Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O6-methylguanine DNA methyltransferase (MGMT).

PURPOSE: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O6-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O6 position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ. METHOD: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer. RESULT: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line. CONCLUSION: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.

Production of Duchenne muscular dystrophy cellular model using CRISPR-Cas9 exon deletion strategy.

Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disorder caused by loss-of-function mutations in the dystrophin gene. Although the search for a definitive cure has failed to date, extensive efforts have been made to introduce effective therapeutic strategies. Gene editing technology is a great revolution in biology, having an immediate application in the generation of research models. DMD muscle cell lines are reliable sources to evaluate and optimize therapeutic strategies, in-depth study of DMD pathology, and screening the effective drugs. However, only a few immortalized muscle cell lines with DMD mutations are available. In addition, obtaining muscle cells from patients also requires an invasive muscle biopsy. Mostly DMD variants are rare, making it challenging to identify a patient with a particular mutation for a muscle biopsy. To overcome these challenges and generate myoblast cultures, we optimized a CRISPR/Cas9 gene editing approach to model the most common DMD mutations that include approximately 28.2% of patients. GAP-PCR and sequencing results show the ability of the CRISPR-Cas9 system to efficient deletion of mentioned exons. We showed producing truncated transcript due to the targeted deletion by RT-PCR and sequencing. Finally, mutation-induced disruption of dystrophin protein expression was confirmed by western blotting. All together, we successfully created four immortalized DMD muscle cell lines and showed the efficacy of the CRISPR-Cas9 system for the generation of immortalized DMD cell models with the targeted deletions.

Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals.

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement.

CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4 µM and the PCR product to 5 µl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1 h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

MicroRNA-96: A therapeutic and diagnostic tumor marker.

Cancer has been always considered as one of the main human health challenges worldwide. One of the main causes of cancer-related mortality is late diagnosis in the advanced stages of the disease, which reduces the therapeutic efficiency. Therefore, novel non-invasive diagnostic methods are required for the early detection of tumors and improving the quality of life and survival in cancer patients. MicroRNAs (miRNAs) have pivotal roles in various cellular processes such as cell proliferation, motility, and neoplastic transformation. Since circulating miRNAs have high stability in body fluids, they can be suggested as efficient noninvasive tumor markers. MiR-96 belongs to the miR-183-96-182 cluster that regulates cell migration and tumor progression as an oncogene or tumor suppressor by targeting various genes in solid tumors. In the present review, we have summarized all of the studies that assessed the role of miR-96 during tumor progression. This review clarifies the molecular mechanisms and target genes recruited by miR-96 to regulate tumor progression and metastasis. It was observed that miR-96 mainly affects tumorigenesis by targeting the structural proteins and FOXO transcription factors.

CFTR mutations causing congenital unilateral absence of the vas deferens (CUAVD) and congenital absence of the uterus (CAU) in a consanguineous family.

Cystic fibrosis (CF) is one of the most common recessive genetic diseases, with a wide spectrum of phenotypes, ranging from infertility to severe pulmonary disease. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are considered the main genetic cause for CF. In this study, we recruited a consanguineous Iranian pedigree with four male patients diagnosed with congenital unilateral absence of the vas deferens (CUAVD), and one female patient diagnosed with congenital absence of the uterus (CAU). Testicular biopsy of one patient was performed, and hematoxylin and eosin (H and E) staining of testis sections displayed the presence of germ cell types ranging from spermatogonia to mature spermatids, indicating obstructive azoospermia. To explore the underlying genetic factor in this familial disorder, we therefore performed whole-exome sequencing (WES) on all available family members. WES data filtration and CFTR haplotype analysis identified compound heterozygous mutations in CFTR among four patients (two CUAVD patients carried p.H949Y and p.L997F, and one CUAVD and the female CAU patient carried p.H949Y and p.I148T). All these mutations were predicted to be deleterious by at least half of the prediction software programs and were confirmed by Sanger sequencing. Our study reported that CFTR compound heterozygous mutations in a consanguineous Iranian family cause infertility in both sexes.

TMEM263: a novel candidate gene implicated in human autosomal recessive severe lethal skeletal dysplasia.

