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The molecular pathogenesis of psoriasis is characterized in part by breakdown of immune tolerance to self-DNA. Recent work demonstrates that the human antimicrobial peptide (AMP) LL37 overexpressed in psoriasis organizes naked DNA into periodic nanocrystals to potently hyperactivate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDCs). Interestingly, a subset of self-DNA in psoriatic lesions remain bound to histones as histone-DNA nucleosome core particles (NCPs) released from neutrophil extracellular traps (NETs). At present, it is unknown how NET components like AMPs interact with NCPs, and whether AMP-NCP complexes form structures compatible with TLR9 activation in psoriasis. Here, we combine synchrotron X-ray scattering, cryo-electron microscopy, and computer simulations to demonstrate that under a broad range of conditions, NCPs stack into columns that present periodically arranged dsDNA ligands like threads on a screw, allowing for optimal interdigitation with clusters of TLR9. Remarkably, simulations and electron microscopy indicate that the superhelical pitch of DNA wrapped around the NCP column relaxes to a value that is well-matched with the steric size of TLR9, which predicts strong immune activation. Taken together, our results suggest that AMPs can remodel the structural organization of nucleosomes from NETs into potent amplifiers of inflammation. Preliminary immune activation experiments will be presented.

Despite extensive studies of the geometry of protein/DNA interfaces the understanding of the affinity and specificity of the protein/DNA interactions remains elusive. We present a novel approach to geometric analysis of protein/nucleic acid interfaces that is based on classification of their local conformations. Protein structures are divided into a series of pentapeptide fragments and each is assigned one of 16 conformers or ‘protein blocks’ [de Brevern et al. Proteins 41, 271 (2000)]. Similarly, each DNA step (unit [base]sugar-phosphate-sugar[base]) is assigned one of ∼20 DNA conformers [Svozil et al. NAR 36, 3690 (2008)]. Significantly, local structures classified into distinct conformers can be represented by symbols so that they can be analyzed more easily than complicated 3D objects. Size of the fragments used for the classification allows analysis of structural features of the interface at a scale intermediate between too detailed interatomic contacts on one side and too crude protein motifs (helix-turn-helix, Zn-finger, ...) on the other. We will present correlations between protein and DNA conformers at their interface from more than 15 hundred protein/DNA crystal structures broken by various criteria of the analyzed structures as protein functional classification, e.g. in GO or Pfam or by crystallographic properties, resolution or overall structure quality.Acknowledgments. This work was supported by Czech-France collaboration Barrande (MEB021032) and Czech Science Foundation (P305/10/2184).

Recombinant DNA vaccines represent a novel method for generating in situ expression of vaccine antigens. Intramuscular injections of naked DNA are able to elicit potent humoral and cellular immune responses but still numerous factors limit the immunogenicity of DNA vaccines. Co-expression of cytokines with antigen encoding genes in DNA vectors can improve the immune responses and modify Th1/Th2 balance. In this study, the immunomodulatory effect of Interleukin 22 (IL-22) as an adjuvant was studied by DNA vaccination with S. Typhi Heat shock protein 60 (HSP60/GroEL) in mice. Further, DNA construct of IL-22 gene fused with GroEL was developed and immunization studies were carried out in mice. DNA vaccination with GroEL alone stimulated humoral and cell-mediated immune responses. Co-immunization (IL-22+GroEL) further resulted in increase in T-cell proliferative responses, antibody titres (IgG, IgG1, IgG2a) and secretion of IFNγ (Th1), IL-1β and Th2 (IL-4, IL-6) cytokines. Co-expression (IL-22-GroEL DNA) also promoted antibody titres and cytokine levels were significantly higher as compared to co-immunized group. A reduction in bacterial load in spleen, liver and intestine was seen in all the immunized groups as compared to control, with least organ burden in fusion DNA construct group (co-expression). Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL-22-GroEL co-expressing DNA vector as compared with plasmid encoding GroEL only (50-60%) or co-immunization group (75-80%). This study thus shows that co-expression of IL-22 and GroEL genes enhances the immune responses and protective efficacy, circumventing the need of any adjuvant.

8-Oxoguanine (oxoG) is an abundant product of oxidative DNA damage. It is removed by repair glycosylases, but exactly how the enzymes recognize oxoG in the large surplus of undamaged bases is not fully understood. The lesion may induce changes in the properties of naked DNA that facilitate the recognition. In this work, we assess the effect of oxoG on DNA structure and mechanical deformability. We performed extensive unrestrained, atomic resolution molecular dynamics simulations to parametrize a nonlocal, rigid base mechanical model of DNA. Our data indicate that oxoG induces unwinding of the base pair step at the 5'-side of the lesion. This brings the damaged DNA closer to its conformation in the initial complex with bacterial glycosylase MutM. The untwisting is partially caused by different BII substate populations and is further enhanced by the base-sugar repulsion within oxoG. On the other hand, our analysis shows that damaged and undamaged DNA have very similar harmonic stiffness. These results suggest an indirect readout component of the MutM-DNA initial complex formation. They also help one to understand the effect of oxoG on the formation of nucleosomes and looped gene regulatory complexes.

