Research Article - DOI: 10.31487/nl.ACO.2020.01.01

ARCHIVE OF CANCER AND ONCOLOGY

Abstract

Objective: one of the essential regulators of carcinogenesis is MicroRNA-21 (miR-21). Yet little light has been shed on its effectiveness as a tumor marker compared to the conventional ones. Comparing the diagnostic value of established tumor markers in breast cancer (BC) such as carcinoembryonic antigen (CEA) and CA153 with circulating level of miR-21 is the aim of this study. Methods: The study included 89 BC patients. Amplification of the circulating levels of miR-21 and miR-16 done using real-time PCR qualitative detection, while electrochemiluminescence assays was used to detect circulating levels of CEA and CA153. The diagnostic sensitivity for BC was compared between the three. Results: the serum miR-21 levels were high significantly BC patients, as the latter had much higher levels (P<0.001). the CA153 and CEA sensitivities were 15.73% and 22.47% respectively, while miR-21 Sensitivity and specificity were 87.6% and 87.3%. Conclusion: in BC patients miR-21 exhibits far higher sensitivity for diagnoses than both CEA and CA153. Thus especially in the early stages of BC, miR-21 can become a potential indicator for diagnosis, albeit the clinical stage, PR and ER statuses were not correlated in this study.

Keywords:
real-time polymerase chain reaction (real-time PCR)
breast cancer (BC)
microRNA-21 (miR-21

Article History:
Received: 4 May, 2020
Accepted: 2 June, 2020
Published: 19 June, 2020

Author:
Harry Lawrany*
University of Keele, School of Medicine, Staffordshire, ST5 5BG, UK

1. Introduction

Cancer is one of the illnesses where the stage at which the disease is diagnosed plays a crucial role patients’ survival and quality of life [1]. A diagnostic indicator that can detect cancer in early stages is of great significance, especially for breast cancer (BC), as it is the most common cancer in woman [2]. One field of interested is that of tumor markers, due to their noninvasive, rapid and simple nature [3].

Despite their low sensitivity and specificity, the carcinoembryonic antigen (CEA) and cancer antigen 153 (CA153) are the commonest markers used. MicroRNAs are found to have close ties with development and formation of tumors and are involved in regulating many cellular processes. They are a class of noncoding RNAs composed of 19-25 nucleotides [4]. miR-21 is involved in oncogenic process and has been demonstrated to be an essential regulator, and due to its involvement in tumor formation, its level is raised in majority of human tumors. The overexpression of miR-21 in BC tissue was noticed by Iorio et al. [1] and suggested it can be an effective marker; however, getting tissue is an invasive procedure. Easy monitoring, little invasiveness and simple collection is an obvious advantage of serum sampling [5, 6]. miR-21 expression was evaluated in 89 BC patients using SYBR-Green as a base and miR-16 as reference for the stem-loop real-time reverse transcription-polymerase chain reaction (RT- PCR) [7]. Considering the hormone receptor status and disease stage, miR-21 expression levels were compared, and then its sensitivity for diagnosing BC was pitted against CEA and CA153.

2. Materials and methods

2.1. Subjects

The study was performed in Kurdistan Hospital and approved by its Ethic committee. All patients agreed to a written informed consent. The BC women confirmed by medical examination were aged between 28 to 60 years, with 50 as a median age and blood samples were collected from March 2011 to April 2011. All the patients had a confirmed diagnosis for primary BC by histology and were undergoing therapy at the time of study, aged between 29 and 40 years, with 36 as a median age. Samples were collected between March 2011 and May 2011.

2.2. miR-21 detection

Serum samples and total RNA preparation

The samples were stored until processing at 80 ℃. Adhering to manufactures instructions, the TRIzol reagent from Invitrogen life technologies was used for extraction of total RNAs from serum.

2.3. Reverse transcription

Each 10 mL RNA sample was mixed with 3 mL stem-loop RT primers of miR-16 and miR-21, and 4 mL of 5× RT Buffer, 1 mL of Moloney murine leukemia virus (M-MLV) reverse transcriptase [Promega (Beijing) Biotech Co., Ltd.], 0.5 mL of dNTPs [Tiangen Biotech (Beijing) Co., Ltd]. ,0.2 mL of RNasin [Tiangen].

Biotech (Beijing) Co., Ltd.]., and 2 μL of 1 mol/L dithiothreitol [DTT; Tiangen Biotech (Beijing) Co., Ltd.] were added (Table 1). The final volume of the mixture was 20.7 L and incubated at various temperatures for different durations, at 61 ℃ for 30 min, 73℃ for 30 min and 170 ℃ for 10 min, and lastly was held at 4 ℃.

2.4. Real-time PCR

1.6 μL cDNA is the product of reverse transcription, and this product is mixed with 10 μL SYBR Green Master (Roche Co., Ltd.) and 1 μL PCR primers along with other PCR reagents (Table 2). The entire reaction was performed in the ABI 700 Fast PCR system, the conditions for the PCR was denaturing for 10 min at 95 ℃, after which 40 cycles of 95 ℃ for 15s is applied, followed by 60 ℃ for 1 min.

2.5. CEA and CA153 detection

Electrochemiluminescence assays was used to calculate CEA and CA153 levels and through Roche E170 MODULAR Immunoassay Analyzer the reaction is carried out.

2.6. Statistical analysis

Using relative change folds, normalization of circulating miR-21 expression was done. The characteristics of miR-21 relative expression levels is their range from 25th to 75th percentile and by their median. The connection of patient’s hormone receptor status with their miR-21is analyzed with Mann-Whitney test, while the association with of their clinical stage with their miR-21 is calculated through Kruskal-Wallis test. Mann-Whitney test was also used to measure miR-21's expression between healthy and BC individuals. The receiver operating characteristic curve (ROC) was used to determine the cut-off value, which was used to identify the specificity and sensitivity values. SPSS 16.0 software was used for all statistical analysis and statistically significant threshold was set as P <0.05.

3. Results

3.1. Target gene amplification

Pure homogenous products of miR-21 and miR-16 from PCR was obtained (Figure 1), as indicated from their melting curves (Figure 2) with narrow peak and sharply defined curves.

