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.

• MeSH
• Genetic Therapy * methods   MeSH
• Genetic Vectors * administration & dosage   MeSH
• Injections, Intraocular MeSH
• Swine, Miniature * MeSH
• Disease Models, Animal MeSH
• Pilot Projects MeSH
• Plasmids administration & dosage   MeSH
• Swine MeSH
• Retina MeSH
• Feasibility Studies * MeSH
• Gene Transfer Techniques * MeSH
• Vitrectomy * methods   MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article 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.

• MeSH
• Equipment Design MeSH
• Electroporation * methods   instrumentation   MeSH
• Embryo, Mammalian MeSH
• Mice MeSH
• Gene Transfer Techniques * instrumentation   MeSH
• Pregnancy MeSH
• Uterus MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Pregnancy MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Posterior Horn Cells MeSH
• Spinal Cord MeSH
• Mice MeSH
• Neuralgia * etiology   therapy   MeSH
• Nociceptors * MeSH
• Swine MeSH
• Gene Transfer Techniques MeSH
• Spinal Cord Dorsal Horn MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural 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
• Cell Line MeSH
• DNA chemistry   ultrastructure   MeSH
• Gene Expression MeSH
• Fluorescent Antibody Technique MeSH
• Genetic Vectors * chemistry   ultrastructure   MeSH
• Cations chemistry   MeSH
• Metal Nanoparticles chemistry   ultrastructure   MeSH
• Humans MeSH
• Microscopy, Atomic Force methods   MeSH
• Molecular Structure MeSH
• Flow Cytometry MeSH
• Genes, Reporter MeSH
• Gene Transfer Techniques * MeSH
• Transfection MeSH
• Cell Survival MeSH
• Ferrous Compounds chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mus musculus is the most commonly used animal model in microRNA research; however, little is known about the endogenous miRNome of the animals used in the miRNA-targeting preclinical studies with the human xenografts. In the presented study, we evaluated the NOD/SCID gamma mouse model for the preclinical study of systemic miR-215-5p substitution with a semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide]-based carrier conjugated with miR-215-5p-mimic via a reductively degradable disulfide bond. Murine mmu-miR-215-5p and human hsa-miR-215-5p have a high homology of mature sequences with only one nucleotide substitution. Due to the high homology of hsa-miR-215-5p and mmu-hsa-miR-215-5p, a similar expression in human and NOD/SCID gamma mice was expected. Expression of mmu-miR-215 in murine organs did not indicate tissue-specific expression and was highly expressed in all examined tissues. All animals included in the study showed a significantly higher concentration of miR-215-5p in the blood plasma compared to human blood plasma, where miR-215-5p is on the verge of a reliable detection limit. However, circulating mmu-miR-215-5p did not enter the human xenograft tumors generated with colorectal cancer cell lines since the levels of miR-215-5p in control tumors remained notably lower compared to those originally transfected with miR-215-5p. Finally, the systemic administration of polymer-miR-215-5p-mimic conjugate to the tail vein did not increase miR-215-5p in NOD/SCID gamma mouse blood plasma, organs, and subcutaneous tumors. It was impossible to distinguish hsa-miR-215-5p and mmu-miR-215-5p in the murine blood and organs due to the high expression of endogenous mmu-miR-215-5p. In conclusion, the examination of endogenous tissue and circulating miRNome of an experimental animal model of choice might be necessary for future miRNA studies focused on the systemic delivery of miRNA-based drugs conducted in the animal models.

• MeSH
• Humans MeSH
• MicroRNAs administration & dosage   genetics   therapeutic use   MeSH
• Disease Models, Animal MeSH
• Mice, Inbred NOD MeSH
• Mice, SCID MeSH
• Mice MeSH
• Drug Carriers MeSH
• Gene Expression Profiling MeSH
• Gene Transfer Techniques * MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Chimeric antigen receptor T-cells (CAR T-cells) represent a novel and promising approach in cancer immunotherapy. According to the World Health Organization (WHO), the number of oncological patients is steadily growing in developed countries despite immense progress in oncological treatments, and the prognosis of individual patients is still relatively poor. Exceptional results have been recorded for CAR T-cell therapy in patients suffering from B-cell malignancies. This success opens up the possibility of using the same approach for other types of cancers. To date, the most common method for CAR T-cell generation is the use of viral vectors. However, dealing with virus-derived vectors brings possible obstacles in the CAR T-cell manufacturing process owing to strict regulations and high cost demands. Alternative approaches may facilitate further development and the transfer of the method to clinical practice. The most promising substitutes for virus-derived vectors are transposon-derived vectors, most commonly sleeping beauty, which offer great coding capability and a safe integration profile while maintaining a relatively low production cost. This review is aimed at summarizing the state of the art of nonviral approaches in CAR T-cell generation, with a unique perspective on the conditions in clinical applications and current Good Manufacturing Practice. If CAR T-cell therapy is to be routinely used in medical practice, the manufacturing cost and complexity need to be as low as possible, and transposon-based vectors seem to meet these criteria better than viral-based vectors.

• MeSH
• Cell Culture Techniques methods   MeSH
• Receptors, Chimeric Antigen genetics   immunology   MeSH
• Genetic Vectors genetics   MeSH
• Immunotherapy, Adoptive methods   MeSH
• Humans MeSH
• Neoplasms immunology   therapy   MeSH
• T-Lymphocytes immunology   transplantation   MeSH
• Gene Transfer Techniques * MeSH
• DNA Transposable Elements genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

• MeSH
• Amyotrophic Lateral Sclerosis genetics   physiopathology   therapy   MeSH
• Atrophy MeSH
• Nerve Degeneration genetics   physiopathology   therapy   MeSH
• Dependovirus metabolism   MeSH
• Interneurons pathology   MeSH
• Humans MeSH
• RNA, Small Interfering administration & dosage   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Spinal Cord diagnostic imaging   pathology   physiopathology   MeSH
• Evoked Potentials, Motor MeSH
• Motor Neurons pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Pia Mater pathology   physiopathology   MeSH
• Swine MeSH
• Primates MeSH
• Disease Progression MeSH
• Gene Expression Regulation MeSH
• Protein Folding MeSH
• Superoxide Dismutase-1 genetics   metabolism   MeSH
• Gene Transfer Techniques * MeSH
• Gene Silencing * MeSH
• Muscle Development MeSH
• Inflammation pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

• MeSH
• Bacteriophages genetics   physiology   ultrastructure   MeSH
• DNA, Bacterial genetics   MeSH
• Cryoelectron Microscopy MeSH
• Gene Transfer, Horizontal MeSH
• Gene Expression Regulation, Bacterial MeSH
• Rhodobacter capsulatus genetics   virology   MeSH
• Siphoviridae genetics   physiology   ultrastructure   MeSH
• Gene Transfer Techniques * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

• MeSH
• Chemical Phenomena MeSH
• CRISPR-Cas Systems * MeSH
• Gene Editing * MeSH
• Gene Expression MeSH
• Fluorescent Antibody Technique MeSH
• HEK293 Cells MeSH
• Colloids MeSH
• Humans MeSH
• Magnetite Nanoparticles * chemistry   ultrastructure   MeSH
• Plasmids genetics   MeSH
• Polyethyleneimine * chemistry   MeSH
• Genes, Reporter MeSH
• Static Electricity MeSH
• Gene Transfer Techniques * MeSH
• Transfection methods   MeSH
• Particle Size MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.

• MeSH
• DNA chemistry   metabolism   MeSH
• Fluorine chemistry   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Methacrylates chemistry   MeSH
• Micelles MeSH
• Muramidase chemistry   metabolism   MeSH
• Plasmids chemistry   metabolism   MeSH
• Polyethylene Glycols chemistry   MeSH
• Polymers chemistry   metabolism   MeSH
• Gene Transfer Techniques MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH