Gene therapy using CRISPR Cas9 technique is rapidly gaining popularity among the scientific community primarily because of its versatility, cost-effectiveness, and high efficacy. While the laboratory-based experiments and findings making use of CRISPR as a gene editing tool are available in ample amounts, the question arises that how much of these findings are actually translatable into measures helping in combating particular disease conditions. In this review, we highlight the important studies and findings done till now in the perspective of lung cancer with an in-depth analysis of various clinical trials associated with the use of CRISPR Cas9 technology in the field of cancer research.
Introduction to lung cancer severity
Characterized by uncontrollable multiplication of cells, cancer when it starts in the cells of the lungs is called lung cancer. As the cancer cells grow, it destroys the healthy tissues surrounding it and ultimately forms a malignant tumor. When cancer originates in cells of the lungs it is called primary lung cancer. Cancer that starts in another part of the body and spreads to the lung is termed lung metastasis, treatment to which is done in a different manner than primary lung cancer. On the histological aspect, there are two major groups of lung cancer—non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), depending on the cell type in which cancer starts.
NSCLC usually starts in glandular cells on the outer part of the lung and is called adenocarcinoma. NSCLC can also commence in squamous cell lining of the bronchi leading to squamous cell carcinoma. Large cell carcinoma is another type of NSCLC, but it is less common. Sarcoma and sarcomatoid carcinoma are other rare types of NSCLC. In SCLC, cells lining the bronchi in the center of the lungs are usually affected. Two major types of SCLC are small cell carcinoma and combined small cell carcinoma (a mixed tumor with squamous or glandular cells). About 14% of all new cancer diagnoses turn out to be lung cancer in which NSCLC being the most dominant type, accounting for 80–85% of all lung cancer diagnoses. Even though being the more dangerous one, SCLC accounts for 10–15% of lung cancer . In India, the prevalence of lung cancer is on the rise, with 57,795 new cases reported in 2010, to reach 67,000 new cases by 2020 . According to a study in a center in Northeast India, 49.1% of the lung cancers were squamous cell carcinoma, the other two centers in northern and western India, adenocarcinoma cases were dominant comprising 39 and 43.8% of lung cancers, respectively [3,4,5]. It has been observed that, worldwide, there is an epidemiologic shift with adenocarcinoma being more prevalent than squamous cell carcinoma .
In NSCLC the most invariable early genetic changes involve loss of genomic regions of chromosomes 3p and 9p, deletions of the chromosomal arm on 5p and mutations in the tumor suppressor proteins like p53 and K-ras . In 90% of SCLCs, there was a loss of 3p alleles. The absence of tumor suppressor genes such as RASSF1A on 3p21 is observed in all SCLCs and in 65% of NSCLCs. Other genes getting lost at the 3p region are RARB , caveolin-1 , FHIT , and β-catenin .
Lung cancer diagnosis and treatment overview
For diagnosing, oncologists make use of many tests including biopsy, imaging tests, sputum cytology tests etc. Combined tests are done to learn if cancer has spread to any other part of the body from where it had originated; for instance, imaging tests can show that cancer has spread, but are never used alone for diagnosing SCLC. A biopsy is the gold standard for confirming cancer, where a small sample of tissue is removed from the lungs and examined under a microscope. Cases in which biopsy is not possible, other tests are incorporated for diagnoses such as bronchoscopy, thoracentesis, and mediastinoscopy. A remarkable role is played by the imaging tests as these tell what is happening within the body in the form of pictures, this involves CT scan, Positron emission tomography (PET) scan, Magnetic resonance imaging (MRI) scan and Bone scan.
