Efficient DNA Delivery Research Articles

GALA-Modified Lipid Nanoparticles for the Targeted Delivery of Plasmid DNA to the Lungs.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.

An X-Band Dielectric Rod Antenna for Subdermal Tumor Heating to Assist Electroporation-Mediated DNA Delivery

An end-fire dielectric rod antenna is proposed as a heating device for integration with an array of electroporation electrodes in order to enable efficient delivery of DNA into the cells that comprise subcutaneous tumors. A 5-7 °C temperature elevation of tumors that are located near the fat-muscle boundary and 3-7 mm below the skin surface can be achieved in a short period of time without damaging surrounding tissues. This capability is demonstrated through a combination of a directional antenna applicator operating at 8 GHz, and utilization of forced air cooling of the outer surface (skin layer). The directionality of the antenna is improved by cladding its high permittivity core with 3D printed, low permittivity dielectric material. Experimental data using a pork skin-fat-muscle tissue show that the desired temperature elevation at the tumor location is obtained after 2.5 W RF illumination for 3 minutes, which is in good agreement with electro-thermal simulations. With the addition of realistic human body model parameters to the same simulation setup, the results indicate that tumors can be uniformly heated with 3 minutes of illumination at 2.5 W input power while keeping the surrounding healthy tissues at a safe temperature.

Endosome-escaping micelle complexes dually equipped with cell-penetrating and endosome-disrupting peptides for efficient DNA delivery into intact plants.

The delivery of DNA to plants is crucial for enhancing their ability to produce valuable compounds and adapt to climate change. Peptides can provide a versatile tool for delivering DNA to a specific target organelle in various plant species without the use of specialized equipment. However, peptide-mediated DNA delivery suffers from endosomal entrapment and subsequent vacuolar degradation of the DNA cargo, which leads to poor transfection efficiency. To overcome the lack of a reliable approach for bypassing vacuolar degradation in plants, we herein present an endosome-escaping micelle. The micelle surface is dually modified with cell-penetrating (CPP) and endosome-disrupting peptides (EDP) and the core is composed of plasmid DNA condensed with cationic peptides. Due to the functions of CPP and EDP, the dual peptide-modified micelles efficiently undergo endocytic internalization and escape from endosomes to the cytosol, thereby achieving significantly enhanced transfection of intact plants with negligible cytotoxicity. The present study offers a robust strategy for efficient intracellular DNA delivery to plants without vacuolar degradation, and can facilitate plant bioengineering for diverse biotechnological applications.

Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer-DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer-pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.

Systematic study of liposomes composition towards efficient delivery of plasmid DNA as potential application of dermal fibroblasts targeting.

The use of non-viral DNA vectors to topically treat skin diseases has demonstrated a high potential. However, vectors applied on the skin face extracellular barriers including the stratum corneum and intracellular barriers such as the endosomal escape and the nuclear targeting of the plasmid DNA. The aim of this study was to develop a formulation suitable for dermal application and effective for delivering plasmid DNA into cells. Different formulations were prepared using different cationic lipids (DOTAP, DC-Chol, DOTMA) and co-lipids (DOPE, DSPE). Lipoplexes were produced by complexing liposomes with plasmid DNA at different pDNA/CL (w/w) ratios. Our results showed that appropriate pDNA/CL ratios allowing total complexation of plasmid DNA differed depending on the structure of the lipid used. The transfection rates showed that (i) higher rates were obtained with DOTMA lipoplexes, (ii) DC-Chol lipoplexes provided a transfection twice as important as DOTAP lipoplexes and (iii) when DSPE was added, the cytotoxicity decreased while transfection rates were similar. We found that formulations composed of DC-Chol:DOPE:DSPE or DOTMA:DOPE were appropriate to complex plasmid DNA and to transfect human primary dermal fibroblasts with efficacy and limited cytotoxicity. Therefore, these formulations are highly promising in the context of gene therapy to treat skin diseases.

Lipid-Encapsulated Silica Nanobowls as an Efficient and Versatile DNA Delivery System

Nonmesoporous Janus silica nanobowls (NBs) are unique in that they possess two different nonporous surfaces per particle for loading biological molecules and can thus be designed with multifunctional properties. Although silica NBs have been successfully employed for both targeted therapeutic and diagnostic applications, their ability to deliver DNA has not yet been fully explored. The purpose of this study was to design and develop an in vitro transfection agent that would exploit the distinct characteristics of the silica NB. First, we determined that the NB surface can be linked to either supercoiled cDNA plasmids or vectorless, linear cDNA constructs. Additionally, the linearized cDNA can be functionalized and chemisorbed on NBs to obtain a controlled release. Second, the successful transfection of cells studied was dependent on lipid coating of the NB (LNBs). Although both NBs and LNBs were capable of undergoing endocytosis, NBs appeared to remain within vesicles as shown by transmission electron microscopy (TEM). Third, fluorescence microscopy and Western blotting assays revealed that transfection of four different cell lines and acutely isolated rat sensory neurons with LNBs loaded with either linear or supercoiled cDNA constructs coding for the fluorescent protein, clover and tdTomato, resulted in protein expression. Fourth, two separate opioid receptor-ion channel signaling pathways were functionally reconstituted in HEK cells transfected with LNBs loaded with three separate cDNA constructs. Overall, these results lay the foundation for the use and further development of LNBs as in vitro transfection agents.

