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Gene editing enables scientists to make precise changes to the genome of an organism using the cell’s own ability to repair damaged DNA using a supplied DNA template. In recent years, gene editing has been applied clinically in the treatment of diseases such as cancer. Gene editing has been used in a type of immunotherapy, known as chimeric antigen receptor‐expressing T cell (CAR‐T) therapy, to restore the body’s ability to find and kill specific cancer cells. For this therapy, viruses are often used to supply the cell with the DNA template used for creating the edit in the target DNA. However, the use of viruses in this context is laborious and costly. Developing non‐viral methods for delivery of DNA templates for gene editing would circumvent these problems, but current methods can have toxic effects on cells and result in low editing efficiency. In a new article published in this issue, Yang et al. describe a novel method for viral‐independent delivery of naked DNA and its incorporation into the genome for engineering cells for CAR‐T therapy., Gene editing can be used to restore the body’s ability to kill specific cancer cells. Viruses are often used to supply the cell with the DNA template used for creating the edit in the target DNA, but this is laborious and costly. In this issue, Yang et al. describe a novel method for viral‐independent delivery of naked DNA for engineering cells for CAR‐T therapy.

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Human immunodeficiency virus type 1 (HIV-1) infection remains one of the worst crises in global health. The prevention of HIV-1 infection is a crucial task that needs to be addressed due to the absence of a licensed vaccine against HIV-1. DNA vaccines present a promising alternative approach to combat HIV-1 infection due to their excellent safety profile, lack of severe side effects, and relatively rapid fabrication. Traditional vaccines composed of a monomeric envelope or peptide fragments have been indicated to lack protective efficacy mediated by inducing HIV-1-specific neutralizing antibodies in clinical trials. The immunogenicity and protection against HIV-1 induced by DNA vaccines are limited due to the poor uptake of these vaccines by antigen-presenting cells and their ready degradation by DNases and lysosomes. To address these issues of naked DNA vaccines, we described the feasibility of CpG-functionalized silica-coated calcium phosphate nanoparticles (SCPs) for efficiently delivering DNA-based HIV-1 trimeric envelope vaccines against HIV-1. Vaccines comprising the soluble BG505 SOSIP.664 trimer fused to the GCN4-based isoleucine zipper or bacteriophage T4 fibritin foldon motif with excellent simulation of the native HIV-1 envelope were chosen as trimer-based vaccine platforms. Our results showed that SCP-based DNA immunization could significantly induce both broad humoral immune responses and potent cellular immune responses compared to naked DNA vaccination in vivo. To the best of our knowledge, this study is the first to assess the feasibility of CpG-functionalized SCPs for efficiently delivering DNA vaccines expressing a native-like HIV-1 trimer. These CpG-functionalized SCPs for delivering DNA-based HIV-1 trimeric envelope vaccines may lead to the development of promising vaccine candidates against HIV-1.

Cancer vaccines that make use of tumor antigens represent a promising therapeutic strategy by stimulating immune responses against tumors to generate long-term anti-tumor immunity. However, vaccines have shown limited clinical efficacy due to inefficient delivery. In this study, we focus on vaccine delivery assisted by nanocomplexes for cancer immunotherapy. Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. Subsequent interaction with nucleic acids led to formation of polymer/nucleic acid nanocomplexes of well-controlled structure. Tumor transfection via FTS-PEI was much more effective than that by PEI, other PEI derivatives, or naked DNA. Significant numbers of transfected cells were also observed in draining lymph nodes (LNs). In vivo delivery of ovalbumin (OVA; a model antigen) expression plasmid (pOVA) by FTS-PEI led to a significant growth inhibition of the OVA-expressing B16 tumor through presentation of OVA epitopes as well as other epitopes via epitope spreading. Moreover, in vivo delivery of an endogenous melanoma neoantigen tyrosinase-related protein 2 (Trp2) also led to substantial tumor growth inhibition. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination., Graphical abstract, Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination.

Background Gold nanocages have been widely used as multifunctional platforms for drug and gene delivery, as well as photothermal agents for cancer therapy. However, their potential as gene delivery systems for cancer treatment has been reported in combination with chemotherapeutics and photothermal therapy, but not in isolation so far. The purpose of this work was to investigate whether the conjugation of gold nanocages with the cancer targeting ligand lactoferrin, polyethylene glycol and polyethylenimine could lead to enhanced transfection efficiency on prostate cancer cells in vitro, without assistance of external stimulation. Methods Novel lactoferrin-bearing gold nanocages conjugated to polyethylenimine and polyethylene glycol have been synthesized and characterized. Their transfection efficacy and cytotoxicity were assessed on PC-3 prostate cancer cell line following complexation with a plasmid DNA. Results Lactoferrin-bearing gold nanocages, alone or conjugated with polyethylenimine and polyethylene glycol, were able to condense DNA at conjugate:DNA weight ratios 5:1 and higher. Among all gold conjugates, the highest gene expression was obtained following treatment with gold complex conjugated with polyethylenimine and lactoferrin, at weight ratio 40:1, which was 1.71-fold higher than with polyethylenimine. This might be due to the increased DNA cellular uptake observed with this conjugate, by up to 8.65-fold in comparison with naked DNA. Conclusion Lactoferrin-bearing gold nanocages conjugates are highly promising gene delivery systems to prostate cancer cells., Graphical Abstract

