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Adeno-associated virus (AAV)–associated gene therapy has been increasingly promising, in light of the drugs progressed to clinical trials or approved for medications internationally. Therefore, scalable and efficient production of recombinant AAV is pivotal for advancing gene therapy. Traditional methods, such as the triple-plasmid transfection of human embryonic kidney 293 cells in suspension culture, have been widely employed but often hampered by low unit yield. In this study, we optimized the cell culture process with high cell density up to 2 × 107 cells/mL by employing a perfusion culture system with centrifugation and medium exchange in shake flasks and perfusion device in bioreactor. Furthermore, we utilized a design of experiments strategy to systematically modulate a series of transfection-related variables including the quantity of plasmid DNA, the DNA-to-polyethylenimine ratio, incubation duration, and the impact of post-transfection feeding strategies on the yield of recombinant AAV (rAAV). Our comprehensive analysis and subsequent optimizations actualized a remarkable unit yield reaching nearly 2 × 1012 vector genomes (vg)/mL. Importantly, the resulting single-cell yield and biological activity were found to be comparable with those obtained from fed-batch cultures, underscoring the efficacy of our approach. Based on these findings, we investigated rAAV yield via high-density suspend culture in bioreactor, particularly focusing on cell aggregation and the use of perfusion technology. Intriguingly, we attempted to elevate the yield of an oversized recombinant coagulation factor VIII AAV843 vector by 3.5-fold, reaching a yield of 1 × 1012 vg/mL. Concurrently, the medium usage rate was only double that of batch feeding, thereby significantly shrinking the upstream cost of rAAV manufacture. In summary, this strategy significantly benefits large-scale AAV production for both commercial and clinical applications. [ABSTRACT FROM AUTHOR]

Background Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture. Objectives As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites). Methods Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested. Results Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation. Conclusions As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs. [ABSTRACT FROM AUTHOR]

Background: Interleukin-21 (IL-21) is a cytokine produced by various cell types, including T cells, natural killer cells, myeloid cells, and B cells, and has a broad range of potential applications in cancer therapy. To improve the therapeutic index, we explored the use of fusion technologies that involved linking other anticancer peptides to the IL-21 gene using specific linkers. Objectives: This study aimed to compare the anticancer potential of IL-21 and IL-21 fusion proteins. Methods: Antimicrobial peptides possessing anticancer properties were fused with IL-21 gene using a flexible linker (-GGGGS-), and the resulting construct was inserted into the pSecTag2a mammalian expression vector. The cassette was transfected into several cancer cell lines including H1 HeLa, HepG2, MCF-7, MDA-MB-231, HCT-116, HCC-1954, HEK-293, and SF-767. The cytotoxic effects of IL-21 and fusion proteins were evaluated using MTT, Caspase-3, LDH, and scratch assays. Results: The IL-21-Tachyplesin I fusion protein had the strongest antiproliferative activity against all tested cancer cells, followed by IL21-LPSBD2 and IL-21. In contrast, IL21-Cop A3, IL21-CSP I-Plus, and IL21-RGD Temporin-Las did not inhibit the viability of cancer cells. Conclusion: Fusion technology is a promising therapeutic technique that can be used to enhance the cytotoxicity and antiproliferative activity of anticancer proteins such as IL-21. [ABSTRACT FROM AUTHOR]