Variations of the shape and polarity of the DNA grooves caused by changes of the DNA conformation play an important role in the DNA readout. Despite the fact that non-canonical trans and gauche- conformations of the DNA backbone angle γ (O5′–C5′–C4′–C3′) are frequently found in the DNA crystal structures, their possible role in the DNA recognition has not been studied systematically. In order to fill in this gap, we analyze the available high-resolution crystal structures of the naked and complexed DNA. The analysis shows that the non-canonical γ angle conformations are present both in the naked and bound DNA, more often in the bound vs. naked DNA, and in the nucleotides with the A-like vs. the B-like sugar pucker. The alternative angle γ torsions are more frequently observed in the purines with the A-like sugar pucker and in the pyrimidines with the B-like sugar conformation. The minor groove of the nucleotides with non-canonical γ angle conformation is more polar, while the major groove is more hydrophobic than in the nucleotides with the classical γ torsions due to variations in exposure of the polar and hydrophobic groups of the DNA backbone. The propensity of the nucleotides with different γ angle conformations to participate in the protein–nucleic acid contacts in the minor and major grooves is connected with their sugar pucker and sequence-specific. Our findings imply that the angle γ transitions contribute to the process of the protein–DNA recognition due to modification of the polar/hydrophobic profile of the DNA grooves.

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

As compared with peptide- or protein-based vaccines, naked DNA vectors and even traditional attenuated or inactivated virus vaccines, virus-like particles (VLPs) are an attractive vaccine platform, as they offer a combination of safety, ease of production and both high-density B-cell epitope display and intracellular presentation of T-cell epitopes that induce potent humoral and cellular immune responses, respectively. Indeed, HPV vaccines based on VLP production by recombinant expression of major capsid antigen L1 in yeast (Gardasil(®), MerckCo., NJ, USA) or insect cells (Cervarix(®), GlaxoSmithKline, London, UK) have been licensed for the prevention of cervical and anogenital infection and disease associated with the genotypes targeted by each vaccine. However, these HPV vaccines have not been demonstrated as effective to treat existing infections, and efforts to develop a therapeutic HPV vaccine continue. Furthermore, current HPV L1-VLP vaccines provide type-restricted protection, requiring highly multivalent formulations to broaden coverage to the dozen or more oncogenic HPV genotypes. This raises the complexity and cost of vaccine production. The lack of access to screening and high disease burden in developing countries has spurred efforts to develop second-generation HPV vaccines that are more affordable, induce wider protective coverage and offer therapeutic coverage against HPV-associated malignancies. Given the previous success with L1-VLP-based vaccines against HPV, VLPs have been also adopted as platforms for many second-generation HPV and non-HPV vaccine candidates with both prophylactic and therapeutic intent. In this article, the authors examine the progress and challenges of these efforts, with a focus on how they inform VLP vaccine design.

Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk. Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 µg DNA per pulse in the first experiment and increments of 10 µg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05. Results: The results showed no significant difference among three first concentrations except for group 1 (10 µg/pulse) and group 3 (30 µg/pulse), but there was a significant difference between these groups and the fourth group (p

Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes. pUMVC3-hIL12 loaded Low Molecular Weight chitosan/β-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line. All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA. LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

The short half-lives due to the enzymatic degradation in blood, the lack of tissue targetability and the incapability to passively diffuse across the plasma membrane and smoothly traffic across the harsh intracelluar environment are the major shortcomings for nucleic acid-based potential therapeutics, such as recombinant plasmid and antisense oligonucleotides or small interferring RNA (siRNA). Plasmid DNA containing a gene of interest could have immense impact as a promising therapeutic drug for treating genetic as well as acquired human diseases at the molecular level with high level of efficacy and precision. Thus both viral and non-viral synthetic vectors have been developed in the past decades to address the aforementioned challenges of naked DNA. While in the viral particles plasmid DNA is integrated into the viral genome, in most non-viral cases the DNA being anionic in nature is electrostatically associated with a cationic lipid or polymer forming lipoplex or polyplex, respectively, or a cationized inorganic gold, silica or iron oxide particle. Due to the potential immunogenicity and carcinogenicity issues with the viral particles, non-viral vectors have drawn much more attention for the clinical evaluation. However, the main concern of using non-biodegradable particles, specially the inorganic ones, is the adverse effects owing to their long term interactions with body components. We have recently developed biodegradable pH-sensitive inorganic nanoparticles of Mg/CaPi and carbonate apatite for efficient transgene delivery to primary, cancer and embryonic stem cells, by virtue of their high affinity binding with the DNA, ability to contact the cell membrane by ionic or ligand-receptor interactions and fast dissolution kinectis in endosomal acidic pH facilitating release of the DNA from the dissolving particles and also from the endosomes.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis.

