Sistema CRISPR-Cas9: uma alternativa terapêutica para neoplasia pulmonar
Amanda S. R. Almeida; Cleide B. Souza
Centro Universitário Lusíada, Santos, São Paulo, Brazil.
J Bras Patol Med Lab. 2021; 57: 1-8.
DOI: 10.5935/1676-2444.20210051
Correspondence Author
Amanda Souza Reis Almeida
ORCID: 000-0001-5171-7405
e-mail: amandasouzareis@icloud.com
First Submission on 02/10/21
Last Submission on 02/11/21
Accepted for publication on 05/20/21
Published on 12/20/21
ABSTRACT
Neoplasms are caused by mutations in deoxyribonucleic acid (DNA) characterized by debilitating treatments and difficult early diagnosis. In 2018, there were 27,200 deaths from lung cancer and 31,270 new cases of the disease. Among the different types, non-small cell lung cancer (NSCLC) accounts for 15% of lung cancer; 25% to 30% of cases are caused by the activating mutation of the epidermal growth factor receptor (EGFR) gene. In this context, through a literature review, this work sought to elucidate the wild-type mechanism of clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system and its applicability in the gene therapy of lung cancer. In vitro experiments have demonstrated that the system promotes tumor suppression by inducing deletion of the mutated gene, presenting high specificity for neoplastic cells. However, it is necessary to improve the technique to reduce possible off-target effects.
Key words: lung cancer; gene therapy; CRISPR-Cas systems; CRISPR-associated protein 9; biotechnology.
RESUMO
Neoplasias são causadas por mutações no ácido desoxirribonucleico (DNA) caracterizadas por tratamentos debilitantes e de difícil diagnóstico precoce. Em 2018, ocorreram 27.200 óbitos por neoplasia pulmonar e 31.270 novos casos da doença. Entre os diversos tipos, o carcinoma pulmonar de células não pequenas (CPNPC) corresponde a 15% das neoplasias pulmonares; 25% a 30% dos casos são causados pela mutação ativadora do gene do receptor do fator de crescimento epidérmico (EGFR). Nesse contexto, por meio de uma revisão da literatura, este trabalho buscou elucidar o mecanismo selvagem do sistema de Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas associadas à proteína 9 (CRISPR-Cas9) e sua aplicabilidade na terapia gênica da neoplasia pulmonar. Experimentos in vitro demonstraram que o sistema promove supressão tumoral por induzir a deleção do gene mutado, apresentando alta especificidade pelas células neoplásicas. Entretanto, é necessário o aprimoramento da técnica visando diminuir possíveis efeitos fora do alvo.
Unitermos: neoplasia pulmonar; terapia genética; sistemas CRISPR-Cas; proteína 9 associada à CRISPR; biotecnologia.
RESUMEN Las neoplasias son causadas por mutaciones en el ácido desoxirribonucleico (ADN) caracterizadas por tratamientos debilitantes y un diagnóstico precoz difícil. En 2018, hubo 27.200 muertes por cáncer de pulmón y 31.270 nuevos casos de la enfermedad. Entre los diferentes tipos, el cáncer de pulmón de células no pequeñas (CPCNP) representa el 15% de los cánceres de pulmón; entre el 25% y el 30% de los casos son causados por la mutación activadora del gen del receptor del factor de crecimiento epidérmico (EGFR). En este contexto, a través de una revisión de la literatura, este trabajo buscó dilucidar el mecanismo salvaje del sistema de repeticiones palindrómicas cortas agrupadas y regularmente espaciadas asociado a la proteína 9 (CRISPR-Cas9) y su aplicabilidad en la terapia génica del cáncer de pulmón. Los experimentos in vitro mostraron que el sistema promueve la supresión tumoral ya
que induce la deleción del gen mutado, presentando una alta especificidad por las células neoplásicas. Sin embargo, es necesario
mejorar la técnica para reducir los posibles efectos fuera de diana.
Palabras clave: cáncer de pulmón; terapia de genes; sistemas CRISPR-Cas; proteína 9 asociada a CRISPR; biotecnología.