Background/Objectives: Bringing small interfering RNA (siRNA) into the cell cytosol to achieve specific gene silencing is an attractive but also very challenging option for improved therapies. The first step for successful siRNA delivery is the complexation with a permanent cationic or ionizable compound. This protects the negatively charged siRNA and enables transfection through the cell membrane. The current study explores the performance of the innovative, ionizable lipid 2-Tetradecylhexadecanoic acid-(2-bis{[2-(2,6-diamino-1-oxohexyl)amino]ethyl}aminoethyl)-amide (T14diLys), in combination with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for siRNA delivery and the impact of the production method (sonication vs. extrusion) on the particle properties. Methods: Liposomes were produced either with sonication or extrusion and characterized. The extruded liposomes were combined with siRNA at different N/P ratios and investigated in terms of size zeta potential, encapsulation efficiency, lipoplex stability against RNase A, and knockdown efficiency using enhanced green fluorescent protein (eGFP)-marked colon adenocarcinoma cells. Results: The liposomes prepared by extrusion were smaller and had a narrower size distribution than the sonicated ones. The combination of siRNA and liposomes at a nitrogen-to-phosphate (N/P) ratio of 5 had optimal particle properties, high encapsulation efficiency, and lipoplex stability. Gene knockdown tests confirmed this assumption. Conclusions: Liposomes produced with extrusion were more reproducible and provided enhanced particle properties. The physicochemical characterization and in vitro experiments showed that an N/P ratio of 5 was the most promising ratio for siRNA delivery. [ABSTRACT FROM AUTHOR]

Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). The knockdown of ATM, ATR, and PDEδ in HEK293 cells increased HIV-1 VLP titers in the supernatant by 3.4-, 2.1-, and 2.2-fold, respectively. Also, possible metabolic synergies between plasmids were investigated by statistical design of experiments (DoE), enabling us to identify the optimal production strategy, that was further demonstrated at lab-scale stirred tank bioreactor operated in perfusion, significantly increasing both VLPs specific and volumetric productivities to 8.3 × 103 VLPs/cellxday and 7.5 × 1012 VLPs/Lxday, respectively. Key points: • ATM, ATR, and PDEδ knockdowns increased VLP production in HEK293 cells. • Knockdown of ATM increased budding efficiency and extracellular vesicle concentration. • ATM knockdown could be intensified to bioreactor scale operated in perfusion. [ABSTRACT FROM AUTHOR]

Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex–membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex–membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology. [ABSTRACT FROM AUTHOR]

Bruck syndrome is a rare autosomal recessive disorder characterized by increased bone fragility and joint contractures similar to those in arthrogryposis and is known to be associated with mutations in the FKBP10 (FKBP prolyl isomerase 10) and PLOD2 (Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2) genes. These genes encode endoplasmic reticulum proteins that play an important role in the biosynthesis of type I collagen, which in turn affects the structure and strength of connective tissues and bones in the body. Mutations are associated with disturbances in both the primary collagen chain and its post-translational formation, but the mechanism by which mutations lead to Bruck syndrome phenotypes has not been determined, not only because of the small number of patients who come to the attention of researchers but also because of the lack of disease models. In our work, we investigated the cellular effects of two forms of the wild-type PLOD2 gene, as well as the PLOD2 gene with homozygous mutation c.1885A>G (p.Thr629Ala). The synthesized genetic constructs were transfected into HEK293 cell line and human skin fibroblasts (DF2 line). The localization of PLOD2 protein in cells and the effects caused by the expression of different isoforms—long, short, and long with mutation—were analyzed. In addition, the results of the transcriptome analysis of a patient with Bruck syndrome, in whom this mutation was detected, are presented. [ABSTRACT FROM AUTHOR]

Objective: The development of systems for targeted delivery of nucleic acids (NAs) is necessary to ensure their selective transport to the site of therapeutic action. The aim of this work was to synthesize carbohydrate-modified amphiphiles containing a spermine residue, required for compaction and binding to NAs, as well as a diglyceride residue for forming lipid aggregates and a carbohydrate residue (lactose or D-mannose) for improving the hydrophilic–lipophilic balance of the molecule. The lactose residue can serve as a targeting ligand for NA delivery into liver hepatocytes, and the D-mannose residue can perform specific NA transport into dendritic cells and macrophages. Methods: New carbohydrate-modified cationic amphiphiles were obtained by organic synthesis, and their aqueous dispersions or cationic liposomes were prepared. Cytotoxicity of the cationic amphiphiles and liposomes was performed using the MTT assay on HEK 293 and BHK cell lines in the absence of fetal bovine serum (FBS). Complexes of the cationic amphiphiles or liposomes with NAs (FITC-ODN, pDNA, and siRNA) were formed at various component ratios (N/P), and the efficiency of transfection in HEK 293 and BHK IR-780 cells was assessed by flow cytometry. Results and Discussion: New cationic amphiphiles containing lactose or D-mannose residues were synthesized. The cationic amphiphiles, whatever the structure of their carbohydrate residue, effectively deliver a short FITC-ODN into HEK293 cells in the presence of FBS, and are nontoxic. The cationic liposome formed by the lactose-containing amphiphile and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) successfully delivers short NAs (FITC-ODN and siRNA) both in the absence and in the presence of serum in the culture media. Conclusions: The obtained carbohydrate-modified cationic amphiphiles, both individually and as component of cationic liposomes, hold promise to be used as systems for the delivery of short nucleic acids in further development of drugs for gene therapy. [ABSTRACT FROM AUTHOR]

