Search Results (55)

Lysosomal storage disorders (LSDs) are inherited metabolic conditions characterized by lysosomal enzyme deficiencies leading to substrate accumulation. As genetic diseases, LSDs can be treated with gene therapies (GT), including the CRISPR/Cas systems. The CRISPR/Cas systems enable precise and programmable genome editing, leading to targeted modifications at specific genomic loci. While the classical CRISPR/Cas9 system has been extensively used to generate LSD disease models and correct disease-associated genetic alterations through homologous recombination (HR), recently described Cas proteins as well as CRISPR/Cas9-derived strategies such as base editing, prime editing, and homology-independent targeted integration (HITI) offer a novel way to develop innovative treatments for LSDs. The direct administration of the CRISPR/Cas9 system remains the primary strategy evaluated in several LSDs; nevertheless, the ex vivo CRISPR/Cas9-based approach has been recently explored, primarily in central nervous system-affecting LSDs. Ex vivo approaches involve genetically modifying, in theory, any patient cells in the laboratory and reintroducing them into the patient to provide a therapeutic effect. This manuscript reviews the molecular aspects of the CRISPR/Cas technology and its implementation in ex vivo strategies for LSDs while discussing novel approaches beyond the classical CRISPR/Cas9 system. Full article

Lung adenocarcinoma exhibits a heterogeneous molecular landscape shaped by key oncogenic drivers and tumor suppressor gene alterations. Mutation frequencies vary geographically, influenced by genetic ancestry and environmental factors. However, the molecular profile of lung adenocarcinoma in Bulgarian patients remains largely uncharacterized. We conducted a prospective study of 147 Bulgarian patients with metastatic lung adenocarcinoma, analyzing clinicopathologic features and somatic mutation frequencies using next-generation sequencing. Key mutations and their prevalence were assessed and compared with published data from other populations. The cohort included predominantly male patients (68.0%) with a median age of 67 years. TP53 mutations were most frequent (41.5%), followed by EGFR alterations (19.0%) and KRAS c.34G>T (p.Gly12Cys) (17.0%). Over half of the patients (51.0%) harbored two or more gene mutations. Mutation frequencies aligned closely with European cohorts, exhibiting a lower prevalence of EGFR mutations compared to East Asian populations. This study characterizes the molecular landscape of lung adenocarcinoma in Bulgaria, highlighting the predominance of TP53 and KRAS mutations. The findings emphasize the need for comprehensive molecular profiling to inform targeted therapies and support precision oncology approaches tailored to the Bulgarian population. Further research is needed to validate these results and improve clinical outcomes. Full article

Encouraging discoveries and excellent advances in the fight against cancer have led to innovative therapies such as photothermal therapy (PTT), photodynamic therapy (PDT), drug targeting (DT), gene therapy (GT), immunotherapy (IT), and therapies that combine these treatments with conventional chemotherapy (CT). Furthermore, 2,041,910 new cancer cases and 618,120 cancer deaths have been estimated in the United States for the year 2025. The low survival rate (<50%) and poor prognosis of several cancers, despite aggressive treatments, are due to therapy-induced secondary tumorigenesis and the emergence of drug resistance. Moreover, serious adverse effects and/or great pain usually arise during treatments and/or in survivors, thus lowering the overall effectiveness of these cures. Although prevention is of paramount importance, novel anticancer approaches are urgently needed to address these issues. In the field of anticancer nanomedicine, carbon nanotubes (CNTs) could be of exceptional help due to their intrinsic, unprecedented features, easy functionalization, and large surface area, allowing excellent drug loading. CNTs can serve as drug carriers and as ingredients to engineer multifunctional platforms associated with diverse treatments for both anticancer therapy and diagnosis. The present review debates the most relevant advancements about the adjuvant role that CNTs could have in cancer diagnosis and therapy if associated with PTT, PDT, DT, GT, CT, and IT. Numerous sensing strategies utilising various CNT-based sensors for cancer diagnosis have been discussed in detail, never forgetting the still not fully clarified toxicological aspects that may derive from their extensive use. The unsolved challenges that still hamper the possible translation of CNT-based material in clinics, including regulatory hurdles, have been discussed to push scientists to focus on the development of advanced synthetic and purification work-up procedures, thus achieving more perfect CNTs for their safer real-life clinical use. Full article