INTRODUCTION
Approximately 7,000 diseases with a known cause are caused by genetic mutation, and only 500 have effective treatments. Over the years, gene therapies have improved the prognosis of individuals with cancer and, when combined with chemotherapy, radiotherapy and surgery, they can help to reduce the number of cancer cells, extending the life of the patient. Gene editing techniques such as zinc-finger nuclease (ZFN) and transcriptional activator-like effector nuclease (TALEN), had limited effectiveness because of the inability of targeting specific targets. However, the clustered regularly interspaced short palindromic repeatsassociated protein 9 (CRISPR-Cas9) system has been shown to be a rising genetic technology in the gene-editing scenario, increasing visibility and accessibility every day due to its simple and effective functioning in editing inherited disease-causing genes. This technique has changed the approach of gene editing when applied in anticancer therapy, as well as in the study of gene expression, in labeling chromosome loci in live-cells, and in genetic screening(1-3).
This system was originally isolated from the adaptive immune response of bacteria. It works from a guide ribonucleic acid (RNA) complementary to the sequence to be edited, responsible for signaling this region to the endonuclease Cas9, which recognizes it and promotes its cleavage, inducing cell repair mechanisms, which can be homologous or non-homologous, causing the cell to lose its mutated version. The most used Cas9 endonuclease in gene therapy originates from Streptococcus spp, belongs to the type II CRISPR system; it is currently used in clinical trials with human cells for the treatment of neoplasms(4, 5).
In 2018 alone, lung cancer caused 27,200 deaths, with an incidence of 31,270 new cases; for this reason, it is considered the leading cause of death from cancer in Brazil in recent years. Today, there is no cure for any kinds of cancer, and treatments often prove debilitating to patients. Thus, the expansion of biotechnology in the health area enabled the search for new therapeutic targets related to the genotype of diseases, highlighting the CRISPR-Cas9 system as one of the main tools in the field of gene therapy. The main basic gene discussed in this work is the epidermal growth factor receptor (EGFR) gene, the oncogene responsible for important characteristics in the neoplastic development in the lung, such as cell proliferation, survival, differentiation, angiogenesis, invasion, and metastasis. Due to drug resistance to medicines used in the treatment of neoplasms, gene therapies demonstrate great results in the search for an effective response of the body to treatments, acting directly on the neoplasm as a promoter of the reduction of tumor proliferation through the deletion of genes responsible for the individual’s disease(1, 6, 7).
OBJECTIVE
This study aimed to describe one of the most frequent mutations in non-small cell lung cancer (NSCLC) cases, such as the activating mutation of the EGRF gene, responsible for the transformation of normal cells into neoplastic cells in most adenocarcinoma, as well as the application of the CRISPR-Cas9 system technique as a therapeutic alternative for lung cancer.
METHOD
This literature review was developed from PubMed, Scholar Google, and Scielo databases. Through these research systems, several studies were found on CRISPR-Cas9 as a therapeutic alternative to the treatment of many diseases, including lung cancer. As descriptors, the following were used: elementos CRISPR; proteínas associadas a CRISPR; terapia gênica; intervenção genética; biotecnologia; neoplasia maligna; neoplasia pulmonar; CRISPR elements; CRISPR-associated proteins; gene therapy; genetic intervention; biotechnology; malignant neoplasm; and lung cancer. National and international works were included, enabling a broad approach to the subject, demonstrating the global importance of this theme. As an exclusion criterion, papers with publication data before 2010 were not accepted.
Neoplasm
According to Maitra (2013)(8), neoplasms are genetic disorders caused in deoxyribonucleic acid (DNA); they are usually acquired spontaneously or expressed by environmental and epigenetic factors. These mutations have hereditary characteristics, which pass from progenitor cells to daughter cells, providing advantages both in their growth and in their survival, with the accumulation of mutations during the proliferation of these cells, which promotes the main characteristics of neoplasms, such as growth factors selfsufficiency, in which their proliferation becomes not dependent on physiological regulation; lack of response to growth-inhibitory signs, tending to increase in size regardless of location; escape from apoptotic pathways, so that this mutated cell remains viable under conditions that induce its cell death; ability to invade nearby or distant tissues, culminating in the spread of their growth; ability to escape the immune response, among other changes that make these cells increasingly proliferative and immortal.
