Grnas that induce apoptosis in cancer cells and a method relating to these grnas

Abstract

The invention relates to a method that enables the specific targeting of tumour cells having amplification and the induction of apoptosis by using CRISPR-Cas9 technology. Particularly in breast cancer cells carrying HER2 gene amplification, gRNA sequences that target only cancer cells while preserving genetic integrity have been designed. This method aims to effectively eliminate cancer cells without damaging healthy cells and can also be adapted to other cancer types in which gene amplification is prevalent.

Description

[0001] DESCRIPTION

[0002] gRNAs THAT INDUCE APOPTOSIS IN CANCER CELLS AND A METHOD RELATING TO THESE gRNAs

[0003] Technical Field

[0004] The invention relates to a genetic treatment method targeting cancer cells having HER2 gene amplification by using CRISPR-Cas9 technology. In this method, gRNAs designed within the scope of the invention, which specifically target amplified genomic regions, create DNA damage in cancer cells and induce apoptosis. This technology, which shows high efficacy particularly in HER2+ breast cancer cells, affects cancer cells with high selectivity by causing minimum damage to cells with diploid HER2 copy number. Furthermore, with its applicability in other cancer types associated with gene amplification, it shows promise for genetic-based cancer therapies. Said method provides a more targeted therapy by minimising the side effects of existing treatments.

[0005] State of the Art

[0006] Cancer is a group of diseases that occur as a result of uncontrolled division and proliferation of somatic cells. While the cycle of growth, division and death operates in a controlled manner in normal cells, cancer cells lose this regulation and proliferate indefinitely, leading to tumour formation. Cancer cells can damage surrounding tissues and can spread to different regions and organs of the body by metastasis. Among cancer types, lung, breast, colon, prostate and haematopoietic system cancers (leukaemia and lymphoma) are the most common. Various methods such as excisional treatment (surgery), chemotherapy, radiotherapy, immunotherapy and targeted therapies are used in cancer treatment. In addition, in recent years, personalised treatment methods have also developed with genetic and biotechnological approaches. The type of treatment is determined depending on the type, stage, phase of the cancer and the general health condition of the patient. Each method has advantages and disadvantages; for example, while chemotherapy and radiotherapy target cancer cells, they can also damage healthy cells, which may lead to treatment-related side effects.In cancer treatment, gene therapy utilises the property of the Cas9 enzyme to create double-strand breaks on DNA without performing gene correction. In tumour cells having gene amplification, the high number of amplified DNA copies allows Cas9 to perform multiple cuts in these regions, and this situation triggers an apoptosis response in cells due to multiple DNA breaks. Gene therapy is generally carried out via vectors; these vectors are viral or non-viral carrier systems used to transport genetic material into cells. Gene editing technologies such as CRISPR-Cas9 have the potential to make significant progress in gene therapy. This technology creates doublestrand breaks in DNA by targeting specific gene regions in cancer cells and induces cell death via apoptosis. However, since this technology is still at the in vitro stage, further research is required for its widespread use in clinical applications.

[0007] There may be some possible problems in the recognition of the genomic region targeted with CRISPR-Cas. First, the efficiency of delivery of Cas9 and gRNA complexes into cells may be low, and there is a risk that Cas9 may cut off-target regions in the genome (regions showing similarity to the target) (off-target effect). This situation may cause damage in healthy cells. Secondly, the vectors used in transporting genetic material to target cells may sometimes cause a response of the immune system. In addition, the high cost of gene therapy and its technical difficulties also limit its widespread use. Finally, the long-term effects and safety of genetic modifications have not yet been fully understood.

[0008] In the study conducted by Wang and Sun in the state of the art, a method aiming to stop proliferation in breast cancer cells by using CRISPR / Cas9 technology targeting the HER2 gene is described. This method presents an innovative mechanism in cancer treatment by suppressing tumour growth with HER2-targeted gene editing. In this study, the CRISPR / Cas9 editing directed to the HER2 gene develops a mechanism based on dominant negative mutations in cases where gene expression cannot be fully reduced. However, in this approach, the accuracy and efficiency of gene editing targeting remain limited by low mutation rates. In particular, deficiencies are observed in terms of additional measures to minimise off-target effects in specific gene targeting and adaptability to clinical applications. In addition, the method requires additional chemical interventions to increase the synergistic effect with PARP inhibitors, which may complicate treatment processes.In the patent application numbered US2021348161A1 in the state of the art, an innovative method is described which targets gene edits in cancer cells using CRISPR-Cas9 technology and selectively eliminates cancer cells by genetic rearrangements. This study aims to perform gene editing by targeting fusion genes specific to cancer cells. The fact that specific gene editing regions are patient-specific reveals the limitations of the method in terms of broad clinical applications. In addition, in this document, the applicability of treatment molecules that cannot be transported outside the cell constitutes an important barrier reducing efficiency. More comprehensive clinical validation is required in order to minimise off-target mutations during genome editing or to demonstrate the effectiveness achieved with multiple genetic targeting. The limitations and insufficiencies of the solutions in the current state of the art, such as low accuracy of gene editing targeting, low mutation rates, inability to minimise off-target mutations, requirement of additional chemical interventions, and lack of amplified DNA sequences that can be selectively targeted in cancer cells, have necessitated making an improvement in this field.