Recombinant adeno‐associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long‐term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled‐to‐total capsids (~1%–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid‐filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream. [ABSTRACT FROM AUTHOR]

In this study, a small library of prednisolone and budesonide conjugated polyethylenimine (PEI) derivatives was prepared. Since nuclear entry is a major barrier for plasmid DNA (pDNA) delivery, it is hypothesized that glucocorticoid-conjugated PEIs that employ their specific receptors to enter the cell nucleus may increase the gene transfer ability of PEI. In this study, prednisolone and budesonide were conjugated onto low and high molecular weight PEI (1.8 and 25 kDa) with different degrees of substitution via different linkers. The glucocorticoid-polyethyleneimine (GC–PEI)/pDNA polyplexes were prepared, and their biophysical characteristics, plasmid delivery, and cell-induced toxicity were evaluated. The polyplexes demonstrated a substantial ability to condense plasmid DNA and the protection of nucleic acids against enzymatic degradation. The polyplexes’ size was in the range of 50–250 nm with zeta potentials between 10 and 30 mV. Interestingly, prednisolone-conjugated low molecular weight PEI could enhance the level of desired protein expression by up to 4 folds with no toxicity compared to the parent unmodified polymer. The results of this study show a promising approach to preparing gene carrier vehicles based on low molecular weight PEI with low toxicity. [ABSTRACT FROM AUTHOR]

Hawraa Ali-Jerman,1 Zainab Al-Quraishi,1 Ashish Muglikar,1 Yvonne Perrie,1 Rothwelle J Tate,1 Margaret Mullin,2 Gayle McNeill,1 Graeme Mackenzie,1 Christine Dufès1 1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0RE, UK; 2Cell Analysis Facility, Medical and Veterinary & Life Sciences Shared Research Facilities, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UKCorrespondence: Christine Dufès, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK, Tel +44 1415483796, Fax +44 1415522562, Email C.Dufes@strath.ac.ukBackground: Gene therapy is a promising therapeutic approach for treating various disorders by introducing modified nucleic acids to correct cellular dysfunctions or introduce new functions. Despite significant advancements in the field, the effective delivery of nucleic acids remains a challenge, due to biological barriers and the immune system’s ability to target and destroy these molecules. Due to their branched structure and ability to condense negatively charged nucleic acids, cationic dendrimers have shown potential in overcoming these challenges. Despite this, standardized scalable production methods are still lacking. This study investigates the use of microfluidics to formulate generation 3-diaminobutyric polypropylenimine (DAB) dendriplexes and compares their characteristics and in vitro gene delivery efficacy to those prepared using conventional manual mixing.Methods: DAB dendriplexes were produced by both microfluidic and manual approaches and characterized. Their cellular uptake and gene expression were evaluated on C6 glioma cancer cells in vitro.Results: Dendriplexes formed using microfluidics at the optimal flow rate and ratio demonstrated enhanced DNA condensation over time, achieving up to 97% condensation at 24 hours. Both preparation methods produced positively charged dendriplexes, indicating stable formulations. However, dendriplexes prepared through hand mixing resulted in smaller particle sizes, significantly higher cellular uptake and gene expression efficacy compared to those prepared by microfluidics. Nonetheless, microfluidic preparation offers the advantage of standardized and scalable production, which is essential for future applications.Conclusion: This study highlights the potential of microfluidic technology to improve precision and scalability in gene delivery, paving the way for future advancements in gene therapy. Our findings suggest that, with further optimization, microfluidic systems could provide superior control over dendriplex formation, expanding their potential use in gene therapy applications. Keywords: polypropylenimine dendrimer, dendriplex, glioma, microfluidics, cellular uptake, transfection

Abstract Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery.

