Fragile X syndrome (FXS) is the most common single gene cause of inherited intellectual disability and autism spectrum disorder (ASD). FMR1, the gene associated with FXS, is located on chromosome X. Accordingly, males with loss-of-function (full) mutations are more severely affected than females. Strategies for therapeutic intervention for this disorder have included behavioral and medication therapy. To date, no management strategies have been shown to be curative. Gene therapy that aims to supply the functional protein product of the gene FMR1 to the brain is an attractive concept for curative treatment. Experiments aimed at activating the mutant FMR1, modifying its abnormal RNA product, or supplying functional FMR1 copies to the CNS have been conducted. The delivery of the FMR1 gene and its product to animal models of FXS have been primarily conducted with intrathecal applications because of the low efficiency of the gene therapy vectors to cross the blood-brain barrier (BBB). This delivery approach is associated with a higher risk of complications and appears to distribute the gene product unevenly across different brain regions. We have explored the efficiency of a recently developed adeno-associated virus (AAV) vector with increased BBB crossing in certain strains of mice to deliver FMR1 with peripheral IV administration. Our experiments demonstrated very high delivery efficiency and also highlighted the risk of oversupplying the brain with FMRP, the protein product of the FMR1 gene. Other AAV vectors with enhanced crossing of the BBB in primates have been developed, providing an attractive option for further experiments involving peripheral administration. Providing the gene product to specific brain cells remains a difficult challenge for future experiments. It may also be important or even necessary to regulate the gene expression to mimic physiological expression patterns since the levels of FMRP change dramatically during development, with maximum levels early in the postnatal period and a decline across early life. In addition, there are 12 identified mouse isoforms of FMRP due to alternative RNA splicing, and an even higher number of isoforms is found in humans. It may thus be a challenge to determine what FMR1 isoform or set of isoforms would have the optimum efficiency in correcting the phenotype. Despite these challenges, the recent developments establish the basis for future research to develop efficient and minimally invasive gene therapy protocols for FXS.
Duffy antigen receptor for chemokines (DARC) is a transmembrane receptor (glycoprotein) expressed on human red blood cells. Sub-Saharan Africa (sSA) individuals, who suffer the most of the global malaria burden, predominantly carry a Duffy negative phenotype. Expression of this gene (found among Duffy-positive individuals) is known to be essential for P. vivax invasion of RBCs. While P. falciparum is the predominant Plasmodium in sSA, the upward trend in P. vivax infection is a major threat to the malaria eradication programme in the region. Since Duffy null individuals (homozygous negative) lack DARC expression, we investigated the DARC gene dynamics in relation to the emerging presence of P. vivax infections in a previously predominant P. falciparum endemic region. A total of 223 DARC genes were retrieved from the NCBI database across various countries, Nigeria, Cameroon, Ethiopia, Madagascar and South Africa and were used for population dynamic analysis using different population genetic metrics. Among these sSA countries, South Africa showed the most haplotype and nucleotide diversity compared to other parts of sSA. Various selection pressures were observed in Western Africa and the Central African Republic. Population structure analysis revealed DARC population clustering of Cameroon, Nigeria and Ethiopia (despite Ethiopia's geographic distance), suggestive of shared ancestry and minimal DARC locus divergence. Conversely, South Africa and Madagascar showed a distinct genetic lineage reflecting differences in evolutionary pressures. Our analysis suggests minimal genetic diversity within sSA with evidence of selection potentially attributed to the recent emergence of P. vivax infections. However, greater diversity was observed in South Africa. Evidence of selection of this gene and detection of P. vivax among Duffy-null individuals in the other regions is truly a public health concern.
