Retroviral gene transfer is the preferred method for stable, long-term integration of genetic material into cellular genomes, commonly used to generate chimeric antigen receptor (CAR)-T cells designed to target tumor antigens. However, the efficiency of retroviral gene transfer is often limited by low transduction rates due to low vector titers and electrostatic repulsion between viral particles and cellular membranes. To overcome these limitations, peptide nanofibrils (PNFs) can be applied as transduction enhancers. Among these, PNFs derived from the 12-mer peptide EF-C are well-investigated and commercially available. EF-C PNFs enhance transduction by forming EF-C PNFs/virus complexes that overcome electrostatic repulsion through their polycationic surface and interaction with cellular protrusions. However, the safe application of PNFs as transduction enhancers in gene therapeutic applications requires a fundamental understanding of their transduction-enhancing mechanisms, uptake, and degradation. In this study, we demonstrate that EF-C PNFs induce plasma membrane invaginations, increasing the membrane surface for viral attachment and reducing the distance to the nuclear membrane, thereby facilitating viral entry and transport to the nucleus. Furthermore, we identified macropinocytosis as the main entry pathway for EF-C PNFs and their subsequent degradation by lysosomal peptidases. The lysosomal degradation of EF-C PNFs prevents their accumulation as amyloid deposits, mitigating potential side effects and supporting their safe use in clinical applications.

• Klíčová slova
• Lysosomal degradation, Macropinocytosis, Peptide nanofibrils, Retroviral transduction, Transduction enhancer,
• MeSH
• endocytóza MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši MeSH
• nanovlákna * chemie   MeSH
• peptidy * chemie   metabolismus   MeSH
• pinocytóza * MeSH
• transdukce genetická metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy * MeSH

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

• Klíčová slova
• Subretinal injection, large animal, non-viral gene vector, pars plana vitrectomy, safety,
• MeSH
• genetická terapie * metody   MeSH
• genetické vektory * aplikace a dávkování   MeSH
• injekce nitrooční MeSH
• miniaturní prasata * MeSH
• modely nemocí na zvířatech MeSH
• pilotní projekty MeSH
• plazmidy aplikace a dávkování   MeSH
• prasata MeSH
• retina MeSH
• studie proveditelnosti * MeSH
• technika přenosu genů * MeSH
• vitrektomie * metody   MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH

MOTIVATION: Lipid nanoparticles (LNPs) are the most widely used vehicles for mRNA vaccine delivery. The structure of the lipids composing the LNPs can have a major impact on the effectiveness of the mRNA payload. Several properties should be optimized to improve delivery and expression including biodegradability, synthetic accessibility, and transfection efficiency. RESULTS: To optimize LNPs, we developed and tested models that enable the virtual screening of LNPs with high transfection efficiency. Our best method uses the lipid Simplified Molecular-Input Line-Entry System (SMILES) as inputs to a large language model. Large language model-generated embeddings are then used by a downstream gradient-boosting classifier. As we show, our method can more accurately predict lipid properties, which could lead to higher efficiency and reduced experimental time and costs. AVAILABILITY AND IMPLEMENTATION: Code and data links available at: https://github.com/Sanofi-Public/LipoBART.

• MeSH
• lipidy * chemie   MeSH
• liposomy MeSH
• messenger RNA metabolismus   MeSH
• nanočástice * chemie   MeSH
• transfekce * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Lipid Nanoparticles MeSH Prohlížeč
• lipidy * MeSH
• liposomy MeSH
• messenger RNA MeSH

BACKGROUND: Electroporation is an effective technique for genetic manipulation of cells, both in vitro and in vivo. In utero electroporation (IUE) is a special case, which represents a fine application of this technique to genetically modify specific tissues of embryos during prenatal development. Commercially available electroporators are expensive and not fully customizable. We have designed and produced an inexpensive, open-design, and customizable electroporator optimized for safe IUE. We introduce NeuroPorator. METHOD: We used off-the-shelf electrical parts, a single-board microcontroller, and a cheap data logger to build an open-design electroporator. We included a safety circuit to limit the applied electrical current to protect the embryos. We added full documentation, design files, and assembly instructions. RESULT: NeuroPorator output is on par with commercially available devices. Furthermore, the adjustable current limiter protects both the embryos and the uterus from overcurrent damage. A built-in data acquisition module provides real-time visualization and recordings of the actual voltage/current pulses applied to each embryo. Function of NeuroPorator has been demonstrated by inducing focal cortical dysplasia in mice. SIGNIFICANCE AND CONCLUSION: The simple and fully open design enables quick and cheap construction of the device and facilitates further customization. The features of NeuroPorator can accelerate the IUE technique implementation in any laboratory and speed up its learning curve.