INTRODUCTION: Skeletal dysplasia is a common, clinically and genetically heterogeneous disorder in the human population. An increasing number of different genes are being identified causing this disorder. We used whole exome sequencing (WES) for detection of skeletal dysplasia causing mutation in a fetus affected to severe lethal skeletal dysplasia. PATIENT: Fetus was assessed by ultrasonography in second trimester of pregnancy. He suffers from severe rhizomelic dysplasia and also pathologic shortening of ribs. WES was applied to finding of causal mutation. Furthermore, bioinformatics analysis was performed to predict mutation impact. RESULTS: Whole exome sequencing (WES) identified a homozygous frameshift mutation in the TMEM263 gene in a fetus with severe lethal skeletal dysplasia. Mutations of this gene have been previously identified in dwarf chickens, but this is the first report of involvement of this gene in human skeletal dysplasia. This gene plays a key role in the growth hormone signaling pathway. CONCLUSION: TMEM263 can be considered as a new gene responsible for skeletal dysplasia. Given the complications observed in the affected fetus, the mutation of this gene appears to produce much more intense complications than that found in chickens and is likely to play a more important role in bone development in human.

Expression and Prognostic Significance of Cancer/Testis Antigens, MAGE-E1, GAGE, and SOX-6, in Glioblastoma: An Immunohistochemistry Evaluation.

BACKGROUND & OBJECTIVE: Glioblastoma is the most common primary malignancy of the brain, the prognosis of which is poor. Immunotherapy with cancer/testis (CT) antigens is a novel therapeutic approach for glioblastoma. This study aimed to investigate the expression rate of MAGE-E1, GAGE, and SOX-6 in glioblastoma tumors using the method of immunohistochemistry (IHC). METHODS: Expression of MAGE-E1, GAGE, and SOX-6 were determined by IHC in 50 paraffin blocks of glioblastoma. The results were compared between variables including age, gender, tumor location, and Karnofsky performance status (Kps) score. Survival analysis was also performed. RESULTS: The expression levels of SOX-6, MAGE-E1, and GAGE were 82%, 78%, and 76%, respectively. The relationship between CT antigens and age, gender, and tumor location was not significant, while the association between MAGE-E1 expression and age was statistically significant (P=0.002). High expression levels of SOX-6 and MAGE-E1 were associated with low Kps scores (P=0.034 and P<0.001, respectively). Survival analysis showed that age >40 and Kps score <80 were associated with significant relationship with shorter survival rate. (P=0.005 and P=0.018, respectively). Expression of MAGE-E1 and GAGE was negatively associated with overall 2-year survival rate (P=0.001 and P=0.021, respectively). CONCLUSION: The expression of all the three CT antigens, especially MAGE-E1 and SOX-6, was high in patients with glioblastoma. It can be concluded that these markers could be ideal targets for immunotherapy in such patients. MAGE-E1 and SOX-6 can be considered as important markers in determining the prognosis of glioblastoma.

Chemokines as the critical factors during bladder cancer progression: an overview.

Bladder cancer (BCa) is one of the most frequent urogenital malignancies which is mainly observed among men. There are various genetic and environmental risk factors associated with BCa progression. Transurethral endoscopic resection and open ablative surgery are the main treatment options for muscle invasive BCa. BCG therapy is also employed following the endoscopic resection to prevent tumor relapse. The tumor microenvironment is the main interaction site of tumor cells and immune system in which the immune cells are recruited via chemokines and chemokine receptors. In present review we summarized the main chemokines and chemokine receptors which have been associated with histopathological features of BCa patients in the world. This review highlights the chemokines and chemokine receptors as critical markers in early detection and therapeutic purposes among BCa patients and clarifies their molecular functions during BCa progression and metastasis.

Investigation of MYO15A and MYO7A Mutations in Iranian Patients with Nonsyndromic Hearing Loss.

BACKGROUND: Hearing loss (HL) is the most common sensory disorder in humans, which affects individuals in both inherited and acquired forms. MYO15A and MYO7A gene mutations have a significant role in the development of deafness. In this study, we assessed the prevalence of MYO15A and MYO7A mutations in one hundred non-relative deaf Iranians. Materials and methods: The existence of MYO15A and MYO7A mutations were assessed using the tetra-primer ARMS-PCR method, High Resolution Melting (HRM) and sequencing method. Results: A heterozygote missense mutation, p.V2135L (c.6403G > T) in the MYO15A gene, was found in a patient using the sequencing method. Conclusion: These results explain the negligible prevalence of selected mutations among Iranian patients. Identifying common mutations in patients of an ethnic group can reduce the financial costs and time needed for identifying the causes of deafness.

Non coding RNAs as the critical factors in chemo resistance of bladder tumor cells.