[0009] Brief Description and Aims of the Invention

[0010] The invention relates to a genetic treatment method that induces apoptosis by targeting cancer cells carrying HER2 gene amplification with CRISPR-Cas9 technology. Said method aims to selectively target cancer cells containing amplification while minimising adverse effects on healthy cells.

[0011] The main aim of the invention is to provide a method that generates an apoptosis response by specifically targeting cancer cells having gene amplification. The CRISPR-Cas9 technology used in the invention performs this process via guide RNAs (gRNAs) that target specific exonic, intronic or amplicon-internal extragenic regions where gene amplifications are present. This approach aims to create double-strand DNA breaks in target cells by utilising genomic copy number differences of cancer cells. When the apoptosis rates of cancer cells with diploid HER2 copy number and HER2+ breast cancer cells are compared, high apoptosis rates are observed in HER2+ cells, whereas low levels of apoptosis are detected in cells with diploid HER2 copy number. These findings indicate the selective targeting potential of the method. The gRNAs used during the targeting process are designed with high specificity. Thus,cancer cells containing amplification are targeted with high selectivity. With this specific targeting, it is aimed to reduce possible side effects in cells that do not contain amplicons and to increase target selectivity. In addition, with its applicability in other cancer types having gene amplification, this approach has a wide field of use. In this way, it provides a significant contribution to the future development of genetic-based therapies.

[0012] Another aim of the invention is to demonstrate that in vitro treatment efficacy can be adapted according to the genomic copy number profile of cells. Said method, which focuses on HER2 gene amplifications, allows gRNA design suitable for copy number variations in different cancer cell lines. For example, the high apoptosis rates obtained in the BT-474 cell line having a high HER2 copy number demonstrate that this technology can be adapted to genetic profile-based treatments. This situation makes it possible to optimise treatment strategies according to different cancer types. By means of said method, specific treatment options can be created for tumours with varying genomic copy number variations. In this way, both treatment efficacy can be increased and unnecessary drug use and therefore costs can be reduced. In addition, the genetic profile-based approach included in the invention increases patients’ response rates to treatment and minimises the risk of side effects. As in said method of the invention, such treatments in which the genetic profile is taken into consideration aim to provide cancer patients with a higher quality of life. Thus, future cancer treatment strategies are ensured to become more effective and reliable. Said method has the potential not only to prolong survival but also to be curative.

[0013] Another aim of the invention is to provide an alternative treatment method in which the side effects caused by conventional cytotoxic agents used in cancer treatment are minimised. Conventional treatments cause serious side effects such as hair loss, nausea and weakening of the immune system by damaging rapidly dividing healthy cells. In said method, cancer cells having gene amplification are targeted with high selectivity, thereby significantly reducing the risk of damaging cells with diploid copy number. This situation has the potential to significantly increase patient comfort as well as increasing treatment efficacy. With the development of this method, which causes minimum damage to cells having diploid genomic copy number, a great advantage is provided especially for patients with weakened immune systems. Thus, the treatment process becomes more manageable by preserving the quality of life of patients duringand after treatment. At the same time, said method also contributes to the prevention of long-term side effects.

[0014] Another aim of the invention is to provide a treatment method applicable to different cancer types depending on gene amplification. This method can also be used in other cancer types containing gene amplification such as breast, neuroblastoma, lung, gastric, colorectal carcinoma and various soft tissue tumours. The flexible structure of CRISPR-Cas9 technology can be customised to target different genomic regions. In this way, the method becomes effective in tumours having various genetic variations. This situation increases the wide-scale application potential of the invention in the biotechnology and pharmaceutical industry. In addition, the adaptation of the method to different cancer types increases success rates by providing an individual approach to treatment. The fact that it yields effective results in cell lines with different genetic profiles demonstrates the potential impact of the invention. Cells having gene amplification can develop resistance mechanisms against chemotherapeutic agents, and this situation reduces treatment efficacy. Double-strand breaks created by CRISPR-Cas9 lead to DNA damage that cannot be repaired in cells. This situation causes cancer cells to undergo apoptosis or necrosis. Targeting resistant cells in this manner may increase treatment efficacy and reduce the likelihood of cancer recurrence. In this way, success rates can be increased even in cancer types that are not responsive to treatment. Therefore, said method presents a new paradigm in the targeting of cancer cells.

