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.

Designing an artificial intelligence system for dental occlusion classification using intraoral photographs: A comparative analysis between artificial intelligence-based and clinical diagnoses.

INTRODUCTION: This study aimed to design an artificial intelligence (AI) system for dental occlusion classification using intraoral photographs. Moreover, the performance of this system was compared with that of an expert clinician. METHODS: This study included 948 adult patients with permanent dentition who presented to the Department of Orthodontics, School of Dentistry, Mashhad University of Medical Sciences, during 2022-2023. The intraoral photographs taken from the patients in left, right, and frontal views (3 photographs for each patient) were collected and underwent augmentation, and about 7500 final photographs were obtained. Moreover, the patients were clinically examined by an expert orthodontist for malocclusion, overjet, and overbite and were classified into 6 groups: Class I, Class II, half-cusp Class II, Super Class I, Class III, and unclassifiable. In addition, a multistage neural network system was created and trained using the photographs of 700 patients. Then, it was used to classify the remaining 248 patients using their intraoral photographs. Finally, its performance was compared with that of the expert clinician. All statistical analyses were performed using the Stata software (version 17; Stata Corp, College Station, Tex). RESULTS: The accuracy, precision, recall, and F1 score of the AI system in the malocclusion classification of molars were calculated to be 93.1%, 88.6%, 91.2%, and 89.7%, respectively, whereas the AI system had an accuracy, precision, recall, and F1 score of 89.1%, 88.8%, 91.42%, and 89.8% for malocclusion classification of canines, respectively. Moreover, the mean absolute error of the AI system accuracy was 1.98 ± 2.11 for overjet and 1.28 ± 1.60 for overbite classifications. CONCLUSIONS: AI exhibited remarkable performance in detecting all classes of malocclusion, which was higher than that of orthodontists, especially in predicting angle classification. However, its performance was not acceptable in overjet and overbite measurement compared with expert orthodontists.

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.

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.

Does Presurgical Nasoalveolar Molding Reduce the Need for Future Bone Grafting in Cleft Lip and Palate Patients? A Systematic Review and Meta-Analysis.

OBJECTIVE: Nasoalveolar molding (NAM) is a technique that is utilized in patients with cleft lip/palate before performing lip surgery. This procedure has been shown to result in a more aesthetic nose with lesser columellar deviation and reduced scaring. The aim of our study was to evaluate the long-term results of NAM and gingivoperiosteoplasty in patients with cleft lip and palate. METHODS AND MATERIALS: An electronic search of databases (ie, PubMed, ISI Web of Science, EMBASE, Scopus, and Google Scholar) from inception to March 2021 was performed and after selecting the eligible studies, relevant data were collected using piloted extraction forms. The success rate of NAM and gingivoperiosteoplasty, and Bergland score were pooled using random-effects inverse variance meta-analysis. RESULTS: Seven studies were included in this meta-analysis and systematic review. The pooled mean success rate of NAM with gingivoperiosteoplasty (GPP) based on the continuity of alveolar bone structure was 71% (95% confidence interval [CI] = 54-85). This means that in 71% of cases NAM + GPP treatment eliminated the need for future bone grafts. Also, no significant difference between the success rate (risk ratio = 1.00, 95% CI = 0.64-1.58) and mean Bergland score (mean difference = 0.64, 95% CI = -1.04 to 2.31) of NAM + GPP and skeletal bone graft was found. CONCLUSIONS: Nasoalveolar molding and gingivoperiosteoplasty was successful in 71% of cases treating patients with cleft lip and palate. This treatment is similar with the secondary alveolar bone graft in both the success rate and the alveolar height that it generates while being less invasive and with lower morbidity.

The significant role of a functional polymorphism in the NF-κB1 gene in breast cancer: evidence from an Iranian cohort.

Aims: Breast cancer (BC) is one of the most common cancers among women. The influence of genetic variations on BC risk has been thus far assessed via genome-wide association studies. NF-κB has been recognized as a major player in BC progression. In this study, the association between rs28362491 and BC was evaluated in a population from northeastern Iran. Materials & methods: This study was conducted on 476 patients with BC and 524 healthy controls. The genotyping method used was an amplification-refractory mutation system. Results: The INS/DEL genotype conferred a statistically significant increased risk in patients in comparison with controls. Additionally, in the recessive model, INS/INS + INS/DEL versus DEL/DEL was statistically significant (OR = 0.34; 95% CI: 0.12-0.96; p = 0.042). Conclusion: This study found that rs28362491, as a susceptibility genetic factor, may affect BC risk in the Iranian population.

Recent advances in CRISPR-Cas systems for colorectal cancer research and therapeutics.

INTRODUCTION: Colon cancer, ranked as the fourth leading global cause of cancer death, exhibits a complex progression marked by genetic variations. Over the past decade, the utilization of diverse CRISPR systems has propelled accelerated research into colorectal cancer (CRC) treatment. AREAS COVERED: CRISPR/Cas9, a key player in this research, identifies new oncogenes, tumor suppressor genes (TSGs), and drug-resistance genes. Additionally, it facilitates the construction of experimental models, conducts genome-wide library screening, and develops new therapeutic targets, especially for targeted knockout in vivo or molecular targeted drug delivery, contributing to personalized treatments and significantly enhancing the care of colon cancer patients. In this review, we provide insights into the mechanism of the CRISPR/Cas9 system, offering a comprehensive exploration of its applications in CRC, spanning screening, modeling, gene functions, diagnosis, and gene therapy. While acknowledging its transformative potential, the article  highlights the challenges and limitations of CRISPR systems. EXPERT OPINION: The application of CRISPR/Cas9 in CRC research provides a promising avenue for personalized treatments. Its potential for identifying key genes and enabling experimental models and genome-wide screening enhances patient care. This review underscores the significance of CRISPR-Cas9 gene editing technology across basic research, diagnosis, and the treatment landscape of colon cancer.

New advancements in CRISPR based gene therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.

Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System.

Generating animal models can explore the role of new candidate genes in causing diseases and the pathogenicity of a specific mutation in the underlying genes. These animals can be used to identify new pharmaceutical or genetic therapeutic methods. In the present experiment, we developed a rpe65a knock out (KO) zebrafish as a retinitis pigmentosa (RP) disease model. Using the CRISPR/Cas9 system, the rpe65a gene was KO in zebrafish. Two specific single-guide RNAs (sgRNAs) were designed for the zebrafish rpe65a gene. SgRNAs were cloned into the DR274 plasmid and synthesized using in vitro transcription method. The efficiency of Ribonucleoprotein (synthesized sgRNA and recombinant Cas9) was evaluated by in vitro digestion experiment. Ribonucleoprotein complexes were microinjected into one to four-celled eggs of the TU zebrafish strain. The effectiveness of sgRNAs in KO the target gene was determined using the Heteroduplex mobility assay (HMA) and Sanger sequencing. Online software was used to determine the percent of mosaicism in the sequenced samples. By examining the sequences of the larvae that showed a mobility shift in the HMA method, the presence of indels in the binding region of sgRNAs was confirmed, so the zebrafish model for RP disease established. Zebrafish is an ideal animal model for the functional study of various diseases involving different genes and mutations and used for evaluating different therapeutic approaches in human diseases. This study presents the production of rpe65a gene KO zebrafish models using CRISPR/Cas9 technology. This model can be used in RP pathophysiology studies and preclinical gene therapy experiments.

Characterization of stability, safety and immunogenicity of the mRNA lipid nanoparticle vaccine Iribovax® against COVID-19 in nonhuman primates.

mRNA-lipid nanoparticle (mRNA-LNP) vaccines have proved their efficacy, versatility and unprecedented manufacturing speed during the COVID-19 pandemic. Here we report on the physicochemical properties, thermostability, immunogenicity, and protective efficacy of the nucleoside-modified mRNA-LNP vaccine candidate Iribovax® (also called SNEG2c). Injection of BALB/c mice, rabbits and nonhuman primates with two doses of SNEG2c induced production of high-titers of SARS-CoV-2 spike-specific and receptor-binding domain (RBD)-neutralizing antibodies in immunized animals. In addition to the strong humoral response, SNEG2c elicited substantial Th1-biased T-cell response. Sera from rhesus macaques immunized with a low dose of the vaccine showed robust spike-specific antibody titers 3-24× as high as those in convalescent sera from a panel of COVID-19 patients and 50% virus neutralization geometric mean titer of 1024 against SARS-CoV-2. Strikingly, immunization with SNEG2c completely cleared infectious SARS-CoV-2 from the upper and lower respiratory tracts of challenged macaques and protected them from viral-induced lung and trachea lesions. In contrast, the non-vaccinated macaques developed moderate to severe pulmonary pathology after the viral challenge. We present the results of repeat-dose and local tolerance toxicity and thermostability studies showing how the physicochemical properties of the mRNA-LNPs change over time and demonstrating that SNEG2 is safe, well tolerated and stable for long-term. These results support the planned human trials of SNEG2c.

Postural sway changes in genu recurvatum deformity during standing with manipulation of visual and proprioceptive systems.

OBJECTIVES: Genu recurvatum deformity shifts the mechanical axis of lower extremity posteriorly which may disturb the balance and lead to injury. The aim of this research was to compare postural sways in genu recurvatum subjects with healthy controls while manipulating visual and proprioceptive systems. METHODS: 10 female subjects with genu recurvatum (20.04 ± 4.64 years) and 11 female healthy controls (20.11 ± 4.64 years) participated in the study. The back knee angle was measured using a goniometer. Dynamic BIODEX balance system was used to measure the total, anteroposterior, and mediolateral postural sways while manipulating the stability of the base of support (stable versus unstable) and visual system (open versus closed eyes). RESULT: During standing on the stable level, in both visual conditions, genu recurvatum subjects demonstrated greater total and anteroposterior postural sways than controls in both standing on the right and left foot (p<0.05). Similarly, when standing on the unstable base of support, in all conditions, the postural sways of genu recurvatum subjects were significantly greater than controls in both right and left foot (p<0.05). The vision had a main effect on postural control of genu recurvatum group (p=0.0001). There was a significant interaction between vision, the stability of the base of support and group factors (p=0.003). CONCLUSION: The results indicated that genu recurvatum subjects have a proprioceptive deficiency and they mainly rely on visual system inputs to control and maintain their balance. Thus, the proprioceptive system and balance control should be considered in assessment and designing an appropriate treatment regimen for genu recurvatum.

Effect of chronic idiopathic low back pain on the kinetic gait characteristics in different foot masks.

Identification of kinetic variables in different masks of foot is important for the evaluation and treatment of chronic low back pain. The aim of this study was to investigate the effect of chronic idiopathic low back pain on kinetic variables of gait in different foot masks. 11 idiopathic chronic low back pain patients and 13 healthy matched controls participated in this study. Using Emed foot-scanner system, the ground reaction force and impulse were measured during barefoot normal walking. Then, the average footprints were divided into 10 masks using the Automask program and the data were extracted using Multimask Evaluation programs. The low back pain disability was measured by Quebec questionnaire. Our results revealed that the ground reaction force and impulse in medial and lateral midfoot and hallux masks of patients were significantly lower than controls. Furthermore, these patients demonstrated greater ground reaction force and impulse in 3-5th metatarsals mask than control group. There was a significant interaction between the low back pain and the foot masks factors. In conclusion, the ground reaction forces and impulses in different areas of foot are affected by low back pain. Therefore, the kinetic gait analysis should be considered as an appropriate tool in evaluation and prescribing proper treatment program in low back pain patients.