discovered in 1994 (Miki et

al., 1994) while BRCA2 was discovered the next year in 1995 (Wooster et al., 1995). The mutations in BRCA1, which cause cancer, occur on exon 13 while in BRCA2 they occur on exon 11. According to WHO statistics in 2020, about 2.26 million cases of breast cancer were reported along with 685,000 deaths. This makes breast cancer the most prevalent cancer in the world. The BRCA1 gene is expressed in a number of tissues, including ovarian and breast tissue. Acting as a tumor suppressor, it is involved in multiple cellular regulatory pathways including gene transcription regulation, ubiquitination, cell-cycle progression and DNA-damage response; the latter being a pathway in which BRCA1 plays a pivotal role. Patients with BRCA1 mutations have a 50–65% lifetime risk of developing breast cancer with a preponderance of an aggressive subtype known as triple negative breast cancer (TNBC) (Zhao et al., 2019). 

Recently a new study conducted by Japanese scientists in the RIKEN Center for Integrative Medical Sciences has discovered that 5 other cancers are also caused by genetic mutations in BRCA1 and BRCA2. These 5 cancers include biliary tract cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, and gastric cancer. These findings further increase the importance of the understanding regarding BRCA1 and BRCA2. Mutations in these cells can be identified by collecting blood or saliva samples. The study had a large sample size collected from 63,828 patients with 14 common cancer types and 37,086 controls that were sourced from a multi-institutional hospital-based registry, Biobank Japan, between April 2003 and March 2018. The data was analyzed between August 2019 and October 2021. The study also grouped each gender, as the mutations can vary depending on the gender of the patient. The result of this study showed that the mean age of diagnosis for breast cancer was merely 56.4 years regardless of gender, which was the second lowest age of occurrence, while the average age of patients suffering from cervical cancer was just 49.7 years. The results displayed that male patients with breast cancer had a higher carrier rate of BRCA2 mutation (18.9%) than BRCA1 mutation (1.89%) by a substantial amount. Patients with ovarian cancer displayed the next highest carrier variation, but in this case the frequency of BRCA1 mutation carrier (4.86 %) was higher than BRCA2 mutation carrier (3.42%). Pathogenic variants in BRCA1 were significantly associated with increased risk of 5 cancer types: ovarian, female breast, biliary tract, gastric, and pancreatic cancers. Pathogenic variants in BRCA2 were associated with increased risk of 7 cancer types: female breast, gastric, ovarian, male breast, pancreatic, prostate, and esophageal cancers (Momozawa et al., 2022). 

This study further illustrates the importance of the new CRISPR Cas9 technology as it can modify BRCA1 and BRCA2 genes, and in this way not only breast and ovarian cancer tumors be suppressed but 5 other cancers linked will also be suppressed. These genes which can be modified will also further diminish the chances of cancer prevalence. 

The prevalence of HER2 positivity has generally been reported as lower among BRCA1 carriers than BRCA2 carriers. The prevalence of HER2 positivity in BRCA1 carriers and BRCA2 carriers has ranged from 0-10% and 0-13% respectively (Whitaker et al., 2020). Targeting of HER2 via CRISPR Cas9 led to inhibition of cell proliferation and carcinogenesis of breast cancer cells. Exons 5, 10 and 12 of HER2 were specifically targeted by selective gRNAs; these exons exist in all HER2 isoforms and are responsible for encoding parts of the extracellular domain. Cas9, along with three gRNA, was introduced into HER2+ breast cancer cell lines BT-474 and SKBR-3 and the HER2- breast cancer cell line MCF-7. In addition, the introduction of Cas9 and gRNAs to a soft agar colony formation assay caused a considerable reduction in colony formation. These results indicate that utilizing CRISPR Cas9 to target HER2 yields could decrease cell proliferation and carcinogenesis, though the effect is limited to HER2 and cell lines (Ahmed et al., 2021). 

The BRCA1 gene is expressed in several tissues, including ovarian and breast tissue. The BRCT domain in BRCA1 functions to regulate interactions of phosphoproteins with BRCA1 as well as facilitate non-phosphoprotein interactions with DNA binding. Mutations of a residue would therefore hinder the role of BRCA1. Patients with BRCA1 mutations have a 50–65% lifetime risk of developing breast cancer with a preponderance of an aggressive subtype known as triple-negative breast cancer. BRCA1 gene knockouts in human adipose-derived stem cells were performed according to published methods using the lentiCRISPRv2 vector system. CRISPR negative controls were generated using non-targeting gRNAs that do not recognize any sequence in the human genome. (TNBC) (Zhao et al. 2019). 

Using the CRISPR Cas9-mediated system, the researchers generated human adipose stem cells (ASC) with stable BRCA1 knockdown models BRCA1-KD ASC. They found 79.1% knockdown expression of the BRCA1 gene in our BRCA1-KD ASCs, compared to CRISPR controls with empty vectors. The study tested the effect of the BRCA1 knockdown on ASC stimulation of breast cancer cell progression and found that BRCA-KD ASC also significantly increased breast cancer cell invasion. The researchers also found that brass from patients with BRCA1 deletion mutation also increased breast cancer cell invasion in the Transwell assay experiment; therefore, the data suggests that BRCA1 mutation in ASCs results in a secretome that promotes in vitro breast cancer cell proliferation and invasion (Zhao et al., 2019).

Conclusion

CRISPR/Cas9 appears effective in treating a range of mutations, namely TP53 and BRCA1/2 in-vitro. 

With regards to TP53, treatment of PC-3 prostate cancer cell lines with CRISPR Cas9 increased p53 mRNA 4-fold, suggesting restoration of wild type TP53. This is in accordance with the result that apoptosis rates remained well-sustained over a period of 72 hours. So far, the use of viral vectors like lentiviral and AAV show promise in improving the efficiency of CRISPR Cas9, through maximizing the induction of HDR. Furthermore, encapsulation of the survivin-promoter gene could be a promising strategy to direct the sgRNA solely towards tumor cells. However, in-vivo studies are required to confirm whether apoptosis significantly contributes to tumor size reduction. Hence, it cannot yet be determined whether CRISPR Cas9 is more effective than drug therapies using APR-246 and COTI-2 that have already undergone clinical trials. Since CRISPR Cas9 has only been carried out in vivo, conclusions cannot be drawn about its efficacy in treating cancer in vivo by reducing tumor size, through the restoration of mutated genes such as TP53 and BRCA1/2. 

Restoration of HER2 via CRISPR Cas9 targeting of exons 5, 10 and 12 resulted in a decrease in cell proliferation in BRCA2 positive cells and a less invasive phenotype, suggesting that CRISPR Cas9 could be used in cancer therapy.

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