The purpose of this study is to evaluate outcomes and complications in developmental dysplasia of the hip (DDH) patients that undergo closed reduction and 3 consecutive months of hip spica casting without a planned 6-week midpoint cast change. A retrospective review was performed of patients diagnosed with DDH treated with closed reduction and spica casting at a single tertiary care pediatric hospital. Clinical documentation was reviewed to identify complications, which were divided into 2 time frames: during the planned casting period and after completion of the planned casting period. Forty-four patients were included. The series consisted of 39 (89%) females and 5 (11%) males with a median age of 9 months and median length of follow-up of 3.2 years. Nine (21%) patients experienced a complication during the casting period. Seven (16%) of these patients experienced a cast-related complication and one patient experienced 2 separate cast complications. These complications included 3 (7%) instances of soiled casts and 5 (11%) patients who outgrew their casts. The median time to cast complication was 36 days. None of the patients with a cast complication experienced further complication after completion of their planned casting period. The remaining 2 patients who experienced a complication during the casting period lost reduction and returned to the OR for open reduction. Nine (21%) patients experienced a total of 14 postcasting period complications. Seven (16%) patients had redislocation of the hip, 5 (11%) patients developed proximal femoral growth disturbance (PFGD), and 2 (5%) patients developed a leg length discrepancy (LLD). There were 20 patients with unplanned return to the OR throughout the study period: 8 in the casting-complication cohort, 7 in the postcasting complication cohort, and 5 patients who did not experience a complication and returned to the OR for separate indications. Closed reduction for DDH with 3 consecutive months of spica casting without a planned cast exchange can yield good outcomes in most patients with acceptable rates of cast-related complication, redislocation, and unplanned return to the OR for cast exchange. Level V.
Acute pancreatitis (AP) is a severe inflammatory disease. Transient receptor potential (TRP) channels have been reported to participate in various pathophysiological processes. However, the role of TRP channels in the AP remains unclear. Here, we investigated the involvement of TRP channels in AP. We identified differentially expressed genes (DEGs) using the public AP datasets GSE3644 and GSE109227 and identified 20 hub genes. We analyzed their correlation with the TRP genes. Four DEGs related to TRP channels were selected, and functional enrichment analyses were conducted. An interaction network involving the transcription factors, microRNAs (miRNAs), and mRNA of the four TRP-associated genes was established. To evaluate the diagnostic effectiveness, receiver operating characteristic (ROC) analysis and machine learning (ML) methods, such as support vector classifiers and random forest, were applied. Furthermore, we analyzed immune cell infiltration and its relationship with the four TRP genes. To confirm these bioinformatics findings, we performed in vitro and in vivo experiments using primary acinar cells and an experimental mouse model of AP. Four TRP-related genes, namely, MCOLN1, MCOLN3, TRPM7, and TRPV4, were selected for the correlation analysis. Functional analyses showed that these four genes are involved in cellular senescence and the calcium signaling pathway. A TF-miRNA-mRNA interaction network was constructed for the four TRP-associated genes. The areas under the ROC curves (AUC) of MCOLN1, MCOLN3, TRPM7, and TRPV4 were 0.833, 0.700, 1.0, and 1.0, respectively. ML algorithms performed well in predicting AP with AUCs ≥ 0.67. TRPM7, TRPV4, and MCOLN3 are associated with immune cell infiltration of AP tissues. Using experimental AP models, we found that the expression of three TRP-related genes, MCOLN1, TRPM7, and TRPV4, was significantly upregulated in mouse AP tissues. Specifically, TRPM7 knockdown or inhibition by its inhibitor 2-APB significantly reduced pancreatic acinar cell damage, inflammatory mediator expression, and mRNA levels of senescence-associated genes. Our study suggests that TRP-related genes play crucial roles in AP and may be promising biomarkers for predicting the pathogenesis of AP.
Ovarian cancer (OC) is still one of the most serious gynecologic malignancies in the world. It is characterized by a significant likelihood of recurrence and resistance to conventional therapies and a lack of efficient screening techniques. MicroRNAs (miRNAs) are small, noncoding RNA molecules that exert pivotal functions in modulating gene expression. miRNAs are improperly regulated in OC, contributing to tumor onset, progression, metastasis, and resistance to chemotherapeutics. As a result, miRNAs are promising therapeutic targets for the treatment of OC. Recently, natural products derived from plants and other sources have drawn more interest because of their potential to modulate miRNA expression. A variety of bioactive substances, such as curcumin, quercetin, and others, have shown the ability to either promote tumor-suppressing miRNAs or suppress tumor-promoting miRNAs. These substances have a great deal of promise for improving the effectiveness of traditional chemotherapy, lowering adverse effects, and providing more individualized treatment plans. Additionally, their capacity to target several miRNAs implicated in cancer-related pathways offers a multimodal strategy for treating OC. We can upgrade the potential therapeutic options for OC and other cancers by exploring novel natural products with miRNA-modulating effects. However, further research is needed to clinically translate miRNA-based therapeutics employing natural compounds, especially in the areas of safety, bioavailability, and drug delivery methods. This review emphasized the implications of miRNAs in OC, the impact of natural products on miRNA regulations, and the potential for incorporating these natural substances into clinical practice for individualized and successful OC treatments.