The neoplasm can be classified according to its behavior (malignant or benign) and its origin, depending on the tissue where cell proliferation started. Benign neoplasms are usually characterized by maintaining a restricted, limited, and slow growing. However, the malignant ones have more aggressive behavior; their neoplastic or transformed cells can destroy nearby structures and spread to other places, characterizing a metastasis. The nomenclature used for this malignant behavior is carcinoma, which often refers to malignant neoplasms that affect the lining epithelium of a given organ. Carcinoma can be further subdivided into adenocarcinoma, which develops into glandular cells, and squamous cell carcinoma, when it produces squamous cells(9, 10).
Lung cancer
Lung cancer has a high incidence; 1.8 million cases are diagnosed every year, characterizing 13% of cases worldwide(6). This neoplasm was declared as the most incident in the world, with approximately 2.1 million cases(11).
For therapeutic and diagnostic purposes, lung cancers are divided into groups and can be classified as small cell lung cancer (SCLC) or NSCLC. NSCLC characterized by a course of aggressive progression and diagnosis in advanced stages of the disease, with low patient survival; it also groups other histological types, such as squamous cell carcinoma, adenocarcinoma, and large cell undifferentiated carcinoma(12) – a category with the highest prevalence of adenocarcinoma(6). The most common mutations found in NSCLC are EGFR gene activators; they are seen in up to 30% of national cases and between 8% and 15% of cases worldwide. This gene is responsible for encoding the EGFR, which is overexpressed and/or in greater quantity on the cell surface, demonstrating an important role in lung carcinogenesis in this group(13).
Genetic basis of lung cancer
The changes that occur in genes responsible for regulating the cell cycle encompass four categories: proto-oncogenes; tumor suppressor genes; the genes that regulate apoptosis; and the genes involved in DNA repair. They can result in the activation and/or inactivation of the gene transcription product, the proteins(8, 9). In NSCLC a mutation occurs in a proto-oncogene, turning it into an oncogene. Proto-oncogenes are responsible for stimulating cell division; it is called a gain-of-function mutation, causing it to increase the proliferative and growth stimulus, transforming a normal cell into a neoplastic cell. In this case, a mutation in the EGFR gene transforms it from a proto-oncogene to an oncogene, due to increased signal transduction in the cell, which enhances proliferation, invasion, differentiation, angiogenesis, metastasis, and resistance to apoptosis(8, 14).
EGFR gene mutations
EGFR is part of a group of protein tyrosine kinases, called ErbB, which play a role in cell physiology, located in the cell membrane with an internal and external domain. These receptors depend on an external ligand for their activation, generating signal transduction, activating cell proliferation, and growth. Within this group, there is the EGFR, composed of an N-terminal portion in the extracellular domain, a transmembrane domain, and a C-terminal located in the cytosolic face. The extracellular domain is responsible for receiving the ligand, while the intracellular domain is able to phosphorylate its structure and adjacent proteins involved in the process of cell division(13, 15).
Therefore, when there is a mutation in the gene that encodes the EGFR, it becomes a mutated protein, capable of sending successive mitogenic signals to the cell, even if there is no ligand mediating its activity. Mutations that increase its expression and the amount in the plasma membrane can also occur(3). According to Almeida (2018)(16), about 15% of the population has the EGFR mutation.
An EGFR activating mutation usually occurs between the first four exons of the gene (18 to 21); exon 21 point mutation (L858R substitution) and exon 19 deletion account for 90% of cases, the former is responsible for 43% of lung adenocarcinoma in Asians. These mutations can permanently activate the receptor, with the presence of a ligand or not, because of an alteration in the adenosine triphosphate (ATP) site in the inner portion, leading to constant signal transduction and activation of growth and proliferation(14, 17, 18).
Considering that EGFR mutations are present in 60% of NSCLC cases, and this corresponds to 85% of lung cancer cases, the investigation of this receptor as a potential therapeutic target has been increasing in cancer research, as it is one of the neoplasms with higher incidence and mortality worldwide(19).
Diagnosis of lung cancer
According to Araujo et al. (2018)(20), in Brazil, NSCLC is usually diagnosed in advanced stages, which influences the low survival rate of patients; 70% of patients have locally advanced disease with metastasis when diagnosed.