Treatment depends on factors such as the patient’s overall health, type of lung cancer and how far it has metastasized. Patients with NSCLC can be treated with surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy or a combination of these methods whereas SCLCs are usually treated with radiation and chemotherapy. Surgeries can remove cancer to an extent but not completely. Apart from that, a large number of people can’t go through resection. So curative resection should be avoided. The currently used chemotherapy for treating lung cancer majorly involves the use of platinum- and cisplatin. This hinders DNA replication by the intrastrand cross-links that bring conformational changes. Also, these have shown to possess cytotoxic outcomes including mitochondrial damage, altered cellular transport mechanisms during the S phase, arresting cell cycle at the G2 phase and decreased ATPase activity. Again, the chemotherapeutic drug docetaxel used for treating NSCLC tampers with mitosis and fades away relatively fast in dividing cancer cells. Chemotherapy affects rapidly dividing cells and as these drugs lack specificity, it destroys a large number of healthy cells too. Other setbacks include cancer cells developing resistance to chemotherapy. This resistance can develop right from the beginning of treatment or it may happen over time. Radiation therapy for lung cancer has been proven to be very effective in destroying cancerous cells and shrinking tumors. This therapy uses X-rays and other high-energy radiations to kill abnormal cells. However, like other treatments, it causes a lot of side effects too. Specifically, when normal healthy cells are exposed to radiation, they can be damaged in the same way as the cancerous cells, leading to adverse effects at the site of exposure. Hence continuing long-term, treatment is not possible. Side effects such as hair loss, nausea, vomiting, hematotoxicity, and gastrointestinal tract disturbances are amongst the common ones seen during the therapies. Most lung cancers can be prevented because they are related to a person’s lifestyle, the major cause being smoking of tobacco. Very less often, exposure to radon or other environmental factors can also lead to lung cancer . The harsh truth being that lung cancer is very difficult to cure and at the time of diagnosis, most lung cancers are found to have been already metastasized.
Inception of crispr
Since the advent of the DNA double helix, contemplating the site-specific changes to the genomes of cells and organisms by researchers and clinicians have been the possibility of making. Many of the early approaches to genome editing was only limited to the principle of site-specific recognition of DNA sequences. The study on bacteria and yeast regarding the natural DNA repair pathways as well as DNA recombination [13, 14] brought into light that these cells have an endogenous machinery to repair double-strand DNA breaks (DSBs) [15, 16]. So for targeted genomic engineering, precise breaks in the DNA at the site where change has to be put in was recognized as a useful strategy.
In a parallel but completely separate area of research, a few microbiologists and bioinformaticians in the mid-2000s began investigating CRISPR (clustered regularly interspaced palindromic repeats). It was described earlier in 1987 by Japanese researchers as a series of short direct repeats interspaced with short . Later on, CRISPRs were detected in various bacteria and archaea , and predictions were made about its role in DNA repair or gene regulation [19, 20]. Researchers got a lead in 2005 with the observation that many spacer sequences within CRISPRs have plasmid and viral origins [21, 22]. CRISPR-associated (Cas) genes encode proteins with putative nuclease and helicase domains [23, 24]. Along with this observation and the fact that CRISPR loci are transcribed , it was proposed that CRISPR-Cas is an adaptive defense mechanism that uses antisense RNAs as memory signatures of past invasions . The first experimental evidence of CRISPR-Cas mediated adaptive immunity  was shown in 2007 by orchestrating infection experiments of the lactic acid bacterium Streptococcus thermophilus with lytic phages. This led to the belief that natural CRISPR-Cas systems exists and was readily incorporated in the dairy industry by culturing the bacteria used in dairy to harness this auto-defense mechanism as a way for immunization against phages—a first successful application of CRISPR- Cas for biotechnological purposes . In 2008, mature CRISPR RNAs (crRNAs) were shown to serve as guides in a complex with Cas proteins to interfere with virus proliferation in E. coli . Later in the same year, the pathogen Staphylococcus epidermidis  showed the DNA targeting activity of the CRISPR-Cas system.
Importance of crispr
During the past few decades, only a few of the gene therapy products were licensed for the treatment of cancer. Gene therapy remains one of the hottest spots in cancer research as cancer is a genetic disease and treating it with gene therapy not only helps it combating cancer from inside the body but also has minimum side effects when compared to the conventionally used treatments.
In this era of cancer genomics, various large-scale cancer genome sequencing has produced a distended catalog of genetic alterations present in human tumors . With the background of driver mutations which directly or indirectly advance the transformation of normal cells into cancer cells via mutational activation of oncogenes and/or inactivation of the tumor-suppressing genes. Passenger mutations are presumed not to directly play a role in the tumorigenic process. Typically oncogenes are activated by gain-of-function mutations whereas tumor-suppressing genes are inactivated by loss-of-function mutations.
Traditional approaches of cDNA overexpression and RNA interference (RNAi)-mediated knockdown have been used to dissect the role of known oncogenes and tumor suppressor genes in cell culture, allograft, xenograft and, in some cases, transgenic mouse models. Despite the fact that these approaches have led to various important findings in cancer biology, there have been various important limitations (Table 1).
Using the CRISPR-Cas9 system for targeted modification of endogenous loci offers a solution for overcoming these limitations. This system not only simplifies the study of oncogenes and tumor-suppressor genes, but it also allows for a quick distinction between driver and passenger mutations.