3.2. The expression of miR-21 in BC

miR-21 expression was evaluated in in 89 BC diagnosed patients. The patients showed high level of miR-21 which was significantly high among BC patients (30.82), resulting in (P < 0.001) with a ratio of 3.39 (Figure 3).

3.3. miR-21 ROC curve

13.22 was the best designated cut-off value, and 92.9% was determined to be the area under ROC curve (ROC-AUC) (95% confidence interval: 88.3%, 97.4%), while 87.6% and 87.3% were sensitivity and specificity values respectively, as shown in (Figure 4).

3.4. Clinical and pathological feature's association with miR021 expression levels

According to status of hormone receptors and clinical stage of the patients, they were classified into groups and miR-21 median expression level is shown according in (Table 3). However, no correlation was observed between patient's clinical stage and hormone receptor statuses against their miR-21(P>0.05).

3.5 miR-21 comparison with traditional tumor markers CEA and CA153

Patients are grouped into different classes according to their clinical stage in (Table 4), and listing their CEA, CA153 and miR-21 median expression levels. Significant difference is detected as the overall sensitivity of CEA and CA153 were merely 15.73% and 22.47% respectively, while miR-21's overall sensitivity was 87.64%. Particularly in early stage (stage I), the sensitivity of CEA and CA153 is only (4.76%), while miR-21 boasts a marked diagnostic sensitivity of (95.24) (Table 4).

4. Discussion

It is 19-25 nucleotides that make up the molecule of miRNAs, Small indeed, but various biological signaling pathways are regulated by them [8]. The close relationship between the many characteristics of tumors including their development, invasion and metastasis and miRNA has been demonstrated by many studies, and in cancer therapy, this could be a bases for entirely new strategies to fight cancer [9]. Many malignant tumors have shown increased miRNA serum expression [10]. And as a diagnostic and prognostic marker, it is been getting increasingly more attention.

It is to no surprise that an early diagnostic indicator for the commonest cancer among woman, that is BC, is of great value, affecting prognosis. Due to their simple and less invasive nature, serum markers are the focus of interest. Despite their low sensitivity and specificity, especially in early stages [11], CEA and CA153 are still commonly used in BC patients monitoring, since relapse is associated with high levels of these markers. Furthermore, CEA cannot be used in diagnosis early stages of BC, since it has a lower positive rate, and it is a nonspecific tumor marker.

Differential expression of some miRNA in normal and BC tissues has been demonstrated recently, such as let-7a (8), miR-21, and miR-145 [12]. In BC cells, they take care of biological process regulation, and various roles in apoptosis and proliferation. The possibility of miRNA acting as a BC tumor marker for diagnostic and therapeutic purposes as been reported recently [13].

miR-21 has independent transcriptional units [10] and is located on 17q23.2. it has shown significant role in colon cancer development [10, 11], lung cancer development [12, 13], as well as stomach cancer [15] and finally BC [13] et al., as it partakes in expressing and regulating numerous tumor suppressor genes. Many studies have used various methods such as Northern blotting , in situ hybridization (ISH), microarray, the profiling method of flow cytometric miRNA expression that is bead based, and finally RT-PCR (20-23) to prove that in BC, miR-21 is up-regulated both in in vitro and in vivo. And a preliminary study about miR-21 overexpression in BC tissue has been taken (1,21,24-26). The conclusion of the study was that miR-21 expression is correlated with pathological and clinical variables in BC tissues, and it is expression was higher than normal breast tissues [12]. Yet the study avoided the discussion of practical value of this finding in diagnosis of BC. In addition to the fact, that breast tissue was the center of previous studies, and little was mentioned about miR-21 serum levels, which is the focus of our study. miR-21 serum expression levels in BC patients was evaluated using stem-loop real-time RT-PCR which is based on SYBR-Green and addressed the application of miR-21 as a diagnostic and monitoring marker in BC patients. The miR-21 expression level was (3.39) times higher in BC patients, which is statistically significant (P<0.001). Moreover, in the diagnosis of BC, the miR-21 has demonstrated sensitivity and specificity of 87.6% and 87.3% respectively, which shines in comparison to traditional marker's sensitivity of CEA and CA153, that were merely 15.73% and 22.47%. our study also showed that there no correlation between clinical stages and miR-21 serum expression, as well as no correlation between hormone receptor statuses (Progesterone receptor and Estrogen receptor) and miR-21 expression. Similar results were reported by other studies [11].

In conclusion, the new serum marker miR-21 topples traditional serum markers like CEA and CA153 in sensitivity, which can improve prognosis of BC by allowing earlier diagnostic sensitivity. miR-21 can be addressed as a potential early stage BC serum tumor marker. We will have a follow up study to this preliminary study, where we will have more in-depth analysis and increase the sample size, hopefully becoming a good basis for miR-21 as a diagnostic tool in BC [9].

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Despite a remarkable increase in the depth of our understanding and management of breast cancer in the past 50 years, the disease is still a major public health problem worldwide and poses significant challenges. The palpability of breast tumors has facilitated diagnosis and documentation since ancient times. The earliest descriptions of breast cancer date back to around 3500 BC. For centuries to follow, theories by Hippocrates (460 BC) and Galen (200 AD), attributing the cause of breast cancer to an “excess of black bile” and treatment options including the use of opium and castor oil, prevailed. Surgical resection was introduced in the 18th century. With the advent of modern medicine, we have come a long way. Yet, at present, breast cancer is the second most common cancer (after lung cancer) in the two sexes combined, and the most common one in women globally, affecting 1 in 7 women (not to forget that men are not spared either). Despite the awareness about this cancer type increasing each day, going by the global statistics, we can safely say that the pink ribbon is here to stay. So, let us take a more detailed look at breast cancer to understand where we presently stand.

What Causes Breast Cancer?

Breast cancer, like other cancers, is a multifactorial disease caused by an interaction between an environmental factor and a genetically susceptible host.