The initial diagnosis is made upon medical suspicion after clinical respiratory presentations or through abnormal radiological findings in tests directed to other purposes. A definitive diagnosis is achieved through histological tests (pleural or pleuropulmonary biopsy). Molecular diagnosis is also important for identifying the EGFR mutation in lung neoplasms with non-squamous histology. After having the malignancy confirmed, the patient undergoes a chest and upper abdomen computed tomography (CT) scan with contrast. In addition, the proton emission tomography (PET) technique is used to estimate the extent of the disease in possibly resectable NSCLC, as it is a more sensitive test in the detection of lymph node involvement (relevant to know if there has been metastasis of the neoplasm, indicating progression) compared to CT(21, 22).
Conventional treatments for lung cancer
According to the Brazilian Ministry of Health (2014)(21), surgery is the best therapy used in NSCLC with curative purposes. The amount of the resection during the procedure is defined by analyzing the tumor extension and the need to preserve patient’s lung functions. It is also necessary to remove the adjacent lymphatic system, in order to prevent metastasis to other organs.
Radiotherapy is a treatment that can be used in palliative and curative care, and may be associated with chemotherapy, depending on patient’s stage and need. External radiation therapy is indicated for NSCLC at any stage; stereotactic ablative radiotherapy, for stage I with no clinical signs and operative need; thoracic irradiation associated with systemic chemotherapy, applied for curative purposes with efficacy in a small portion of patients with nonresectable NSCLC. Systemic chemotherapy can be used for palliative purposes, through the use of different drugs, and it is essential to assess patient’s functional capacity and the choice of procedures for therapy, as there may be several side effects, such as fatigue, hair loss, bruises, hemorrhages, among others(20, 23).
The response to cytotoxic chemotherapy for NSCLC cases varies from 20% to 30%, and it gives only 8-10 months of life to the patient undergoing this treatment. Fortunately, the molecular differentiation strategies of neoplasms help in choosing the therapy, making it possible to identify the mutations involved in the pathophysiology of the disease, showing new therapeutic targets. Therefore, the most recent treatment applied for NSCLC is the use of tyrosine kinase inhibitors in pre-identified oncogenic agents, such as the mutated version of EGFR, to inhibit the constantly active receptor in large numbers, preventing it from continuing to promote signal transduction, leading to a decrease in cell proliferation and growth(3, 13). However, the literature describes cases of drug resistance that reduce the effectiveness of the treatment in a portion of those affected(16, 24).
CRISPR-CAS9 SYSTEM
Mechanism of action and origin
CRISPR is a system found in prokaryotic organisms; it comprises clustered regularly interspaced short palindromic repeats, and is responsible for recognizing and preventing the action of exogenous DNA, whether from bacteriophages or plasmids, acting as a mechanism of the adaptive immune system of bacteria, preventing specific infections. According to the functioning of CRISPR-associated (Cas) proteins, they can be divided into class I, in which numerous Cas proteins are required to act, or class II, in which a single Cas protein is active. The CRISPR-Cas9 system is classified as class II, and its effector is the Cas 9 protein, considered one of the most used systems currently in genomic editing(5, 25).
Approximately 40% of bacterial and 90% of archaeal species have this system that works by a sequence of guide RNA and DNA segments encoded by the CRISPR loci, which has about 21 to 72 base pairs (bp), together with the Cas9 protein(14, 26).
In the immunization phase, the bacteria are exposed to an exogenous genetic material, which is processed by Cas endonuclease, transforming it into short fragments called protospacer; then, they are integrated as new CRISPR repeat spacers within the bacterial chromosome, that is, the exogenous genetic material fragment will intersperse the repeated DNA palindromic regions of the bacterium, providing a genetic record of the invaders, acting to prevent future infection of the same agent(3).
When there is a new attempt at infection, as shown in Figure 1, the biogenesis phase occurs, in which the CRISPR loci is transcribed, generating a long precursor CRISPR RNA (pre-crRNA), which hybridizes to a trans-activating crRNA (tracrRNA), responsible for assisting in the maturation of this pre-crRNA, as then, RNAse III cleaves this hybrid to produce short mature CRISPR RNAs (crRNAs). At its 5’ end, the crRNA will have a short DNA segment complementary to the invader’s exogenous DNA (spacer) and, at the 3’ end it will contain a portion of the CRISPR repeat sequence. Transcription of Cas genes will encode CRISPR-associated proteins, specifically the cas9 gene, which encodes a nuclease capable of inactivating exogenous genetic material complementary to the spacer. Thus, a complex formed by Cas9, crRNA linked to post-maturation tracrRNA is obtained, which will search for the DNA corresponding to the spacer. This binding requires a protospacer adjacent motif (PAM), a region close to the sequence corresponding to the crRNA in the invading DNA, which acts as a site where the Cas9 protein can bind and begin its editing mechanism. When there is complementarity between the crRNA spacer and the target genetic material, Cas9 promotes the destruction of exogenous DNA or RNA.