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock‐in approaches using homology‐directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene‐edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double‐stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator‐like effector nuclease (TALEN)‐mediated gene editing in primary T cells to improve nonviral gene knock‐in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single‐stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time‐controlled two‐step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T‐cell receptor alpha constant region (TRAC) locus to generate ‘off‐the shelf’ CAR‐T cells., With understanding of TALEN‐mediated gene editing kinetics, we developed a rapid and efficient gene insertion strategy with naked DNA as template. We demonstrated that the time‐controlled two‐step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells and achieved ˜ 30% integration frequency when inserting a 2 kb cassette into the T‐cell genome.

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.

International audience; Mucosa are the routes of entry of most pathogens into animals' organisms. Reducing the important global burden of mucosal infectious diseases in livestock animals is required in the field of veterinary public health. For veterinary respiratory pathogens, one possible strategy is the development of intranasal (IN) DNA vaccination. The aim of this study was to assess the feasibility of IN DNA vaccination in pigs, an important species in livestock production industry, and a source of zoonotic diseases. To achieve this goal, we used a DNA vaccine against pseudorabies virus (PrV) encoding the immunogenic glycoprotein B (pcDNA3-gB plasmid). When pigs were inoculated with the naked DNA vaccine through the IN route, PrV-specific IgG and IgA type antibodies were detected in porcine sera. Interestingly, mucosal salivary IgA antibodies against PrV were also detected, at similar levels to those measured following intramuscular injection (positive controls). Furthermore, the IN delivery of pcDNA3-gB combined with PLGA-PEI nanoparticles resulted in similar levels of antibodies but was associated with an increase in the duration of detection of mucosal IgA for 2 out of 3 pigs. Our results suggest that there is room to improve the efficacy of IN DNA vaccination in pigs through optimization of IN inoculations, for example by using nanoparticles such as PLGA-PEI. Further studies will be dedicated to optimizing and testing the protective potential of IN DNA vaccination procedures against PrV.

Monosaccharides are added to the hydrophilic face of a self‐assembled asymmetric FeII metallohelix, using CuAAC chemistry. The sixteen resulting architectures are water‐stable and optically pure, and exhibit improved antiproliferative selectivity against colon cancer cells (HCT116 p53+/+) with respect to the non‐cancerous ARPE‐19 cell line. While the most selective compound is a glucose‐appended enantiomer, its cellular entry is not mainly glucose transporter‐mediated. Glucose conjugation nevertheless increases nuclear delivery ca 2.5‐fold, and a non‐destructive interaction with DNA is indicated. Addition of the glucose units affects the binding orientation of the metallohelix to naked DNA, but does not substantially alter the overall affinity. In a mouse model, the glucose conjugated compound was far better tolerated, and tumour growth delays for the parent compound (2.6 d) were improved to 4.3 d; performance as good as cisplatin but with the advantage of no weight loss in the subjects., Asymmetric triplex metallohelices can be conjugated with sugars using post‐assembly CuAAC chemistry, to give optically pure architectures with anti‐cancer activity and marked selectivity with respect to non‐cancerous cell lines. Glucose‐appended structures exhibit improved nuclear uptake in vitro and supress tumour growth in vivo.

Intestinal mucosal immunity plays a vital role against Vibrio mimicus infection because it is an enteric pathogen causing serious vibriosis in fish. In the previous studies, we developed an oral double-targeted DNA vaccine of V. mimicus and demonstrated that the vaccine could elicit significantly higher intestinal mucosal immune response than did naked DNA vaccine. But, little is known underlying regulatory molecular mechanisms of the enhanced intestinal mucosal immunity. Here the transcriptome and proteome in the intestines of the grass carps immunized or not with the double-targeted DNA vaccine were investigated by using RNA-seq and iTRAQ-coupled LC-MS/MS. Compared with the control group, a total of 5339 differentially expressed genes (DEGs) and 1173 differentially expressed proteins (DEPs) were identified in the immunized fish intestines. Subsequently, the integrated analysis between transcriptome and proteome data revealed that 250 DEPs were matched with the corresponding DEGs (named associated DEPs/DEGs) at both transcriptome and proteome levels. Fifty of all the associated DEPs/DEGs were immune-related and mainly enriched in phagosome, antigen-processing and presentation, complement and coagulation cascades, NLRs and MAPK signaling pathways via Gene Ontology and KEGG pathway analyses, which suggested the coordination of the five activated pathways was essential to the enhanced intestinal mucosal immune response in the immunized fish. The protein-protein interaction analysis showed that 60 of the 63 immune-related DEPs to form an integrated network. Additionally, randomly selected DEGs and DEPs were respectively validated by quantitative real-time RT-PCR and multiple reaction monitoring (MRM) assay, indicating that the both RNA-Seq and iTRAQ results in the study were reliable. Overall, our comprehensive transcriptome and proteome data provide some key genes and their protein products for further research on the regulatory molecular mechanisms underlying the enhanced intestinal mucosal immunity.

In this study, we evaluated DNase activity of rainbow trout oocyte using an in vitro and in vivo study. First, synthetic single strand and natural double strand DNA from Eukaryotic and prokaryotic sources as well as naked DNA were in vitro incubated with the oocyte cytoplasm. Results showed that the DNase activity of rainbow trout oocyte is strong enough to degrade any type of DNA at the onset of the incubation. Then, we evaluated if similar to the mammalian species, dead spermatozoa from rainbow trout can protect exogenous DNA from oocyte DNases. A series of dead spermatozoa was incubated with pDB2, carrying EGFP transgene, for 30 min followed by the ooplasm treatment for an additional 30 min. Not only did oocyte DNases completely degrade the exogenous DNA, but also it degraded the compact genome of spermatozoa. In conclusion, in vitro results clearly showed that strong DNase activity of ooplasm could degrade any types of foreign DNAs including oligonucleotides and intensively compact sperm genome. The strong DNase activity of rainbow trout ooplasm could be a stumbling block for genetic modification using plasmids in salmonids.

BackgroundHuman Papillomavirus type 16 (HPV16) has been associated with a subset of head and neck cancers. Two HPV encoded oncogenic proteins, E6 and E7, are important for the malignant progression of HPV-associated cancers. A spontaneous HPV16 E6/E7-expressing oral tumor model in human HLA-A2 (AAD) transgenic mice will be important for the development of therapeutic HPV vaccines for the control of HPV-associated head and neck cancers.MethodsIn the current studies, we characterized the HLA-A2 restricted HPV16 E7-specific CD8 + T cell mediated immune responses in the HLA-A2 (AAD) transgenic mice using a therapeutic naked DNA vaccine encoding calreticulin (CRT) linked to a mutated E7(N53S). We also employed oncogenic DNA plasmids that encoded HPV16E6/E7/Luc, NRasG12V, and sleeping beauty transposase for the transfection into the submucosal of oral cavity of the transgenic mice with electroporation to create a spontaneous oral tumor. Furthermore, we characterized the therapeutic antitumor effects of CRT/E7(N53S) DNA vaccine using the spontaneous HPV16 E6/E7-expressing oral tumor model in HLA-A2 (AAD) transgenic mice.ResultsWe found that CRT/E7(N53S) DNA vaccine primarily generated human HPV16 E7 peptide (aa11-20) specific CD8 + T cells, as compared to the wild-type CRT/E7 vaccine, which primarily generated murine H-2Dbrestricted E7 peptide (aa49-57) specific CD8 + T cell responses. We also observed transfection of the oncogenic DNA plasmids with electroporation generated spontaneous oral tumor in all of the injected mice. Additionally, treatment with CRT/E7(N53S) DNA vaccine intramuscularly followed by electroporation resulted in significant antitumor effects against the spontaneous HPV16 E6/E7-expressing oral tumors in HLA-A2 (AAD) transgenic mice.ConclusionsTaken together, the data indicated that the combination of HPV16 E6/E7-expressing DNA, NRasG12VDNA and DNA encoding sleeping beauty transposase is able to generate spontaneous oral tumor in HLA-A2 (AAD) transgenic mice, which can be successfully controlled by treatment with CRT/E7(N53S) DNA vaccine. The translational potential of our studies are discussed.

Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH-cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs(−/–)) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs(−/–) mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 μM before injection to 176 μM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.

We have carried out a series of experiments on the ultra-low-energy ion beam bombardment of naked DNA to study the final stages of interaction between energetic ions and biological molecules and atoms. A carbon ion beam (carbon is the ion species usually used for cancer therapy) was used to bombard naked plasmid DNA pGFP to fluences of 1 × 1015, 2 × 1015 and 4 × 1015 ions/cm2 at the ultra-low energy of 50, 100 and 300 eV, or ∼10 eV/amu. Modification of the DNA in terms of topological form changes was investigated using gel electrophoresis. We find that there is a threshold of ion beam conditions for occurrence of the linear form indicating DNA double strand breakage (DSB), which is the dominant course for cell mutation or lethality. DNA DSBs can be brought about by carbon ion bombardment at the lowest ion energy of 50 eV and fluence of 4 × 1015 ions/cm2, or at the higher energy of 100 or 300 eV and the lowest fluence of 1 × 1015 ions/cm2. We discuss the origin of these threshold conditions using a simplified DNA double strand model as well as in terms of the chemical activity of carbon.

Vibrio mimicus (V. mimicus) is an enteric pathogen causing serious vibriosis in aquatic animals, and intestinal mucosal immunity elicited by oral DNA vaccines plays a vital role in the resistance to the pathogen. Yet, the immunogenicity of oral naked DNA vaccine is poor due to the degradation in the digestive tract. Therefore, it is necessary to use targeted DNA delivery strategy for improving the potency of vaccine. In our previous studies, the epitopes of OmpU and VMH proteins from V. mimicus have been identified. Here, a double-targeted DNA vaccine based on the multi-epitope peptide (named OVepis) of V. mimicus was developed by using E. coli DH5α bacterial ghosts (BGs) and invariant chain-like protein (Iclp) of grass carps as exogenous and endogenous targeting delivery carriers, respectively, and then the efficacy was evaluated in grass carps via oral gavage administration. The results showed that the loading efficiency and capacity of DH5α-BGs to pcDNA3.1-Iclp-OVepis were 89.91% and 16.6 μg per milligram BGs, respectively. DH5α-BGs loaded with pcDNA3.1-Iclp-OVepis targeted efficiently RAW264.7 cells and mediated effective gene expression. Higher transcription levels of the Iclp-OVepis gene were detectable in the different tissues of fish in the double-targeted DNA vaccine (DH5α-BGs/pcDNA3.1-Iclp-OVepis) group than that in the endogenous-targeted DNA vaccine (pcDNA3.1-Iclp-OVepis) group at 21 dpv. The double-targeted DNA vaccine significantly enhanced SOD activity, LZM activity and C3 contents in the serum and intestinal mucus of immunized fish. Specific antibody levels in the serum and intestinal mucus were markedly increased in the double-targeted DNA vaccine group compared to the other DNA vaccine groups. at each examined time point. Likewise, the proliferative activities of peripheral blood and intestinal intraepithelial lymphocytes of fish in the double-targeted DNA vaccine group were also significantly higher at 28 dpv. Importantly, the vaccine conferred high protection against V. mimicus challenge with 81.11% RPS. This study suggested that the oral double-targeted DNA vaccine might be used as a promising vaccine candidate to control V. mimicus infection.

Nucleosomes, the fundamental organizing units of eukaryotic genomes, contain ∼146 base pairs of DNA wrapped around a histone H3–H4 tetramer and two histone H2A–H2B dimers. Converting nucleosomes into hexasomes by removal of a H2A–H2B dimer is an important regulatory event, but its regulation and functional consequences are not well-understood. To investigate the influence of hexasomes on DNA accessibility, we used the property of the Widom-601 Nucleosome Positioning Sequence (NPS) to form homogeneously oriented hexasomes in vitro. We find that DNA accessibility to transcription factors (TF) on the hexasome H2A–H2B distal side is identical to naked DNA, while the accessibility on the H2A–H2B proximal side is reduced by 2-fold, which is due to a 2-fold reduction in hexasome unwrapping probability. We then determined that a 23 bp region of the Widom-601 NPS is responsible for forming homogeneously oriented hexasomes. Analysis of published ChIP-exo data of hexasome containing genes identified two DNA sequence motifs that correlate with hexasome orientation in vivo, while ExoIII mapping studies of these sequences revealed they generate homogeneously oriented hexasomes in vitro. These results indicate that hexasome orientation, which is influenced by the underlying DNA sequence in vivo, is important for modulating DNA accessibility to regulate transcription.

The RNA polymerase II (RNApII) transcription cycle consists of multiple steps involving dozens of protein factors. Here we describe a useful approach to study the dynamics of initiation and early elongation, comprising an in vitro transcription system in which complexes are assembled on immobilized DNA templates and analyzed by quantitative mass spectrometry. This unbiased screening system allows quantitation of RNApII complex components on either naked DNA or chromatin templates. In addition to transcription, the system reproduces co-transcriptional mRNA capping and multiple transcription-related histone modifications. In combination with other biochemical and genetic methods, this approach can provide insights into the mechanistic details of gene expression by RNApII.