Recent studies reveal that a suboptimal vaginal microbiome (VMB), including the enrichment of anaerobic bacteria associated with multiple female genital disorders, is linked to adverse pregnancy and birth outcomes in pregnant people. Problematically, however, the majority of the available data, to date, is biased toward highly developed, Global North countries, leaving underrepresented populations like the Democratic Republic of the Congo (DRC) poorly characterized. Here, we investigate the VMB from a cohort of 82 pregnant people living with human immunodeficiency virus (PLWH) on antiretroviral therapy (ART) from the DRC. Specifically, we explore the associations between the VMB via 16S rRNA gene sequencing and maternal peripheral immune factors. Additionally, we compare the VMB of pregnant PLWH-ART from DRC with publicly available VMB data (5 studies, 1861 samples) in a meta-analysis to elucidate the impact of HIV on the VMB. Combined, these analyses revealed the differences in community structure and predicted function of the microbiota between pregnant PLWH-ART and pregnant people without HIV (PWoH). Taxonomically, the VMB of DRC PLWH-ART were enriched for Lactobacillus iners-dominated VMBs (53%) or a diverse, polymicrobial VMB, that is, bacterial vaginosis (BV) (43%). Functional predictions made from these taxa suggested that protein-coupled receptors, amino sugar and nucleotide sugar metabolism, fatty acid metabolism, and polycyclic aromatic hydrocarbon degradation pathways were differentially abundant between the communities. Correlation with host plasma immune factors revealed putative links between some VMB metrics (e.g., alpha diversity and species abundance) that have been linked to adverse pregnancy and birth outcomes. Human immunodeficiency virus (HIV) remains prevalent in sub-Saharan Africa, where it has been linked to adverse birth outcomes. Suboptimal vaginal microbiomes (VMBs) have shown similar links. This pilot study fills critical gaps in understanding how HIV interacts with the pregnant VMB in populations underrepresented in microbiome research, like the Democratic Republic of the Congo (DRC). We identified maternal systemic immune factors associated with suboptimal VMBs that have been linked to poor birth outcomes. In a global meta-analysis, we found significant taxonomic and functional differences in the VMBs between pregnant people living with and without HIV, revealing potential biomarkers that increase the risk of adverse birth outcomes. These findings provide crucial insights into VMB features that may influence pregnancy health in PLWH-ART, guiding future research and tailored interventions to support safer pregnancies in the DRC and similar populations.This study is registered with NCT03048669.
Tertiary lymphoid structures (TLSs) are lymphoid structures found in non-lymphoid tissues, which have been involved in the pathogenesis of multiple chronic diseases including tumors. TLSs are associated with better prognosis and response to immunotherapy in most types of tumors. However, what their associations with prognosis and the exact roles of TLS-related genes in gallbladder cancer (GBC) are still unclear. Here, we investigated the associations of TLS with the prognosis in 85 patients with GBC. Our results showed that the presence of intra-tumoral TLS was negatively associated with T stage (p = 0.035) and vascular invasion (p = 0.048) and predicted a higher rate of overall survival (p = 0.004) and a decreased risk of early recurrence (p = 0.002). Moreover, using integrative analysis of single-cell RNA sequencing, bulk RNA sequencing, and deep machine learning, we screened six TLS-related candidate genes in GBC, including CTSG, FLNC, CCNB1, HSPB8, NR4A1, and MYLK. Furthermore, through the evaluation of biofunctions and clinical significance, we found that CTSG, FLNC, CCNB1, and HSPB8 played an important role in immune infiltration, diagnosis, and prognosis of GBC. Finally, cell-based experimental validation indicated that either knockdown of CTSG or overexpression of HSPB8 promoted CD8+ T cell apoptosis and exhaustion. In conclusion, TLS had a protective effect on human GBC, and its related genes, played a potential role in diagnosis, prognosis, immune infiltration, and metastasis of GBC. Our findings provided a new insight for the research on clinical biomarkers of GBC and development of its therapeutic targets.