Nuclease Cas9 contains two endonuclease sites: the HNH and the RuvC, which cleave the complementary and noncomplementary exogenous DNA strand, forming blunt ends; they are also described as double-strand breaks (DSB), with approximately three nucleotides (nt) upstream of the PAM sequence. Binding to PAM is important for the functioning of the system as it characterizes its specificity, it is directed to the invading DNA since the host does not have this sequence(4, 25, 27).
Mechanism of CRISPR-Cas9 gene-editing of lung cancer
One of the main applications of the CRISPR-Cas9 system is gene editing, as it makes it possible to edit a DNA segment from the Cas9 nuclease driven by a single-guide RNA (sgRNA), a molecule that combines trRNA and crRNA into a single RNA transcript, improving the specificity of the system. Several types of Cas9 have been described in different species of bacteria, such as Streptococcus spp and E. coli; in nature, most of these variants need a crRNA and a tracrRNA to be targeted, however, the emergence of sgRNA facilitated the performance of the system since it is easily designed(1, 4).
The process works by producing a programmable crRNA for the target sequence and a fixed trRNA, which are fused to form a sgRNA, usually containing 20 nt complementary to the sequence that is desired to edit, serving as a guide for Cas9, which, through its endonuclease action, generates a double-strand break (DSB) – 3 bp upstream of the PAM –, which induces the activation of the cell’s DNA repair systems. The specificity and efficiency of the method depend, in large part, on the sgRNA, in which the first 10 to 12 nt at the 3’ end of the guide RNA are complementary to the PAM of the target gene; this sequence is called the seed, as this is where the endonuclease begins its activity(25, 27). However, using a short PAM can increase the possibility of generating off-target mutations and unwanted mutations compared to using a longer PAM(26).
As shown in Figure 2, DSBs can be repaired by two distinct pathways, either HDR (homology-directed repair) or NHEJ (non-homologous end joining). Generally, the most activated mechanism is the NHEJ, predominant in the G1, S, and G2 phases of the cell cycle. It incorporates random nt into the break region, making it an error-prone system due to random insertions that often cause indels (insertions and deletions) mutations. For this reason, this system can be used to eliminate gene expression. Because of the indels mutations, the NHEJ promotes extreme alterations in the reading phase of the genetic sequence (knockout). On the other hand, the HDR repair system uses a designing donor DNA (approximately 400 bp for plasmid or 25 to 65 bp for oligodeoxynucleotide) as a template for the synthesis of a new DNA segment in the break region, thus characterizing its mechanism of homologous recombination. This system can be used for introducing transgenes or for extremely precise editing of the genome, as the template DNA is designed with a specific genetic sequence. Thus, when a template DNA is inserted along with the CRISPR system, the HDR is activated and can serve as a basis for the insertion of a homologous sequence; but, in the absence of this template, the NHEJ system is operational. This strategy can be essential when the neoplasm originates from an oncogene, which can be deleted by the CRISPR-Cas9 system(17-19).
Therefore, the CRISPR-Cas9 technology enables the study of therapeutic targets in different types of solid neoplasms, such as lung cancer. Within several mechanisms in which the disease can be expressed, EGFR mutations are one of the main alterations with regards to NSCLC. When altered, this gene activates the tyrosine kinase consecutively, leading to oncogene-mediated transformation of lung epithelial cells. Mutations usually have a single point characteristic, which means an amino acid substitution (leucine to arginine) at codon 858(9, 18).
Based on this principle, the use of the CRISPR-Cas9 system with a sgRNA complementary to the mutated sequence of the EGFR gene induces the endonuclease to cleave it from the neoplastic cell, causing gene suppression, preventing it from encoding an altered tyrosine kinase protein, and decreasing cell proliferation. Thus, it allows to correct an altered gene with replacement of the wild version when editing by CRISPR is accompanied by a homologous DNA fragment to trigger the HDR(17).