Abstract Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs. Graphical Abstract

Macrophages are unique immune cells attracting growing attention as a potential candidate for cell‐based therapy for infectious diseases and cancer. Strategies that can reprogramme or gene‐edit macrophages hold potential across a spectrum of acute and chronic conditions. Herein, lipid nanoparticles (LNPs) are developed containing the ionizable lipid SM‐102, helper lipid monoolein which is known for self‐assembly in aqueous solutions into the inverse cubic lyotropic liquid crystalline mesophase, and cholesterol as an mRNA nanocarrier. The immortalized alveolar macrophage cell line (MH‐S cells) is utilized to investigate how cholesterol concentration impacts on mRNA delivery which is further validated using primary mouse alveolar macrophages isolated from the bronchoalveolar compartment and human monocyte derived macrophages. By using high‐throughput synchrotron small angle X‐ray scattering (SAXS), an acidification‐induced non‐ordered to ordered internal nanostructure transition of the formulated LNPs is observed, following the transition sequence of inverse micellar to hexagonal to cubic mesophase in the pH range from 7 to 4. Cholesterol is identified as another crucial component for superior mRNA transfection in macrophages, contributing to nanostructure transition and protein corona variation. Successful ex vivo mRNA transfection is also achieved in primary macrophages, highlighting the prospectivity of reprogramming macrophages as a cell therapy for lung diseases. [ABSTRACT FROM AUTHOR]

In this study, tocopherol‐derived ionizable lipids were synthesized to create functional lipid nanoparticles for mRNA delivery. Tocopherol succinate is considered a biocompatible material because it has anti‐cancer effects and degrades into vitamin E in the body. Two types of new ionizable tocopherol derivative lipids (DMT and AIT) were synthesized by introducing ionizable functional groups. The synthesized tocopherol lipids were used to make lipid nanoparticles with helper lipid, cholesterol, and PEG‐DMG. The DMT LNP showed high mRNA encapsulation efficiency. Using HeLa cell lines, it was confirmed that gene transfection efficiency was increased approximately 2‐fold in DMT LNP‐treated cells compared to that of MC3 LNP. The two types of tocopherol derivative lipids exhibited high cell viability of over 90%, confirming very low levels of toxicity. The results of this study confirmed that lipid nanoparticles using newly developed ionizable tocopherol derivatives have potential as biocompatible and effective mRNA delivery vehicles. [ABSTRACT FROM AUTHOR]

Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualized using live high-content fluorescence microscopy, followed by higher-resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2, we established that cellular membranes in PCLS are permeable in the initial 24 h of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 h after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared with other LNPs, with detection possible as low as 25 nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs. NEW & NOTEWORTHY: RNA-based therapeutics hold significant promise for disease treatment; however, limited research exists on miRNA transfection specifically within PCLS. miRNA transfection has thus far required custom functionalization for stabilization and internalization. We aimed to optimize a transfection protocol for rapid screening approaches of miRNA sequences. We show that transfecting miRNA in PCLS is possible using lipid nanoparticles. In addition, we show that 25 nM of TYE665-miRNA is sufficient for detection in a high-content imaging system. [ABSTRACT FROM AUTHOR]

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Transfection of mammalian and human cell lines in medical research both are key technologies in molecular biology and genetic engineering. A vast variety of techniques to facilitate transfection exists including different chemical and nanoparticle-based agents as mediators of nucleic acid uptake, with nanoparticles composed of the lipids DOSPA/DOPE belonging to the established type of agents. We show that inverse-nanoemulsion-derived protein nanohydrogels (NanoTrans-gels), prepared by a simple synthesis protocol, are suited to transfect two model cancer cell lines (MCF7 and A549) with high efficiency. The transfection efficiency was analyzed in comparison to the DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories. [ABSTRACT FROM AUTHOR]