Developmental dysplasia of the hip (DDH), a morphological abnormality of the hip joint, is a well-recognized risk factor for hip osteoarthritis (OA). Much remains unknown about the genetic factors of DDH and its subtypes. To further understand its genetic basis, we conducted genome-wide association studies (GWASs) using a total of 1 085 Japanese DDH cases (including 788 hip dysplasia cases without dislocation and 297 cases with dislocated hip) and 24 000 controls. Additionally, we meta-analyzed with United Kingdom (UK) DDH GWAS and the largest hip OA GWAS to date. We identified three genome-wide significant novel loci, COL11A2, CALN1 and TRPM7, associated with hip dysplasia without dislocation. None of these signals were significant in dislocated hips, and additionally two of the signals had an opposite direction of association, suggesting distinct genetic architectures between the subtypes. The Japanese DDH GWAS identified five associated loci (VEGF-C, FOXC1, SMC2, SLC38A4, and TRPM7), and the trans-ancestry meta-analysis with UK revealed two loci (COL11A1 and GDF5) supported by strong trans-ancestry genetic correlation (r = 1.0). In total, nine loci were identified for DDH and its subtypes, with hip dysplasia without dislocation showing distinct genetic signals from hip dislocation. The meta-analysis of DDH and hip OA identified five novel signals for hip OA. Susceptibility loci and heritability enrichment analyses implicated pathways involving bone formation, collagen type XI trimer, and chondrocyte development, as well as their gene regulation, in DDH. These findings enhance understanding of the genetic architecture and biological pathways underlying DDH, providing new insights into its relationship with OA.
Chronic granulomatous disease (CGD) is a rare inborn error of immunity caused by defects in components of the NADPH oxidase that impair the elimination of infectious microorganisms. Individuals affected by CGD become more susceptible to recurrent and severe infections. Six male patients from Southern Brazil were clinically and genetically analyzed through data collection from medical records and massively parallel sequencing by a panel for the following genes: CYBB, CYBA, NCF1, NCF2, and NCF4 and whole genome sequencing analysis. The gene-scan technique was used to identify the GT deletion in NCF1. The most common affected organs were the lungs, skin, and lymph nodes; the most common clinical manifestations were recurrent pneumonia, cutaneous involvement, lymph node manifestations, and failure to thrive. Four patients were identified with variants in CYBB: p.Cys257Ser, which is novel; p.Cys257Arg; p.Arg157Ter; and p.Trp483Ter. Both missense variants damage the loop E in gp91phox, a region with functional and structural relevance for the protein. Functional studies show the expression absence of the protein in patients with the variant p.Arg157Ter. The variant p.Trp483Ter is predicted to undergo nonsense mRNA-mediated decay. The GT deletion in NCF1 was identified in two siblings from consanguineous parents: one homozygous and the other apparently heterozygous for the deletion, both with a clinical diagnosis of CGD. Variant analysis in this gene is particularly challenging due to the presence of pseudogenes. A hypothesis for this genotypic discrepancy is the occurrence of a second type of pseudogene lacking the GT deletion, which may have arisen in one parent and been transmitted to the patient observed as heterozygous, being misinterpreted in the analyses as a functional NCF1 sequence.
Cardia carcinoma (CC) is a highly heterogeneous cancer with an increasing incidence worldwide. Gastroesophageal reflux disease has been identified as a risk factor for CC, and patients with Sjögren's syndrome (SS) are often reported to have esophageal motility disorders. This study aimed to identify potential hub genes and molecular processes for CC with SS. Four datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) analysis and weighted gene coexpression network analysis (WGCNA) were conducted to identify shared genes between CC and SS. Functional enrichment analysis and protein-protein interaction (PPI) network construction were performed on these genes. Four machine learning algorithms, including random forest (RF), least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE), and extreme gradient boosting (XGBoost), were applied to screen hub genes. Then, a nomogram predicting the risk of CC in SS patients was constructed and validated by the receiver operating characteristic (ROC) curve and calibration curve. Additionally, we analyzed the transcriptional regulatory relationships, coexpression networks, and correlations between the hub genes and immune infiltration. By intersecting DEGs and module genes identified by WGCNA, we screened 60 shared genes that were mainly enriched in cell cycle, response to xenobiotic stimulus, and p53 signaling pathways. Based on machine learning algorithms, three hub genes were identified and used to construct a nomogram with high predictive performance (the AUC for the training cohort and validation cohort were 0.991 and 0.978, respectively). Furthermore, the immune infiltration results suggested that T cells, mast cells, macrophages, and B cells play an important role in both diseases, and the hub genes were significantly associated with T cells and B cells. This study identified three hub genes (E2F3, CHIA, and SCNN1B) and established a nomogram that could effectively predict the risk of CC. The unbalanced immune response may be the common pathogenesis of these two diseases, which provides novel insights into the diagnosis and therapy of CC with SS.