Traditional methods for addressing chronic wounds focus on correcting dysfunction by controlling extracellular elements. This review highlights technologies that take a different approach - enhancing chronic wound healing by genetic modification to wound beds. Featured cutaneous transduction/transfection methods include viral modalities (ie adenoviruses, adeno-associated viruses, retroviruses and lentiviruses) and conventional non-viral modalities (ie naked DNA injections, microseeding, liposomal reagents, particle bombardment and electroporation). Also explored are emerging technologies, focusing on the exciting capabilities of wound diagnostics such as pyrosequencing as well as site-specific nuclease editing tools such as CRISPR-Cas9 used to both transiently and permanently genetically modify resident wound bed cells. Additionally, new non-viral transfection methods (ie conjugated nanoparticles, multi-electrode arrays, and microfabricated needles and nanowires) are discussed that can potentially facilitate more efficient and safe transgene delivery to skin but also represent significant advances broadly to tissue regeneration research.

Naked plasmid DNA and DNA/liposome complexes are currently being considered as gene therapy treatments for cystic fibrosis (CF) pulmonary disease. Current methods of gene delivery to the airways result only in transient correction of the CF ion transport defect, and disease treatment is likely to require repeated administrations of vector. However, it is unclear if repeat administration will be tolerated by CF individuals. Technologies including TaqMan (Applied Biosystems) real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) can be used to determine the efficacy of gene transfer formulations. TaqMan RT-PCR assays were designed and optimised to detect plasmid vector-derived and endogenous gene expression. Subsequently, these assays were used to quantify vector-derived mRNA after delivery of naked DNA and DNA/liposome formulations expressing human and murine cystic fibrosis transmembrane conductance regulator (CFTR) to the mouse airways. Vector-derived mRNA was detected in samples following the delivery of naked DNA or DNA/liposomes to the mouse airways, and no reduction in vector-derived mRNA was observed upon repeat administration, a finding that is consistent with the murine and human CFTR being tolerated by the mouse. Although it remains to be seen if CF patients can tolerate long-term expression of wild-type CFTR, these data demonstrate that TaqMan RT-PCR is an effective tool to accurately quantify transgene expression in the airways.

Development of a vaccine against human immunodeficiency virus type 1 (HIV-1) is the main hope for controlling the acquired immunodeficiency syndrome pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell-mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. This review focuses on the stimulation of HIV-specific T cells and discusses in the context of the current 'state-of-art' of DNA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development.

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-γ response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-γ response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-γ responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-γ response was mainly due to CD4+ T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8+ T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.

Following many years of research using isolated human tissues and animal models, sufficient knowledge concerning rheumatoid arthritis has accumulated so that novel immunotherapies have been proposed. Biological agents are being tested in clinical trials and include antibodies to T cells and cytokines. Currently the most promising of these is intravenously administered neutralizing anti-TNF antibody. In order to establish disease modification, however, therapy needs to be delivered continuously over the long term. The prospect of delivering cytokine inhibitors as genetic material (naked DNA), viruses or in engineered autologous cells is considered as one option for achieving this goal. We compare two strategies, firstly, using immobile cells such as fibroblasts, myoblasts or keratinocytes, and secondly, the migratory cells of the immune system. The former provides a reservoir of systemic delivery of the therapeutic protein whereas the latter provides targeted delivery determined by the antigen specificity of the immune cells. Early validation has begun in animal models of rheumatoid arthritis.

Naked nuclear DNA is easily sheared. Two general methods are described for preparing intact DNA in a stable form that can be pipetted without breaking it. Cells are encapsulated in agarose microbeads and then lysed in a non-ionic detergent (i.e., Triton X-100) and 2 M NaCl or an ionic detergent (e.g., sodium or lithium dodecyl sulphate) in low salt. Most cellular protein and RNA then diffuse out through pores in the beads to leave encapsulated and naked DNA which is nevertheless accessible to enzymes and other probes. Remarkably, considerable structure is preserved since the DNA is supercoiled and chromosomes retain their shape.

More than 75 million people has been infected HIV and it is responsible for nearly 36 million deaths on a global scale. As one of the deadliest infectious diseases, HIV is becoming the urgent issue of the global epidemic to tackle. In order to settle this problem from the source, some effective prevention strategies should be developed to control the pandemic of HIV. Vaccines, especially DNA vaccines, could be the optimal way to control the spread of HIV due to the unparalleled superiority that DNA vaccines could generate long-term humoral and cellular immune responses which could provide protective immunity for HIV. But the naked DNA could hardly enter into cells and is easily degraded by DNases and lysosomes, so designing effective delivery system is a promising strategy. Since delivery system could be constructed to promote efficient delivery of DNA into mammalian cells, protect them from degradation, and also could be established to be a target system to arrive at certain position of expectation. The current review discusses the potential of various nano-delivery vehicles/adjuvants such as polymer, lipid, liposome, peptide and inorganic material in improving efficiency of diverse modalities available for HIV DNA vaccines.