Lysosomal storage disorders (LSDs) constitute a group of monogenic systemic diseases resulting from deficiencies in specific lysosomal enzymes that cause the intralysosomal accumulation of non- or partially degraded substrates, leading to lysosomal dysfunction. In some cases of LSDs, the bone is more severely affected, thus producing skeletal manifestations in patients. Current therapies, such as enzyme replacement therapy (ERT) and gene therapy (GT), show limited efficacy in correcting skeletal abnormalities. Increasing evidence suggests that microenvironmental disturbances also contribute significantly to disease pathogenesis. Therefore, therapeutic strategies targeting lysosomal dysfunction and microenvironmental dysregulation are needed. Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) are emerging as promising candidates in regenerative medicine due to their immunomodulatory, pro-regenerative, and paracrine properties. MSC-EVs have shown potential to modulate the microenvironment and favor tissue repair in bone-related disorders such as osteoarthritis and osteoporosis. Interestingly, MSC-EVs can be engineered to reach the bone and carry therapeutics, including ERT- and GT-related molecules, enabling targeted delivery to hard-to-reach bone regions. This review describes the main features of MSC-EVs and discusses the therapeutic potential of MSC-EVs as a potential cell-free strategy for bone-affected LSDs. Full article

Osteogenesis imperfecta type VI is a rare autosomal recessive disorder characterized by bone fragility and defective mineralization, caused by pathogenic variants in the SERPINF1 gene. This study aimed to expand the understanding of OI type VI by analyzing clinical, radiological, and molecular findings in four patients from three unrelated families. Genotyping revealed two novel SERPINF1 variants, c.185G>T (p.Gly62Val) and c.992_993insCA (p.Glu331Asnfs), in a compound heterozygous state in one patient, and a known pathogenic variant, c.261_265dup (p.Leu89Argfs26), in a homozygous form in three patients. Clinical manifestations included early-onset fractures, severe skeletal deformities, impaired mobility, and growth failure. Radiological assessments revealed multilevel and multiplanar bone deformities and metaphyseal widening. RNA analysis demonstrated that the c.992_993insCA variant results in a truncated PEDF protein without triggering nonsense-mediated decay. Population screening identified a carrier frequency of 0.0044 for the c.261_265dup variant, suggesting a founder effect in the Tuvinian population. These findings expand the mutational spectrum of the SERPINF1 gene and provide new insights into the phenotypic variability of OI type VI. Our results highlight the importance of genetic screening in isolated populations and emphasize the need for further research to develop more effective therapeutic approaches for patients with limited response to bisphosphonate therapy. Full article

Cystic Fibrosis (CF) is a common genetic disease in the United States, resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators, particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI), have significantly improved clinical outcomes for patients with CF. However, many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study, we report that a patient carrying rare complex CFTR mutations, c.1680-877G>T and c.3067_3072delATAGTG, showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly, patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics, including lung function, body mass index (BMI), and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. Full article