Preeclampsia (PE) is a common complication during pregnancy. Trophoblast cells are the main cell type of the placenta, and abnormalities in proliferation and differentiation cause anterior placental accretion and lead to the development of PE. This study will investigate the function and related mechanism of N6-methyladenosine (m6A) reader YTH N6-methyladenosine RNA binding protein F3 (YTHDF3) in PE and provide a rationale for the therapeutic treatment of PE. In the study, YTHDF3 and suppressor of cytokine signaling 1 (SOCS1) expression were significantly upregulated within PE placental tissue samples. YTHDF3 knockdown promoted the capacity of hypoxia-treated trophoblast cells to proliferate, migrate, invade, and undergo epithelial-mesenchymal transition (EMT) and inhibited apoptosis. The JAK1/STAT3 pathway is aberrantly activated in trophoblast cells of PE. YTHDF3 binds m6A-modified SOCS1 mRNA to enhance its stability and translation. YTHDF3/SOCS1 promotes disease progression in PE by inhibiting the JAK1/STAT3 signaling pathway. In conclusion, YTHDF3 promotes the disease progression of PE by enhancing the m6A modification of SOCS1 mRNA and suppressing the JAK1/STAT3 signaling pathway. YTHDF3 has been identified as an underlying target for the clinical treatment of PE.
Chronic tendon injuries, characterized by persistent pain, reduced flexibility, and impaired function, pose a significant clinical challenge. Current therapeutic strategies for these injuries are limited. This study highlighted the crucial role of OXPHOS in maintaining tendon homeostasis and suggested potential therapeutic strategies targeting the OXPHOS pathway. This study utilized both bulk-sequencing (bulk-seq) and single-cell RNA sequencing (scRNA-seq) to analyze the heterogeneity in tenocytes, vascular endothelial cells, tendon-derived stem cells, adipocytes, and neurons from both non-lesional and lesional tendons. Key oxidative phosphorylation (OXPHOS)-related genes, such as COX15, COX4I1, COX5B, COX7A1, COX8A, NDUFA12, NDUFA5, NDUFB10, NDUFB3, NDUFC1, NDUFS1, and NDUFS4, were found to be significantly downregulated in lesional tendons compared with non-lesional ones, indicating impaired energy metabolism. This reduction in OXPHOS activity may contribute to increased necroptosis in chronic tendon injuries. Furthermore, bisphenol A and valproic acid were found to activate OXPHOS-related genes. The findings highlighted the crucial role of OXPHOS in maintaining tendon homeostasis and demonstrated potential therapeutic strategies targeting the OXPHOS pathway, such as bisphenol A and valproic acid, to enhance healing in chronic tendon conditions. The crucial role of OXPHOS in maintaining tendon homeostasis underscores its potential as a therapeutic target, reflecting that strategies aimed at modulating the OXPHOS pathway may provide promising treatment options. Chronic tendon injuries present a major clinical challenge with limited treatments. This study investigated the molecular mechanisms underlying these injuries using bulk and single-cell RNA sequencing. We identified significant downregulation of oxidative phosphorylation (OXPHOS)-related genes in lesional tendons across multiple cell types, contributing to increased necroptosis. In vitro experiments and molecular docking revealed that valproic acid activates OXPHOS and inhibits necroptosis. These findings highlight the critical role of mitochondrial function in tendon homeostasis and suggest valproic acid as a promising therapeutic candidate for treating chronic tendon injuries by restoring OXPHOS activity.