The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520±12nm to 464±25nm) while the PDI increased after lyophilization (0.094±0.017 to 0.220±0.004). In addition. The mean particle size of vesicles increases when DNA is incorporated to the liposomes (673±27nm). According to the Scanning Electron Microscopy (SEM) and transmission electron microscopy (TEM) images, the spherical shape of liposomes confirmed their successful preservation and reconstitution from the powder. It was found that liposomal formulation has enhanced transfection considerably compared to the naked DNA as negative control. Finally, liposomal formulation in this research had a better function than Lipofectamine® 2000 as a commercialized product because the cellular activity (cellular protein) was higher in the prepared lipoplex than Lipofectamine® 2000.

Background Idarubicin (IDA) is one of clinically important anticancer drugs belonging to the anthracycline antibiotic family. The aim of this study was to examine DNA damage induced by NADPH cytochrome P450 reductase (CPR)-activated IDA in human sensitive MCF7 and multidrug resistant MCF7/DOX500 (overexpressing P-gp) breast adenocarcinoma cells. Methods The evaluation of DNA fragmentation caused by single strand breaks (SSB) and double strand breaks (DSB) was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) test. Additionally, DSB formation was examined using H2AX histone phosphorylation assays. Results It was found that IDA alone and CPR-activated used at IC90 caused a higher level of DNA strand breaks in sensitive MCF7 cells detected by TUNEL assessments (p = 0.0011 for IDA alone and p = 0.0109 for IDA reductively activated, Kruskal-Wallis test) and γ-H2AX-positive staining (p = 0.0003 for IDA alone and p = 0.0193 for IDA reductively activated, Kruskal-Wallis test) than in multidrug resistant MCF7/DOX500 cells. However, no changes were observed in the percentage of TUNEL-positive and DSB-positive cells for MCF7 as well as MCF7/DOX500 cells in the case of IDA alone and the drug pretreated in the presence of the activating system. Conclusions The obtained results suggest that CPR-activation of IDA does not significantly change the cellular DNA damage response of studied sensitive MCF7 and multidrug resistant MCF7/DOX500 breast cancer cells, even if the results concerning the interaction of IDA undergoing CPR activation with naked DNA showed the important differences in comparison with the drug alone (non-activated).

Viral and non-viral vectors have been used as methods of delivery in gene therapy for many CNS diseases. Currently, viral vectors such as adeno-associated viruses (AAV), retroviruses, lentiviruses, adenoviruses and herpes simplex viruses (HHV) are being used as successful vectors in gene therapy at clinical trial levels. However, many disadvantages have risen from their usage. Non-viral vectors like cationic polymers, cationic lipids, engineered polymers, nanoparticles, and naked DNA offer a much safer option and can therefore be explored for therapeutic purposes.This review discusses different types of viral and non-viral vectors for gene therapy and explores clinical trials for CNS diseases that have used these types of vectors for gene delivery. Highlights include non-viral gene delivery and its challenges, possible strategies to improve transfection, regulatory issues concerning vector usage, and future prospects for clinical applications.Transfection efficiency of cationic lipids and polymers can be improved through manipulation of molecules used. Efficacy of cationic lipids is dependent on cationic charge, saturation levels, and stability of linkers. Factors determining efficacy of cationic polymers are total charge density, molecular weights, and complexity of molecule. All of the above mentioned parameters must be taken care for efficient gene delivery.

Many transition metal complexes have unique physicochemical properties that can be efficiently exploited in medicinal chemistry for cancer treatment. Traditionally, double-stranded DNA has been assumed to be the main binding target; however, recent studies have shown that nucleosomal DNA as well as proteins can act as dominant molecular binding partners. This has raised new questions about the molecular determinants that govern DNA versus protein binding selectivity, and has offered new ways to rationalize their biological activity and possible side effects. To address these questions, molecular simulations at an atomistic level of detail have been used to complement, support, and rationalize experimental data. Herein we review some relevant studies—focused on platinum and ruthenium compounds—to illustrate the power of state-of-the-art molecular simulation techniques and to demonstrate how the interplay between molecular simulations and experiments can make important contributions to elucidating the target preferences of some promising transition metal anticancer agents. This contribution aims at providing relevant information that may help in the rational design of novel drug-discovery strategies. MOE (Min. of Education, S’pore) Published version

To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B2 to duplex DNA could advance its design as an antitumor agent.