Background: This study examines the feasibility and effects of introducing microRNA mimic into red blood cells (RBCs) at the initial phases of Plasmodium falciparum 3D7 (Pf3D7) infection. The aim is to determine the correlation between increased expression of miR-451a and parasitaemia. Methods: In this study miR-mimic-451a labelled with Cy3 and transfected into control and infected RBCs using lipofectamine and analysed using the fluorescence microscopy and flow cytometry. The study demonstrated the efficacy of miR-451a by treating pre-and post-transfected control RBCs and Pf3D7-infected RBCs with miR-mimic-451a. We also examined its impact on % growth inhibition of Pf3D7, oxidative stress markers (Luminometry, LPO, SOD, CAT, GSH and GPx). Additionally, determination of pH, haemoglobin (Hb), and proteomic profile performed using SDS-PAGE. Results: Modified expression level of mir-451a has the potential to change the progression of the infection and yielded a 50% decrease in parasitaemia within 48 h. Moreover, transfected samples were shown to be efficacious in counteracting the oxidative stress-induced alterations during Pf3D7 infection and enable to return the cells towards the normalcy. Modified proteomic profile of transfected iRBCs demonstrates the correlation between overexpression of miRNA and protein expression. where, the major changes were observed in the heavy molecular weight proteins more than 57 kDa. Conclusion: The study reveals promising effects of miR-mimic-451a enrichment during RBC stages of Pf3D7, offering insights into potential malaria therapeutic strategies and potential biomedical research implications. [ABSTRACT FROM AUTHOR]

Eplet 52SK is unique in the HLA eplet registry as targeting the whole family of DQA1*01 alleles. It is proposed as an antibody‐verified eplet but has not been validated enough to deserve this label. Especially, confusion can occur with reactivity targeting the 52PQ eplet which is present on the DQB1*05 and DQB1*06 alleles families, as DQ molecule stability imposes DQA1*01 to selectively associate with these DQ‐β families only. Using two Luminex single antigen (LSA) assays from two vendors, beads bearing DR‐α/DQ6 heterodimers, a special build LSA panel of additional DQ beads, and an adsorption/elution strategy relying on cells from deceased donors or recombinant cells solely expressing one DQ antigen, we definitely established the antibody‐verified status of eplet 52SK using patients' sera reacting only against the DQ5 and DQ6 beads of the One Lambda LSA panel in routine patients' follow up. We also show that reactivity against this eplet is not a rare event among anti‐DQ1 immunisation. This study further strengthens the importance of considering the DQA1 locus in immunological studies of HLA and in organ allocation strategies. [ABSTRACT FROM AUTHOR]

The invasive ascomycete Hymenoscyphus fraxineus is the causative agent for ash dieback on the European species Fraxinus excelsior and Fraxinus angustifolia, and there is concern that it is going to replace the native, closely related and nonpathogenic Hymenoscyphus albidus. Fungal management in forests is limited, and alternative approaches for control are needed. Within the scope of the project "FraxForFuture", several strategies are being investigated. One idea comprises the use of a viral hyperparasite, which can induce a reduced virulence in the fungal host H. fraxineus in an antagonist-like system. This phenomenon, the reduction of fungal virulence by a viral infection, is known as hypovirulence, and a similar method has already been established to control the Chestnut Blight in Europe. We examined 34 isolates of H. fraxineus for both their virulence and presence of a viral infection. Although a predominant number of isolates were found to be infected with Hymenoscyphus mitovirus 1 (HfMV1), no additional viruses were detected, and our data did not indicate a link to reduced virulence. The search for a viral infection was extended to one isolate of H. albidus in which we found and characterized a novel mycovirus. Based on phylogenetic analysis and sequence properties, it was assigned to the genus Victorivirus in the family of Totiviridae and was tentatively denominated as Hymenoscyphus albidus victorivirus 1. This novel and native mycovirus might be suitable for inducing hypovirulence in H. fraxineus as a biocide. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs. [ABSTRACT FROM AUTHOR]