Permanent Cas9 mediated modification of single or multiple endogenous loci can be done by stable delivery of CRISPR components. The process involves transfection of plasmid DNA encoding Cas9 and sgRNAs [30,31,32] or Cas9–sgRNA ribonucleoprotein complexes (RNPs) [33, 34]. Retroviruses or lentiviruses can also stably deliver the CRISPR components [35, 36]. Engineering loss-of-function mutations depend on non-homologous end joining (NHEJ), which creates indels (insertions and deletions) near the Cas9 cleavage site that lead to frameshift mutations. Engineering gain-of-function mutations require the inclusion of an HDR (homology-dependent recombination) template in the form of ssDNA or dsDNA carrying the desired mutation. The advantage of transient expression of the CRISPR components offers a “hit-and-run” strategy which allows unlimited serial editing of endogenous genes without the need for multiple viral integrations or continuous expression of CRISPR components. With the help of a series of cell-based and in vivo assays, cell lines carrying one or more targeted mutations can be tested to examine the cancer-associated phenotypes created by the mutation (or mutations). Moreover, by sequential or multiplex gene editing this technology allows for systematic analysis of epistatic interactions and comprehensive dissection of oncogenic signaling pathways. Besides allowing the functional characterization of true cancer genes, such studies rule out the functional effect of a passenger mutation on cancer initiation and progression. This approach can be used to study established cancer cell lines and primary cell lines of a mouse or human origin, as well as on organoid cultures and patient-derived xenografts.
The studies on lung cancer using gene editing tools have been very prominent and the onset of CRISPR based techniques have enabled researchers to go one step further with discovering the unexplored areas in cancer research.
Crispr CAS9 variants
Apart from being an effective option for genome engineering, CRISPR/Cas9 also finds its use in ample other techniques as well. A few tweaks to the basic structure of the CRISPR system has given rise to many different variants such as the sgRNA scaffold [37, 38], dead Cas9 (dCas9) [37, 39,40,41], RNA targeting Cas9 (RCas9)  and dead RCas9.
The sgRNA scaffolds or scRNA have RNA scaffolds attached to the sgRNA [37, 38]. These scaffolds function by recruiting specific RNA binding proteins fused with effector molecules to perform specific modulations to the activity of the gene and its synergistic activity makes it an ideal candidate for genomic screening and functional interrogation .
The dCas9 variant has its RuvC and HNH domains removed and replaced with other effectors like fluorescent proteins for tagging a specific sequence. This is particularly useful for determining the exact genomic loci of a particular sequence . Epigenetic modulations are also achievable by fusing the modulation effector molecules with the dCas9 molecule. A specific instance is the fusion of transcriptional activators like VP64  or inhibitors like KRAB  with dCas9 to execute specific transcriptional activation or suppression of a gene.
The absence of a Protospacer Adjacent Motif (PAM) in ssRNA renders it uneditable by wtCas9  but the addition of an exogenous PAM to serve as the opposite DNA strand can make the RNA editable using the conventional CRISPR/Cas9 technique . This variant is known as the RCas9. Removal of the RuvC and HNH domains from the RCas9 molecule turns it to dead RCas9 which in turn can be used to perform investigations similar to that of dCas9 on ssRNA molecules.
Crispr-CAS9 in lung cancer modelling
Lung cancer modelling has always been a daunting task and the new wave of techniques associated with CRISPR has enabled researchers to create more customized and accurate models for research purposes. Knockout of tumor suppressor proteins has resulted in modelling of carcinomas functionally and structurally resembling a natural tumor. NHEJ (Non-Homologous End Joining) based knockout of genes using CRISPR has been very effective. Studies done by albers et al.  show the knockout of 3 major tumor suppressor genes TRP53, PTEN, and VHL by a Lentiviral vector using a polycistronic system consisting of a library of sgRNAs for inactivation of the above genes. CRISPR has also been used in combination of other gene editing techniques including Cre recombinase. Sanchez-Rivera et al.  showed that activation of KrasG12D using a Cre dependent mechanism followed by CRISPR based editing of major tumor suppressor genes can lead to the formation of functionally accurate adenocarcinomas in the mice lung (Table 2).
The deletion of AMPK α1 AND α2 through a CRISPR Cas9 mediated editing in murine lung adenocarcinoma cells (KRASG12D p53f/f mediated non small cell lung carcinoma cells) showed a substantial decrease in the lung tumor size .