Normal cells undergo programmed cell death when they are no longer needed. Until then, they are protected by several protein clusters and pathways, like the PI3K/AKT pathway and the RAS/MEK/ERK pathway. Sometimes the genes along these pathways are mutated, leading to continuous cell proliferation and cancer. Also, mutation in the gene that encodes the PTEN protein that normally turns off the PI3K/AKT pathway can lead to cancer.

An important aspect is that of hereditary breast-ovarian cancer syndrome, a familial tendency to develop these two cancers. The most common of these are related to the BRCA (BRCA1 and BRCA2) mutations, which confer a lifetime risk of breast cancer of 60-85%. These mutations can be inherited or occur after birth.

Mutations that can lead to breast cancer have been experimentally linked to estrogen exposure. Also, the loss of GATA-3, a transcription factor which directly controls the expression of estrogen receptor (ER) and other genes related to epithelial differentiation, leads to loss of cell differentiation and poor prognosis.

The risk factors are diverse, including lifestyle (e.g., consumption of alcohol, high-fat diet, tobacco, sitting for prolonged periods, late childbirth, exposure to radiation and some chemicals, etc.), genetics, and some other medical conditions.

Are there Different Types of Breast Cancer?

Well, breast cancer can be categorized based on multiple factors, viz. histopathology, grade, stage, receptors status. These classifications greatly influence the prognosis.

• Histopathology – Based on its tissue type, breast cancer can be classified into ductal or lobular carcinoma, and carcinoma in situ or invasive carcinoma.

• Grade – Considering that cancer cells increasingly lose differentiation that is required for normal functioning, they can be categorized as low-grade (well-differentiated), intermediate-grade (moderately differentiated), and high-grade (poorly differentiated).

• Stage – This is determined based on the TNM staging system (size of tumor, whether it has spread to the lymph nodes, and whether it has metastasized) –

 Stage 0: precancerous or marker condition.

 Stages 1-3: within the breast or lymph nodes.

 Stage 4: metastatic cancer.

• Receptor Status – The presence or absence of some hormone receptors on the surface, cytoplasm or nucleus of the breast cancer cells have consequence for the prognosis, viz. estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2). The cancer type that lacks all three are called triple negative breast cancer (TNBC).

How is Breast Cancer Diagnosed?

Together, physical examination of the breasts, mammography, and fine-needle aspiration can be used to diagnose breast cancer with a good degree of accuracy. Other options for biopsy include a core biopsy or vacuum-assisted breast biopsy or an excisional biopsy. Where available, imaging studies may be employed as part of the staging process in select cases to look for signs of metastatic cancer. However, in cases of breast cancer with low risk for metastasis, the risks associated with PET scans, CT scans, or bone scans outweigh the possible benefits, as these procedures expose the person to a substantial amount of potentially dangerous ionizing radiation. With respect to regular screening, Cochrane states that, due to recent improvements in breast cancer treatment, and the risks of false positives from breast cancer screening leading to unnecessary treatment, "it therefore no longer seems beneficial to attend for breast cancer screening" at any age.

How is Breast Cancer Treated?

Various treatment modalities are available for breast cancer management, with a more advanced cancer requiring more aggressive treatment. In general, these are surgery, radiation, and medication.

• Surgery – The standard surgery options include mastectomy (removal of the whole breast), quadrantectomy (removal of one-quarter of the breast), and lumpectomy (removal of a small part of the breast including the lump). Post tumor removal, a person can opt for breast reconstruction surgery or prosthesis.

• Radiation – Radiotherapy can be given externally or internally during or after the surgery to target the microscopic tumor cells that may escape surgery, thus reducing the probability of recurrence.

• Medication – Different kinds of medications may be used as adjuvant (during or after surgery) or neoadjuvant (before surgery) therapy. The currently used types are –

 Hormonal therapy: Some breast cancers which require hormones like estrogen to grow have their receptors which can be detected. They are treated with either receptor blockers (tamoxifen) or hormone production blockers like aromatase inhibitors (e.g., anastrozole). These are taken for long durations.

 Chemotherapy: These are mostly used for stage 2-4 breast cancer and ER- breast cancer. The medications are administered in combinations like “AC”, “CAT”, “CMF”, etc. for a period of 3-6 months.

 Targeted therapy: The increasing amounts of information about the cancers have led to the design of targeted therapies, like monoclonal antibodies (e.g., herceptin), antibody-drug conjugates (e.g., kadcyla) and kinase inhibitors (e.g., tykerb) for HER2+ breast cancer, CDK4/6 inhibitors (e.g., ibrance), mTOR inhibitor (e.g., afinitor) and PI3K inhibitor (e.g., piqray) for hormone receptor-positive breast cancer, olaparib and talazoparib for BRCA gene mutations, and antibody-drug conjugate (e.g., trodelvy) for HER2- and hormone receptor-negative breast cancer.

 Immunotherapy: These stimulate the person’s own immune system to recognize and destroy cancer cells more effectively, like immune checkpoint inhibitors (PD-L1 inhibitors, e.g., tecentriq).

Another important aspect of management of this disease is post treatment follow-up care, which may include regular tests, rehabilitation programmes and improvements in lifestyle.

Current Breast Cancer Research

Breast cancer research is a very active field with various aspects. The recent research focuses on the causes and risk factors, newer, safer, and more effective screening and diagnostic methods, and improvements in each modality of treatment.

 Several studies are looking at the effect of exercise, weight gain or loss, and diet on risk.

 Studies on the best use of genetic testing for breast cancer mutations continue.

 Scientists are exploring how common gene variations (small changes in genes that are not as significant as mutations) may affect breast cancer risk. Gene variants typically have only a modest effect on risk, but when taken together they could possibly have a large impact.

 Possible environmental causes of breast cancer have also received more attention in recent years. While much of the science on this topic is still in its earliest stages, this is an area of active research.