Introduction: Pathology of organs of the male reproductive system remains one of the most urgent problems of modern medicine, which is associated not only with patients’ impaired quality of life, but also with high rates of male infertility, which tends to increase. The aim: To evaluate the efficacy of apoE gene transfection on the morphofunctional state of rat testes in experimental atherosclerosis. Materials and methods: The study was conducted on 30 sexually mature male rats weighing 150-170 grams. Experimental animals were divided into 3 groups. Group 1 - intact animals. Group 2 – animals with experimental atherosclerosis; Group 3-animals with experimental atherosclerosis, which were intramuscularly injected with the apolipoprotein E gene at a dose of 50 μg of DNA per animal on the first day of modeling atherosclerosis. To study the efficiency of apolipoprotein E gene transfection on morphofunctional changes in testes, the following research methods were used: 1) histological; 2) biochemical; 3) reverse transcriptase PCR diagnostic method. Results and discussion. Experimental cholesterol loading leads to impaired lipid metabolism, decreased serum testosterone levels, degenerative and dystrophic changes in the testis of experimental rats, which is manifested by luminal occlusion of blood vessels, overgrowth of connective tissue in the organ interstitium, decreased number and qualitative change of Sertoli and Leydig cells, as well as spermatogenic epithelium thinning and inhibition of gamete maturation process in it. Conclusions: intramuscular transfection of the apoE gene inhibits lipid metabolism disorders and has a positive effect on the morphofunctional state of the testes of experimental animals.

Nucleic acid-based therapeutics are a common approach that is increasingly popular for a wide spectrum of diseases. Lipid nanoparticles (LNPs) are promising delivery carriers that provide RNA stability, with strong transfection efficiency, favorable and tailorable pharmacokinetics, limited toxicity, and established translatability. In this review article, we describe the lipid-based delivery systems, focusing on lipid nanoparticles, the need of their use, provide a comprehensive analysis of each component, and highlight the advantages and disadvantages of the existing manufacturing processes. We further summarize the ongoing and completed clinical trials utilizing LNPs, indicating important aspects/questions worth of investigation, and analyze the future perspectives of this significant and promising therapeutic approach.

CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.

Objective(s): Gene therapy is a hopeful approach for treatment of a wide range of life threatening disease from infectious and inherited diseases to cancer. Multifunctional Envelope-type Nano Device (MEND) is a new carrier as non-viral genetic vector. Moreover, associating peptide structures with the nuclear localization signals (NLSs), which contains various functional groups enables them to condense DNA and specifically transfer genetic material to the nucleus.Materials and Methods: In this study, two forms of low molecular weight protamine (LMWP) were used for preparation of MEND carrier. The MEND carriers were then targeted with GE11 ligand to obtain T-MEND structure. The size distribution of the resulting nanoparticles, as well as their transfection efficiency and cytotoxicity, were investigated on the A549 cell line.Results: Results demonstrated that the size of polyplex carrier’s formulation by both peptides (VV45 and VV32) was below 200 nm and MEND formulations were between 200-300 nm. T-MEND formulations contained VV32 and VV45 peptides showed slightly higher transfection than similar MEND formulations. Also, MEND formulation showed increased transfection efficiency compared to similar PD complexes. The result of metabolic activity test showed that MEND lipopolyplex did not represent any remarkable cytotoxicity.Conclusion: It can be concluded that multifunctional carriers designed based on LMWP are considered as the safe carrier for gene delivery. Presence of protamine and targeted ligand in the nanoparticulate structure did not increase the risk of cytotoxicity of carriers. So, MEND and T-MEND lipoplexes showed low cytotoxicity and acceptable transfection efficiency at the level of PEI 25 kDa.

Epitope tagging represents a powerful strategy for expedited identification, isolation, and characterization of proteins in molecular biological studies, including protein–protein interactions. We aimed to improve the reproducibility of epitope-tagged protein expression and detection by developing a range of plasmids as positive controls. The pJoseph2 family of expression plasmids functions in diverse cellular environments and cell types to enable the evaluation of transfection efficiency and antibody staining for epitope detection. The expressed green fluorescent proteins harbor five unique epitope tags, and their efficient expression in Escherichia coli, Drosophila Schneider's line 2 cells, and human SKOV3 and HEK293T cells was demonstrated by fluorescence microscopy and western blotting. The pJoseph2 plasmids provide versatile and valuable positive controls for numerous experimental applications.