Background Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer as well as subsets of anogenital and oropharyngeal cancers. The two HPV viral oncoproteins, E6 and E7, are uniquely and consistently expressed in all HPV infected cells and are therefore promising targets for therapeutic vaccination. Both recombinant naked DNA and protein-based HPV vaccines have been demonstrated to elicit HPV-specific CD8+ T cell responses that provide therapeutic effects against HPV-associated tumor models. Here we examine the immunogenicity in a preclinical model of priming with HPV DNA vaccine followed by boosting with filterable aggregates of HPV 16 L2E6E7 fusion protein (TA-CIN). Results We observed that priming twice with an HPV DNA vaccine followed by a single TA-CIN booster immunization generated the strongest antigen-specific CD8+ T cell response compared to other prime-boost combinations tested in C57BL/6 mice, whether naïve or bearing the HPV16 E6/E7 transformed syngeneic tumor model, TC-1. We showed that the magnitude of antigen-specific CD8+ T cell response generated by the DNA vaccine prime, TA-CIN protein vaccine boost combinatorial strategy is dependent on the dose of TA-CIN protein vaccine. In addition, we found that a single booster immunization comprising intradermal or intramuscular administration of TA-CIN after priming twice with an HPV DNA vaccine generated a comparable boost to E7-specific CD8+ T cell responses. We also demonstrated that the immune responses elicited by the DNA vaccine prime, TA-CIN protein vaccine boost strategy translate into potent prophylactic and therapeutic antitumor effects. Finally, as seen for repeat TA-CIN protein vaccination, we showed that the heterologous DNA prime and protein boost vaccination strategy is well tolerated by mice. Conclusions Our results provide rationale for future clinical testing of HPV DNA vaccine prime, TA-CIN protein vaccine boost immunization regimen for the control of HPV-associated diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0080-z) contains supplementary material, which is available to authorized users.

A fluorophore displaying aggregation-induced emission was introduced at the terminus of branched polyethylenimine (PEI). The formulated polyplex not only demonstrated an improved safety profile and preserved transfection activity but also importantly indicated that the uncomplexed naked DNA rather than the polyplexes translocated into the nucleus.

Gene therapy is a newer approach in the twenty-first century which developed through the advancement of molecular biology and Human Genome Project. It has emerged as a key future strategy to cure both T1DM and T2DM. Gene therapy is accomplished by introducing DNA into cells, which can be carried out by several methods. Viral vectors (recombinant viruses) and non-viral vectors (naked DNA or DNA complexes) are mostly used in such process. Gene therapy usually targets the β cells of pancreatic islet or the insulin sensitivity toward the peripheral tissues. Introduction of DNA into target cells artificially should be stable and functional; it is also necessary that the target cells should accept the new DNA properly for natural reproduction function. Usually, ex vivo engineering and in vivo delivery methods are used in gene therapy. In ex vivo method, a cell will be isolated from the host and used as a transgene factory, and genetic manipulation is carried out in a laboratory. Cell expansion may or may not be part of it. This strategy is mostly employed in gene therapy. On the other hand, in vivo gene transfer method genetic modification is carried out in situ using vector-mediated delivery of genetic material into specific target organ. It is a difficult method due to selection of gene delivery vehicle that becomes an issue (Welsh 2000; Giannoukakis and Trucco 2003; Creusot and Fathman 2004; Bertolaso et al. 2010).

Purpose: We have clinically evaluated a DNA fusion vaccine to target the HLA-A*0201–binding peptide CAP-1 from carcinoembryonic antigen (CEA605–613) linked to an immunostimulatory domain (DOM) from fragment C of tetanus toxin. Experimental Design: Twenty-seven patients with CEA-expressing carcinomas were recruited: 15 patients with measurable disease (arm-I) and 12 patients without radiological evidence of disease (arm-II). Six intramuscular vaccinations of naked DNA (1 mg/dose) were administered up to week 12. Clinical and immunologic follow-up was up to week 64 or clinical/radiological disease. Results: DOM-specific immune responses demonstrated successful vaccine delivery. All patients without measurable disease compared with 60% with advanced disease responded immunologically, while 58% and 20% expanded anti-CAP-1 CD8+ T cells, respectively. CAP-1–specific T cells were only detectable in the blood postvaccination but could also be identified in previously resected cancer tissue. The gastrointestinal adverse event diarrhea was reported by 48% of patients and linked to more frequent decreases in CEA (P < 0.001) and improved global immunologic responses [anti-DOM responses of greater magnitude (P < 0.001), frequency (P = 0.004), and duration] compared with patients without diarrhea. In advanced disease patients, decreases in CEA were associated with better overall survival (HR = 0.14, P = 0.017). CAP-1 peptide was detectable on MHC class I of normal bowel mucosa and primary colorectal cancer tissue by mass spectrometry, offering a mechanistic explanation for diarrhea through CD8+ T-cell attack. Conclusions: Our data suggest that DNA vaccination is able to overcome peripheral tolerance in normal and tumor tissue and warrants testing in combination studies, for example, by vaccinating in parallel to treatment with an anti-PD1 antibody. Clin Cancer Res; 22(19); 4827–36. ©2016 AACR.

Chagas disease is an important neglected disease affecting thousands of people in the Americas. Novel strategies for prophylactic and therapeutic vaccines against the etiological agent, the intracellular protozoan Trypanosoma cruzi, are urgently needed. Vaccines based on attenuated virus and bacteria as a foreign DNA delivery system represent a strong advantage over naked DNA-based vaccines. Here we describe the use of attenuated Salmonella carrying a eukaryotic expression plasmid encoding a T. cruzi antigen. The main advantages of the methodology are the oral administration of the Salmonella-based vaccine and the induction of a strong humoral and cell-mediated immune response at both mucosal and systemic level, favored by the adjuvant effect elicited by the bacteria pathogen-associated molecular patterns.

Gene therapy refers to any therapy in which the therapeutic entity is a nucleic acid. Gene therapy most commonly delivers a DNA sequence, the transcription of which produces a protein to perform a therapeutic function. Other approaches have delivered RNA, short interfering RNA (siRNA), RNA aptamers, nucleases (e.g., to knock out host cell genes or to facilitate “editing” of genomic DNA), or sequences to express antigens that produce specific immunization. Considered most broadly, there are two approaches to introduce therapeutic genes. The first is to directly introduce the nucleic acid sequences of interest into the target tissue in vivo. Transfer of the nucleic acid may or may not employ a delivery vehicle, referred to as a vector. Naked DNA or RNA transfer is in general inefficient unless mediated by a virus or other vector. In nature, viruses have evolved mechanisms to enter cells and deliver their viral DNA (or RNA) to the nucleus of cells, where they hijack the host cell’s machinery to transcribe and translate the virus’ genetic payload. Dozens of viruses have been investigated as potential gene delivery vectors. The second approach is to introduce the gene of interest ex vivo into cells (whether allogeneic or autologous) in culture, expand if desired the cells that express the gene efficiently, and then introduce the cells with their transgenic payload into the host. The goal of correcting hemophilia, the most common severe bleeding disorder, has been approached using multiple variations of in vivo vectors and ex vivo cell-based strategies over the last quarter century, ultimately leading to partial correction of hemophilia B in a human clinical trial (St. Louis and Verma 1988; Palmer et al. 1989; Nathwani et al. 2011).

17 páginas, 6 figuras. En la versión online contiene 3 figuras y 1 tabla en información suplemetaria, INTRODUCTION: Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. MATERIAL AND METHODS: A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. RESULTS: Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p, This study has been supported by a project from the Spanish Ministry of Economy and Competitiveness (SAF2011-27002).

Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen.

Gene therapy is the therapeutic delivery of nucleic acids for treatment of genetic diseases into patient’s cells. They might be either expressed as proteins, interfere with the expression of proteins, or possibly even correct genetic mutations. But this may be approached by two different ways: (1) ex vivo, which is when cells are modified outside the body and then transplanted back again. Cells from the patient’s blood or bone marrow are removed and grown in the laboratory to be genetically modified by different molecular biology techniques on a dish. This also involves the so-called cell therapy which is now widely used. (2) But also gene therapy could be approached by in vivo therapy, in which the gene is transferred into the cells inside the patient's body. However, an efficient transfer of the genetic material into a cell is necessary to achieve the desired therapeutic effect. The therapeutic molecule is packaged into vehicles called vectors, but the “vector” is used to carry the nucleic acid inside the cells within the body, facilitating the transfer of genetic information into a cell. The simplest way to perform gene therapy involves the use of therapeutic naked DNA that encodes a functional gene to replace a mutated version of the defective gene. In most cases, a relatively large piece of genetic material (>1 kb) is required which includes the promoter sequences that activate the expression of the gene, the coding sequences that direct the production of a specific protein, and signaling sequences that direct RNA processing such as polyadenylation. In this way, no vector would be required but the efficiency of this methodology was demonstrated to be low. Vectors can be divided into viral and nonviral delivery systems. The two major methods are those that use biological nanoparticles (mainly recombinant viruses or also called viral vectors, although recently exosomes are taking a place among the biological vectors for genetic transfer) and those that use chemical nanoparticles on DNA complexes or naked DNA (nonviral methods). In this chapter, we will review both categories and their applications in clinical trials.

The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual's cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer.

Cationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose (QHEC) derivatives were studied as gene carriers for their efficient DNA binding abilities. All QHECs could form stable QHEC/DNA complexes and resist the degradation of DNase I. And the dynamic light scatter (DLS) results showed that all QHEC/DNA complexes could form compact particles. These QHEC/DNA complexes exhibited effective transfection abilities in comparison to the naked DNA. The cytotoxicities of QHEC and QHEC/DNA complexes were also evaluated in four cell lines which were relatively low compared with 25 kDa bPEI. All results indicated that these quaternized hydroxyethylcelluloses could be used as potential gene delivery vectors.

Surra, caused by Trypanosoma evansi, is an economically important veterinary disease of the tropics. Lack of effective drugs or vaccines have made surra a severe economic burden particularly in Asia and sub-Saharan Africa. In this study, a naked DNA construct encoding full length T. evansi beta (β) tubulin gene was used to immunize mice, to elicit a T. evansi β tubulin protein specific humoral immune response, delineated by ELISA. The serum cytokine profile post immunization, as determined by flow cytometry bead based assay, showed a predominant T helper cell Type 1 (Th1) response with significant increase in levels of IFNγ and TNFα. Lethal challenge with T. evansi blood-form trypomastigotes post immunization generated a β tubulin specific recall response and a stronger Th1 type serum cytokine profile which correlated with an extended survival and better control of parasitemia in the immunized mice.