• Reducing Breast Cancer Risk –

 Estrogen blocking drugs are typically used to help treat breast cancer, but some might also help prevent it. Tamoxifen and raloxifene have been used for many years to prevent breast cancer.  More recent studies with another class of drugs called aromatase inhibitors (exemestane and anastrozole) have shown that these drugs are also very effective in preventing breast cancer

 Other clinical trials are looking at non-hormonal drugs for breast cancer reduction. Drugs of interest include drugs for diabetes like metformin, drugs used to treat blood or bone marrow disorders, like ruxolitinib, and bexarotene, a drug that treats a specific type of T-cell lymphoma.

• New Lab Tests –

 Circulating tumor cells (CTCs) are cancer cells that break away from the tumor and move into the bloodstream. Circulating tumor DNA (ctDNA) is DNA that is released into the bloodstream when cancer cells die. Researchers are investigating tests that measure the amounts of CTCs and ctDNA in the blood of women with breast cancer. Identifying and testing the CTCs and ctDNA in the blood is sometimes referred to as a “liquid biopsy.” This type of biopsy may offer an easier and less expensive way to test the tumor than a traditional needle biopsy, which comes with risks such as bleeding and infection.

 Some studies have shown that in women with metastatic (Stage 4) breast cancer, a high level of CTCs might predict a poorer outcome compared to women with a lower level.  

• New Imaging Tests –

 Scintimammography (molecular breast imaging)

 Positron emission mammography (PEM)

 Electrical impedance imaging (EIT)

 Elastography

 New types of optical imaging tests

• Chemotherapy –

 In 2019, the immunotherapy drug Atezolizumab (Tecentriq), was approved along with the chemotherapy drug nab-paclitaxel (Abraxane) for use in women with advanced triple negative breast cancer that makes the PD-L1 protein. Other potential targets for new breast cancer drugs have been identified in recent years. Drugs based on these targets, such as kinase inhibitors, are now being studied to treat triple-negative breast cancers, either by themselves, or in combination with chemotherapy. One example is the AKT inhibitor ipatasertib, which, when used with paclitaxel, shows promising results in treating women with TNBC as the first treatment. Another AKT inhibitor, capivasertib, is also showing encouraging results when given with paclitaxel.

• Combating Drug Resistance –

 Research is on to tackle this common hurdle of cancer therapy.

• Supportive Care –

 There are trials looking at different medicines to try and improve memory and brain symptoms after chemotherapy. Other studies are evaluating if certain cardiac drugs, known as beta-blockers, can prevent the heart damage sometimes caused by common breast cancer drugs such as doxorubicin and trastuzumab.

Breast cancer remains a serious public health issue worldwide. However, appreciable growth in our understanding of breast cancer in the past century has led to remarkable progress in the early detection, treatment, and prevention of the disease. The clinical focus is shifting more towards tailored therapy as more targets are characterized and novel highly innovative approaches are developed. The increasing focus on tailored therapy and the integration of cancer stem cell-based targeted therapies and immune therapies, together with existing therapeutic methods, hold promise for the cure of breast cancer. With news like CRISPR being able to hold back TNBC in mice, we are surely moving towards more hopeful days.

The RNA World Hypothesis suggests that prebiotic life revolved around RNA instead of DNA and proteins. Many discoveries such as housekeeping RNAs (rRNA, tRNA, etc.) supported the messenger RNA (mRNA) model that is the pillar of the central dogma of molecular biology, which was first devised in the late 1950s. Thirty years later, the first regulatory non-coding RNAs (ncRNAs) were initially identified in bacteria and then in most eukaryotic organisms. A few long ncRNAs (lncRNAs) such as H19 and Xist were characterized in the pre-genomic era but remained exceptions until the early 2000s. Indeed, when the sequence of the human genome was published in 2001, studies showed that only about 1.2% encodes proteins, the rest being deemed "non-coding." It was later shown that the genome is pervasively transcribed into many ncRNAs, but their functionality remained controversial. Since then, regulatory lncRNAs have been characterized in many species and were shown to be involved in processes such as development and pathologies, revealing a new layer of regulation in eukaryotic cells. This newly found focus on lncRNAs, together with the advent of high-throughput sequencing, have led to the rapid discovery of many novel transcripts which were further characterized and classified according to specific transcript traits. LncRNAs are certainly revolutionizing the field of molecular biology, inching towards playing a part in RNA therapeutics. Targeting lncRNAs is quite difficult because of their large size and heterogeneous mode of action, which may explain why their evaluation is not as advanced as that of miRNAs. Nevertheless, they have significant potential as future therapeutic targets.

So, What Exactly are the Long Non-Coding RNAs?

Long non-coding RNAs or lncRNAs are RNAs over 200 nucleotides long that are not translated into proteins. They are found in different places within the cell, including chromatin, nucleus, cytoplasm, and exosomes. Most of them (78%) are tissue-specific, compared to just 19% for mRNAs. Also, their abundance in cells is ~10 fold lower than mRNAs. In 2018, a comprehensive integration of exiting databases and published literature placed their number at 2,70,044 in humans.

Functions of Long Non-Coding RNAs

LncRNAs can originate from the locus that they regulate, usually from the antisense strand, and regulate their target in cis (Natural antisense Transcripts (NATs), or they can map to entirely different genomic regions form their targets (introns, pseudogenes, and non-coding DNA) and cause regulation in trans. They can also be associated with promoters, enhancers or other regulatory regions and do not have a homogeneous mode of action. They can activate or repress their targets and can work by a number of mechanisms. Their functions may be categorized based on their location with respect to the cell (nucleus, cytoplasm, or exosome), the molecular pathways they are part of, or the pathophysiological processes they have roles in.

LncRNAs function as parts of various processes in cells or tissues in multiple ways–

• Transcriptional regulation

• Post transcriptional modification, like splicing

• Translational regulation

• Post translational regulation

• Epigenetic regulation

• Cell cycle regulation

Besides, lncRNAs play diverse roles in physiological processes, like aging and disease.

• Aging: Aging-associated lncRNAs are highly tissue-specific but are expressed with a common pool of protein coding genes and are associated with similar functional categories. Aging-associated lncRNAs are associated with immune system processes as well as with signal transduction, transcription, and translation.

• Disease: Recent research has implicated lncRNAs in a range of diseases, starting from different cancers to neurological, cardiovascular, and other disorders.