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

Cancer poses a significant health challenge, with traditional treatments like surgery, radiotherapy, and chemotherapy often lacking in cell specificity and long‐term curative potential. Chimeric antigen receptor T cell (CAR‐T) therapy,utilizing genetically engineered T cells to target cancer cells, is a promising alternative. However, its high cost limits widespread application. CAR‐T manufacturing process encompasses three stages: cell isolation and activation, transfection, and expansion.While the first and last stages have straightforward, commercially available automation technologies, the transfection stage lags behind. Current automated transfection relies on viral vectors or bulk electroporation, which have drawbacks such as limited cargo capacity and significant cell disturbance. Conversely, micro and nano‐tool methods offer higher throughput and cargo flexibility, yet their automation remains underexplored.In this perspective, the progress in micro and nano‐engineering tools for CAR‐T transfection followed by a discussion to automate them is described. It is anticipated that this work can inspire the community working on micro and nano transfection techniques to examine how their protocols can be automated to align with the growing interest in automating CAR‐T manufacturing. [ABSTRACT FROM AUTHOR]

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non‐palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non‐viral materials, while offering safer and non‐cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled‐coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C‐terminal decahistidine tags on α‐helical secondary structure. Cross‐correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency. [ABSTRACT FROM AUTHOR]

We describe in this work an evaluation of bundles of hollow mesoporous silicon nanotubes to facilitate transfection of HeLa cells using small interfering RNA designed to knock down expression of Enhanced Green Fluorescent Protein (eGFP). These experiments entail direct visualization of the nanotube bundles associated with the cells using both scanning electron microscopy (SEM) and confocal fluorescence imaging. These nanotube bundles are generated by surface modification of nanotube arrays with aminopropyl‐triethoxysilane (APTES), followed by their ultrasonication in water, to create the amine‐terminated structures capable of electrostatic conjugation of siRNA at an efficiency of 23%–50% (depending on initial siRNA concentration). Delivery and transfection to HeLa cells are verified by quantification of fluorescence imaging; an average percent knockdown of ∼50% eGFP is achieved. As nanoscale drug delivery vehicles are expected to be resorbed in clinical use, we also assess SiNT bundle degradation during the above in vitro timescale using scanning and transmission electron (TEM) microscopies. We conclude with a brief discussion of challenges and opportunities in future experiments involving this platform. [ABSTRACT FROM AUTHOR]

The enormous therapeutic potential of selective ribonucleic acid (RNA) interference has recently been manifested by the approval of several small interfering RNA (siRNA)‐based drugs. However, the efficacy of siRNA delivery is still limited, and an extensive search for alternative and highly effective delivery approaches is ongoing. With this aim, three generations of non‐viral vectors based on modified nanodiamonds (NDs) have been gradually developed in the past decade. They show great promise due to the negligible toxicity of the ND core. Here, a robust methodological approach is presented to enable the evaluation of new vector nanomaterials. Using a new type of third‐generation ND vector coated with a copolymer with tunable charge density, variables such as colloidal stability, surface electrostatic properties, the molecular composition of the copolymer, and the mode of complexation with siRNA are optimized. Using an innovative data processing strategy, the results are related to biological potency, toxicity, and cell proliferation. Finally, the optimized composition of a coating copolymer consisting of a cationic component, 2‐dimethylaminoethyl methacrylate, and an electroneutral biocompatible component, N‐(2‐hydroxypropyl) methacrylamide, is evaluated. The optimized NDs vectors are colloidally and biologically stable siRNA delivery tools with broad potential for RNA interference‐based therapeutics. [ABSTRACT FROM AUTHOR]

