Search Results (164)

Recent studies have demonstrated the clinical potential of nucleic acid therapeutics (NATs). However, their efficient and scalable delivery remains a major challenge for both ex vivo and in vivo gene therapy. Microfluidic platforms have emerged as a powerful tool for overcoming these limitations by enabling precise intracellular delivery and consistent therapeutic carrier fabrication. This review examines microfluidic strategies for gene delivery at the cellular level. These strategies include mechanoporation, electroporation, and sonoporation. We also discuss the synthesis of lipid nanoparticles, polymeric particles, and extracellular vesicles for systemic administration. Unlike conventional approaches, which treat ex vivo and in vivo delivery as separate processes, this review focuses on integrated microfluidic systems that unify these functions. For example, genetic materials can be delivered to cells that secrete therapeutic extracellular vesicles (EVs), or engineered cells can be encapsulated within hydrogels for implantation. These strategies exemplify the convergence of gene delivery and carrier engineering. They create a single workflow that bridges cell-level manipulation and tissue-level targeting. By synthesizing recent technological advances, this review establishes integrated microfluidic platforms as being fundamental to the development of next-generation NAT systems that are scalable, programmable, and clinically translatable. Full article

Considering the high plasticity of FoxP3⁺ regulatory T (Treg) cells and Interleukin (IL)-17-producing Th17 cells, we hypothesized that a Th17 inflammatory milieu may impair the functional properties of Treg cells in chronic inflammatory arthritides. Therefore, a cross-sectional explorative analysis was set up in patients with psoriatic arthritis (PsoA), rheumatoid arthritis, or spondyloarthritis to investigate the features of Th17 and Treg cells. T cell subpopulation counts, FOXP3 mRNA expression, CpG methylation of the FOXP3 gene, and the suppressive capacity of isolated Treg cells were determined. Ex vivo analysis of PsoA-derived peripheral blood lymphocytes showed a Th17-mediated inflammation. It was accompanied by demethylation of the FOXP3 promotor and Treg-specific demethylated region (TSDR) in Treg cells which, however, resulted neither in elevated FOXP3 mRNA expression nor in increased suppressive Treg cell capacity. To clarify this conundrum, in vitro stimulation of isolated Treg cells with Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGFβ), recombinant IL-17, or the anti-IL-17A antibody secukinumab was performed, demonstrating that cell culture conditions polarizing towards Th17, but not IL-17 itself, impair the suppressive function of Treg cells, accompanied by diminished FOXP3 mRNA expression due to hypermethylation of the FOXP3 promotor and TSDR. This potential causal relationship between Th17 inflammation and impaired Treg cell function requires attention regarding the development of immunomodulatory therapies. Full article

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

Background: Malaria remains a significant global health burden, particularly in sub-Saharan Africa, accounting for high rates of illness and death. The growing resistance to frontline antimalarial therapies underscores the urgent need for novel drug targets and therapeutics. Bromodomain-containing proteins, which regulate gene expression through chromatin remodeling, have gained attention as potential targets. Plasmodium falciparum bromodomain protein 1 (PfBDP1), a 55 kDa nuclear protein, plays a key role in recognizing acetylated lysine residues and facilitating transcription during parasite development. Methods: This study investigated ex vivo PfBDP1 gene mutations and identified potential small molecule inhibitors using computational approaches. Malaria-positive blood samples were collected. Genomic DNA was extracted, assessed for quality, and amplified using PfBDP1-specific primers. DNA sequencing and alignment were performed to determine single-nucleotide polymorphism (SNP). Structural modeling used the PfBDP1 crystal structure (PDB ID: 7M97), and active site identification was conducted using CASTp 3.0. Virtual screening and pharmacophore modeling were performed using Pharmit and AutoDock Vina, followed by ADME/toxicity evaluations with SwissADME, OSIRIS, and Discovery Studio. GROMACS was used for 100 ns molecular dynamics simulations. Results: The malaria prevalence rate stood at 12.24%, and the sample size was 165. Sequencing results revealed conserved PfBDP1 gene sequences compared to the 3D7 reference strain. Virtual screening identified nine lead compounds with binding affinities ranging from −9.8 to −10.7 kcal/mol. Of these, CHEMBL2216838 had a binding affinity of −9.9 kcal/mol, with post-screening predictions of favorable drug-likeness (8.60), a high drug score (0.78), superior pharmacokinetics, and a low toxicity profile compared to chloroquine. Molecular dynamics simulations confirmed its stable interaction within the PfBDP1 active site. Conclusions: Overall, this study makes a significant contribution to the ongoing search for novel antimalarial drug targets by providing both molecular and computational evidence for PfBDP1 as a promising therapeutic target. The prediction of CHEMBL2216838 as a lead compound with favorable binding affinity, drug-likeness, and safety profile, surpassing those of existing drugs like chloroquine, sets the stage for preclinical validation and further structure-based drug design efforts. These findings are supported by prior experimental evidence showing significant parasite inhibition and gene suppression capability of predicted hits. Full article

Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their clinical application remains constrained by several inherent limitations. This study explores a cell-free alternative by investigating the therapeutic potential of exosomes derived from bone marrow (BMSCs), adipose tissue (ADSCs), and umbilical cord (UMSCs) MSCs in mitigating OA pathogenesis, utilizing both in vitro and ex vivo models. Exosomes from each MSC source were isolated and characterized through nanoparticle tracking analysis, transmission electron microscopy, and Western blotting to confirm their identity and purity. Subsequently, their chondroprotective, anti-inflammatory, and regenerative properties were systematically assessed through evaluations of cell viability, expression profiles of inflammatory and chondroprotective markers, and chondrocyte migration assays. The results demonstrate that all three types of MSC-derived exosomes (MSC-Exos) exhibit low cytotoxicity while significantly suppressing proinflammatory markers and enhancing the expression of chondroprotective genes. Notably, BMSC-Exos and UMSC-Exos displayed superior efficacy in attenuating inflammation, promoting cartilage protection, and inhibiting chondrocyte apoptosis. Furthermore, all MSC-Exos markedly enhanced chondrocyte motility, a critical component of cartilage repair. Collectively, these findings support the therapeutic promise of MSC-Exos, particularly those derived from BMSCs and UMSCs, as a targeted, cell-free approach for the treatment of OA compared to ADSCs. By modulating inflammation, promoting cartilage regeneration, and preventing chondrocyte apoptosis, MSC-Exos may serve as a viable and scalable alternative to current MSC-based therapies for this widespread degenerative disease. Full article

Lentiviral vectors (LVs) have become a fundamental tool in gene therapy due to their unique ability to transduce both dividing and non-dividing cells, transfer large genes of up to 10 kb, and facilitate stable, long-term expression of therapeutic genes into target cells. A key application of LVs is the ex vivo genetic modification of patient-derived cells, such as the production of CAR-T cells by transducing isolated T cells with LVs to express the CAR gene, enabling them to target and destroy cancer cells once infused back into the patient. However, these ex vivo gene therapy drugs are often dismally unaffordable due to the complex procedures involved, including cell isolation, genetic modification, and expansion, along with the significant risks associated with immune conditioning to ensure successful engraftment. To overcome these barriers, direct in vivo transgene delivery to physiologically relevant cells has been explored, bypassing the need for ex vivo manipulations and reducing costs. Yet, a major challenge in this approach is engineering LV cell tropism to ensure the precise targeting of specific cells while avoiding off-target effects. Recent advances in modifying LV surface proteins have shown promise, including the successful in vivo generation of CAR T cells and ensuing clinical trials. This review is aimed at providing an up-to-date account of the progress in engineering LV tropism, covering the utility of different heterologous viral envelopes and their engineering to achieve cell-type-specific delivery and host immune evasion, and highlighting the potential of in vivo gene therapy to improve the affordability and accessibility of life-saving treatments. Full article

Background: Mesenchymal stem cell-based therapy may be indicated in ischaemic heart disease. The use of autologous adipose-derived mesenchymal stromal cells (AdMSCs) offers regenerative potential due to their paracrine effects. The aim of this study was to expand and characterise adult human thymus-derived MSCs harvested during open heart surgery with respect to their stem cell and paracrine properties. Methods: Enzymatically and non-enzymatically isolated human thymic AdMSCs (ThyAdMSCs) were cultured in xeno-free media containing pooled human platelet lysate (pPL). MSC characterisation was performed. Ex vivo expanded ThyAdMSCs were differentiated into three lineages. Proliferative capacity and immunomodulatory properties were assessed by proliferation assays and mixed lymphocyte reaction, respectively. Gene expression analysis was performed by qPCR. Results: Both isolation methods yielded fibroblast-like cells with plastic adherence and high proliferation. Flow cytometry revealed distinct expression of MSC markers in the absence of haematopoietic cell surface markers. Ex vivo expanded ThyAdMSCs could be differentiated into adipocytes, osteocytes, and chondrocytes. Activated peripheral blood mononuclear cells were significantly reduced when co-cultured with ThyAdMSCs, indicating their ability to inhibit immune cells in vitro. Gene expression analysis showed significantly less IFNγ and TNFα, indicating an alteration of the activated and pro-inflammatory state in the presence of ThyAdMSCs. Conclusions: These results demonstrate an efficient method to generate AdMSCs from human thymus. These MSCs have a strong immunomodulatory capacity and are, therefore, a promising cell source for regenerative medicine. The culture conditions are crucial for cells to proliferate in culture. Further research could explore the use of ThyAdMSCs or their secretome in surgical procedures. Full article

Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah^-/- graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver. Full article

This study aimed to develop an ex vivo retinal model to examine inflammatory processes in diabetic retinopathy (DR) without animal testing. Porcine eyes were collected from a local abattoir, dissected, and cultivated for four days in five experimental groups: control group (Co), 25 mM and 50 mM mannitol groups (Man25, Man50) as osmotic controls, and 25 mM and 50 mM glucose groups (Glc25, Glc50) as diabetic groups. A TUNEL assay was used to determine relative cell death. Immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect inflammatory markers. An increase in the cell death rate in Man50 (30%), Glc25 (36%) and Glc50 (37%) compared to Co (12%) (p < 0.01, p < 0.001, p < 0.001, respectively) and between Glc25 and Man25 (21%) (p < 0.01) was found. Immunofluorescence staining and qRT-PCR analysis revealed a TNF-α increase in Glc25 compared to Man25 and Co. iNOS was increased in Glc25 vs. Man25 but not in Co vs. Glc25. iNOS gene expression was upregulated with Glc25 treatment compared to Co and Man25 groups. Expression levels of IL-6 and CD31 were significantly higher in Glc25 than in Co and Man25. Glucose treatment increased cell death and inflammation, prompting us to present a DR model for better understanding DR and testing new therapies. Full article

Bacteriophage therapy is a promising approach for targeting antibiotic-resistant bacteria and modulating gut microbiota in metabolic diseases such as obesity. This study evaluated the impact of a two-phage cocktail on an ex vivo colonic simulation model of gut microbiota derived from obese individuals, both in its normalized state and after enrichment with Enterobacter cloacae, an obesity-related bacteria. Microbiological analyses confirmed that the phage cocktail remained active throughout the colonic regions over three digestion cycles and effectively reduced enterobacterial populations in the enriched microbiota. Metabarcoding of the 16S rRNA gene revealed that phage therapy did not significantly alter the abundance of dominant genera, but selectively reduced E. cloacae across all colonic regions. Alpha diversity was significantly affected only in the enriched microbiota, while beta diversity analysis indicated significant compositional shifts during therapy, with reduced dispersion in the final treatment stage. Short-chain fatty acid profiling demonstrated region- and group-specific metabolic responses, with increased lactic and butyric acid concentrations in the ascending colon of the enriched microbiota following phage treatment. This study provides the first ex vivo evidence that a two-phage cocktail can selectively eliminate E. cloacae while preserving overall microbiota structure and functionality. These findings establish a foundation for future in vivo studies exploring the role of phage therapy in reshaping gut microbial communities and metabolic profiles, highlighting its potential as a precision tool for managing gut dysbiosis in metabolic disorders. Full article

Background/Objectives: Parkinson’s disease (PD) is a rapidly growing neurological disorder in the developed world, affecting millions over the age of 60. The decline in motor functions occurs due to a progressive loss of midbrain dopaminergic neurons, resulting in lowered dopamine levels and impaired muscle function. Studies show defective mitochondrial autophagy (or “mitophagy”) links to PD. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are serine/threonine kinases, and their inhibition can enhance neuroprotection in PD by promoting mitophagy. Methods: We examine the effects of ROCK inhibitor SR3677, delivered via macrophage-derived small extracellular vesicles (sEVs) to Parkin Q311X(A) PD mouse models. sEVs with SR3677, administered intranasally, increased mitophagy gene expression, reduced inflammatory factors, and elevated dopamine levels in brain tissues. Results: ROCK2 expression decreased, showing the drug’s inhibitory effect. sEV-SR3677 treatment was more effective than treatment with the drug alone, although sham EVs showed lower effects. This suggests that EV-SR3677 not only activates mitochondrial processes but also promotes the degradation of damaged mitochondria through autophagy. Mitochondrial functional assays and oxygen consumption in ex vivo glial cultures revealed that sEV-SR3677 significantly improved mitochondrial respiration compared to that in untreated or SR3677-only treated cells. Conclusion: We demonstrated the efficacy of ROCK2 inhibition on mitochondrial function via sEV-SR3677 in the PD mouse model, necessitating further studies to explore design challenges and mechanisms of sEV-SR3677 as mitochondria-targeted therapy for PD Full article