CLN2 disease (neuronal ceroid lipofuscinosis type 2) is an ultra-rare lysosomal storage disorder caused by mutations in the TPP1/CLN2 gene, resulting in impaired tripeptidyl peptidase 1 (TPP1) activity. The timely initiation of enzyme replacement therapy is pivotal for attenuating progressive and irreversible neurodegeneration. This study aimed to benchmark the performance of Oxford Nanopore long-read sequencing (ONT-LRS) for targeted TPP1 mutation detection in a Turkish CLN2 cohort and to assess its concordance with orthogonal validation methods, including Sanger sequencing and enzymatic activity assays. Using a custom-designed primer panel, the entire TPP1 gene (6846 bp) was sequenced on the Oxford Nanopore (ONT) MinIon platform in seven clinically confirmed CLN2 index patients and sixteen unaffected family members. Detected variants were validated via Sanger sequencing and correlated with TPP1 enzyme activity in leucocytes and dried blood spots. Four pathogenic or likely pathogenic TPP1 variants were identified: c.622C>T (p.Arg208*), c.857A>G (p.Asn286Ser), c.1204G>T (p.Glu402*), and c.225A>G (p.Gln75=), along with fourteen additional benign variants. Variant allele frequencies were 50% for c.622C>T, 28.6% for c.1204G>T, 14.3% for c.857A>G, and 7.1% for c.225A>G. Notably, this is the first report to document the homozygous state of the c.857A>G variant and the compound heterozygous configuration of the c225A>G and c.622C>T variants in CLN2 patients, thereby expanding the known mutational landscape. In contrast, the globally common variant c.509-1G>C was not observed, suggesting regional variation in TPP1 mutation patterns. Consistent with the prior Turkish studies, c.622C>T (p.Arg208*) was the most prevalent variant, followed by c.1204G>T (p.Glu402*). TPP1 enzymatic activity was significantly reduced in all affected individuals (p < 0.0001), supporting the functional relevance of the identified variants. ONT-LRS offers a robust, cost-effective platform for high-resolution analysis of the TPP1 gene. Integrating molecular and biochemical data improves diagnostic precision and supports timely, targeted interventions for CLN2 disease, particularly in regions with high consanguinity and limited diagnostic infrastructure. Full article

Variants in the GBA1 gene, encoding the lysosomal enzyme glucosylceramidase beta 1 (GCase), are linked to Parkinson’s disease (PD) and Gaucher disease (GD). Heterozygous variants increase PD risk, while homozygous variants lead to GD, a lysosomal storage disorder. Some GBA1 variants impair GCase maturation in the endoplasmic reticulum, blocking lysosomal transport and causing glucosylceramide accumulation, which disrupts lysosomal function. This study explores therapeutic strategies to address these dysfunctions. Using Site-directed Enzyme Enhancement Therapy (SEE-Tx^®), two structurally targeted allosteric regulators (STARs), GT-02287 and GT-02329, were developed and tested in GD patient-derived fibroblasts with relevant GCase variants. Treatment with GT-02287 and GT-02329 improved the folding of mutant GCase, protected the GCase_Leu444Pro variant from degradation, and facilitated the delivery of active GCase to lysosomes. This enhanced lysosomal function and reduced cellular stress. These findings validate the STARs’ mechanism of action and highlight their therapeutic potential for GCase-related disorders, including GD, PD, and Dementia with Lewy Bodies. Full article

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. Full article

Hereditary Tyrosinemia Type-1 (HT1), an inherited error of metabolism caused by a mutation in the fumarylacetoacetate hydrolase gene, is associated with liver disease, severe morbidity, and early mortality. The use of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) has almost eradicated the acute HT1 symptoms and childhood mortality. However, patient outcomes remain unsatisfactory due to the neurocognitive effects of NTBC and the requirement for a strict low-protein diet. Gene therapy (GT) offers a potential single-dose cure for HT1, and there is now abundant preclinical data showing how a range of vector-nucleotide payload combinations could be used with curative intent, rather than continued reliance on amelioration. Unfortunately, there have been no HT1-directed clinical trials reported, and so it is unclear which promising pre-clinical approach has the greatest chance of successful translation. Here, to fill this knowledge gap, available HT1 preclinical data and available clinical trial data pertaining to liver-directed GT for other diseases are reviewed. The aim is to establish which vector-payload combination has the most potential as a one-dose HT1 cure. Analysis provides a strong case for progressing lentiviral-based approaches into clinical trials. However, other vector-payload combinations may be more scientifically and commercially viable, but these options require additional investigation. Full article