Developmental dysplasia of the hip (DDH) is a common orthopedic disorder characterized by abnormal development of the hip joint, which can lead to pain, instability, and early-onset osteoarthritis if left untreated. Its etiology is multifactorial, involving both genetic and environmental factors. This study investigated the association between selected single-nucleotide polymorphisms (SNPs) related to joint and bone development and the occurrence of DDH. It assessed potential copy number variations (CNVs) in key skeletal genes using MLPA. A total of 125 individuals were examined, including 43 patients with DDH and 82 healthy controls. Six SNPs were genotyped using real-time PCR with TaqMan assays: TGFB1 (rs1800470), CX3CR1 (rs3732378, rs3732379), GDF5 (rs143384), COL1A1 (rs113647555), and MMP24 (rs12479765). Allele and genotype distributions were compared between cases and controls, and CNVs in COL1A1, COL2A1, LRP5, DKK1, FZD4, and NDP genes were analyzed using Multiplex Ligation-Dependent Probe Amplification. Among the examined variants, only GDF5 rs143384 showed a nominally significant association with DDH (p = 0.040), with the A allele more common in affected individuals. However, after correcting for multiple testing, this result no longer remained significant. No significant associations were detected for TGFB1, CX3CR1, COL1A1, or MMP24. Although CX3CR1 rs3732378 allele frequencies differed slightly from international reference data, no link to DDH was confirmed. MLPA analysis did not identify pathogenic CNVs in the analyzed loci, which indicates that the studied genes have no association with DDH in the Slovak population. Similarly, SNPs in the studied genes yielded no significant results, apart from rs143384 in GDF5, which requires further investigation to confirm our findings.
Secondary angiosarcoma of the breast is a rare and aggressive malignancy, with characteristic MYC amplification. It typically arises in the field of prior radiation for breast cancer treatment, or rarely, in patients with a history of radical mastectomy with axillary lymph node dissection. Concurrent angiosarcoma and carcinoma in the same breast is exceedingly rare. In this report, we describe a rare occurrence of concurrent MYC-amplified angiosarcoma in the breast skin and recurrent carcinoma in the breast parenchyma in a patient without any history of breast radiation or axillary lymph node dissection. In addition, both angiosarcoma and carcinoma were MYC amplified, but with distinct MYC amplification patterns. The angiosarcoma demonstrated a 5' MYC only amplification with a breakpoint between the 5' and 3' MYC probes, while the carcinoma harbored a large amplification on chromosome 8q24.21, encompassing both 5' MYC and 3' MYC. This case report highlights the occurrence of secondary angiosarcoma arising in a patient without a history of prior radiotherapy or an axillary dissection, as well as the rare concurrence of angiosarcoma and carcinoma in the same breast. More intriguingly, the distinct MYC gene amplification patterns in the concurrent angiosarcoma and carcinoma reflect the complexity of the MYC gene in the tumorigenesis of different types of malignancies and may explain the different levels of MYC protein expression on immunohistochemistry.
Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.
Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.
Background & objectives Sickle cell anaemia (SCA) is a serious inherited blood disorder caused by mutations in the β-globin gene, leading to abnormal haemoglobin (HbS). Understanding the genetic diversity of SCA is important for improving diagnosis, treatment, and public health planning. Our aim was to systematically review and summarise the genetic variations associated with SCA in various populations, and to explore how these differences affect clinical outcomes and inform public health responses. Methods A systematic search was conducted across databases, including PubMed, Scopus, Cochrane, and Science Direct, for studies published between 1990 and 2025. A total of 62 studies were included, covering populations with a high prevalence of haemoglobinopathies. Results Significant genetic heterogeneity was identified. Common coinherited conditions included α- and β-thalassaemia, particularly in Saudi Arabia, Iran, and Sub-Saharan Africa, influencing haemoglobin levels and disease severity. Specific βS haplotypes (e.g. Benin, Bantu, Senegal) were regionally dominant, with some (e.g. Senegal) linked to higher foetal haemoglobin levels and milder symptoms. Genetic modifiers such as BCL11A and MYH9 variants were also found to affect disease expression. Public health screening programmes in countries like the UAE and India have achieved high coverage, but diagnostic and treatment challenges persist due to ongoing genetic and environmental variation. The Quantitative findings include regional dominance of βS haplotypes: Benin (29%), Bantu (3%), Senegal (1%), with the Senegal haplotype linked to higher foetal haemoglobin (HbF) levels (average 14.6%) and the Arab Indian haplotype (6.7%). Co-inheritance of β-thalassaemia was notably common in Saudi Arabia, Iran, and Sub-Saharan Africa. Interpretations & conclusions Tailored, genomically informed public health strategies are needed to address the diverse genetic landscape of SCA. Clinicians should incorporate genetic profiling and culturally appropriate counselling to improve care in affected populations. Variability in study design, sample size, and genetic reporting limited the ability to perform direct comparisons across regions.