Treating and interrogating lung tumorigenesis with crispr
Apart from knockin and knockouts, CRISPR is also capable of performing various complex chromosomal recombinations. One such instance can be picked up from the work done by Maddalo et al. , in which they were able to achieve Eml4-Alk inversion by inducing double strand breaks in both Eml4 and Alk genes (Table 2). This model was utilized to interrogate the functional aspects of Eml4-Alk gene rearrangements  detected in cases of Non Small cell Lung Carcinomas (NSCLC) . Svensson et al.  interrogated the metabolic aspects of tumorigenesis in the KRAS-P53 and KRAS-LKB1 mouse models (Table 2) and reported that the knockout of acetyl Co-A carboxylase in NSCLCs is deleterious for growth of the tumor. Wang et al.  showed through similar mechanisms that the knockdown of CCL20 in A549 lung cancer cell line decreases the proliferative capacity of the cells while also inhibiting the A549 cell migration.
CRISPR based interrogations to reveal the processes behind the evolution of the tumors are not only helping in joining the unconnected dots but also to find new breakthroughs in understanding the tumorigenesis process. An exemplar to this is the deletion of gene 33 using CRISPR/Cas9. Park et al.  showed that gene33 deletion not only decreases cell proliferation but also increases cell migration. The deletion also leads to the upregulation of a deubiquitination enzyme, UCHL1 associated with early stages of lung epithelial cell transformation and tumorigenesis. Similarly, deletion of PTPN2 (Protein Tyrosine Phosphatase-2) enhanced the efficiency of immunotherapy by improving the interferon ƴ mediated effect on growth suppression and antigen presentation .
The study by li et al. 2018 identified the progression of lung cancer metastasis through the repression of tumor suppressor miR584-3p by FECR (FLI1 Exonic Circular RNAs) in SCLC and NSCLC tissues. CRISPR Cas9 based knock-out of FLI1 gene resulted in a halt of tumor progression through the inhibition of miR584-ROCK1 pathway .
Assessment of the role of PCDH7 in progression of lung cancer done by zhou et al. 2018 by CRISPR Cas9 based knockout of PCDH7 in KRASLSL-G12D; TP53f/f (KP Mice) showed not only a significant reduction in the tumor size in the lung but also prolonged survival of the mice and less ERK1/2 expression in cells . Another study being conducted by the University of Pennsylvania, uses specially engineered autologous T-cells called NYCE T-Cells, to act as an intervention for Multiple Myeloma, Synovial Sarcoma, Myxoid/Round Cell Liposarcoma, and Melanoma (NCT03399448). The preclinical in vivo studies for this clinical trial were conducted against a Human Lung Cancer cell line A549-ESO-CBG (which was HLA-A2+ and NY-ESO-1+) transplanted into genetically modified NOD Scid Gamma (NSG) mice to assess the effectiveness of this treatment. Successful in the in vivo stage, it was transferred to treat Multiple Myeloma, Synovial Sarcoma, Myxoid/Round Cell Liposarcoma, and Melanoma, in the clinical trial stage. (Baylis F., & McLeod M.)
Target compound validation is another way in which the molecular interrogation is being carried out using CRISPR. Wanzel et al.  showed that the effectiveness of p53 reactivating compounds is highly dependent on the resistance of the drug for the patient and that this can be resolved by validating the target using CRISPR-Cas9. The dependence of nutlin-a drug inhibiting the Mdm2-p53 binding on the presence of a functional p53 can be considered as an instance to this as shown in the same literature.
Drug resistance is a huge difficulty often faced by clinicians during treating a patient with lung cancer. An example to combat this was put forward as CRISPR Cas9 based knockout of the receptor uPAR (Urokinase plasminogen activator receptor) which resulted in a decrease of resistance of lung cancer cells towards drugs like 5-FU, Doxorubicin, cisplatin and docetaxel. This knockout also resulted in inhibition of cancer cell proliferation, invasion and migration .
CRISPR based diagnosis of lung cancer is another emerging field. The work done by Gootenberg et al.  shows the use of Cas13 to selectively preamplify and detect very minute amounts of DNA or RNA. This was done using a platform called SHERLOCK (Specific High-Sensitivity Enzymatic Reporter Unlocking). A rapid paper based test was employed to detect mutations in the liquid biopsies of patients affected by Non Small Cell Lung Carcinoma. SHERLOCK was designed to detect either the EGFR mutations or isolated cell free DNA (cf DNA), or exon 19 deletion in the patients. These mutations were successfully detected with both- a fluorescence based and lateral flow assay based readout. The fluorescence based readout while was able to detect different common EGFR mutations, lateral flow readout was combined with both csm6 and cas13 to reduce the occurrences of false-positives and to make the process more robust.
Crispr-based gene therapy clinical trials for lung cancer
CRISPR/Cas9 is a very powerful and potential genome-editing tool [60,61,62,63]. Due to its high potency, very few projects have acquired an approval to conduct human clinical trials using CRISPR/Cas9 genome editing. Thus far, CRISPR as a tool has mostly been used for editing the genomes of cells in vitro. Till date, there are 13 documented clinical trials all over the world, which have been listed under CRISPR as an intervention for Cancer Treatment (Source: clinicaltrials.gov). Of these 13 listed trials, only one study pertains to in vivo Cancer cell gene editing by the administration of CRISPR/Cas9 Systems directly into the human body. The other 12 studies use in vitro CRISPR/Cas9 gene editing to modify immune cells and transfuse them into the human body as an intervention against various Cancers. In the ex vivo gene editing studies, two approaches have commonly been followed—approach (A) attempts to increase the effectiveness of T-cells by knocking out certain genes in T-Lymphocytes that decrease their targeting efficiency; the second approach (B) attempts to attach Chimeric Antigen Receptors—that are specific to antigens on cancerous cells—to the surfaces of T-Lymphocytes in order to increase their targeting specificity and in turn their targeting efficiency.
CRISPR/Cas9 as an intervention for lung cancer has only been implemented in one clinical trial thus far. This trial is currently underway and is being conducted by Sichuan University, and evaluates the safety of PD-1 knockout engineered T cells in treating metastatic non-small cell lung cancer (Trial ID: NCT02793856). This study falls under category (A), as mentioned above. The basis of this study lies in the function of the PD-1 gene, a Programmed Cell Death promoting gene, that has been shown to express only in activated T-cells and functions as an immune checkpoint. When PD-1 binds to PD-1 ligands, T-cell apoptosis is initiated. PD-1 ligands are commonly found on antigen presenting cells. The PD-1 pathway inhibits T-cell responses by interfering with T cell receptor signaling [64,65,66,67,68,69,70,71,72]. Hence, knocking out the PD-1 gene would enhance the lifespan of the T-cells and also prevent T-cell death upon activation by disrupting the T-cell cycle checkpoint inhibitor . This would, in turn, increase the activated T-cell count in the blood, hence increasing the vulnerability of the tumor.
In the above study, the investigators would collect T-cells of autologous origin from the peripheral blood and use a CRISPR/Cas9 system to specifically knock out the PD-1 gene ex vivo. Upon achieving this, the researchers would further expand and select PD-1 knockout T-lymphocytes of autologous origin. The treatment would start with the administration of Cyclophosphamide 3 days before T-cell infusion. The patients would be divided into three test groups, each of which would receive 1 × 107/kg PD-1 Knockout T cells, 2 × 107/kg PD-1 Knockout T cells, 4 × 107/kg PD-1 Knockout T cells per cycle respectively. The completion of treatment would entail two cycles in total. Upon the completion of treatment, the study would continue to evaluate the response of the patients to the treatment and any adverse effects.
Conclusion and author’s perspective
Gene therapy based techniques have the potential to bring about breakthroughs in cancer research and CRISPR being a very targeted and efficient tool, has the potential to turn these into usable therapies. CRISPR has been shown to possess a lot of advantages over the other prominent gene therapy techniques like ZFN and TALENs (Table 3). While other treatment options available now are temporary to the body and fade out with time, gene therapy being genetic, can be made permanent and is like hard coding the treatment directly into the body. This has the potential to create vaccines and permanent cures (Fig. 1). Apart from therapeutic applications, CRISPR is also an excellent tool for understanding the processes happening in our cells (Fig. 1). The variants like dead Cas9 and dead nCas9 are excellent for interrogating and finding the pathways and processes (Fig. 1) associated with growth and proliferation of cancer. While the amount of research done and going on with respect to lung cancer based on gene therapy techniques have been staggering, the number of treatment measures available for lung cancer is still very low; mainly because of the ethical concerns associated with the implementation of these techniques. With governments introducing new laws and norms, the translatability of the research in gene therapy is expected to increase; paving way for the inception of a new wave of therapies coming into action against cancer.
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DS is supported by a start-up fund (SEED grant) from VIT, Vellore. DS is also supported by a Indian Council of Medical Research (ICMR) Funded Project (Sanction Order No.NCD/Ad-hoc/66/2016-17), a “Fast Track Young Scientist” grant (YSS/2014/000027) from the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India and an extramural grant (BT/PR19625/MED/30/1703/2016) from the Department of Biotechnology, Government of India.
Conflict of interest
The authors declare that they have no conflict of interest.
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