[0015] Description of the Drawings

[0016] Figure 1: Dot plot graph of morphological differentiation and Annexin V-PE-7AAD results of the MCF-7 cell transfected with PX458-gRNA1, PX458-gRNA2 and PX458-gRNA1+gRNA2 by electroporation (A: changes in cell size and granularity after transfection of MCF-7 cells with PX458 without gRNA cloning, B: transfection efficiencies of MCF-7 cells after transfection with PX458 without gRNA cloning, C: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 without gRNA cloning, D: changes in cell size and granularity after transfection of MCF-7 cells with PX458 cloned with gRNA1, E: transfection efficiencies of MCF-7 cells aftertransfection with PX458 cloned with gRNA1, F: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 cloned with gRNA1, G: changes in cell size and granularity after transfection of MCF-7 cells with PX458 cloned with gRNA2, H: transfection efficiencies of MCF-7 cells after transfection with PX458 cloned with gRNA2, J: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 cloned with gRNA2, K: changes in cell size and granularity after transfection of MCF-7 cells with PX458 cloned with gRNA1+gRNA2, L: transfection efficiencies of MCF-7 cells after transfection with PX458 cloned with gRNA1 +gRNA2, M: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 cloned with gRNA1 +gRNA2). Figure 2: Statistical analysis results of apoptosis and necrosis data detected as a result of transfection of MCF-7 cells with PX458-gRNA1, PX458-gRNA2 and PX458-gRNA1+gRNA2 (A: statistical analysis results obtained by One-way ANOVA from apoptosis rates obtained as a result of transfection of MCF-7 cells with empty PX458, PX458 containing gRNA1, PX458 containing gRNA2 and gRNA1 PX458+gRNA2 PX458, * p < 0.1, ** p < 0.05, *** p < 0.01 and B: statistical analysis results obtained by One-way ANOVA from necrosis rates obtained as a result of transfection of MCF-7 cells with empty PX458, PX458 containing gRNA1, PX458 containing gRNA2 and gRNA1 PX458+gRNA2 PX458, * p < 0.1, ** p < 0.05, *** p < 0.01).

[0017] Figure 3: Dot plot graph of morphological differentiation and Annexin V-PE-7AAD results of the BT-474 cell transfected with PX458-gRNA1, PX458-gRNA2 and PX458-gRNA1+gRNA2 by electroporation (A: changes in cell size and granularity after transfection of BT-474 cells with PX458 without gRNA cloning, B: transfection efficiencies of BT-474 cells after transfection with PX458 without gRNA cloning, C: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 without gRNA cloning, D: changes in cell size and granularity after transfection of BT-474 cells with PX458 cloned with gRNA1, E: transfection efficiencies of BT-474 cells after transfection with PX458 cloned with gRNA1, F: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 cloned with gRNA1 , G: changes in cell size andgranularity after transfection of BT-474 cells with PX458 cloned with gRNA2, H: transfection efficiencies of BT-474 cells after transfection with PX458 cloned with gRNA2, J: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 cloned with gRNA2, K: changes in cell size and granularity after transfection of BT-474 cells with PX458 cloned with gRNA1+gRNA2, L: transfection efficiencies of BT-474 cells after transfection with PX458 cloned with gRNA1 +gRNA2, M: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 cloned with gRNA1+gRNA2). Figure 4: Statistical analysis results of apoptosis and necrosis data detected as a result of transfection of BT-474 cells with PX458-gRNA1, PX458-gRNA2 and PX458-gRNA1+gRNA2 (A: statistical analysis results obtained by One-way ANOVA from necrosis rates obtained as a result of transfection of BT-474 cells with empty PX458, PX458 containing gRNA1 , PX458 containing gRNA2 and gRNA1 PX458+gRNA2 PX458. * p < 0.1, ** p < 0.05, *** p < 0.01, B: statistical analysis results obtained by One-way ANOVA from necrosis rates obtained as a result of transfection of BT-474 cells with empty PX458, PX458 containing gRNA1, PX458 containing gRNA2 and gRNA1 PX458+gRNA2 PX458. * p < 0.1, ** p < 0.05, *** p < 0.01).

[0018] Figure 5: Comparison of A: apoptosis and B: necrosis rates among cell groups.

[0019] Figure 6: Dot plot graph of morphological differentiation and Annexin V-PE-7AAD results of the MCF-7 cell transfected with PX458, PX458-intron 7 and PX458-intron 15 by electroporation (A: changes in cell size and granularity after transfection of MCF-7 cells with PX458 without gRNA cloning, B: transfection efficiencies of MCF-7 cells after transfection with PX458 without gRNA cloning, C: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 without gRNA cloning, D: changes in cell size and granularity after transfection of MCF-7 cells with PX458 cloned with intron 7 gRNA, E: transfection efficiencies of MCF-7 cells after transfection with PX458 cloned with intron 7 gRNA, F: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 cloned with intron 7 gRNA, G: changes in cell size and granularity after transfection of MCF-7 cells with PX458 cloned with intron 15 gRNA, H: transfection efficiencies ofMCF-7 cells after transfection with PX458 cloned with intron 15 gRNA, J: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of MCF-7 cells after transfection with PX458 cloned with intron 15 gRNA). Figure 7: Dot plot graph of morphological differentiation and Annexin V-PE-7AAD results of the BT-474 cell transfected with PX458, PX458-intron 7 and PX458-intron 15 by electroporation (A: changes in cell size and granularity after transfection of BT-474 cells with PX458 without gRNA cloning, B: transfection efficiencies of BT-474 cells after transfection with PX458 without gRNA cloning, C: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 without gRNA cloning, D: changes in cell size and granularity after transfection of BT-474 cells with PX458 cloned with intron 7 gRNA, E: transfection efficiencies of BT-474 cells after transfection with PX458 cloned with intron 7 gRNA, F: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 cloned with intron 7 gRNA, G: changes in cell size and granularity after transfection of BT-474 cells with PX458 cloned with intron 15 gRNA, H: transfection efficiencies of BT-474 cells after transfection with PX458 cloned with intron 15 gRNA, J: viability (lower left), early apoptosis (lower right), late apoptosis (upper right) and necrosis (upper left) responses of BT-474 cells after transfection with PX458 cloned with intron 15 gRNA).

[0020] Detailed Description of the Invention

[0021] The invention describes an innovative CRISPR-Cas9-based treatment method that induces apoptosis by creating DNA damage in cancer cells having HER2 gene amplification. It provides high efficacy and selectivity in cancer treatment while preserving healthy cells.

[0022] gRNAs that induce apoptosis in cancer cells having HER2 amplification are;

[0023] • gRNA1 having a forward primer with the nucleotide sequence of SEQ ID NO:

[0024] 1 and a reverse primer with the nucleotide sequence of SEQ ID NO: 2, and / or• gRNA2 having a forward primer with the nucleotide sequence of SEQ ID NO: 3 and a reverse primer with the nucleotide sequence of SEQ ID NO: 4, which target the exonic region of cancer cells,

[0025] and / or

[0026] • lnt7-gRNA having a forward primer with the nucleotide sequence of SEQ ID NO: 5 and a reverse primer with the nucleotide sequence of SEQ ID NO: 6, and / or

[0027] • lnt15-gRNA having a forward primer with the nucleotide sequence of SEQ ID NO: 7 and a reverse primer with the nucleotide sequence of SEQ ID NO: 8,

[0028] which target the intronic region of cancer cells.

[0029] The method for inducing apoptosis in cancer cells having HER2 amplification in which said gRNAs are used comprises the process steps of;

[0030] i. adding the prefix “caccg” to the initial sequence of the forward primers SEQ ID NO: 1 and / or SEQ ID NO: 3 and adding the suffix “aaac” to the terminal sequence, in accordance with the Bbsl restriction enzyme cut site present in the PX458 plasmid, and cloning the reverse primers SEQ ID NO: 2 and / or SEQ ID NO: 4 into the plasmid, and obtaining a plasmid having the nucleotide sequence of SEQ ID NO: 9 as a result of this cloning,

[0031] ii. performing colony PCR in order to verify after cloning studies,

[0032] iii. performing transformation into the DH5alpha E. coli strain in order to amplify the plasmid into which gRNA is cloned and producing bacteria in ampicillin- containing lysogeny broth (LB broth) medium,

[0033] iv. subsequently isolating the plasmids from E. coli bacteria,

[0034] v. performing transfection of the isolated plasmids containing the target locus into cancer cells by electroporation with parameters of 1600v 20ms 1 pulse, and vi. occurrence of apoptosis or necrosis in cancer cells due to a large number of irreparable double-strand breaks in cells carrying the plasmid 48 hours after transfection.