Cancer: LncRNAs demonstrably play roles in the incidence of various cancers like lung cancer, breast cancer, leukemia, carcinomas, epithelial cancer, gastrointestinal stromal tumors and others (shown in the image below). Currently, cancer therapy is greatly hampered by many difficulties, e.g., specific targeting of cancer cells without interfering with normal tissue function, specific delivery of antitumor drugs, and, in case of carcinoma of unknown primary, exact characterization of the malignancy. Here, long ncRNAs could offer a number of advantages, both as diagnostic and prognostic markers but also as novel specific therapeutic targets. The latter, of course, first requires detailed knowledge about the tumor-specific ncRNA function and its requirement for essential cancer cell properties.

Neurological disease: The dysregulation of lncRNAs could result in neurological diseases like schizophrenia, autism spectrum disorder, Parkinson’s, Huntington’s and Alzheimer’s diseases. This can be brought about by a variety of mechanisms.

Cardiovascular disease: LncRNAs play various roles in cardiac afflictions like cardiac hypertrophy and myocardial infarction.

Other disorders: LncRNAs also play critical roles in the development of many other disorders like obesity, diabetes, depression, asthma, muscular dystrophy, psoriasis, fragile X syndrome, etc. A study has even found its role in antiviral innate responses

Thus, the budding field of the once-called “junk” matter continues to throw new surprises to us at an astonishing pace. Their promise as biomarkers and therapeutics certainly makes the upcoming days very exciting.

CRISPR (pronounced “crisper”) as a genome-editing tool is currently one of the hottest topics of research in the biological sciences. This is very much evident in the fact that in 2011, there were fewer than 100 published papers on CRISPR; but in 2019, there were more than 30,000 and counting, including various applications, refinements to CRISPR, new techniques for manipulating genes, improvements in precision, and more. So, let us today take a look at Science’s choice for Breakthrough of the year 2015.

History and Origin

The discovery of clustered DNA repeats occurred independently in three parts of the world. The first description came from Osaka University researcher Yoshizumi Ishino and his colleagues while studying Escherichia coli in 1987. In 1993, researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in that bacterium. They recognized the diversity of the sequences that intervened the direct repeats among different strains of M. tuberculosis. At the same time, repeats were observed in the archaeal organisms of Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. By 2000, Mojica performed a survey of scientific literature and one of his students performed a search in published genomes with a program devised by himself. They identified interrupted repeats in 20 species of microbes as belonging to the same family. In 2001, Mojica and Ruud Jansen, who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature. In 2002, Tang et al. showed evidence that CRISPR repeat regions from the genome of Archaeoglobus fulgidus were transcribed into long RNA molecules that were subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. A major addition to the understanding of CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Researchers have also discovered that there are numerous CRISPRs. When people talk about CRISPR, they are usually referring to the CRISPR/Cas9 system. In recent years, researchers have found other types of CRISPR proteins that also work as gene editors.

Function in the Wild

The function of these CRISPR sequences was mostly a mystery until 2007, when food scientists studying the bacteria Streptococcus thermophilus used to make yogurt showed that these odd clusters actually served a vital function: They’re part of the bacteria’s acquired immune system.

It works like this: The bacteria are under constant assault from viruses; so, they produce enzymes to fight off viral infections. Whenever a bacterium’s enzymes manage to kill off an invading virus, other little enzymes will come along, scoop up the remains of the virus’s genetic code and cut it into tiny bits. The enzymes then store those fragments in CRISPR spaces in the bacterium’s own genome. The CRISPR spaces act as a “most wanted” gallery for viruses, and bacteria use the genetic information stored in these spaces to fend off future attacks. When a new viral infection occurs, the bacteria produce special attack enzymes, known as Cas9, that carry around those stored bits of viral genetic code like a mug shot. When these Cas9 enzymes come across a virus, they see if the virus’s RNA matches what is in the mug shot. If there is a match, the Cas9 enzyme starts chopping up the virus’s DNA to neutralize the threat.

CRISPR in the Lab

For a while, CRISPR was not of much interest to anyone except microbiologists, who marvelled at the sophistication of the system in the humble bacteria. But soon enough, scientists discovered that they could fool the Cas9 protein by feeding it artificial RNA, i.e., a fake mug shot. Now, the enzyme would search for anything with that same code, not just viruses, and start chopping. In a landmark 2012 paper, Jennifer Doudna of the University of California Berkeley, Emmanuelle Charpentier of Umeå University in Sweden , and Martin Jinek showed they could use this CRISPR/Cas9 system to cut up any genome at any place they wanted. The unbelievable implications opened the floodgates of research in this field. Further advances followed when Feng Zhang, a scientist at the Broad Institute in Boston, co-authored a paper in Science in February 2013 showing that CRISPR/Cas9 could be used to edit the genomes of cultured mouse cells or human cells. In the same issue of Science, Harvard’s George Church and his team showed how a different CRISPR technique could be used to edit human cells.

Progress Using CRISPR Gene-Editing

I. Disease models – Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Successful in vivo genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including bacteria (Escherichia coli), yeasts (Saccharomyces cerevisiae, Candida albicans), nematode (Caenorhadbitis elegans), plants (Arabidopsis spp.), fish (Danio rerio), and animal (Mus musculus). CRISPR has also been utilized to create human cellular models of disease, polycystic kidney disease and focal segmental glomerulosclerosis.

II. Treatment of disorders with genetic causes – Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases, including cancer, beta-thalassemia, sickle cell disease, hemophilia, cystic fibrosis, Duchenne's muscular dystrophy, Huntington's disease, heart disease, and deafness.

III. Gene activation/ inactivation – Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.

IV. Reversing diabetes in mice

V. Elimination of cardiovascular disease in embryo

VI. Development of molecular recorder

VII. Killing superbugs

VIII. Successful targeting of cancer – It targeted the “command center” of cancer – called the hybrid fusion – which leads to abnormal tumor growths. It also helped to slow the spread.