• Klíčová slova
• Current-limited, Electroporator, Focal cortical dysplasia, Gene transfer, In utero electroporation, NeuroPorator, Pulse generator,
• MeSH
• design vybavení MeSH
• elektroporace * metody   přístrojové vybavení   MeSH
• embryo savčí MeSH
• myši MeSH
• technika přenosu genů * přístrojové vybavení   MeSH
• těhotenství MeSH
• uterus MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

• Klíčová slova
• 6-cys-e gene knockout, Babesia divergens, GFP-expression, bidirectional promoter, erythrocytes pre-loading, gene targeting, transfection system,
• MeSH
• Babesia * genetika   MeSH
• babezióza * parazitologie   MeSH
• erytrocyty parazitologie   MeSH
• genový targeting MeSH
• lidé MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

• MeSH
• aktiny genetika   metabolismus   MeSH
• cytomegalovirové infekce * genetika   metabolismus   MeSH
• Dependovirus genetika   metabolismus   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Parvovirinae * genetika   MeSH
• retinální gangliové buňky metabolismus   MeSH
• transdukce genetická MeSH
• transgeny MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• zelené fluorescenční proteiny MeSH

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

• Klíčová slova
• GAD65 and VGAT, long-lasting pain reversal, neuropathic pain, precision spinal vector delivery, subpial injection technique, subpial non-human primates, subpial pig, targeted AAV9 delivery,
• MeSH
• buňky zadních rohů míšních MeSH
• mícha MeSH
• myši MeSH
• neuralgie * etiologie   terapie   MeSH
• nociceptory * MeSH
• prasata MeSH
• technika přenosu genů MeSH
• zadní rohy míšní MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

• MeSH
• aziridiny MeSH
• DNA * chemie   MeSH
• myši MeSH
• plazmidy genetika   MeSH
• polyethylenimin chemie   MeSH
• polymery * chemie   MeSH
• technika přenosu genů MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aziridine MeSH Prohlížeč
• aziridiny MeSH
• DNA * MeSH
• polyethylenimin MeSH
• polymery * MeSH

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

• MeSH
• buněčné linie MeSH
• DNA chemie   ultrastruktura   MeSH
• exprese genu MeSH
• fluorescenční protilátková technika MeSH
• genetické vektory * chemie   ultrastruktura   MeSH
• kationty chemie   MeSH
• kovové nanočástice chemie   ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil metody   MeSH
• molekulární struktura MeSH
• průtoková cytometrie MeSH
• reportérové geny MeSH
• technika přenosu genů * MeSH
• transfekce MeSH
• viabilita buněk MeSH
• železnaté sloučeniny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• kationty MeSH
• železnaté sloučeniny MeSH

Transfection is based on nonviral delivery of nucleic acids or proteins into cells. Viral approaches are being used; nevertheless, their translational capacity is nowadays decreasing due to persistent fear of their safety, therefore creating space for the field of nanotechnology. However, nanomedical approaches introducing static nanoparticles for the delivery of biologically active molecules are very likely to be overshadowed by the vast potential of nanorobotics. We hereby present a rapid nonviral transfection of protein into a difficult-to-transfect prostate cancer cell line facilitated by chemically powered rectangular virus-sized (68 nm × 33 nm) nanorobots. The enhanced diffusion of these biocompatible nanorobots is the key to their fast internalization into cells, happening in a matter of minutes and being up to 6-fold more efficient compared to static nanorobots in a nonfueled environment. The Au/Ag plasmonic nature of these nanorobots makes them simply traceable and allows for their detailed subcellular localization. Protein transfection mediated by such nanorobots is an important step forward, challenging the field of nanomedicine and having potential in future translational medical research.

• Klíčová slova
• metallothionein, nanorobots, protein delivery, protein transfection, transfection,
• MeSH
• nanočástice * chemie   MeSH
• nanomedicína MeSH
• nanotechnologie MeSH
• nukleové kyseliny * MeSH
• transfekce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleové kyseliny * MeSH