According to Cheung et al. (2018)(18), the targeting of CRISPR to the mutant version of the EGFR (L858R) had a relevant result in the reduction of tumor proliferation, since it is selective to neoplastic cells, both in vitro and in vivo.
Koo et al. (2017)(14) used CRISPR-Cas9 technology to differentiate the allele with a single nt missense mutation in the EGFR gene located in exon 21, and produced a sgRNA that hybridizes to the mutant allele, composed of extra guanine at the 3’ end which binds to the U6 promoter, as well as more 19 nt of DNA complementary to the sequence to be edited upstream of the PAM sequence recognized by SpCas9 (Cas protein from Streptococcus spp). Delivery was made through an adenovirus incapable of replication, resulting in its interruption with high specificity. Furthermore, upon cleavage of this oncogene, we observed a high rate of neoplastic cell death leading to tumor reduction in an animal model of a mouse with human lung cancer. Indels mutations were also detected in one of the cell lines used in the study, demonstrating the effectiveness in vitro of the system.
However, according to Khan et al. (2016)(17), the selectivity of the system is still questionable; it is necessary to improve the technique so that the off-target effect is minimized, which is supported, in part, by the method of delivery and the PAM specificity in the target genome, as the sequence may be similar in undesirable regions. In large genomes, such as eukaryotic ones, many regions may have homology to the target sequence complementary to the guide RNA used in CRISPR-Cas9, which leads to undesired mutations along the host genome(26). Therefore, the delivery of the system in neoplastic cells is listed as a challenge to be faced when applying the method in vivo. It should be considered that the components of the CRISPR-Cas9 system are macromolecules that do not easily pass through the cell plasma membrane, let alone by the nuclear membrane. In general, many methodologies have been, and still are tested, as viral vectors (plasmids) and nonviral vectors via. Viral vectors work by incorporating a plasmid with genes encoding the Cas9 protein and its associated guide RNA into a viral lineage, which naturally injects this plasmid into the host cell. The most used strains are integrase-defecient lentivirus (IDLV) and adeno-associated virus (AAV)(3).
However, even viral delivery methodologies have their limitations, that is the reason why non-viral deliveries have been developed, which include the production of synthetic vehicles that encapsulate the CRISPR-Cas9 system for insertion into the target cell. Among them can be included: electroporation, hydrodynamic injection, microinjection, and self-assembly of nanoparticles (NPs), however, they are not as efficient, depending on the tissue, due to stability or biocompatibility, which makes their application in vivo experiments difficult (1).
DISCUSSION
According to Gupta et al. (2019)(2), the effectiveness and simplicity of the CRISPR-Cas9 system has expanded its use for various purposes in research laboratories, especially when it is proposed to analyze the gene expression of endogenous cells, aiding in the identification and research of possible therapeutic targets.
In this context, the problem of lung cancer is not restricted to the baseline issue related to the pathophysiology of the disease (such as proliferation and metastasis), but also to evidence of resistance to the drugs currently used, as reported by Leite et al. (2013)(24) about the tyrosine kinase inhibitor in EGFR geneactivating neoplasms. Based on this principle, according to the CRISPR-Cas9 mechanism described by Zhang et al. (2018)(5), Lander (2016)(25), and Jiang et al. (2017)(4), we can apply the mechanism as an effective gene therapy, in which, considering its high specificity, compared to current clinical treatments, CRISPRCas9 is less likely to affect other patient’s organs and systems.
As for the effectiveness of the CRISPR-Cas9 system in editing tumor genes, the experiments carried out by Koo et al. (2017)(14), were analyzed; the authors highlight the CRISPR-Cas9 system as an effective tool to target specific neoplastic cells, capable of leading to the suppression of tumor growth without extreme side effects due to nonspecific action on wild cells. However, Khan et al. (2016)(17) highlighted that the specificity of the system, when used in large genomes, is still questionable, since it may contain binding sequences from the CRISPR-Cas9 system, generating undesirable interactions and possibly causing off-target mutations. It is also necessary to consider that the experiments by Koo et al. (2017)(14) used animal and non-human research, so the results can be encouraging. But there is still a lot to improve before the system is introduced into clinical practice as a treatment, because, according to Liu et al. (2019)(3), one of the problems faced by researchers is to find an effective delivery mechanism that can be totally safe in vivo application.