BACKGROUND: Bladder cancer (BCa) is the ninth frequent and 13th leading cause of cancer related deaths in the world which is mainly observed among men. There is a declining mortality rates in developed countries. Although, the majority of BCa patients present Non-Muscle-Invasive Bladder Cancer (NMIBC) tumors, only 30% of patients suffer from muscle invasion and distant metastases. Radical cystoprostatectomy, radiation, and chemotherapy have proven to be efficient in metastatic tumors. However, tumor relapse is observed in a noticeable ratio of patients following the chemotherapeutic treatment. Non-coding RNAs (ncRNAs) are important factors during tumor progression and chemo resistance which can be used as diagnostic and prognostic biomarkers of BCa. MAIN BODY: In present review we summarized all of the lncRNAs and miRNAs associated with chemotherapeutic resistance in bladder tumor cells. CONCLUSIONS: This review paves the way of introducing a prognostic panel of ncRNAs for the BCa patients which can be useful to select a proper drug based on the lncRNA profiles of patients to reduce the cytotoxic effects of chemotherapy in such patients.

Role of tyrosine kinases in bladder cancer progression: an overview.

BACKGROUND: Bladder cancer (BCa) is a frequent urothelial malignancy with a high ratio of morbidity and mortality. Various genetic and environmental factors are involved in BCa progression. Since, majority of BCa cases are diagnosed after macroscopic clinical symptoms, it is required to find efficient markers for the early detection. Receptor tyrosine-kinases (RTKs) and non-receptor tyrosine-kinases (nRTKs) have pivotal roles in various cellular processes such as growth, migration, differentiation, and metabolism through different signaling pathways. Tyrosine-kinase deregulations are observed during tumor progressions via mutations, amplification, and chromosomal abnormalities which introduces these factors as important candidates of anti-cancer therapies. MAIN BODY: For the first time in present review we have summarized all of the reported tyrosine-kinases which have been significantly associated with the clinicopathological features of BCa patients. CONCLUSIONS: This review highlights the importance of tyrosine-kinases as critical markers in early detection and therapeutic purposes among BCa patients and clarifies the molecular biology of tyrosine-kinases during BCa progression and metastasis. Video abstract.

Biallelic MADD variants cause a phenotypic spectrum ranging from developmental delay to a multisystem disorder.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.

Role of extra cellular proteins in gastric cancer progression and metastasis: an update.

BACKGROUND: Gastric cancer (GC) is one of the most common cancers in the world with a high ratio of mortality. Regarding the late diagnosis, there is a high ratio of distant metastasis among GC cases. Despite the recent progresses in therapeutic modalities, there is not still an efficient therapeutic method to increase survival rate of metastatic GC cases. MAIN BODY: Apart from the various intracellular signaling pathways which are involved in tumor cell migration and metastasis, the local microenvironment is also a critical regulator of tumor cell migration. Indeed, the intracellular signaling pathways also exert their final metastatic roles through regulation of extra cellular matrix (ECM). Therefore, it is required to assess the role of extra cellular components in biology of GC. CONCLUSION: In the present review, we summarize 48 of the significant ECM components including 17 ECM modifying enzymes, seven extracellular angiogenic factors, 13 cell adhesion and cytoskeletal organizers, seven matricellular proteins and growth factors, and four proteoglycans and extra cellular glycoproteins. This review paves the way of determination of a specific extra cellular diagnostic and prognostic panel marker for the GC patients.

Genetics of blood malignancies among Iranian population: an overview.

BACKGROUND: Blood malignancies are among the leading causes of cancer related deaths in the world. Different environmental and genetic risk factors are involved in progression of blood malignancies. It has been shown that the lifestyle changes have affected the epidemiological patterns of these malignancies. Hematologic cancers are the 5th common cancer among Iranian population. It has been observed that there is a rising trend of blood malignancies incidences during the recent decades. Therefore, it is required to design novel diagnostic methods for the early detection of such malignancies in this population. MAIN BODY: In present review we have summarized all of the significant genes which have been reported among Iranian patients with blood malignancies. The reported genes were categorized based on their cell and molecular functions to clarify the molecular biology and genetics of blood malignancies among Iranian patients. CONCLUSION: It was observed that the epigenetic and immune response factors were the most frequent molecular processes associated with progression of blood malignancies among Iranian population. This review paves the way of introducing a population based panel of genetic markers for the early detection of blood malignancies in this population.

Genetic and molecular biology of bladder cancer among Iranian patients.

