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Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

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The mammalian cerebral cortex undergoes a strictly regulated developmental process. Detailed in situ visualizations, imaging of these dynamic processes, and in vivo functional gene studies significantly enhance our understanding of brain development and related disorders. This review introduces basic techniques and recent advancements in in vivo electroporation for investigating the molecular mechanisms underlying cerebral diseases. In utero electroporation (IUE) is extensively used to visualize and modify these processes, including the forced expression of pathological mutants in human diseases; thus, this method can be used to establish animal disease models. The advent of advanced techniques, such as genome editing, including de novo knockout, knock-in, epigenetic editing, and spatiotemporal gene regulation, has further expanded our list of investigative tools. These tools include the iON expression switch for the precise control of timing and copy numbers of exogenous genes and TEMPO for investigating the temporal effects of genes. We also introduce the iGONAD method, an improved genome editing via oviductal nucleic acid delivery approach, as a novel genome-editing technique that has accelerated brain development exploration. These advanced in vivo electroporation methods are expected to provide valuable insights into pathological conditions associated with human brain disorders.

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Glioma is the most prevalent type of neurological malignancies. Despite decades of efforts in neurosurgery, chemotherapy and radiation therapy, glioma remains one of the most treatment-resistant brain tumors with unfavorable outcomes. Recent progresses in genomic and epigenetic profiling have revealed new concepts of genetic events involved in the etiology of gliomas in humans, meanwhile, revolutionary technologies in gene editing and delivery allows to code these genetic "events" in animals to genetically engineer glioma models. This approach models the initiation and progression of gliomas in a natural microenvironment with an intact immune system and facilitates probing therapeutic strategies. In this review, we focus on recent advances in in vivo electroporation-based glioma modeling and outline the established genetically engineered glioma models (GEGMs).

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The delivery of bispecific antibodies (BsAbs) targeting B-cell maturation antigen (BCMA) and CD3 using the gene therapy approach is a promising alternative for BsAb administration in patients with multiple myeloma (MM). In the present study, we evaluated the efficacy of this approach using a xenograft model. Tumour growth was significantly delayed in mice treated with single electroporation-enhanced intramuscular injection of plasmid DNA encoding BCMA/CD3 BsAb in contrast to the vehicle control-treated group. Limited toxicity was observed following treatment. This study demonstrates that the gene therapy-based approach for the delivery of BCMA/CD3 BsAb is effective and safe for the treatment of MM.

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Supergenes are sets of genes and genetic elements that are inherited like a single gene and control complex adaptive traits, but their functional roles and units are poorly understood. In Papilio polytes, female-limited Batesian mimicry is thought to be regulated by a ∼130 kb inversion region (highly diversified region: HDR) containing 3 genes, UXT, U3X, and doublesex (dsx) which switches non-mimetic and mimetic types. To determine the functional unit, we here performed electroporation-mediated RNAi analyses (and further Crispr/Cas9 for UXT) of genes within and flanking the HDR in pupal hindwings. We first clarified that non-mimetic dsx-h had a function to form the non-mimetic trait in female and only dsx-H isoform 3 had an important function in the formation of mimetic traits. Next, we found that UXT was involved in making mimetic-type pale-yellow spots and adjacent gene sir2 in making red spots in hindwings, both of which refine more elaborate mimicry. Furthermore, downstream gene networks of dsx, U3X, and UXT screened by RNA sequencing showed that U3X upregulated dsx-H expression and repressed UXT expression. These findings demonstrate that a set of multiple genes, not only inside but also flanking HDR, can function as supergene members, which extends the definition of supergene unit than we considered before. Also, our results indicate that dsx functions as the switching gene and some other genes such as UXT and sir2 within the supergene unit work as the modifier gene.

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Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

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Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, making them an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We evaluated this possibility by in vivo electroporation of Semliki Forest virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. Optimization of saRNA electroporation conditions in tumors was performed using an SFV vector coding for luciferase. Then we evaluated the therapeutic potential of this approach using an SFV saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine with potent antitumor effects. Delivery of SFV-IL-12 saRNA by electroporation led to improvement in tumor control and higher survival compared with mice treated with electroporation or with SFV-IL-12 saRNA alone. The antitumor efficacy of SFV-IL-12 saRNA electroporation increased by combination with systemic PD-1 blockade. This therapy, which was also validated in a hepatocellular carcinoma tumor model, suggests that local delivery of saRNA by electroporation could be an attractive strategy for cancer immunotherapy. This approach could have easy translation to the clinical practice, especially for percutaneously accessible tumors.

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New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

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As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

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BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 µM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

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To generate a mouse glioblastoma model by genome editing, we introduced Cas9 protein and guide RNAs specific for Nf1, Pten, and Trp53 into the neonatal mouse forebrain by electroporation. We found a high incidence (approximately 90%) of glial tumor development, including glioblastomas, 15 weeks later. The histological features of the tumors were similar to those of diffuse gliomas and, in some cases, similar to human glioblastomas, with microvascular proliferation (glomeruloid structure). In addition, unlike glial fibrillary acidic protein (GFAP)-positive glioblastomas generated using a similar method in a previous model, the majority of tumor cells were positive for oligodendrocyte lineage transcription factor 2, but negative for GFAP and neurofilaments. One base pair insertions identical to those seen in a previous model were found around the target sequences in Nf1, Pten, and Trp53, and additional deletions were found only in Pten. Considering that the histological characteristics were different from those seen in the previous model, our new model provides an additional research tool to investigate the early stages of glioblastoma development.

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Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome-engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome-editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low-cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i-GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats.

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Preventing surgical flaps necrosis remains challenging. Laser Doppler imaging and ultrasound can monitor blood flow in flap regions, but they do not directly measure the cellular response to ischemia. The study aimed to investigate the efficacy of synergistic in-vivo electroporation-mediated gene transfer of interleukin 10 (IL-10) with either hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) on the survival of a modified McFarlane flap, and to evaluate the effect of the treatment on cell metabolism, using label-free fluorescence lifetime imaging. Fifteen male Wistar rats (290-320 g) were randomly divided in three groups: group-A (control group) underwent surgery and received no gene transfer. Group-B received electroporation mediated hIL-10 gene delivery 24 h before and VEGF gene delivery 24 h after surgery. Group-C received electroporation mediated hIL-10 gene delivery 24 h before and hHGF gene delivery 24 h after surgery. The animals were assessed clinically and histologically. In addition, label-free fluorescence lifetime imaging was performed on the flap. Synergistic electroporation mediated gene delivery significantly decreased flap necrosis (P = 0.0079) and increased mean vessel density (P = 0.0079) in treatment groups B and C compared to control group-A. NADH fluorescence lifetime analysis indicated an increase in oxidative phosphorylation in the epidermis of the group-B (P = 0.039) relative to controls. These findings suggested synergistic in-vivo electroporation-mediated gene transfer as a promising therapeutic approach to enhance viability and vascularity of skin flap. Furthermore, the study showed that combinational gene therapy promoted an increase in tissue perfusion and a relative increase in oxidative metabolism within the epithelium.

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The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and nonhuman primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.

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Eosinophils are differentiated in the bone marrow and transit through the blood circulation to home into tissues primarily under the regulation of IL-5. Because the number of eosinophils in the peripheral blood is relatively low under normal conditions, in vivo functional studies of eosinophils remain extremely difficult. Increasing their numbers in vivo might be useful for assessing eosinophil activities during parasite infections, allergic inflammation, and so on. Here, we provide a method for eosinophil expansion using IL-5 gene transfer by electroporation in vivo.

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OBJECTIVES: As a common model for adverse early experience and depression, maternal separation (MS) is always used to investigate the psychological disease. Despite extensive and strong evidence verified the depression-like state induced by MS, little is known about the specific mechanism of MS. Therefore, the present study aimed to investigate the neurobiology mechanism of the MS-induced depression-like state. METHODS: To verify the depression-like behaviors of offspring induced by MS, a series of behavioral tests were performed. Then, in vivo electroporation and three-dimensional reconstruction, combining with immunohistochemistry and BrdU labeling, were mainly used to explore the neurogenesis and synaptogenesis in postnatal dentate gyrus. RESULTS: Prolonged MS indeed induced the depression-like behaviors of offspring in adulthood. Surprisingly, learning and memory were enhanced by prolonged MS. Further investigation indicated that prolonged MS inhibited the proliferation of neural stem cells, impaired the survival, and altered the fate decision of newborn cells, whereas the total length and terminal tips of dendrite, and the spine density, especially thin spine, were significantly increased in prolonged MS mice. CONCLUSIONS: Our results elucidated that prolonged MS induced the depression-like state by impairing postnatal neurogenesis of dentate gyrus. Importantly, our results emphasized that prolonged MS increased the spine density, especially thin spine, by increasing the total length and number of terminal tips of dendrite, thereby enhancing learning and memory.

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Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350-400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare's serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.

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An effective prophylactic vaccine targeting HIV must induce a robust humoral response and must direct the bulk of this response to the mucosa-the primary site of HIV transmission. The chemokine, CCL28, is secreted by epithelial cells at mucosal surfaces and recruits' cells expressing its receptor CCR10. CCR10 is predominantly expressed by IgA + ASCs. We hypothesized that co-immunization with plasmid DNA encoding consensus envelope antigens with plasmid-encoded CCL28 would enhance anti-HIV IgA responses at mucosal surfaces. Indeed, animals receiving pCCL28 and pEnvA/C had significantly increased HIV-specific IgA in fecal extract. Surprisingly, CCL28 co-immunization induced a significant increase in anti-HIV IgG in the serum in mice compared to those receiving pEnvA/C alone. These robust antibody responses were not associated with changes in the frequency of germinal center B cells but depended upon the expression of CCR10, as these responses we abolished in CCR10-deficient animals. Finally, immunization with CCL28 led to increased frequencies in HIV-specific CCR10 + and CCR10 + IgA + B cells in the small intestine and Peyer's patches of vaccinated animals as compared to those receiving pEnvA/C alone. These data indicate that CCL28 administration can enhance antigen-specific humoral responses systemically and at mucosal surfaces.

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Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves. A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers. Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury. Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy. At 12 and 18 hours, and 1, 2, 3, 4, 5, and 6 days of injury, lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images. Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay. We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia. Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique. Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing. The results facilitate direct time course analysis of peripheral nerve axon regeneration. This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University, China (approval No. GLMC201503010) on March 7, 2014.