IX. Creating plants more resistant to disease and other stresses

X. Editing countless genes at once

Concerns with CRISPR

Despite all the promise, CRISPR is not a perfect tool, at least not yet. Scientists have recently learned that the approach to gene editing can inadvertently wipe out and rearrange large swaths of DNA in ways that may imperil human health. That follows recent studies showing that CRISPR-edited cells can inadvertently trigger cancer. That’s why many scientists argue that experiments in humans are premature: The risks and uncertainties around CRISPR modification are extremely high. However, this has only made scientists more determined to improve this invaluable tool. One way of doing that is through editing RNA instead of DNA. Another technique, discovered recently, is prime editing. It does not rely on the ability of a cell to divide to help make the desired changes in DNA. Also, it does not cut both strands of the DNA double helix, minimizing the chances of making unintended changes that could be dangerous.

However, there is something else that makes CRISPR controversial – its potential for human germline modification. This aspect raises several questions with respect to the ethics of human enhancement and that of heritable genetic modification. The waters have been muddled further by the shocking news declared in November 2018 about the world’s first CRISPR-edited babies by a Chinese scientist. The work was widely condemned as unethical, dangerous, and premature, and an international group of scientists called for a global moratorium on genetically editing human embryos.

Future Applications

Even amidst the legal battles regarding the ownership of CRISPR, one thing is clear from the current speed of progress in CRISPR research – despite the misgivings, CRISPR will continue to impact our world more and more in the upcoming days. It is presently thought to have the potential to help big issues like climate change (with genetically engineered phytoplanktons), energy crisis (with more sustainable biofuels), eradication of vectors and rodents (through gene drive), food security (with more nutritious crops), antibiotic resistance, genetic diseases, other diseases like AIDS, diagnostics (yes, it may be used to scale up coronavirus testing!), and even bringing back extinct organisms! Other prospects exist with respect to freedom from allergies, pet breeding, more nutritious fish, faster racehorses, and much more. For biologists in general, the most exciting prospect, however, probably is exploring how genomes work, that can in turn open up unending avenues for critical discoveries.

Although dreaming in the lines of Gattaca right now would be a stretch to say the least, with the ongoing improvements in the field (e.g., the recent proof of safety of CRISPR’d cells in humans, the invention of Cas-CLOVER – a cleaner, more precise alternative of CRISPR/Cas, and better targeting using light-controlled guide RNA), it would not be an exaggeration to say humanity is standing at a crossroads in the history of time.

The thoughts of another pandemic while already in the grips of one sends chills down our spines! So, without further ado, let us see if we can nip it in its bud. Although not contagious, unlike COVID-19, there is indeed reason to tout it as a pandemic in the foreseeable future, at least according to the leading Dutch neurologist Dr. Bastiaan R. Bloem. Researchers explain that this condition is communicable via new types of vectors – namely, social, political, and economic trends.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, mainly affecting the aged population. Although primarily a movement disorder, patients also suffer from non-motor symptoms, such as depression, anxiety, fatigue, and sleep disorders. The cause of this disorder is mostly unknown, and a subject of active research. The current mode treatment primarily involves medication like levodopa and others (e.g., dopamine agonists, monoamine oxidase-B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, anticholinergics, glutamate antagonists, and medication against non-motor symptoms), most often in combination with levodopa. Along with these, supportive therapy, including physiotherapy, occupational therapy, speech and language therapy and changes in diet, are frequently prescribed. In cases where medication is ineffective, surgery (mostly deep brain stimulation, and rarely, lesioning surgery) is prescribed.

PD is the fastest growing neurological disorder globally, and the spike has been attributed to the increasing human lifespan, the reduction in the number of smokers, and an increase in the number of industrial pollutants like the herbicide paraquat in the recent decades. Researchers believe that the key to transforming this seemingly inevitable rise in PD is activism, e.g., raising awareness, amassing funds, improving treatments, and changing policy. Stopping the production and use of the chemicals that may increase the risk of PD is essential. Also, crucial, as ever, is financial backing. More research is needed to understand why the condition appears and how it progresses, and this type of scientific investigation is never cheap. The most effective therapy remains levodopa, which is 50 years old and not without its issues, including both psychological and physical side effects.

Two hundred years since its first description, the scientists’ understanding of PD has made unimaginable progress, but there is hardly much in terms of treatment to show for it. PD is driven by the loss of dopamine-containing neurons in the brain, particularly those in the substantia nigra. For decades, researchers mimicked this pattern of cell death in animals using toxins, but the insights gleaned did not translate to humans, leading to a raft of failed clinical trials. Many failed clinical trials later, pharmaceutical company investment has withered as PD is deemed too high risk. In response, researchers are getting back to basics: re-examining their animal models, monitoring PD symptoms over years to better understand the different ways the disease unfolds, looking for early signs of the disease, and refining clinical trials so that effective therapies will not be missed. With a better understanding of the challenges, a new generation of treatment ideas is now in clinical trials, some of which aim to stall progression of the disease. A new study casts PD as an autoimmune disorder, with evidence that the immune system mistakenly attacks neurons and the alpha-synuclein protein (the normal physiological function of the α-synuclein protein involves roles in compartmentalization, storage, and recycling of neurotransmitters, and mutations in the SNCA gene that encodes it facilitates its aggregation and the Parkinson pathology). The emerging complexity of PD biology suggests that future treatment may involve multiple targets.

To quote Dr. Bloem, “From 1990 to 2015, the number of people with Parkinson disease doubled to over 6 million. Driven principally by aging, this number is projected to double again to over 12 million by 2040. Additional factors, including increasing longevity, declining smoking rates, and increasing industrialization, could raise the burden to over 17 million. For most of human history, Parkinson has been a rare disorder. However, demography and the by-products of industrialization have now created a Parkinson pandemic that will require heightened activism, focused planning, and novel approaches.” This year, a promising molecule has offered hope for a new treatment that could stop or slow Parkinson's, something no treatment can currently do. Also, the link with environmental toxicants is a trending topic of research.

So, worrying as the recent analysis is, there is reason to keep the hope alive in our hearts that the Parkinson pandemic is preventable, not inevitable.