Background/Objectives: Mesenchymal stromal cells (MSCs) hold the potential for tumor-targeted gene delivery due to their ex vivo manipulability, low immunogenicity, scalability, and inherent tumor-homing properties. Despite the widespread use of viral vectors for MSC genetic modification, safety concerns have prompted interest in non-viral alternatives, such as gene electrotransfer (GET). This study aimed to optimize GET parameters for MSCs transfection, assess MSCs biodistribution after in vivo administration, and evaluate the therapeutic potential of interleukin-12 (IL-12)-modified MSCs in a mouse melanoma model. Methods: Human MSCs were isolated from umbilical cords under ethically approved protocols. GET protocols were optimized using a fluorescent reporter gene to evaluate transfection efficiency and cell viability. MSC biodistribution was examined following intravenous and intratumoral injections in murine tumor models using luminescent reporter gene. The therapeutic efficacy of IL-12-modified MSCs was assessed in a syngeneic mouse melanoma model. Results: Optimized GET protocols achieved a transfection efficiency of 80% and a cell viability of 90%. Biodistribution studies demonstrated effective tumor retention of MSCs following intratumoral injections, whereas intravenous administration resulted in predominant cell localization in the lungs. IL-12-modified MSCs injected intratumorally significantly inhibited tumor growth, delaying tumor progression by five days compared to controls. Conclusions: Optimized GET conditions enabled high-efficiency, high-viability MSCs transfection, facilitating their use as effective vehicles for localized cytokine delivery. While the innate tumor tropism of MSCs was not conclusively demonstrated, the study highlights the potential of GET as a reliable non-viral gene delivery platform and underscores the therapeutic promise of IL-12-modified MSCs in tumor-targeted gene therapy. Full article

Age-related macular degeneration (AMD) is a predominant cause of vision loss, posing significant challenges in its management despite advancements such as anti-vascular endothelial growth factor (anti-VEGF) therapy. Nanomedicine, with its novel properties and capabilities, offers promising potential to transform the treatment paradigm for AMD. This review reports the significant advancements in the use of diverse nanoparticles (NPs) for AMD in vitro, in vivo, and ex vivo, including liposomes, lipid nanoparticles, nanoceria, nanofibers, magnetic nanoparticles, quantum dots, dendrimers, and polymer nanoparticles delivered in forms such as gels, eye drops, intravitreally, or intravenously. Drug delivery was the most common use of NPs for AMD, followed by photodynamic therapy dose enhancement, antioxidant function for nanoceria, biomimetic activity, and immune modulation. Innovative approaches arising included nanotechnology-based photodynamic therapy and light-responsive nanoparticles for controlled drug release, as well as gene therapy transfer. Nanomedicine offers a transformative approach to the treatment and management of AMD, with diverse applications. The integration of nanotechnology in AMD management not only provides innovative solutions to overcome current therapeutic limitations but also shows potential in enhancing outcomes and patient quality of life. Full article

Ex vivo regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which ex vivo regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies. Full article

Recombinant therapeutic and vaccine proteins have revolutionized healthcare, but there remain challenges, as many are awaiting development due to their slow development speed and high development cost. Cell-free in vivo ribosomes offer one choice, but they come with similar constraints. The validation of in vivo messenger RNA (mRNA) technology has been accomplished for COVID-19 vaccines. The bioreactors inside the body, the ribosomes, deliver these proteins at a small cost, since these are chemical products and do not require extensive analytical and regulatory exercises. In this study, we test and validate the final product. A smaller fraction of the recombinant protein cost is needed, removing both constraints. Although thousands of in vivo mRNA products are under development, their regulatory classification remains unresolved: do they qualify as chemical drugs, biological drug, or gene therapy items? These questions will soon be resolved. Additionally, how would the copies of approved in vivo mRNA protein products be brought in, and how would they be treated: as new drugs, generic drugs, or new biological drugs? Researchers are currently working to answer these questions. Regardless, these products’ cost of goods (COGs) remains much smaller than that of ex vivo mRNA or recombinant products. This is necessary to meet the needs of the approximately 6.5 billion people around the world who do not have access to biological drugs; these products will indeed serve the dire needs of humanity. Given the minor cost of establishing the manufacturing of these products, it will also prove financially attractive to investors. Full article