Ovarian cancer (OC) is a highly heterogeneous malignancy, often characterized by complex genomic alterations that drive tumor progression and therapy resistance. In this paper, we report a novel de novo BRCA2 germline variant NM_000059.3:c.(8693_8695delinsGT) associated with early-onset OC that featured two regions with differential MMR (Mismatch Repair) gene expression. To date, only six cases of de novo BRCA2 variants have been reported, none of which were associated with early-onset high-grade serous OC. The immunohistochemical analysis of MMR genes revealed two distinct tumor areas, separated by a clear topographic boundary, with the heterogeneous expression of MLH1 and PMS2 proteins. Seventy-five percent of the tumor tissue showed positivity, while the remaining 25% exhibited a complete absence of expression, underscoring the spatial variability in MMR gene expression within the tumor. Integrated comparative spatial genomic profiling identified several tumor features associated with the genetic variant as regions of loss of heterozygosity (LOH) that involved BRCA2 and MLH1 genes, along with a significantly higher mutational tumor burden in the tumor area that lacked MLH1 and PMS2 expression, indicating its further molecular evolution. The following variants were acquired: c.6572C>T in NOTCH2, c.1852C>T in BCL6, c.191A>T in INHBA, c.749C>T in CUX1, c.898C>A in FANCG, and c.1712G>C in KDM6A. Integrated comparative spatial proteomic profiles revealed defects in the DNA repair pathways, as well as significant alterations in the extracellular matrix (ECM). The differential expression of proteins involved in DNA repair, particularly those associated with MMR and Base Excision Repair (BER), highlights the critical role of defective repair mechanisms in driving genomic instability. Furthermore, ECM components, such as collagen isoforms, Fibrillin-1, EMILIN-1, Prolargin, and Lumican, were found to be highly expressed in the MLH1/PMS2-deficient tumor area, suggesting a connection between DNA repair deficiencies, ECM remodeling, and tumor progression. Thus, the identification of the BRCA2 variant sheds light on the poorly understood interplay between DNA repair deficiencies and ECM remodeling in OC, providing new insights into their dual role in shaping tumor evolution and suggesting potential targets for novel therapeutic strategies. Full article

Background/Objectives: Fabry disease is an X-linked disorder caused by lysosomal accumulation of glycosphingolipids due to the deficiency of α-Galactosidase (α-GAL), which leads to pathology in multiple organ systems. The standard of care is enzyme replacement therapy (ERT) with recombinant native α-GAL protein. Shortcomings of the native α-GAL include low stability, a short circulating half-life, and inadequate uptake by affected tissues that limits the efficacy of ERT and could potentially reduce AAV gene therapy (GT) benefits. Cross-correction by secreted α-GAL is essential for liver-targeted as well as ubiquitous AAV GT due to poor transduction and/or short half-life of some of the significantly affected cell types. Methods: To overcome potential limitations of AAV GT delivering native α-GAL, we used an engineered GLA transgene product to improve enzyme stability and reduce predicted immunogenicity. Results: The stabilized α-GAL variant, Eng-C, had an extended circulatory half-life, allowing for enhanced distribution and efficient uptake by target organs. AAV gene therapy with Eng-C demonstrated significantly greater substrate reduction in the severe Fabry G3Stg/GlaKO mouse model across all affected tissues. Efficacy of the Eng-C AVV GT was equal to or greater than the efficacy of the higher doses of the AAV GT with native α-GAL. Furthermore, this study is the first to demonstrate that the pre-existing pathology in some tissues in G3Stg/GlaKO mice can be reversed with efficient treatment. Conclusions: Our findings demonstrate that an AAV-based gene therapy expressing an engineered α-GAL with improved stability and lower immunogenicity could be effective at lower doses than other AAV GTs, potentially offering lower safety risks typically associated with high AAV vector doses. Full article

Background/Objectives: to report a novel KRT3 Meesmann corneal dystrophy (MECD) mutation and its clinical findings in a Spanish family, thus completing the international database. Case series study. Methods: Two generations of three family members were studied. The clinical ophthalmologic evaluation was made including best-corrected visual acuity (BCVA), biomicroscopy with and without fluorescein, fundoscopy, Schirmer test I, non-invasive break-up time (NiBUT), and esthesiometry. In vivo confocal microscopy (IVCM), anterior segment optical coherence tomography (AS-OCT) with an epithelial map, and genetic analysis were also performed. Results: A novel heterozygous mutation in the KRT3 gene c.1527G>T (p. Glu509Asp) was identified. Biomicroscopy revealed bilateral multiple corneal intraepithelial cysts. IVCM showed numerous and relatively small microcysts (12–32 µm), hyperreflective materials, subepithelial nerve and Bowman’s layer alterations. AS-OCT scan revealed diffuse hyperreflectivity and the epithelial map displayed thickening of the corneal epithelium in the interpalpebral zone (proband: 52–68 µm and father’s proband: 55–71 µm) with a slightly thinned cornea. Conclusions: We identified a new mutation in the KRT3 gene–c.1527G>T (p. Glu509Asp) in a Spanish family with MECD. A comprehensive characterization of the clinical signs, using different techniques, especially an epithelial map, could be useful to diagnose and monitor epithelial changes by quantitative measures. Epithelial map changes provide better understanding of MECD differential epithelial behavior and its progression changes. Larger studies will be necessary to better understand these specific patterns and clinically evaluate new therapies. Full article