Quantification of DNA Delivery Efficiency Labeled with Fluorescent Dye in Digital Electroporation System

Quantification of DNA Delivery Efficiency Labeled with Fluorescent Dye in Digital Electroporation System

Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection

Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efficient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7 ± 2.5% and 52 ± 7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confirmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efficient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.

Carbon Dots Enable Efficient Delivery of Functional DNA in Plants.

Genetic engineering is an important technology in plant sciences. The current delivery techniques, such as Agrobacterium-mediated gene transfer and gene gun instant gene expression system, always need a very complicated process or have host-range limitations due to the inhibition of the cell wall structure. Here, we demonstrate that a nanocomposite (CDP) prepared by combining polyethyleneimine (PEI) with carbon dot (CD) was confirmed as a good vector for gene transfection in both animal and plant cells, especially in intact plants. Efficient DNA delivery and strong gene expression without DNA integration are accomplished in rice (Oryza Sativa japonica), wheat (Triticum aestivum), and mung bean (Phaseolus radiatus) leaves and rice roots. Notably, the CDP successfully deliver the hydamycin resistance gene into the rice and induce hygromycin resistance in rice. Also, CDP can achieve the β-glucuronidase gene delivery and expression in mature rice embryo induced callus. The CDP not only facilitates plasmid transport into cells but also protects DNA from DNase degradation. Our work provides a highly efficient DNA delivery system for instant gene expression, which could be a useful and fast gene engineering tool for functional research in plant biology.

Highly efficient gene transfer in the mouse gut microbiota is enabled by the Incl2 conjugative plasmid TP114

The gut microbiota is a suspected hotspot for bacterial conjugation due to its high density and diversity of microorganisms. However, the contribution of different conjugative plasmid families to horizontal gene transfer in this environment remains poorly characterized. Here, we systematically quantified the transfer rates in the mouse intestinal tract for 13 conjugative plasmids encompassing 10 major incompatibility groups. The vast majority of these plasmids were unable to perform conjugation in situ or only reached relatively low transfer rates. Surprisingly, IncI2 conjugative plasmid TP114 was identified as a proficient DNA delivery system in this environment, with the ability to transfer to virtually 100% of the probed recipient bacteria. We also show that a type IV pilus present in I-complex conjugative plasmids plays a crucial role for the transfer of TP114 in the mouse intestinal microbiota, most likely by contributing to mating pair stabilization. These results provide new insights on the mobility of genes in the gut microbiota and highlights TP114 as a very efficient DNA delivery system of interest for microbiome editing tools.

Cell-based high-throughput screening of cationic polymers for efficient DNA and siRNA delivery

Development of non-viral gene vectors which can efficiently and safely transfect plasmid DNA and siRNA into cells is of great importance for gene therapy. Despite lots of efforts spent, it is still imperative to develop suitable gene vectors with better transfection efficiency and low cytotoxicity. To this end, we successfully designed, synthesized and screened a library of 120 polymers (via nucleophilic substitution reaction between dihalides and amines). With cell-based transfection screening assays, 120 polymers were tested to evaluate their transfection efficiency of transporting DNA and siRNA into cells. Our results indicated that hydrophobic modification could greatly enhance cationic polymers’ transfection efficiency, and polymers with long linkers usually showed better transfection performance, especially for polymers with the linker of 1, 12-dibromododecane (L3 linker). Besides, polyalkylamines exhibited better transfection efficiency with the polymer particle size around 200 nm and the zeta potential in the range of + 40 mV to +50 mV. Interestingly, polymer particles made from N15HL3 not only exhibited better DNA transfection efficiency in HEK 293T cells but also showed higher siRNA transfection efficiency in U87 Luc-GFP cells together with low cell toxicity than Lipofectamine 2000 (one of commercial transfection reagents). Therefore, it is hoped that our study here not only provides promising gene vector candidates for further evaluation in gene therapy, but also provides valuable insights for better understanding of the relationship between the chemical structures and gene transfection efficiency to rationally design better non-viral gene vectors for gene therapy in the future.

Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts.