While political leaders across the globe have been closing their borders, scientists have been transcending theirs, creating an unprecedented global collaboration. Never have so many experts in so many countries focused simultaneously on a single topic and with such urgency. Nearly all other research has come to a grinding halt. Usual imperatives like academic credit have taken a backseat. Online repositories are making studies available months ahead of journals. Researchers have identified and shared hundreds of genome sequences of the viral pathogen. More than 200 clinical trials have been launched, amassing global endeavours. It is thought that the closest comparison to this moment might be the height of the AIDS epidemic in the 1990s, when scientists and doctors locked arms to combat the disease. But the two can hardly be compared considering today’s technology and pace of information-sharing three decades later. This openness is very much reflected on the servers of medRxiv and bioRxiv, two online archives that share academic research before it is reviewed and published in journals. They have been flooded with coronavirus research from across the globe. And, despite the nationalistic tone set by the Chinese government, even Chinese researchers have contributed a sizeable portion of the coronavirus research available in the archives. So, nearly five months into the pandemic, with over 1.5 lakh people already dead and no signs of slowing of the spread, let us look at the round-up on some of the vital aspects of COVID-19 research.

Therapy

• The combination of lopinavir-ritonavir features prominently in the SOLIDARITY trial launched by the World Health Organization (WHO), both alone and in combination with interferon-β.

• To date, over 380 trials for COVID-19 have been posted on ClinicalTrials.gov, ranging from repurposed antiviral drugs to novel diagnostic imaging techniques.

• Antibody- and convalescent plasma-based approaches have dominated the news. The FDA just approved a plasma therapy trial at Johns Hopkins University. Takeda has announced a polyclonal hyperimmune antigen-purified antibody concentrate. The process used to recover antibodies from patients, already approved for the treatment of other infectious diseases, could lead to fast-track approval. Regeneron is pursuing a monoclonal antibody strategy using its humanized mouse antibody screening platform to produce an antibody cocktail for both therapy and prophylaxis.

• Although hopes for antibody-based immunity are high, currently, there is hardly any data available on whether human populations develop immunity to SARS-CoV-2. The WHO has announced a large-scale effort (named SOLIDARITY II) to collate serological data from different countries and post results from the initiative within the next few months.

• Some studies have found a correlation between the serum levels of interleukin-6 (IL-6) and severity of COVID-19 symptoms. Additionally, a preprint suggests that treatment of 20 severe or critical COVID-19 patients with the anti-IL-6 receptor drug tocilizumab could have been effective. Roche announced the launch of a trial of tocilizumab, recruiting 330 participants with severe COVID-19. Preliminary results are expected in the summer. Sanofi and Regeneron have expanded the testing in an existing clinical trial of their own anti-IL-6 receptor monoclonal antibody in rheumatoid arthritis to include severe or critically ill COVID-19 patients.

• Novartis announced plans for a phase III trial of its Janus kinase 1 (JAK1) and JAK2 inhibitor ruxolitinib in patients suffering from COVID-19-associated cytokine storms.

• Gilead reported the outcomes of patients who received a repurposed RNA polymerase inhibitor, remdesivir, on a compassionate basis in the New England Journal of Medicine. 36 out of 53 patients showed some sign of improvement, including 17 out of 30 patients on mechanical ventilation who were extubated. However, several limitations include the lack of a primary endpoint, a target for patient recruitment and a control group. The study also did not collect information on viral load, making it impossible to correlate the results with direct measures of the drug’s antiviral activity.

• A phase IIb trial of 81 patients in Manaus, Brazil, treated with azithromycin-chloroquine, was reported in MedRxiv. The study found no significant benefits of chloroquine–azithromycin and highlighted some safety concerns, with the high-dose arm terminated early due to the incidence of QT interval prolongation. A Chinese multicenter open-label randomized control study of COVID-19 patients treated with hydroxychloroquine alone, also in MedRxiv, found no effect on its primary endpoint, the negative conversion rate, but did exhibit a moderate reduction in lymphopenia and C-reactive protein levels.

Vaccines

• No vaccines are expected to reach the market within the next year. Currently, the WHO is curating a list of potential vaccines, of which two are currently under clinical evaluation: an adenoviral vector-based approach by CanSino Biological Inc. and the Beijing Institute of Biotechnology, and an RNA product by Moderna Inc. and the National Institute of Allergy and Infectious Diseases.

• Inovio Pharmaceuticals launched a phase I clinical trial of INO-4800, its DNA vaccine for COVID-19. Previously Inovio had reportedpartial positive results using a similar strategy in a phase I trial of its Middle Eastern respiratory syndrome (MERS) DNA vaccine.

• Shenzhen Geno-Immune Medical Institute (SGIMI) has begun phase I trials of LV-SMENP-DC, a cellular vaccine made up of dendritic cells (DCs) transduced with SARS-CoV-2 spike, membrane, nucleocapsid, envelope and protease (SMENP) minigenes along with immunomodulatory genes using a lentiviral vector. SGIMI also announced a second phase I trial testing artificial antigen-presenting cells modified with lentiviral vectors to express multiple SARS-CoV-2 minigenes and immunomodulatory genes.

• Pfizer announced jointly with BioNTechSE from Germany to bring its COVID-19 mRNA vaccine into phase I trials by the end of April.

Diagnostics and Serology

• A detailed analysis of 9 mild cases of COVID-19 in Germany detected SARS-CoV-2 in oro- and naso-pharyngeal samples that peaked at day 5 of symptoms. Live replicating SARS-CoV-2 was found in the throat, unlike that described for SARS-CoV. Both the viral RNA and the live virus were detected in the sputum, which may lead to simpler sample-collection protocols. Although high viral RNA concentrations were found in the stool, the authors were not able to isolate live virus from it. No urine or blood samples had viral RNA.

• Ju et al. used labeled SARS-CoV-2 spike protein receptor-binding domain (RBD) as a probe to sort antigen-specific B cells from eight infected patients in Shenzhen, China. From these sorted B cells, they produced 206 monoclonal antibodies with confirmed RBD binding. The capacity of monoclonal antibodies to compete with the receptor ACE2 for RBD binding was the best predictor of their neutralizing activity. Interestingly, the study found both germline clones and somatically mutated clones with high virus-neutralizing capacity.