Generally, as shown by Cheung et al. (2018)(18), it is correct to state that the application of the system for lung cancer promoted advances in tumor suppression and in the identification of several potential therapeutic targets. In vivo and in vitro studies have shown promising results in its use in reducing distant metastases (major cause of death) and in reaching specific oncogenes, such as EGFR, characterizing a major step in cancer gene therapy and an important role in the cancer treatment system.
CONCLUSION
CRISPR-Cas9 system is a promising tool for editing genes responsible for the emergence of cancer in specific genes. Its application in lung cancer as a target disease proved to be effective since tumor proliferation was reversed through the deletion of the mutated version of the EGFR gene. Its simple mechanism contributes to its easy application in the gene therapy scenario, representing a promising therapeutic alternative for this disease, and the possibility of reducing patient suffering and weakness, since this method does not require prolonged treatments or invasive surgery to be effective. It will still be necessary to improve several issues related to the specificity and means of delivery of the system to the target cell, however, the applicability of the therapy worthwhile the investment in research. Therefore, this method does not only represent the treatment of the neoplasm in question but possibly for other diseases caused by mutations, highlighting the importance of biotechnology developments in the health area in gene therapies, in which the CRISPR-Cas9 system stands out.
ACKNOWLEDGEMENTS
Professors are the basis for academic development, especially in the area of research, as they are the ones who shape future researchers. My sincere thanks to Prof. Doctor Cleide Barbieri de Souza, who believed in my potential and improved my work with extreme impetus and competence.
REFERENCES
1. Luther DC, Lee YW, Nagaraj H, Scaletti F, Rotello VM. Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges. Expert Opin Drug Deliv [Internet]. 2018; 15(9): 905-13. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295289.
2. Gupta D, Bhattacharjee O, Mandal D, et al. CRISPR-Cas9 system: a new-fangled dawn in gene editing. Life Sci [Internet]. 2019; 232. Available at: https://www.sciencedirect.com/science/article/abs/pii/S0024320519305624?via%3Dihub.
3. Liu B, Saber A, Haisma HJ. CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment. Drug Discov Today [Internet]. 2019; 24: 955-70. Available at: https://www.sciencedirect.com/science/article/pii/S1359644618305245.
4. Jiang F, Doudna JA. CRISPR-Cas9 estruturas e mecanismos. Annu Rev Biophys [Internet]. 2017; 46: 505-29. Available at: https://doi.org/10.1146/ annurev-biophys-062215-010822.
5. Zhang C, Quan R, Wang J. Development and application of CRISPR/Cas9 technologies in genomic editing. Hum Mol Genet [Internet]. 2018; 27: 79- 88. Available at: https://academic.oup.com/hmg/article/27/R2/R79/4962527.
6. Oliveira AC, Silva AV, Alves M, et al. Perfil molecular do carcinoma pulmonar de células não pequenas no Nordeste brasileiro. J Bras Pneumol [Internet]. 2019; 45: 1-7. Available at: https://www.scielo.br/pdf/jbpneu/v45n3/pt_1806-3713-jbpneu-45-03-e20180181.pdf.
7. Costa GJ, Mello MJ, Bergmann A, Ferreira CG, Thuler LC. Estadiamento tumor-nódulo-metástase e padrão de tratamento oncológico de 73.167 pacientes com câncer de pulmão no Brasil. J Bras Pneumol [Internet]. 2020; 46(1): 1-8. Available at: http://www.jornaldepneumologia.com.br/ detalhe_artigo.asp?id=3108.
8. Maitra A. Neoplasia. In: Kumar V, Abbas AK, Aster JC, editors. Robbins patologia básica. 9. ed. Rio de Janeiro: Elsevier; 2013. pp. 161-213.
9. Cooper WA, Lam DC, O’Toole SA, Minna JD. Molecular biology of lung cancer. J Thorac Dis [Internet]. 2013; 5: 479-90. Available at: https://pubmed. ncbi.nlm.nih.gov/24163741/. 10. Instituto Nacional do Câncer (BR). Como surge o câncer? Brasília: Ministério da Saúde; 2019. Available at: https://www.inca.gov.br/como-surge-ocancer. [Accessed on: Nov 20, 2020].