Gene electrotransfer (GET) has gained significant momentum as a non-viral gene delivery method for various clinical applications, primarily in the cancer immunotherapy and vaccine development space. Preclinical studies have demonstrated exogenous gene delivery and expression in various tissues, including the liver, skin, cardiac muscle, and skeletal muscle. However, protein replacement applications of this technology have yet to be fully actuated. Plasmid DNA skeletal muscle delivery has been shown to maintain expression for up to 18 months. In the current study, we evaluated localized skeletal muscle delivery for protein replacement applications. We developed localized in vivo electro gene therapy (liveGT) protocols utilizing mono- and biphasic pulse sequences for localized pulse delivery directly to skeletal muscle with a custom monopolar platinum electrode. Plasmid DNA encoding human insulin and human glucokinase were chosen for this study to evaluate the liveGT platform for protein replacement potential. Initial in vitro GET was performed in mouse myoblasts to evaluate human insulin and glucokinase co-delivery. This was followed by liveGT-mediated reporter gene delivery in the skeletal muscle of Sprague–Dawley rats for pulse sequence selection. Protein replacement potential was evaluated in healthy (non-diabetic) rats with liveGT-mediated human insulin and glucokinase co-delivery to skeletal muscle. Human and rat insulin levels were measured via ELISA over the course of 3 months. Fed-state blood glucose measurements were monitored in correlation with serum human insulin levels. LiveGT-mediated skeletal muscle reprogramming successfully produced physiological levels of human insulin in serum over the course of 3 months. Hypo- and hyperglycemic events were not observed. Therefore, liveGT is a safe and viable platform for potential protein replacement therapies. Full article

Background/Objectives: Vitamin D (VD) has immunoregulatory properties, generating interest in its potential to influence therapeutic outcomes in inflammatory bowel disease (IBD), other than affecting the expression of genes encoding enzymes and transporters involved in drug metabolism and transport. This study investigated VD-related single nucleotide polymorphisms (SNPs) as predictors of clinical responses in patients with Crohn’s disease (CD) and ulcerative colitis (UC) treated with vedolizumab (VDZ) or ustekinumab (UST) after 3 (T3) and 12 months (T12), as well as the achievement of fecal calprotectin (FC) levels < 250 mg/kg, a marker of mucosal healing. Methods: In this prospective study, 103 patients (67 CD, 36 UC) were enrolled, 40 receiving VDZ and 63 receiving UST. SNPs in the genes CYP24A1, GC, CYP27B1, and VD receptor (VDR) were analyzed via polymerase chain reaction (PCR) and associated with clinical and laboratory outcomes. Results: UST therapy demonstrated a higher clinical response rate at T12 compared to VDZ (p = 0.03). A correlation was found between response at T3 and T12 (p = 0.0002). GC 1296 AC polymorphism negatively predicted response at T12, with 63.6% of non-responders carrying this genotype. CYP24A1 8620 AG was a negative predictor for achieving FC < 250 mg/kg (p = 0.045). CYP24A1 22776 CT and VDR Cdx2 GG increased the likelihood of presenting CD over UC (OR 3.40, p = 0.009 and OR 3.74, p = 0.047, respectively). Additionally, CYP27B1 −1260 GT and +2838 CT increased the likelihood of non-ileal CD (OR 3.13, p = 0.054; OR 7.02, p = 0.01). Conclusions: This study reveals associations between VD-SNPs, clinical response to VDZ and UST, and IBD phenotype and localization, supporting the development of personalized IBD treatment and warranting further validation. Full article