• A study surveying levels of neutralizing antibodies (NAbs) to the SARS-CoV-2 spike protein in the plasma of 175 patients who had recovered from COVID-19 in one health centers in Shanghai, China, published in medRxiv, shed light on the development of natural immunity to the virus. Although patients in this study were classified as mild cases, antibody levels correlated positively with C-reactive protein and inversely with lymphocyte counts (NAb levels also tended to be higher in older subjects). A troublesome aspect of the study was that approximately 30% of recovered patients showed low NAb titers, including 10 patients with NAbs below the detection limit of the assay.

• A screen of Epstein-Barr virus-immortalized memory B cells derived from a patient who recovered from SARS in 2003 identified eight monoclonal antibodies that cross-reacted with SARS-CoV-2, and one, S309, showed potent neutralizing activity. Though S309 recognizes an epitope on the SARS-CoV-2 spike glycoprotein, it does not target the receptor-binding domain and is not predicted to block angiotensin-converting enzyme 2 (ACE2) binding. The work, led by David Veesler (University of Washington, USA) and Davide Corti (Humabs Biomedical, Switzerland), was reported as a preprint.

Pathophysiology

As the number of confirmed cases surges past 2.2 million globally, clinicians are uncovering new information about the spread of this disease in the body of an individual every day. The fast-evolving understanding of how the virus attacks cells around the body could be a crucial help for the doctors on the front lines providing treatment.

Animal Models

Establishing animal models of COVID-19 is a critical step toward understanding its pathophysiology and developing novel therapies.

• In Cell Host & Microbe, ferrets have been shown to mimic important aspects of human SARS-CoV-2 infection, including viral replication, fever and ferret-to-ferret transmission (although there were no fatalities). Infected ferrets also shed virus in nasal washes, saliva, urine and feces like humans.

• A preprint in bioRxiv also reported viral replication and transmission in domestic cats, but not in dogs, pigs, chickens or ducks.

• Also, a tiger in the Bronx zoo, USA has tested positive for SARS-CoV-2.

Epidemiology

• Modelling work by Ferretti et al. suggests that digitizing contact tracing through a mobile phone app may be able to suppress the epidemic sustainably. The app would help build “a memory of proximity contacts” and eliminate the delay in notifying contacts of infected people. The authors, however, caution that their models rely on a basic reproduction number (R0) derived using the Chinese data, which may not be accurate for the fast-spreading European epidemic.

• An analysis of 1,591 patients in 72 regional hospitals in Lombardy, Italy, published in JAMA, reports that mortality in the intensive care unit was 26%. Among the patients studied, the majority were male (82%) and had extensive comorbidities, confirming previous reports that these factors may play a part in the severity of the disease. The most frequent comorbidity was hypertension (49% overall and 62% of deaths).

• Among those patients whose files had respiratory support data, 88% were put on mechanical ventilation.

• A brief communication, published in Nature Microbiology, reports evidence of community spread of SARS-CoV-2 within the city of Wuhan, China. The researchers re-analysed 640 throat swabs collected for influenza-like illness in Wuhan between October 2019 and January 2020. Nine of the swabs tested positive for SARS-CoV-2; the first positive sample was collected in the first week of 2020.

• A modeling study led by Marc Lipsitch at Harvard’s T. H. Chan School of Public Health, published in Science, concludes that “prolonged or intermittent social distancing may be necessary into 2022”. The model assumes that immunity to SARS-CoV-2 will resemble what is observed for the related human coronaviruses OC43 and HKU1 — an assumption that remains to be tested. Based on serological samples from approximately 1,000 inhabitants of the German town of Gangelt (population of 12,529 people), an early COVID-19 epicenter, Bonn University researchers estimate an infection rate of 14% and a fatality rate of 0.37% (44 reported deaths in the town) in this non-peer reviewed report (in German). Correspondence in the New England Journal of Medicine shows results from the systematic screening of 214 mothers admitted into Columbia University Irving Medical Center’s labor and delivery unit in New York City. The letter reports a 13.7% frequency of asymptomatic carriers of SARS-CoV-2 (along with four symptomatic cases, or 1.9%).

• The New England Journal of Medicine published a large-scale COVID-19 diagnostic testing effort in Iceland, which found that 43% of positive cases had reported no symptoms at the time of testing. The study also found very low rates of infection in children under 10 years of age. A brief communication in Nature Medicine reinforces the importance of asymptomatic carriers. The study analyzed 414 throat swabs from 94 SARS-CoV-2-positive mildly or moderately ill patients in Guangzhou Eighth People’s Hospital, China. They found the highest SARS-CoV-2 viral load in the first days after symptom onset, which gradually declined to the detection limit around day 21 after onset. The authors also analyzed publicly available data on 77 infector-infectee pairs and arrived at an estimate of 44% of pre-symptomatic transmission. The study estimates that peak infectivity occurs between days 0 and 2 of symptom onset.

Viral Origin and Structure

• Andersen et al. presented a detailed analysis of the origin of SARS-CoV-2. A couple of papers report additional crystal structures for the SARS-CoV-2 RBD bound to ACE2. Lan et al. inferred convergent evolution of SARS-CoV and SARS-CoV-2 RBDs, indicative of selection in the passage to humans. Shang et al. used surface plasmon resonance to show that the RBD from SARS-CoV-2 bound more strongly to human ACE2 than did the RBD from SARS-Co-V. Interestingly, Shang et al. proposed that a related bat coronavirus, RaTG13, might also use ACE2 to enter human cells – a finding with worrying implications for the ability of bat coronaviruses to directly invade human hosts. In another report, a probable link to pangolin was identified with respect to the origin of SARS-CoV-2.

• A Nature study led by Haitao Yang at Shanghai Tech University, China, has solved the crystal structure of the SARS-CoV-2 main protease (Mpro).

Here’s looking forward to many more such discoveries lighting up our gloomy hearts in the upcoming days!