11. Instituto Nacional do Câncer. Estimativa 2020. Brasília: Ministério da Saúde; 2020. Available at: https://www.inca.gov.br/estimativa/ introducao#:~:text=O%20c%C3%A2ncer%20de%20pulm%C3%A3o%20%C3%A9,47%25)%20de%20casos%20novos. [Accessed on: Nov 20, 2020].
12. Barros RL. Validação do uso da proteína cofilina como biomarcador preditivo do prognóstico de carcinoma de pulmão de não pequenas células [dissertation]. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2010. Available at: https://www.lume.ufrgs.br/bitstream/ handle/10183/26938/000762398.pdf?sequence=1&locale-%20attribute=en.
13. Lopes GL, Vattimo EF, Junior GC. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer. J Bras Pneumol [Internet]. 2015; 41: 365-75. Available at: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1806- 37132015000400365&lng=en&tlng=en.
14. Koo T, Yoon AR, Cho HY, Bae S, Yun CO, Kim JS. Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression. Nucleic Acids Res [Internet]. 2017; 45(13): 7897-908. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570104/.
15. Shea M, Costa DB, Rangachari D. Management of advanced non-small cell lung cancers with known mutations or rearrangements: latest evidence and treatment approaches. Ther Adv Respir Dis [Internet]. 2016; 10(2): 113-29. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933559/.
16. Almeida DV. Nova opção de terapia alvo para tratamento do câncer de pulmão metastático [Internet]. 2018. Available at: https://mocbrasil.com/ blog/noticias/nova-opcao-de-terapia-alvo-para-tratamento-do-cancer-de-pulmao-metastatico/.
17. Khan FA, Pandupuspitasari NS, Chun-Jie H, et al. CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases. Oncotarget [Internet]. 2016; 7: 52541-52. Available at: https://www.oncotarget.com/article/9646/text/.
18. Cheung AH, Chow C, Zhang J, et al. Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9. Lab Invest [Internet]. 2018; 98: 968-76. Available at: https://www.nature.com/articles/s41374-018-0056-1#citeas.
19. Silva ID. Uso da detecção de mutações no gene EGFR. Revista Médica [Internet]. 2015; (4): 1. Available at: https://www.fleury.com.br/medico/ artigos-cientificos/uso-da-deteccao-de-mutacoes-no-gene-egfr-revista-medica-ed-4-2015.
20. Araujo LH, Baldotto C, Castro G, et al. Câncer de pulmão no Brasil. J Bras Pneumol [Internet]. 2018; 44(1): 55-65. Available at: https://www.scielo. br/pdf/jbpneu/v44n1/pt_1806-3713-jbpneu-44-01-00055.pdf.
21. Ministério da Saúde. Brasil. Portaria nº 957, de 26 de setembro de 2014. Aprova as diretrizes diagnósticas e terapêuticas do câncer de pulmão. Brasília: Ministério da Saúde; 2014.
22. Montella T, Carvalho B, Jacob R, Ferreira CG. Câncer de pulmão: células não pequenas: doença metastática. Oncologia D’or [Internet]. 2017; 24. Available at: https://sboc.org.br/images/Pulmao_nao_pequenas_celulas_meta.pdf.
23. Evans M. Lung cancer: needs assessment, treatment and therapies. Br J Nurs [Internet]. 2014; 22. Available at: https://www.magonlinelibrary.com/ doi/abs/10.12968/bjon.2013.22.Sup17.S15?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org.
24. Leite CA, Costa JV, Callado RB, Torres JN, Júnior RC, Ribeiro RA. Receptores tirosina-quinase: implicações terapêuticas no câncer. Rev Bras Oncol Clin [Internet]. 2012; 8: 130-42. Available at: https://www.sboc.org.br/app/webroot/Site_RBOC_OFICIAL/pdf_edicao_29/artigo4.pdf.
25. Lander ES. The heroes of CRISPR. Cell [Internet]. 2016; 164: 18-28. Available at: http://www.sciencedirect.com/science/article/pii/S0092867415017055.
26. Zhang F, Wen Y, Guo X. CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet [Internet]. 2014; 23: 40-6. Available at: https://academic.oup.com/hmg/article/23/R1/R40/2900693.
27. Hryhorowicz M, Lipiński D, Zeyland J, Słomski R. CRISPR/Cas9 Immune system as a tool for genome engineering. Arch Immunol Ther Exp [Internet]. 2017; 65(3): 233-40. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434172/.