[0035] Said gRNAs of the invention exhibit a therapeutic effect particularly towards cancer types having HER2 amplification. Gene amplification refers to the condition in whichcertain genes are excessively replicated in some cancers; and these genetic alterations constitute an important factor supporting the growth and proliferation of cancer cells. Such amplifications constitute an important target in cancer treatment. For example, HER2 gene amplification is a common condition particularly in certain cancer types such as breast cancer. gRNAs specific to amplicon regions present in each tumour type can be designed for different tumour types having gene amplification (such as lung cancer, colorectal cancer, gastric cancer, neuroblastoma). These gRNAs, as part of the CRISPR-Cas9 system, are capable of recognising and cutting specific DNA sequences. gRNAs are targeted to DNA sequences in genetic regions exhibiting amplification and the Cas9 enzyme cuts the target DNA sequence. This cutting property of Cas9 activates DNA repair mechanisms in the cell; however, these repair processes are generally erroneous and a large number of DNA breaks occur in the cell. In this case, the survival of the cells becomes difficult and the apoptosis (cell death) process is triggered. For example, in cancer cells having MYCN amplification, gRNAs specific to DNA sequences containing MYCN amplification can be designed and Cas9 can be enabled to cut these sequences. This leads to the occurrence of apoptosis by increasing DNA damage in cells having MYCN amplification. In this way, cancer cells become unable to repair themselves due to genetic damage and die. Said method of the invention can be adapted not only to a single amplification type such as HER2, but also to different gene amplification types. That is, by designing gRNAs specific to amplicon regions unique to each cancer type, Cas9 can be enabled to perform cutting specific to target regions. This provides great flexibility in genetic targeting and enables its use as an effective method in the treatment of different cancer types. In summary, in cancer cells exhibiting gene amplification, DNA breaks can be created with Cas9 by using gRNAs specific to amplicon regions, and this process can direct cancer cells to apoptosis.

[0036] In the first step of said method of the invention, gRNAs with high on-target and off-target scores are designed via bioinformatics databases for intronic genomic loci containing amplicons. In accordance with the Bbsl restriction enzyme cut site present in the PX458 plasmid (Plasmid ID: 48138), the prefix “caccg” is added to the initial sequence of the forward primers; and the terminal sequence is provided with the suffix “aaac”. After cloning studies, colony PCR is performed in order to verify. In order to amplify the plasmid into which gRNA is cloned, transformation is performed into theDH5alpha E. coli strain and bacteria are produced in ampicillin-containing LB broth medium. Subsequently, the plasmids are isolated. After the isolated plasmid is confirmed to contain the target locus, transfection is performed into the cell. Since the highest transfection efficiency is obtained by electroporation, electroporation is used as the transfection method. The highest transfection efficiency is obtained at electroporation parameters of 1600v20ms 1 pulse. Forty-eight hours after transfection, the rates at which the cells carrying the plasmid undergo apoptosis and necrosis are measured by Annexin V in a flow cytometry device. One of the reasons for selecting 48 hours in this process step is that in transient transfection, the expression level of Cas9 reaches its maximum at 6-12 hours after entering the cell, and subsequently apoptosis due to multiple DNA breaks reaches its maximum at 24-72 hours. Another reason is that transfection efficiency is highest within 48 hours after transfection. Finally, in analyses within 48 hours, the early apoptosis response is more detectable. It is generally stated in protocols and studies that apoptosis measurements can be taken 24-72 hours after transfection. Normally, Cas9 is maximally expressed between 6-12 hours after transfection and DNA breaks occur. Between 48-72 hours, cells in the early apoptosis stage that are not necrotic become detectable. Based on the principle that the Cas9 enzyme creates double-strand breaks in DNA, targeting amplicons having high copy number such as gene amplification with Cas9 leads to the breakage of a large number of DNA double strands. A large number of irreparable double-strand breaks lead to apoptosis or necrosis of the cells. Thus, cell death occurs in cancer cells and cancer treatment is achieved.

[0037] gRNAs having the sequences SEQ ID NO: 1-7 in the invention do not target cells containing gene amplification. These gRNAs are specific to the targeted gene region and include exonic and / or intronic loci that specifically target gene amplification. The designed gRNAs are cloned into a carrier plasmid. Plasmids containing gRNAs are transferred into cells using an appropriate in vitro transfection method such as lipofection and electroporation. The transfected plasmids enable the formation of the gRNA and Cas9 complex within the cell. This complex, guided by the gRNAs, creates irreparable double-strand breaks in the targeted HER2 gene region. The double-strand breaks created by Cas9 cause the cell to undergo apoptosis. The states of cells undergoing apoptosis and necrosis are analysed in a flow cytometry device using Annexin V after transfection. This analysis is used to evaluate morphological changesoccurring in cells and cell death. In order to test the specificity of the system, transfection of gRNAs is performed in cells in which gene amplification is not observed. If the ratio of apoptotic cells not containing amplicons is significantly lower than that of apoptotic cells containing amplicons, the effectiveness of the system is confirmed. In the invention, the primers used to direct tumour cells containing gene amplification specifically to apoptosis have the nucleotide sequences SEQ ID NO: 1-7. In the invention, both exon and intron targeting have been performed, and additionally, with the intron targeting strategy, it is aimed to generate an apoptosis response in the cell while preserving gene integrity. Said method of the invention is important in terms of specifically directing tumour cells to apoptosis while preserving the genomic integrity of regions expressed in normal cells. In addition, the design and selection of said gRNAs of the invention have been optimised to increase the operational efficiency and specificity of the system.

[0038] In the invention, introns are targeted with specific gRNA design. Specific gRNAs targeting gene amplification direct cells exhibiting copy number variation to apoptosis while causing minimum damage to cells having diploid copy number. In order to minimise damage to off-target regions, it is important to design nucleotide sequences that are not present in another region of the genome. Targeting intron regions in order to direct the cell to apoptosis without disrupting gene integrity carries a lower risk of genomic instability compared to conventional exon targeting. In the invention, apoptosis rates depending on high gene copy number are obtained. The fact that higher apoptosis rates are obtained in the BT-474 cell line due to higher HER2 copy number indicates that treatment efficacy can be adapted according to the genetic profile of cells and points to an effective treatment potential.

[0039] In the invention, gene amplification in cancer cells is specifically targeted by using the CRISPR-Cas system. Targeting amplified gene regions leads to apoptosis of cancer cells. In this way, considering the genomic heterogeneity of tumours, it enables the targeting of various oncogenes in which gene amplification is important. In the invention, targeting gene amplification with CRISPR-Cas9 is not limited to HER2+ cells since it targets tumour cells depending on gene amplification, and said method of the invention can also be used in the treatment of cancer types having other amplified oncogenes apart from HER2 amplification. At this point, the main phenomenon arisesfrom the quantity of the targeted sites. The fact that the number of double-strand breaks is numerically very high due to amplification likely causes the activation of apoptosis pathways. In other words, since the invention targets gene amplification, it can perform a broader genomic intervention and can be adapted according to the molecular profile of cancer cells.

[0040] Within the scope of the invention, base sequences related to the HER2 gene were obtained from the Ensembl database (Transcript ID: ENST00000269571.10 ERBB2-201) in order to design sgRNA. A 120-base region targeting exon 8 was selected as the target sequence. The IDT.DNA database was used to design sgRNA containing the target sequence. The target sequence of the HER2 gene was entered into the IDT.DNA database in FASTA format. While designing sgRNA, the database performs analysis based on the content of guanine and cytosine bases (GC content) in the target sequence, their positions in the sequence, the PAM motif and the Tm value, and presents the sequences most suitable for these conditions. Thus, it attempts to reduce the possibility of the gRNA targeting another region within the genome (off-target effect). According to the algorithms of the IDT.DNA database, a diagram was created regarding possible gRNA sequences belonging to exon 8 of the HER2 gene, for which on-target and off-target efficiency were calculated. Among the gRNA sequences numbered based on on-target and off-target scores in clusters, the sequences in cluster number 3 were considered suitable for the study.

[0041] Table 1. Sequences of exonic gRNA primers

[0042] Forward Primer Reverse Primer gRNA1 SEQ ID NO:1 SEQ ID NO:2

[0043] gRNA2 SEQ ID NO:3 SEQ ID NO:4

[0044]

[0045] Table 2. Sequences of intronic gRNA primers

[0046] Forward Primer Reverse Primer Intron7 SEQ ID NO:5 SEQ ID NO:6

[0047] Intron 15 SEQ ID NO:7 SEQ ID NO:8

[0048]

[0049] The most important advantage of said method of the invention in HER2+ breast cancers is that, unlike cytotoxic agents frequently used in routine practice that create DNA damage, it specifically directs tumour cells to apoptosis while preserving the genomic integrity of regions expressed in normal cells. The fact that DNA doublestrand breaks formed depending on the nuclease activity of Cas9 direct cells exhibiting copy number variation to apoptosis at a higher rate also indicates that this method may be effective in various cancers containing gene amplification such as neuroblastoma, lung, gastric, ovarian and colorectal cancers. In other words, said method of the invention is not limited only to breast cancers but is a method that can be adapted to many cancer types exhibiting gene amplification. Said gRNAs of the invention and the cancer treatment method in which these gRNAs are used can also be applied in other cancer types containing gene amplification such as breast, neuroblastoma, lung, gastric, colorectal carcinoma and various soft tissue tumours. In order to demonstrate the effectiveness of the invention, tests were performed based on the induction of apoptosis in HER2+ breast cancer cells. In the invention, both exon and intron targeting have been performed, however, the main novelty arises from the approach of inducing cell death while preserving gene integrity by means of the intron targeting strategy. Said method is important in terms of specifically directing tumour cells to apoptosis while preserving the genomic integrity of regions expressed in normal cells. In addition, the design and selection of gRNAs have been optimised to increase the operational efficiency and specificity of the system.