Osteoporosis and osteosarcoma share certain molecular pathways, and identifying common hub genes could provide new insights into their pathogenesis. This study aimed to identify and validate hub genes involved in both osteoporosis and osteosarcoma and explore their potential as biomarkers and therapeutic targets. We retrieved three publicly available datasets: GSE13850 (osteoporosis), GSE16088, and GSE12865 (osteosarcoma). Differentially expressed genes (DEGs) were identified using the limma package in R, and common DEGs were determined through Venn analysis. The hub genes were identified using the STRING database and Cytoscape software. Reverse transcription quantitative PCR was used to validate gene expression in osteosarcoma cell lines and normal bone tissue cell lines. Functional assays including cell proliferation, colony formation, and wound healing were conducted after CLTA and SMAD3 knockdown via small interfering RNA. Gene set enrichment, promoter methylation analysis, and genetic alteration studies were conducted using publicly available databases. The hub genes identified, CLTA, MSR1, MTF2, and SMAD3, were highly expressed in osteosarcoma compared to normal controls, and their expression was validated across osteosarcoma tissue samples. Survival analysis showed that high expression of these genes correlated with poor prognosis in osteosarcoma patients. Gene enrichment analysis suggested that these genes play crucial roles in osteosarcoma development through various signaling pathways. CLTA and SMAD3 silencing in MG63 and U2OS cells led to significant reductions in cell proliferation, colony formation, and migration. This study identified CLTA, MSR1, MTF2, and SMAD3 as potential biomarkers and therapeutic targets for osteoporosis and osteosarcoma. However, further studies are needed before determining clinical implications.
Nonviral gene therapy holds promise as a potential treatment for lung cystic fibrosis (CF). However, the transient expression of the CF transmembrane conductance regulator (CFTR) protein has limited its clinical application. To circumvent this challenge, a CpG-depleted CFTR plasmid was developed. The CpG-depleted CFTR plasmid could be compacted into DNA nanoparticles and modified with the addition of highly branched poly(β-amino ester)s (HPAEs), leading to an improved and sustained CFTR protein expression. Using a CpG-depleted and codon-optimized CFTR sequence, around 20-fold increase in CFTR protein production was achieved 48 h after treatment, compared with healthy human bronchial epithelial cells (16HBE14o-). To evaluate the duration of CFTR protein expression induced by the plasmid based on human elongation factor 1α (hEFIα) and cytomegalovirus (CMV) promoters, a time course study was conducted in human CF bronchial epithelial (CFBE14o-) cells. hEFIα promoter revealed a remarkable 2.26-fold increase in CFTR protein expression at 7 days after transfection compared with 16HBE14o- cells. This level of CFTR protein expression outperformed the commonly used CMV promoter. The in vitro results demonstrated that CpG-depleted CFTR plasmid could be used to achieve high efficacy in subsequent in vivo evaluations, which, if validated, may provide promising prospects for the development of a novel and effective treatment for lung cystic fibrosis.
BACKGROUND Primary ciliary dyskinesia (PCD) is an uncommon autosomal recessive disease resulting from dysfunction of motile cilia that causes impaired mucociliary clearance and abnormal embryonic left-right axis differentiation. The CCDC39 gene is a known cause of PCD, which is most commonly associated with inner dynein arm defects and microtubular disorganization. Although heterotaxy-related congenital heart defects are well described in PCD, their presence in patients with CCDC39 mutations, particularly those with congenitally corrected transposition of the great arteries (ccTGA), has not been previously described. CASE REPORT We report a female neonate was born to consanguineous Saudi parents with prenatal findings of dextrocardia, abdominal situs inversus, and ccTGA. Postnatal evaluation confirmed these findings, including a significant ventricular septal defect and moderate tricuspid regurgitation. She presented with early-onset respiratory symptoms of copious secretions and pneumonia requiring oxygen support and hospitalization. The whole-exome sequencing identified a novel homozygous frameshift variant in CCDC39 (c.2230_2233del p.Gln744Aspfs*17) and thus validated the diagnosis of PCD. Despite multidisciplinary management, the patient had cardiopulmonary arrest secondary to sepsis at 4 months of age. CONCLUSIONS This is the first report describing the relationship between CCDC39-related PCD and ccTGA, thereby expanding the phenotypic spectrum of CCDC39 mutations. This report emphasizes the pivotal role of motile cilia in cardiac morphogenesis and underscores the importance of considering PCD in neonates with laterality anomalies and complex congenital heart defects. Early genetic testing and a multidisciplinary approach are critical to timely diagnosis and management.