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

Cationic cross-linked polymers containing labile disulfide and boronic ester linkages for effective triple responsive DNA release

Disruption of DNA carriers triggered by intracellular bio-stimulants has been broadly considered as most convenient strategy for efficient DNA delivery. In this direction, we have designed and synthesized pH, redox and ATP responsive cationic cross-linked polymers (CLPs) having disulfide and reversible boronic ester linkages. These CLPs also contain folate groups that are known for their targeting capability towards cancer cells. Biophysical studies showed that these cationic CLPs exhibited more effective DNA condensation in comparison to cationic linear polymers resulting in the formation of nano-sized polyplexes with sufficient positive zeta potentials and good colloidal stability at neutral pH (∼7.4). More interestingly, the polyplexes prepared from these CLPs have the ability to selectively release complexed DNA under conditions similar to those prevalent in cancer cells such as acidic pH, ATP rich surroundings or presence of glutathione, as revealed by ethidium bromide exclusion assay, agarose gel electrophoresis, AFM measurements, etc. Therefore, these cross-linked polymers have high potential of being effective non-viral gene delivery vehicles.

Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System.

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

CRISPR/dCas9 (D10A) -Mediated Hb CS Gene Editing and Identification of Genetically Modified Fibroblasts

Background: Alpha-thalassemia (α-thalassemia) is an inherited hemolytic disease caused by complete or absent synthesis of α-globin chains due to alpha-globin chain synthesis disorders and is one of the most common genetic diseases in the world. At present, the most commonly used strategies in the gene therapy research of thalassemia, the nonhomologous end joining (NHEJ) apparatus, is only suitable for the study of beta thalassemia and sickle-type anemia. The DNA double strand breaks have a high risk to induce frameshift mutations, leading to more serious clinical consequences. Therefore, it is still necessary to study a safer genetic repair program for alpha-thalassemia. Methods: The donor we chose to repair was from a patient with a genotype of -SEA/αCSα, whose mutation is one of the most common thalassemia alleles in Longyan city, and the HBA1 gene is normal. Due to the deletion of a copy of the HBA2 gene, only one copy of the mutation needs to be repaired, making it a suitable candidate for the study of alpha thalassemia gene therapy. To minimize the risk of non-specific mutations introduced into the locus of interest, we used the single-base editing apparatus, dCas9 (dead Cas9), which only cleaves one strand of DNA and precisely modifies one base, effectively improve the specificity of target cleavage and reduce the risk of off-target. The recognition sequence on the homologous arm in the template was lengthened to reduce the risk of off-target.(see Figure 1 ) Result We used the pmaxGFP plasmid as a positive control for optimizing electroporation conditions. More than 80% transfection efficiency was obtained (see Figure 2 ) Fig 2 pmaxGFP transfection to verify the transfection efficiency of fibroblasts ( 100x ) To investigate the effectiveness of sgRNA and donor plasmids for mutated gene repair, we analyzed the repair efficiency of fibroblasts. Primers were designed near the repair site and PCR sequences were amplified by PCR . After amplification of the genomic fragments, BamH1 digestion assay was performed. Fig 3 PCR and Bam HI digested results using primers for the gene of interest, showed the gene editing efficiency was 4~10%. The sequencing results showed that three clonals of α- thalassaemia mutations were successfully repaired. Fig 4 Hb CS gene mutation target sequence repair and sequencing results The sequencing results of Fig. 5 show that no mutation was detected at the six potential off-target sites. Fig 5 off-target effect detection The use of base editing is limited by off-target activity and DNA delivery efficiency to cells. Filtration into fibroblasts by plasmid electroporation, CRISPR/Cas9-mediated editing efficiency is usually 3~10%, and we achieve a gene editing efficiency of 4~10% (Figure 4). Conclusions Our data show that the gene transfer of normal HBA2 gene by electroporation the single-base editor CRISPR/dCas9 (D10A) and donor into cells can successfully repair Hb CS mutations. Disclosures No relevant conflicts of interest to declare.

Carbon nanotube-mediated DNA delivery without transgene integration in intact plants.

Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species-independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.

Transdermal Delivery of Nucleic Acid Mediated by Punching and Electroporation.

Transdermal delivery of gene medicine holds a great promise in gene therapy, and electroporation-mediated method is a high-efficiency drug transfer technique. Traditional skin electroporation approaches require high voltage that may cause severe injure. In this chapter, we provide a detailed protocol of a novel skin electroporation approach via combination of a microneedle roller and a flexible interdigitated electroporation array (FIEA) for efficient delivery of DNA and siRNA into mouse skin. This electroporation protocol assisted by punching with the microneedle roller represents significant advantages over treatment with electroporation alone, allowing successful nucleic acid transportation at low voltage, with ideal safety outcomes. We describe the details of fabrication process of the FIEA, experiment preparation, and mouse thigh skin electroporation.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost‐efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non‐lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1β, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2posCD172Apos CD163int and CD163neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.