[0050] The data regarding Annexin V measurement results obtained as a result of transfection of the PX458 plasmid containing gRNAs belonging to exon 8 are presented in Figure 1. Said measurement is an Annexin V measurement in the MCF-7 cell line; empty PX458 plasmid, PX458-gRNA1, PX458-gRNA2 and PX458-gRNA1+gRNA2 plasmids were transfected into the MCF-7 cell. The analysis results obtained using the Kaluza Analysis program are shown in Figure 1 in the form of a dot plot graph. The total apoptosis rate obtained from the analysis results was calculated as the sum of early apoptosis and late apoptosis rates. The necrosis rates of the cells were evaluated as a separate parameter. Statistical analyses and post hoc data are shown in Figure 2. The obtained results show that cell viability is between 92.2% and 80% in the transfection of empty or gRNA-containing plasmids. In this group, it was observed that the cells undergoing apoptosis the most were those transfected with gRNA2. Nosignificant difference was observed between the apoptosis rates of cells as a result of gRNA1-PX458 and gRNA1+gRNA2-PX458 transfection (p=0.094). In multiple comparisons of the other groups, a significant difference was observed in all groups (p<0.001). When the rates of necrotic cells were examined, it was observed that the cells undergoing necrosis the most were those transfected with gRNA2. In multiple comparisons of the groups, a significant difference was observed in all groups (p<0.001). Empty PX458 plasmid, gRNA1-PX458, gRNA2-PX458 and gRNA1+gRNA2-PX458 plasmids were transfected into the BT-474 cell. The analysis results obtained using the Kaluza Analysis program are shown in Figure 3 in the form of a dot plot graph. The total apoptosis rate obtained from the analysis results was calculated as the sum of early apoptosis and late apoptosis rates. The necrosis rates of the cells were evaluated as a separate parameter. Statistical analyses and post hoc data are shown in Figure 4. The obtained results showed that cell viability was between 65.5% and 5.8% as a result of transfection of empty or gRNA-containing plasmids. In this group, it was observed that the cells undergoing apoptosis the most were those transfected with gRNA2 at a rate of 94.09%. When the apoptosis rates in transfection of gRNA-containing plasmids were compared with PX458 transfection, it was observed that the apoptosis rates of gRNA-containing plasmids increased significantly compared to empty PX458 (p<0.001). No significant difference was observed between the apoptosis rates of cells as a result of gRNA2-PX458 and gRNA1+gRNA2-PX458 transfection (p=0.596). In multiple comparisons of other apoptotic cell groups, a significant difference was observed between all groups (p<0.001). When the rates of necrotic cells were examined, it was observed that there was no significant difference between the groups undergoing necrosis (p<0.386). Since the significance condition was not met, no post hoc test was performed.

[0051] In order to evaluate the significance of the difference between cell groups, the apoptosis and necrosis rates of each group were compared separately. In statistical analyses, One Way ANOVA was performed in R programming. In the transfection of empty PX458 plasmid, when the MCF-7 cell was compared with the BT-474 cell, it was observed that BT-474 underwent apoptosis at a higher rate than MCF-7 (p<0.001). When the MCF-7 cell was compared with BT-474 cells, it was observed that MCF-7 underwent necrosis at a higher rate than BT-474 (p<0.001).In the transfection of PX458 plasmid containing gRNA1 , the highest apoptotic cell rate belongs to BT-474. When the MCF-7 cell is compared with BT-474 cells, it is observed that BT-474 undergoes apoptosis at a higher rate than MCF-7 (p<0.001). In multiple comparisons of the groups, a significant difference was observed between all groups (p<0.001). When MCF-7 and BT-474 were compared in the transfection of PX458 plasmid containing gRNA1, it was observed that there was no significant difference between MCF-7 and BT-474 in terms of necrosis rates (p=0.980).

[0052] In the transfection of PX458 plasmid containing gRNA2, the highest apoptotic cell rate belongs to BT-474. When the MCF-7 cell is compared with BT-474 cells, it is observed that BT-474 undergoes apoptosis at a higher rate than MCF-7 (p<0.001). In multiple comparisons of the groups, a significant difference was observed between all groups (p<0.001).

[0053] In the transfection of PX458 plasmid containing gRNA1+gRNA2, the highest apoptotic cell rate belongs to BT-474. When the MCF-7 cell is compared with BT-474 cells, it was observed that BT-474 undergoes apoptosis at a higher rate than MCF-7 (p<0.001). In multiple comparisons of the groups, a significant difference is observed between all groups (p<0.001). In the transfection of PX458 plasmid containing gRNA1+gRNA2, the highest necrotic cell rate belongs to MCF-7. A significant difference was observed between MCF-7 and BT-474 in terms of necrosis rates (p<0.001).

[0054] In the invention, HER2(+) breast cancer cell lines were targeted using CRISPR Cas9 technology and apoptotic responses of the cells were evaluated. The aim of the invention is to determine whether cells are directed to apoptosis by targeting the HER2 gene and the effects of this process on cell viability. When the HER2 gene is targeted using the CRISPR Cas9 system in the invention, significant levels of apoptosis were detected in HER2(+) breast cancer cell lines. In particular, when the effects of doublestrand breaks created by CRISPR Cas9 on cell viability were examined in cell lines having different HER2 copy numbers, significantly higher apoptosis rates were detected in the BT-474 cell line having a high copy number. These findings support our hypothesis that a large number of irreparable double-strand breaks will trigger apoptosis or necrosis in the cell, while emphasising the potential of said method of the invention as a therapeutic strategy that disrupts survival mechanisms of HER2(+)breast cancer cells and induces cell death by stimulating apoptosis. Thus, it has also been demonstrated that in vitro treatment efficacy can be adapted according to the genetic copy number profile of cells and more effective results can be obtained.

[0055] It is shown that targeting the HER2 gene via target-specific designed gRNAs induces cell death and disrupts survival mechanisms of tumour cells. The most important advantage of said method of the invention in HER2+ breast cancers is that, unlike cytotoxic agents, it specifically directs tumour cells to apoptosis while preserving the genomic integrity of regions expressed in normal cells. Specific gRNAs targeting gene amplification direct only cells exhibiting copy number variation to apoptosis, while not damaging healthy cells. The fact that the apoptosis rate of MCF-7 cells is significantly lower than that of BT-474 indicates that this method can be used as a more specific and effective method compared to other DNA-damaging agents used in HER2+ breast cancer treatment. The high selectivity of said method increases treatment efficacy by creating DNA damage only in targeted cancer cells and inducing cell death, while reducing the risk of damaging genetically normal cells. In this way, cancer cells can be effectively eliminated without damaging healthy cells.

[0056] As a result, the invention demonstrates that creating DNA breaks using CRISPR Cas9, particularly in HER2(+) breast cancer cells, is effective in inducing apoptosis by targeting tumour cells with high selectivity. The obtained findings support that CRISPR Cas9 used in said method of the invention can be used as a potential tool in the treatment of HER2(+) breast cancer.References

[0057] [1] Wang H, Sun W. “CRISPR-mediated targeting of HER2 inhibits cell proliferation through Q4 a dominant negative mutation”. Cancer Letters (2016), 1 -7.

Claims

CLAIMS1. gRNAs that induce apoptosis in cancer cells having HER2 amplification, wherein said gRNAs are;• gRNA1 having a forward primer with the nucleotide sequence of SEQ ID NO:1 and a reverse primer with the nucleotide sequence of SEQ ID NO: 2, and / or• gRNA2 having a forward primer with the nucleotide sequence of SEQ ID NO:3 and a reverse primer with the nucleotide sequence of SEQ ID NO: 4, which target the exonic region of cancer cells,and / or• lnt7-gRNA having a forward primer with the nucleotide sequence of SEQ ID NO: 5 and a reverse primer with the nucleotide sequence of SEQ ID NO: 6, and / or• lnt15-gRNA having a forward primer with the nucleotide sequence of SEQ ID NO: 7 and a reverse primer with the nucleotide sequence of SEQ ID NO: 8,which target the intronic region of cancer cells.

2. Method for inducing apoptosis in cancer cells having HER2 amplification in which gRNAs according to claim 1 are used, comprising the process steps of;i. adding the prefix “caccg” to the initial sequence of the forward primers SEQ ID NO: 1 and / or SEQ ID NO: 3 and adding the suffix “aaac” to the terminal sequence in accordance with the Bbsl restriction enzyme cut site present in the PX458 plasmid, and cloning the reverse primers SEQ ID NO: 2 and / or SEQ ID NO: 4 into the plasmid, and obtaining a plasmid having the nucleotide sequence of SEQ ID NO: 9 as a result of this cloning, ii. performing colony PCR in order to verify after cloning studies,iii. performing transformation into the DH5alpha E. coli strain in order to amplify the plasmid into which gRNA is cloned and producing bacteria in ampicillin- containing lysogeny broth (LB broth) medium,iv. subsequently isolating the plasmids from E. coli bacteria,v. performing transfection of the isolated plasmids containing the target locus into cancer cells by electroporation with parameters of 1600v 20ms 1 pulse, andvi. occurrence of apoptosis or necrosis in cancer cells due to a large number of irreparable double-strand breaks in cells carrying the plasmid 48 hours after transfection.

3. gRNAs according to claim 1 for use in the treatment of cancers having HER2 amplification, comprising nucleotide sequences SEQ ID NO: 1-8.

4. gRNAs according to claim 3 that induce apoptosis in cancer cells having HER2 amplification, wherein said cancer cells are breast, neuroblastoma, lung, colorectal carcinoma or gastric cancer cells.

5. gRNAs according to claim 4 that induce apoptosis in cancer cells having HER2 amplification, wherein the cancer cell line is the MCF-7 (breast cancer) or BT-474 (breast cancer) cell line.

6. gRNAs according to any one of claims 3-5 that induce apoptosis in cancer cells having HER2 amplification, wherein they induce apoptosis in cancer cells during cancer treatment.

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