Polydopamine‐based materials, notably Polydopamine‐polyethyleneimine (PDA‐PEI), have gained considerable interest for surface‐mediated transfection. However, the high laser power density required to achieve high transfection efficiency poses a significant challenge in preserving cell viability. An organic photosensitizer CRO32TMI was developed to improve the photothermal conversion capability of PDA‐PEI. The modified PDA‐PEI‐CRO32TMI exhibited remarkable photothermal and photostability properties upon NIR irradiation, enabling it to achieve better transfection efficiency at lower laser power density as compared to the traditional solution or lipid‐based transfection methods. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) represent a groundbreaking advancement in stem cell research, offering new avenues for regenerative therapy and disease modeling. This study explores the application of calcium phosphate (CaP) nanoparticles in gene transfer studies involving iPSCs. By meticulously analyzing the characteristics of CaP nanoparticles—such as mean particle size and zeta potential—using dynamic light scattering (DLS), the research provides insights into their potential application in iPSC-related research. In addition, in vitro assessments were conducted on L929 mouse fibroblast cells to evaluate the biocompatibility of CaP nanoparticles, further demonstrating their potential utility. The results from the MTT assay indicate no significant toxic effects across various concentrations (ranging from 5 to 25 µg/µL), highlighting their safety profile and supporting their use in iPSC-related studies. Additionally, the CaP nanoparticle group exhibited lower total oxidant levels, suggesting potential antioxidant properties or the ability to mitigate oxidative stress. This reduction in oxidant levels may contribute to maintaining cell health and normal cellular functions, complemented by higher total antioxidant levels observed in the CaP group. Moreover, increased levels of cyclin D1 in the CaP group indicate enhanced cellular activity in proliferation and division processes, particularly significant during the G1 phase of the cell cycle. In summary, calcium phosphate nanoparticles play a multifaceted role in iPSC-related research, showcasing antioxidant properties, supporting cell proliferation, and potentially enhancing cell survival by inhibiting apoptosis. This research underscores their efficacy as non-viral carriers for genetic studies and sheds light on their application in gene transfer experiments. Successful transfection of L929 cells with a selected plasmid encoding Oct4-Sox2-Klf4-Myc-GFP demonstrates the effectiveness of CaP nanoparticles in delivering genetic material into cells. With a transfection efficiency persisting at 85% (± 3.4%) over a 72-h observation period, coupled with significant expression levels of key genes OCT-4 and SSEA-4 detected via flow cytometry (88%), CaP nanoparticles emerge as a promising tool for facilitating cellular reprogramming. [ABSTRACT FROM AUTHOR]

Objective(s): Gene therapy is a hopeful approach for treatment of a wide range of life threatening disease from infectious and inherited diseases to cancer. Multifunctional Envelope-type Nano Device (MEND) is a new carrier as non-viral genetic vector. Moreover, associating peptide structures with the nuclear localization signals (NLSs), which contains various functional groups enables them to condense DNA and specifically transfer genetic material to the nucleus. Materials and Methods: In this study, two forms of low molecular weight protamine (LMWP) were used for preparation of MEND carrier. The MEND carriers were then targeted with GE11 ligand to obtain T-MEND structure. The size distribution of the resulting nanoparticles, as well as their transfection efficiency and cytotoxicity, were investigated on the A549 cell line. Results: Results demonstrated that the size of polyplex carrier's formulation by both peptides (VV45 and VV32) was below 200 nm and MEND formulations were between 200-300 nm. T-MEND formulations contained VV32 and VV45 peptides showed slightly higher transfection than similar MEND formulations. Also, MEND formulation showed increased transfection efficiency compared to similar PD complexes. The result of metabolic activity test showed that MEND lipopolyplex did not represent any remarkable cytotoxicity. Conclusion: It can be concluded that multifunctional carriers designed based on LMWP are considered as the safe carrier for gene delivery. Presence of protamine and targeted ligand in the nanoparticulate structure did not increase the risk of cytotoxicity of carriers. So, MEND and T-MEND lipoplexes showed low cytotoxicity and acceptable transfection efficiency at the level of PEI 25 kDa. [ABSTRACT FROM AUTHOR]