BACKGROUND: Bladder cancer (BC) is the sixth common cancer among Iranians. Various risk factors such as smoking, body mass index, chronic infection, age, and genetic factors are associated with BC progression. METHODS: It has been shown that a significant ratio of patients have tumors with muscle bladder layer invasion and poor prognosis at the time of diagnosis. Therefore, the early detection of tumors is required to reduce the mortality rate of BC cases. Since there is a wide geographical incidence variation in BC in Iran, it seems that the ethnic and genetic factors can be the main risk factors among Iranian BC patients. RESULTS: For the first time, in present review we have summarized all of the reported genes among Iranian BC patients until now which were significantly associated with tumorigenesis. Moreover, we categorized all of the reported genes based on their cell and molecular functions to clarify the genetic and molecular biology of BC among Iranian population. CONCLUSION: This review paves the way of determination of a population-based genetic panel markers for the early detection of BC in this population.

Long non-coding RNAs as the critical factors during tumor progressions among Iranian population: an overview.

BACKGROUND: Cancer is associated with various genetic and environmental risk factors. Beside the mutations or aberrant expression of protein-coding genes, the genetic deregulation of non-coding RNAs has also an important role during tumor progression and metastasis. Long non-coding RNAs (lncRNAs) are a class of ncRNAs larger than 200 nucleotides that may function as tumor-suppressor or oncogene. MAIN BODY: There is a raising trend of cancer incidence among Iranian population during the last decades. Therefore, it is required to prepare a general population specific panel of genetic markers for the early detection of cancer in this population. The tissue-specific expression characteristics and high stability in body fluids highlight the lncRNAs as efficient diagnostic and prognostic noninvasive biomarkers in cancer. In present review we summarized all of the lncRNAs which have been reported until now in different tumors among Iranian patients. CONCLUSIONS: This review paves the way of introducing a population based noninvasive diagnostic panel of lncRNAs for the early detection of tumor cells among Iranian population.

Cardiomyogenic differentiation of human adipose-derived mesenchymal stem cells transduced with Tbx20-encoding lentiviral vectors.

Ischemic heart disease often results in myocardial infarction and is the leading cause of mortality and morbidity worldwide. Improvement in the function of infarcted myocardium is a main purpose of cardiac regenerative medicine. One possible way to reach this goal is via stem cell therapy. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types but display limited cardiomyogenic differentiation potential. Members of the T-box family of transcription factors including Tbx20 play important roles in heart development and cardiomyocyte homeostasis. Therefore, in the current study, we investigated the potential of Tbx20 to enhance the cardiomyogenic differentiation of human adipose-derived MSCs (ADMSCs). Human ADMSCs were transduced with a bicistronic lentiviral vector encoding Tbx20 (murine) and the enhanced green fluorescent protein (eGFP) and analyzed 7 and 14 days post transduction. Transduction of human ADMSCs with this lentiviral vector increased the expression of the cardiomyogenic differentiation markers ACTN1, TNNI3, ACTC1, NKX2.5, TBX20 (human), and GATA4 as revealed by RT-qPCR. Consistently, immunocytological results showed elevated expression of α-actinin and cardiac troponin I in these cells in comparison to the cells transduced with control lentiviral particles coding for eGFP alone. Accordingly, forced expression of Tbx20 exerts cardiomyogenic effects on human ADMSCs by increasing the expression of cardiomyogenic differentiation markers at the RNA and protein level.

Cardiogenic effects of characterized Geum urbanum extracts on adipose-derived human mesenchymal stem cells.

Stem cell therapy is considered as a promising treatment for cardiovascular diseases. Adipose-derived mesenchymal stem cells (ADMSCs) have the ability to undergo cardiomyogenesis. Medicinal plants are effective and safe candidates for cell differentiation. Therefore, the aim of our study was to investigate cardiogenic effects of characterized (HPLC-UV) extracts of Geum urbanum on ADMSCs of adipose tissue. The methanolic extracts of the root and aerial parts of G. urbanum were obtained and MTT assay was used for studying their cytotoxic effects. Then, cells were treated with 50 or 100 µg/mL of the extracts from root and aerial parts of G. urbanum. MTT assay showed that the extracts of G. urbanum did not have any toxic effects on ADMSCs. Immunostaining results showed increase in the expression of α-actinin and cardiac troponin I (cTnI), and quantitative real-time reverse-transcription PCR data confirmed the upregulation of ACTN, ACTC1, and TNNI3 genes in ADMSCs after treatment. According to HPLC fingerprinting, some cardiogenic effects of G. urbanum extracts are probably due to ellagic and gallic acid derivatives. Our findings indicated that G. urbanum extracts effectively upregulated some essential cardiogenic markers, which confirmed the therapeutic role of this plant as a traditional cardiac medicine.