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken l-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery.

Background Foot and mouth disease (FMD) can be controlled by regular vaccination and restriction of the movement of infected animals in the endemic countries. Although presently used, tissue culture inactivated vaccine gives protection, it has several limitations, including a short duration of immunity. DNA vaccine delivered through microparticles could comprise an alternative approach to conventional vaccine when aiming to circumvent these limitations. Methods We constructed the expression plasmid (pVAC-1D) containing 1D gene FMD virus serotype Asia 1. Poly(d,l-lactide-co-glycolide) (PLG) microparticles were prepared and coated with the pVAC-1D plasmid. Guinea pigs were vaccinated with PLG-coated and naked DNA vaccine constructs intramuscularly. The humoral response was measured by an enzyme-linked immunosorbent assay (ELISA) and the serum neutralization test (SNT). Analysis of the persistence and the expression of pVAC-1D plasmid construct was carried out by quantitative polymerase chain reaction (qPCR). Results The humoral response lasted for 1 year, as measured by ELISA and SNT. Analysis of the persistence and the expression of pVAC-1D plasmid construct by qPCR has shown that pVAC-1D expression was seen for a longer duration compared to the naked DNA vaccine. Microparticles coated plasmid DNA-injected guinea pigs were protected when challenged with FMD virus. Conclusions The present study has shown that the delivery of plasmid coated on cationic PLG microparticles enhance the duration of immunity of the DNA vaccine constructs. Copyright © 2012 John Wiley & Sons, Ltd.

Aim: To evaluate the safety of compacted DNA nanoparticles (NPs) in retinal pigment epithelial (RPE) cells. Materials & methods: Enhanced GFP expression cassettes controlled by the RPE-specific vitelloform macular dystrophy promoter were constructed with and without a bacterial backbone and compacted into NPs formulated with polyethylene glycol-substituted lysine 30-mers. Single or double subretinal injections were administered in adult BALB/c mice. Expression levels of enhanced GFP, proinflammatory cytokines and neutrophil/macrophage mediators, and retinal function by electroretinogram were evaluated at different time-points postinjection. Results: Immunohistochemistry and real-time PCR demonstrated that NPs specifically transfect RPE cells at a higher efficiency than naked DNA and similar results were observed after the second injection. At 6 h postinjections, a transient inflammatory response was observed in all cohorts, including saline, indicating an adverse effect to the injection procedure. Subsequently, no inflammation was detected in all experimental groups. Conclusion: This study demonstrates the safety and efficacy of NP-mediated RPE gene transfer therapy following multiple subretinal administrations.

Gene therapy of the heart has been attempted in a number of clinical trials with the injection of naked DNA, although quantitative information on myocellular transfection rates is not available. The present study aimed to quantify the efficacy of electropulsing protocols that differ in pulse duration and number to stimulate transfection of cardiomyocytes and to determine the impact on myocardial integrity.

DNA conformation change or damage induced by low-energy ion irradiation has been of great interest owing to research developments in ion beam biotechnology and ion beam application in biomedicine. Mechanisms involved in the induction of DNA damage may account for effect from implanting ion charge. In order to check this effect, we used both ion beam and neutralized beam at keV energy to bombard naked DNA. Argon or nitrogen ion beam was generated and extracted from a radiofrequency (RF) ion source and neutralized by microwave-driven plasma in the beam path. Plasmid DNA pGFP samples were irradiated with the ion or neutralized beam in vacuum, followed by gel electrophoresis to observe changes in the DNA conformations. It was revealed that the ion charge played a certain role in inducing DNA conformation change. The subsequent DNA transfer into bacteria Escherichia coli ( E. coli ) for mutation analysis indicated that the charged ion beam induced DNA change had high potential in mutation induction while neutralized beam did not. The intrinsic reason was attributed to additional DNA deformation and contortion caused by ion charge exchange effect so that the ion beam induced DNA damage could hardly be completely repaired, whereas the neutralized beam induced DNA change could be more easily recoverable owing to absence of the additional DNA deformation and contortion.

The effects of acyl chain length and unsaturation on physicochemical characteristics and transfection efficiency of novel nanomicelles of N -acyl-substituted low-molecular-weight chitosan ( N -acyl LMWC) were studied. After transfection optimization, 18-carbon chain length grafts were selected, and N -acyl LMWCs were prepared with increasing unsaturation (18:1–18:3 carbon acyl grafts). N -acyl LMWCs were characterized using infrared spectroscopy and elemental analysis. The effect of DNA addition on size and zeta potential of N -acyl LMWCs was determined by dynamic light scattering. N -acyl LMWC–plasmid DNA (pDNA) polyplex stability was confirmed using gel electrophoresis. Transfection efficiency of the derivative polymers was visualized in human embryonic kidney cells using a plasmid encoding green fluorescent protein by confocal fluorescence microscopy and was quantified using therapeutic plasmids encoding for interleukin-4 and interleukin-10. N -acyl LMWCs could form cationic nanomicelles with average hydrodynamic size between 73 and 132 nm. DNA addition to nanomicelles led to minimal increase in the size. N -acyl LMWC–pDNA polyplexes showed excellent stability on storage and could protect DNA from enzymatic degradation. The transfection efficiencies of N -acyl LMWCs with 18:1 and 18:2 grafts were comparable with FuGENE® HD but were approximately eightfold and 35-fold greater as compared with LMWC and naked DNA, respectively. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association.