Background DNA-launched vaccine is “manufactured” in vaccinated individuals and does not require traditional vaccine manufacturing facility and technology. Goals. Using yellow fever 17D vaccine, we have provided proof-of-concept evidence that these vaccine can be launched from DNA and induce specific immune responses against pathogenic virus causing yellow fever. The infectious DNA vaccine technology is based on the transcription of the full-length genomic RNA of the live-attenuated virus from plasmid DNA in vitro and in vivo. A few ng of infectious DNA encoding the fulllength genomic RNA are required to initiate the replication of the vaccine virus in vitro. The in vivo-generated viral RNA initiates limited replication of the vaccine virus, which in turn leads to efficient immunization. Electroporation in vivo has induced specific immune responses against pathogenic virus and protected mice against fatal disease. Here we describe a novel infectious DNA vaccine technology which combines advantages of naked DNA vaccination and live-attenuated vaccine efficacy. Conclusions If successful in further testing, this technology can dramatically change the way we make vaccines as well as vaccination practice.

Naked plasmid DNA electrotransfer offers advantages over viral-based gene delivery, including being regulatory permissive, but factors influencing expression efficiency and cell fate impact on translational utility. This study compared co-expression of red and green fluorescence reporter plasmids with differing promoters in HEK293 cells and in vivo in guinea pig cochlear mesenchymal cells using Bionic array-Directed Gene Electrotransfer (BaDGE®). A functional plasmid copy number of ∼64 was established in HEK293 cells by co-transfecting with separate CMV-actin-globin (CAGp) promoter-driven mCherry and green fluorescent protein (GFP) reporters, where cell division diluted plasmids toward discrete red or green channels from 100% co-expression to 10% over 24 days (∼17 cell cycles). Cross-talk between promoters was identified by interchanging a cytomegalovirus promoter (CMVp)-driven GFP plasmid for the CAGp-GFP plasmid. Here, expression of the CMVp-GFP plasmid dominated, while a dual CAGp-based reporter plasmid cocktail showed persistent co-expression beyond 2 weeks. In contrast, in vivo, cochlear mesenchymal cells co-transduced with CAGp-mCherry and CMVp-GFP plasmids showed stable co-expression at ∼50%, while the total transfectant numbers diminished over 2 weeks. This is consistent with a lack of mitosis in the cochlear mesenchymal cells and shows that cell type is a factor in plasmid interaction.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25–20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3 h with the liposome-DNA complexes. The samples were collected after 1 h and after 3 h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1 µg of DNA and (5) incubation with liposomes and 10 µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P

Eukaryotic DNA-binding proteins operate in the context of chromatin, where nucleosomes are the elementary building blocks. Nucleosomal DNA is wrapped around a histone core, thereby rendering a large fraction of the DNA surface inaccessible to DNA-binding proteins. Nevertheless, first responders in DNA repair and sequence-specific transcription factors bind DNA target sites obstructed by chromatin. While early studies examined protein binding to histone-free DNA, it is only now beginning to emerge how DNA sequences are interrogated on nucleosomes. These readout strategies range from the release of nucleosomal DNA from histones, to rotational/translation register shifts of the DNA motif, and nucleosome-specific DNA binding modes that differ from those observed on naked DNA. Since DNA motif engagement on nucleosomes strongly depends on position and orientation, we argue that motif location and nucleosome positioning co-determine protein access to DNA in transcription and DNA repair.

Background DNA vaccine is one of the research hotspots in veterinary vaccine development. Several advantages, such as cost-effectiveness, ease of design and production, good biocompatibility of plasmid DNA, attractive biosafety, and DNA stability, are found in DNA vaccines. Methods In this study, the plasmids expressing bovine herpesvirus 1 (BoHV-1) gB, gC, and gD proteins were mixed at the same mass ratio and adsorbed polyethyleneimine (PEI) magnetic beads with a diameter of 50 nm. Further, the plasmid and PEI magnetic bead polymers were packaged into double carboxyl polyethylene glycol (PEG) 600 to use as a DNA vaccine. The prepared DNA vaccine was employed to vaccinate mice via the intranasal route. The immune responses were evaluated in mice after vaccination. Results The expression of viral proteins could be largely detected in the lung and rarely in the spleen of mice subjected to a vaccination. The examination of biochemical indicators, anal temperature, and histology indicated that the DNA vaccine was safe in vivo. However, short-time toxicity was observed. The total antibody detected with ELISA in vaccinated mice showed a higher level than PBS, DNA, PEI + DNA, and PBS groups. The antibody level was significantly elevated at the 15th week and started to decrease since the 17th week. The neutralizing antibody titer was significantly higher in DNA vaccine than naked DNA vaccinated animals. The total IgA level was much greater in the DNA vaccine group compared to other component vaccinated groups. The examination of cellular cytokines and the percentage of CD4/CD8 indicated that the prepared DNA vaccine induced a strong cellular immunity. Conclusion The mixed application of plasmids expressing BoHV-1 gB/gC/gD proteins by nano-carrier through intranasal route could effectively activate long-term humoral, cellular, and mucosal immune responses at high levels in mice. These data indicate PEI magnetic beads combining with PEG600 are an efficient vector for plasmid DNA to deliver intranasally as a DNA vaccine candidate.

Stimuli-responsive nanocarriers have become increasingly important for nucleic acid and drug delivery in cancer therapy. Here, we report the synthesis, characterization and evaluation of disulphide-linked, octadecyl (C18 alkyl) chain-bearing PEGylated generation 3-diaminobutyric polypropylenimine dendrimer-based vesicles (or dendrimersomes) for gene delivery. The lipid-bearing PEGylated dendrimer was successfully synthesized through in situ two-step reaction. It was able to spontaneously self-Assemble into stable, cationic, nanosized vesicles, with low critical aggregation concentration value, and also showed redox-responsiveness in presence of a glutathione concentration similar to that of the cytosolic reducing environment. In addition, it was able to condense more than 70% of DNA at dendrimer: DNA weight ratios of 5 : 1 and higher. This dendriplex resulted in an enhanced cellular uptake of DNA at dendrimer: DNA weight ratios of 10 : 1 and 20 : 1, by up to 16-fold and by up to 28-fold compared with naked DNA in PC-3 and DU145 prostate cancer cell lines respectively. At a dendrimer: DNA weight ratio of 20 : 1, it led to an increase in gene expression in PC-3 and DU145 cells, compared with DAB dendriplex. These octadecyl chain-bearing, PEGylated dendrimer-based vesicles are therefore promising redox-sensitive drug and gene delivery systems for potential applications in combination cancer therapy. This journal is

Recently, a cryo-electron microscopy study has captured different stages of nucleosome breathing dynamics that show partial unwrapping of DNA from histone core to permit transient access to the DNA sites by transcription factors. In practice, however, only a subset of transcription factors named pioneer factors can invade nucleosomes and bind to specific DNA sites to trigger essential DNA metabolic processes. We propose a discrete-state stochastic model that considers the interplay of nucleosome breathing and protein dynamics explicitly and estimate the mean time to search the target DNA sites. It is found that the molecular principle governing the search process on nucleosome is very different compared to that on naked DNA. The pioneer factors minimize their search times on nucleosomal DNA by compensating their nucleosome association rates by dissociation rates. A fine balance between the two presents a tradeoff between their nuclear mobility and error associated with the search process.

The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability.; The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability.

Delivery of therapeutic genes into retina is proving to reverse degeneration and restore vision, however, viral vector-based gene delivery is prone to immunorejection, inflammatory/immune-response and non-targeted. Here, we report non-viral gene delivery and expression of opsin encoding genes in mouse retina in-vitro and in-vivo by use of pulsed femtosecond laser microbeam. In-vitro patch clamp recording of the opsin-sensitized retinal cells and visually evoked in-vivo electrical recording from laser-transfected eye of mouse with degenerated retina showed functional response. The ultrafast laser based naked gene delivery showed minimal damage and reliable expression of therapeutic opsin in cell membrane of the selected cells and in targeted retinal region. Laser based "naked DNA gene therapy" in a spatially targeted manner will pave the way for treatment of inherited retinal diseases. This article is protected by copyright. All rights reserved.

Intradermal administration of naked DNA with a conventional needle syringe is a simple and inexpensive method to expose an encoded antigen to the dermal immune system. We aimed to enhance intradermal gene expression with a pyro-drive jet injector using pig skin, which is similar in structure and biomechanical properties to human skin. When Cy3-labeled plasmid (pCy3) was applied to pig skin with the jet injector, pCy3 was distributed preferentially in the intradermal tissue. Precise localization analysis revealed that pCy3 was also detected in the intracellular nucleus, and the frequency was substantially higher with the jet injector than with a needle syringe. When a luciferase expression plasmid (pLuc) was injected transdermally, the luciferase activity was 100-fold higher with the jet injector than with a needle syringe. Furthermore, immunohistochemistry analysis showed that the epidermis was positive for luciferase protein expression. These data indicate that the jet injector facilitates stable intradermal administration, resulting in more efficient gene expression compared to that with conventional syringe methods. Thus, intradermal administration of an antigen-expression plasmid with the pyro-drive jet injector may provide a clinically viable method for future gene therapy.

Herpesvirus genomes enter the eukaryotic nucleus as large linear double stranded DNA molecules that are free of any proteins (naked DNA). Once inside the nucleus, the HSV-1 genomes immediately associate with proteins that will be instrumental in the organization and regulation of these genomes. These initial interactions are thought to determine the fate of the infecting genomes. In general, the host cell has evolved several mechanisms to suppress viral genomes and induce latent or abortive infections. On the other hand, the virus has evolved to use viral and cellular factors to promote lytic infection. Recent findings suggest that not all viral genomes in the infected nucleus will develop progeny and that not all genetically identical cells will support successful virus propagation. Thus, the decision between different fates of infection is determined at both single-cell and single-genome levels. Here we summarize current knowledge on the conditions and interactions that lead to each outcome and discuss the unknown determinants.

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes., eLife digest Genome editing is a powerful tool used across a wide range of biomedical research. There are several different techniques used, depending on the type of edit being made, and one known as homology-directed repair – or HDR for short – is a common technique for precisely inserting large sections of DNA, such as those needed to make desired proteins in cells. HDR takes advantage of the cell’s mechanisms for repairing damage to DNA if both strands of the DNA double helix are broken. The mechanism relies on a DNA template to stitch the strands back together. To insert or replace a new DNA sequence, scientists can add a customized piece of DNA of their choosing to the cell so that it might be incorporated into the genome. However, HDR is not very efficient, and the success rate is often less than a few percent. In HDR gene editing, the DNA template is typically added as purified, or ‘naked’, DNA. However, the natural form of DNA in cells, known as chromatin, is where the DNA helix is wrapped around a cluster of proteins known as histones. Cruz-Becerra and Kadonaga tested the idea that DNA in the form of chromatin might be more effective as a template for HDR than naked DNA. The two approaches were compared to see which was better at inserting a sequence at three different locations in the genome of lab-grown human cells. In these experiments, the chromatin templates were 2.3- to 7.4-fold more efficient than the naked DNA. Also, the DNA in human cells is organized as pairs of chromosomes, and chromatin was better than naked DNA for editing both copies of the chromosome pairs rather than only one of them. In addition, the chromatin is potentially less toxic to the cells. Cruz-Becerra and Kadonaga hope that this will be useful for increasing the success rate of HDR experiments and potentially other methods of gene editing in the future.

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.

Finding and exploiting safe and high-efficiency gene carriers have always been critical tasks for gene therapy. In this work, novel GSH-triggered degradable organosilica nanoparticles grafted with guanidinated-fluorinated α-polylysine (o-SiNP-GF) are prepared to be studied as gene carriers. The organosilica matrix of o-SiNP-GF is synthesized through the hydrolysis and condensation of 1,2-bis(triethoxysilyl)ethane (BTSE) and bis[3-(triethoxysilyl)propyl]tetrasulfide (BTSPTS). The o-SiNP-GF nanoparticles have a size of about 20 nm. They possess a positive zeta potential of 42 mV in PBS (pH 7.4) and can be disintegrated in the presence of GSH. The cytotoxicity and DNA-binding ability of o-SiNP-GF, as well as in vitro gene transfection performance of DNA/o-SiNP-GF complexes, have been investigated using enhanced green fluorescent protein plasmid (pEGFP) as the DNA model. MTT assay shows that the cytotoxicity of o-SiNP-GF is very low even at a concentration up to 800 μg mL-1. The o-SiNP-GF nanoparticles can effectively bind to pEGFP through a complex coacervation method. The in vitro transfection efficiency of pEGFP/o-SiNP-GF complexes in 293T cells is up to 94.7% at the N/P ratio of 10, much higher than that of pEGFP/PEI complexes. Luciferase gene and fibroblast growth factor (FGF2) gene are also used as the DNA models to study the in vivo gene transfection performance of the o-SiNP-GF carrier by bioluminescence imaging and the evaluation of the healing rate of a mouse wound, respectively. Compared with naked DNA and DNA/PEI complexes, DNA/o-SiNP-GF complexes show much higher in vivo transfection efficiency. This work not only provides a way to prepare novel GSH-triggered degradable organosilica nanoparticles of size less than 50 nm, but also proves that the modification of guanidinated-fluorinated α-polylysine is an effective method to improve the efficiency of gene carriers.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA’s helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2’s methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.

Presently, more than 3400 genes have been associated with diseases [1], some of these pathologies are debilitating, mortal, and without any effective therapeutic options, and this number is expected to increase in the next decade as genomic studies advance. Gene therapy is a therapeutic strategy that seeks to target the genes behind the disorders in order to cure patients. Thus, this approach holds the promise of establishing therapies that treat the causes, rather than the symptoms, by manipulating deficient genes, removing or silencing pathologic genes, or adding missing genes. However, these ambitious strategies have been hampered by several problems, such as the lack of safety and efficiency of some of the developed approaches, which can be explained by the very nature of most gene-manipulating tools. Naked DNA plasmids, for example, are rapidly degraded in biological fluids, they are unable to efficiently cross the cellular membranes and therefore reach the target cells and they activate the immune system, which is programmed to identify and eliminate vehicles containing foreign genetic information [2]. Thus, although a large number of preclinical and clinical studies have been performed using naked nucleic acids, the use of delivery vectors yields better results, namely because they protect the nucleic acids from nuclease degradation and increase intracellular delivery. Still, despite the big efforts made in the last decades with gene therapy strategies, very few gene therapy-based drugs or therapeutic products have reached the market, revealing that much still has to be done in the field of vectors for gene therapy.

DNA is constantly attacked by different damaging agents; therefore, it requires frequent repair. On the one hand, the base excision repair (BER) system is responsible for the repair of the most frequent DNA lesions. On the other hand, the formation of poly(ADP-ribose) is one of the main DNA damage response reactions that is catalysed by members of the PARP family. PARP1, which belongs to the PARP family and performs approximately 90% of PAR synthesis in cells, could be considered a main regulator of the BER process. Most of the experimental data concerning BER investigation have been obtained using naked DNA. However, in the context of the eukaryotic cell, DNA is compacted in the nucleus, and the lowest compaction level is represented by the nucleosome. Thus, the organization of DNA into the nucleosome impacts the DNA-protein interactions that are involved in BER processes. Poly(ADP-ribosyl)ation (PARylation) is thought to regulate the initiation of the BER process at the chromatin level. In this review, we focus on the mechanisms involved in BER in the nucleosomal context and the potential effect of PARylation, which is catalysed by DNA-dependent PARP1, PARP2 and PARP3 proteins, on this process.

International audience; The Deinococcus radiodurans protein HU (DrHU) was shown to be critical for nucleoid activities, yet its functional and structural properties remain largely unexplored. We have applied atomic force microscopy (AFM) imaging to study DrHU binding to pUC19-DNA in vitro and analyzed the topographic structures formed at the nanoscale. At the single-molecule level, AFM imaging allows visualization of super-helical turns on naked DNA surfaces and characterization of free DrHU molecules observed as homodimers. When enhancing the molecular surface structures of AFM images by the Laplacian weight filter, the distribution of bound DrHUs was visibly varied as a function of the DrHU/DNA molar ratio. At a low molar ratio, DrHU binding was found to reduce the volume of condensed DNA configuration by about 50%. We also show that DrHU is capable of bridging distinct DNA segments. Moreover, at a low molar ratio, the binding orientation of individual DrHU dimers could be perceived on partially "open" DNA configuration. At a high molar ratio, DrHU stiffened the DNA molecule and enlarged the spread of the open DNA configuration. Furthermore, a lattice-like pattern could be seen on the surface of DrHU-DNA complex, indicating that DrHU multimerization had occurred leading to the formation of a higher order architecture. Together, our results show that the functional plasticity of DrHU in mediating DNA organization is subject to both the conformational dynamics of DNA molecules and protein abundance.

Nucleic acid immunization has recently exhibited a great promise for immunotherapy of various diseases. However, it is now clear that powerful strategies are imminently needed to improve their efficiency. In this regard, whole bacteriophage particles have been described as efficient DNA vaccine delivery vehicles, capable of circumventing the limitations of naked DNA immunization. Moreover, phage particles could be engineered to display specific peptides on their surfaces. Given these inherent characteristics of phages, we have designed a novel hybrid phage-DNA immunization vector using both M13 and pAAV plasmid elements. Following the construction and in vitro confirmation of the designed vectors, they were used for comparative mice immunization, carrying the same DNA sequence. The results indicated the efficacy of the designed hybrid phage particles, to elicit higher humoral immunity, in comparison to conventional DNA-immunization vectors (pCI). In light of these findings, it could be concluded that using adeno-associated virus (AAV) expression cassette along with displaying TAT peptide on the surface of the phage particle could be deemed as an appealing strategy to enhance the DNA-immunization and vaccination efficacy.

Stenostomum are tiny planarians of the phylum Platyhelminthes that reproduce asexually. We transfected these worms using plasmids containing a gfp reporter gene. Here we show that they can express genes present in plasmids carried by bacteria and those that are encoded by naked DNA, such as plasmids or PCR fragments, transfected by electroporation; they can also express genes taken up during feeding. The microbiome associated with worm maintenance was evaluated, and the results indicated that when a plasmid is maintained in the microbiome, gfp gene expression is stable. When genes originate from naked DNA or bacteria not maintained in the microbiome, GFP expression is transient. Therefore, changes in the microbiome can modify the ability of worms to express foreign genes. In stable GFP-expressing worms, NSG showed that the gfp gene was maintained in the plasmid and was not integrated into the chromosome. These results suggest that, at least for some organisms such as flatworms, the expression of genes provided by the microbiome or the environment can be considered among the potential sources of phenotypic plasticity, which can have implications for evolvability.