Background Although known for its success in ameliorating muscle disorders, ultrasound (US)-mediated gene transfer in skeletal muscle has recently shown potential for treating a wide range of therapeutic applications. The disadvantages of the technique, however, including its relatively low transfer efficiency and toxic effects resulting in tissue damage, have impeded clinical progress. We describe a novel syringe-focused US device of our design that combines simultaneous DNA injection and US irradiation to resolve these issues, achieving improved DNA uptake for high expression and lowering the incidence of tissue injury. Methods The novel, syringe-focused US device was used to introduce purified plasmid DNA encoding human neprilysin (hNEP) into mouse hindlimb skeletal muscle concurrent with US protocol (1.7 MHz, 1 W/cm2, 1-min exposure time). hNEP expression was measured in local skeletal muscle, blood serum and brain by immunoassay at different post-injection time points. The incidence of muscle fiber damage was subsequently assessed by Evans Blue dye (EBD) uptake using fluorescence microscopy. Results The syringe-US device at the first time enabled simultaneous combination of DNA transfer and US stimulation gives rise to two months of gene expression of the encoded polypeptide as it is measured, at the same time as withstanding minor tissue damage. In addition, DNA injection without US treatment failed to induce hNEP expression. Conclusions This new technique provides a simple, safe and efficient nonviral gene delivery approach in skeletal muscle and shows promising applications for gene therapy of human disease, such as Alzheimer's disease. Copyright © 2012 John Wiley & Sons, Ltd.

Background Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external ‘artificial kidney’. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery. Materials and methods To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation. Results Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation. Conclusions These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods. Copyright © 2011 John Wiley & Sons, Ltd.

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.

Patients with malignant non-Hodgkin's lymphomas (NHL) of B-cell lineages relapse despite initial anti-tumor response to chemotherapy or antibody treatments. Failure to eliminate the tumor is often because of inadequate priming, low cell numbers and suboptimal phenotype of effector T cells. Here we describe a new biomaterial-based controlled-release paradigm to treat weakly immunogenic NHLs by in-situ amplifying the number of functional, antigen-specific T-helper 1 (Th1) cells following immunotherapy. An injectable, synthetic immune priming center (sIPC) consisting of an in-situ crosslinking, chemokine-carrying hydrogel and both DNA- and siRNA dual-loaded microparticles, is reported. This sIPC chemo attracts a large number of immature dendritic cells (DCs) at the site of administration and efficiently co-delivers both DNA antigens and interleukin-10 (IL10)-silencing siRNA to those cells. Using a murine model of A20 B cell lymphoma, we demonstrate that combination of DNA-antigen delivery and IL10 silencing, synergistically activate recruited immature DCs and cause a strong shift towards Th1 response while suppressing Th2 and Th17 cytokines. sIPC-based immunotherapy showed 45% more CD8+ cytotoxic T cell (CTL) response and 53% stronger CD4+ CTL activity compared to naked DNA vaccine. In addition, in-vivo sIPC immunization induced significant protection (p

Background The therapeutic potential of pCK-HGF-X7, a naked DNA designed to express two isoforms of hepatocyte growth factor (HGF723 and HGF728), was studied in the rat ischemic heart disease model. Methods First, the kinetics of gene expression was examined by injecting pCK-HGF-X7 DNA into the rat heart. Second, the cardioprotective effects were compared between the two naked DNA constructs, expressing a single (HGF728) or both isoforms (HGF728 and HGF723) of HGF, in the rat ischemic heart disease model. The ischemic injury to the rat heart was created by ischemia–reperfusion in the anterior descending artery. The respective naked DNA constructs were injected into the anterior wall of the rat heart with the ischemia–reperfusion injury. Cardiac function, capillary density and anti-fibrotic activity were compared between the two naked DNA constructs. Results The intramyocardial administration of pCK-HGF-X7 resulted in transient and localized HGF expression for 3 weeks. At its peak, approximately 678 pg (per mg of tissue protein) of HGF was produced in the injected heart without an increase of HGF protein level in other tissues, and serum. pCK-HGF-X7 more efficiently improved the left ventricular ejection fraction and the systolic anterior wall thickness, increased the capillary density, and inhibited myocardial fibrosis, in a statistically significant manner, compared to the identical vector encoding HGF728 only. Conclusions These results demonstrate that transfer of the genomic-cDNA hybrid expressing both isoforms of the HGF gene might provide higher therapeutic effects than the cDNA sequence producing HGF728 alone in the treatment of ischemic heart disease. Copyright © 2011 John Wiley & Sons, Ltd.

In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization.