Naked Plasmid Research Articles

A DNA Prime Immuno-Potentiates a Modified Live Vaccine against the Porcine Reproductive and Respiratory Syndrome Virus but Does Not Improve Heterologous Protection.

The porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus inducing abortion in sows and respiratory disease in young pigs, is a leading infectious cause of economic losses in the swine industry. Modified live vaccines (MLVs) help in controlling the disease, but their efficacy is often compromised by the high genetic diversity of circulating viruses, leading to vaccine escape variants in the field. In this study, we hypothesized that a DNA prime with naked plasmids encoding PRRSV antigens containing conserved T-cell epitopes may improve the protection of MLV against a heterologous challenge. Plasmids were delivered with surface electroporation or needle-free jet injection and European strain-derived PRRSV antigens were targeted or not to the dendritic cell receptor XCR1. Compared to MLV-alone, the DNA-MLV prime- boost regimen slightly improved the IFNγ T-cell response, and substantially increased the antibody response against envelope motives and the nucleoprotein N. The XCR1-targeting of N significantly improved the anti-N specific antibody response. Despite this immuno-potentiation, the DNA-MLV regimen did not further decrease the serum viral load or the nasal viral shedding of the challenge strain over MLV-alone. Finally, the heterologous protection, achieved in absence of detectable effective neutralizing antibodies, was not correlated to the measured antibody or to the IFNγ T-cell response. Therefore, immune correlates of protection remain to be identified and represent an important gap of knowledge in PRRSV vaccinology. This study importantly shows that a naked DNA prime immuno-potentiates an MLV, more on the B than on the IFNγ T-cell response side, and has to be further improved to reach cross-protection.

The application of naked DNA plasmid (DrZP3) and recombinant adenovirus (Ad-rZP3) in rat animal model to determine comparative efficacy of ZP3-Immunocontraceptive vaccines

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.

Modification effects of low-energy (∼10 eV/amu) carbon ion beams on naked DNA

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.

Dual-Plasmid Bionic Array-Directed Gene Electrotransfer in HEK293 Cells and Cochlear Mesenchymal Cells Probes Transgene Expression and Cell Fate.

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.

Preparation and characterization of a nanovaccine of pVAX1-NH36 for leishmaniasis

Leishmaniasis is an endemic disease mainly in very poor countries. This disease may cause only a cutaneous lesion but also a fatal visceral disease. Vaccines of plasmid DNA (pDNA) are an alternative of conventional treatments of the disease since are considered safer, cheaper and easier to produce than conventional vaccines. However, it is being shown that naked plasmids are very susceptible to degradation and do not enter the cells efficiently. Liposomes may be used for pDNA administration due to their biocompatibility, biodegradability and low immunogenicity. However, they also present problems in reaching the correct cells, such as opsonization, aggregation and phagocytosis. Pegylation of liposomes has proven to increase circulation time and reduce cytotoxicity. This work aimed at the development of a process to prepare lipidic nanoparticles (NPs) loaded with plasmid pVAX1-NH36 for application as leishmaniasis nanovaccine. NPs were prepared by thin film technique using l-α-phosphatidylcholine, octadecylamine and cholesterol in a molar ratio of 7:2:1. An experimental setup was used to analyze the effect of PEG% and molar charge ratio (R) on the physicochemical properties and pDNA carry capacity of the NPs. In the preparation of the nanovaccines 0, 2.5 or 5 mol% of PEG2000-PE was used. The molar charge ratio of pVAX1 to phospholipids was varied between 1 and 11 for each type of nanovaccine. The initial experiments showed that R = 1 and R = 5 nanovaccines present instability and were discarded for further studies. Dynamic light scattering (DLS) analysis showed pDNA-NPs size with an average diameter of 120–160 nm depending on R and PEG2000-PE% and a polydispersity index (PDI) between 0.06-0.16. The Z potentials determined by electrophoretic light scattering were in the range of 15–40 mV. The PEGylated particles showed a PDI and Z potential values more adequate for nanovaccine preparation. The PEGylated and non-PEGylated nanovaccines were visualized by transmission electron microscopy (TEM) and presented a rather spherical shape which is also a desired property. To determine the stability of the nanovaccines their pDNA content was estimated in samples obtained from the reaction solutions at days 0, 1, 2, 7 and 14, measuring the pDNA concentration in the solution by high-performance liquid chromatography on a hydrophobic interaction column (HPLC-HIC). All the samples presented a stability greater than 84% in the period of study. A mixed order lumped parameter model was successfully fitted (with r2 > 0.92 in all the preparations) to the kinetics of plasmid liberation as initial approach to model the phenomena.

In vivo genome editing of mucopolysaccharidosis I mice using the CRISPR/Cas9 system

Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha-L-iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/−1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients.

Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs.

Gene therapy is a breakthrough treatment strategy against several intractable and lethal diseases that previously lacked established treatments. Viral and nonviral vectors have been studied to realize higher gene transfection efficiencies and to suppress the degradation of gene by nucleolytic enzymes in vivo. However, it is often the case that the addition of a vector results in adverse effects. In this study, we identified formulations of dry naked plasmid DNA (pDNA) powders with no vector showing significantly higher gene expression than pDNA solutions including vectors such as polyethylenimine (PEI) in the lungs of mice. We prepared the naked pDNA powders by spray-freeze-drying with various excipients. The gene expression of naked pDNA powders exceeded those of pDNA solutions containing PEI, naked pDNA solution, and reconstituted pDNA powder. Gene expression of each naked pDNA powder was dependent on the composition of excipients. Among them, the mice that were administered the pDNA powder composed of low-molecular-weight hyaluronic acid (LHA) as an excipient showed the highest gene expression. The lactate dehydrogenase activity and concentration of inflammatory cytokines in bronchoalveolar lavage fluid were comparable to those caused by ultrapure water. The results suggest that useful dry naked nucleic acid powders for inhalation could be created by optimizing the excipients, offering new insights into the development of pulmonary gene therapy.

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) “conventional” tail-vein injections, (2) “primed” injections, (3) “hydrodynamic” injections, or (4) direct “intrahepatic” injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse.

Pre-clinical pharmacology and mechanism of action of SG3199, the pyrrolobenzodiazepine (PBD) dimer warhead component of antibody-drug conjugate (ADC) payload tesirine

Synthetic pyrrolobenzodiazepine (PBD) dimers, where two PBD monomers are linked through their aromatic A-ring phenolic C8-positions via a flexible propyldioxy tether, are highly efficient DNA minor groove cross-linking agents with potent cytotoxicity. PBD dimer SG3199 is the released warhead component of the antibody-drug conjugate (ADC) payload tesirine (SG3249), currently being evaluated in several ADC clinical trials. SG3199 was potently cytotoxic against a panel of human solid tumour and haematological cancer cell lines with a mean GI50 of 151.5 pM. Cells defective in DNA repair protein ERCC1 or homologous recombination repair showed increased sensitivity to SG3199 and the drug was only moderately susceptible to multidrug resistance mechanisms. SG3199 was highly efficient at producing DNA interstrand cross-links in naked linear plasmid DNA and dose-dependent cross-linking was observed in cells. Cross-links formed rapidly in cells and persisted over 36 hours. Following intravenous (iv) administration to rats SG3199 showed a very rapid clearance with a half life as short as 8 minutes. These combined properties of cytotoxic potency, rapid formation and persistence of DNA interstrand cross-links and very short half-life contribute to the emerging success of SG3199 as a warhead in clinical stage ADCs.

Mechanisms of G0/G1 phase arrest induced by longevity-assurance homologue 2 in Hepa1-6 hepatoma cells

Objective To investigate the effects of longevity-assurance homologue gene 2 (Lass2) on the cell cycle ofmouse hepatic carcinoma cell line Hepa1-6 and the related mechanism. Methods Lass2 recombinant eukaryotic expression vector has been successfully constructed in the early stage, and were transfected in Hepa1-6 hepatoma cell line 48 h by liposome 2000. The cell cycle of each group (negative control, empty load, experimental group) was detected by flow cytometry (PI). The relative expression levels of Lass2, Cyclin D1, nuclear factor-κB (NF-κB) p65 mRNA and protein were detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting; In order to further verify and clarify the mechanism, the C57BL/6J mouse hepatic carcinoma in situ model was constructed by injecting Hepa1-6 hepatic carcinoma cell suspension directly into the liver parenchyma of C57BL/6J mice. The naked plasmid was injected into the tail vein separately by hydrodynamics, and the total RNA and total protein in liver tissue of each group were extracted after transfection. The relative expression levels of Lass2, Cyclin D1, NF-κB p65 mRNA and protein in mice liver were detected by FQ-PCR and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting. Results The relative expression levels of Lass2 mRNA and protein in Hepa1-6 hepatoma cells were significantly higher than those in negative control group and blank control group (F=1 486.895, all P=0.000; protein: F=55.964, all P=0.000), and the percentage of cell cycle G0/G1 phase increased significantly (F=239.867, all P=0.000), significantly decreased in G2/M phase and S phase (FG2/M=13.462, PG2/M=0.003, 0.005; FS=46.207, all P=0.000), at the same time, the levels of NF-κB p65, CyclinD1 mRNA and protein in the experimental group were decreased (mRNA: FNF-κB p65=29.993, FCyclin D1=113.260, all P=0.000; protein: FNF-κB p65=17.679, P=0.003, 0.002; FCyclin D1=176.357, all P=0.000), the phosphorylation of NF-κB p65 were significantly downregulated (FNF-κB p-p65=33.817, P=0.000). Conclusion Lass2 gene blocks the cell cycle of Hepa1-6 cells in G0/G1 phase, and its mechanism may be related to inhibits NF-κB p65 activation and expression and down-regulation of Cyclin D1 transcription and protein expression. Key words: Longevity-assurance homologue gene 2; Hepatocellular carcinoma; Cell cycle; Nuclear factor-κB p65; Cyclin D1

Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos)

A simple, safe, targeted, and efficient invivo DNA delivery system is necessary for clinical-grade liver-targeted gene therapy in humans. Intravascular hydrodynamic gene delivery has been investigated in large animal models, but translation to humans has been hampered by its technical challenges, invasiveness, and potential for significant cardiovascular adverse events. We posited that intrabiliary delivery of DNA plasmids via ERCP-guided hydrodynamic injection could overcome these obstacles. Twelve pigs (40-50 kg) were divided into 3 groups (4 per group) and survived 21, 30, or 60 days. ERCP was performed by inflating a balloon catheter in the common hepatic duct and creating a closed space between it and the liver parenchyma. Last, a solution composed of plasmid/sleeping beauty (SB) mix was injected under pressure through the catheter into the closed space. Swine were killed at the 3 different time points and liver tissue harvested. Plasmid DNA expression and functional translated protein expression were assessed. ERCP-guided hydrodynamic delivery of naked plasmid DNA facilitated by pCytomegalovirus-Sleep Beauty (pCMV-SB) transposons was technically feasible and devoid of cardiovascular and local adverse events in all 12 pigs. Furthermore, plasmid DNA (both single and combination) was successfully transferred into swine hepatocytes in all 12 pigs. Additionally, stable integration of the DNA constructs in hepatocyte genomic DNA was reliably noted at all 3 time points. In the 4 swine that were kept alive to 60 days, successful genomic integration and subsequent protein expression was observed in the targeted liver tissue. ERCP-guided hydrodynamic delivery of gene therapy may usher in the next chapter in gene therapy with the potential to impact a variety of single-gene, complex genetic, and epigenetic liver diseases. It also raises the possibility that other nucleic acid therapeutics (microRNA, lncRNA, siRNA, shRNA) could similarly be delivered.

Prolonged release of bone morphogenetic protein-2 in vivo by gene transfection with DNA-functionalized calcium phosphate nanoparticle-loaded collagen scaffolds

In the combination of scaffolds immersed in growth factor solutions, the release of growth factors mainly depends on scaffold degradation. However, the release of bone morphogenetic protein (BMP)-2 at an appropriate concentration during the stage of tissue regeneration would enhance bone regeneration. To achieve this condition, the present study was performed to investigate the effects of scaffolds combined with gene transfection using non-viral vectors. Nanohydroxyapatite-collagen (nHAC) scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or ascorbic acid/copper chloride, and a collagen scaffold (Terdermis®) were prepared, loaded with BMP-2-encoding plasmid DNA-functionalized calcium phosphate nanoparticles (CaP), naked plasmid DNA, or BMP-2 solution, and implanted in rats. The yield of released BMP-2 and its releasing period, respectively, were larger and longer from the scaffolds loaded with CaP than from those incubated with BMP-2 solution. In addition, the alkaline phosphatase activity induced by the CaP-loaded scaffolds was higher. Histological analysis showed that released BMP-2 could be observed on the macrophages or multinuclear giant cells surrounding the nHAC fragments or collagen fibres. TRAP-positive or OCN-positive sites were observed in all groups and a mineralization area was observed in the Terdermis®/CaP sample. The present study demonstrates that gene transfection by scaffold loaded with CaP gene transfer vectors induces a larger yield of BMP-2 for a longer period than by scaffolds loaded with BMP-2 solution or naked plasmid.

Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) gene therapy could offer a disease-modifying treatment for Parkinson's disease (PD). Here, we report that plasmid DNA nanoparticles (NPs) encoding human GDNF administered intranasally to rats induce transgene expression in the brain and protect dopamine neurons in a model of PD. To first test whether intranasal administration could transfect cells in the brain, rats were sacrificed 1week after intranasal pGDNF NPs or the naked plasmid. GDNF ELISA revealed significant increases in GDNF expression throughout the brain for both treatments. To assess whether expression was sufficient to protect dopamine neurons, naked pGDNF and pGDNF DNA NPs were given intranasally 1week before a unilateral 6-hydroxydopamine lesion in a rat model of PD. Three to four weeks after the lesion, amphetamine-induced rotational behavior was reduced, and dopaminergic fiber density and cell counts in the lesioned substantia nigra and nerve terminal density in the lesioned striatum were significantly preserved in rats given intranasal pGDNF. The NPs afforded a greater level of neuroprotection than the naked plasmid. These results provide proof-of-principle that intranasal administration of pGDNF DNA NPs can offer a non-invasive, non-viral gene therapy approach for early-stage PD.

New microRNA biotechnology as an in vivo therapeutic molecule to inhibit cancer

MicroRNAs (miRs) are biomarkers of cancer development and progression, they regulate cell fate, stemness and embryonic development. It is well-known that miRs play a role in cancer and are associated with numerous types of cancer as either oncogenes or tumor-suppressor genes. We developed the Plasmid-based microRNA Inhibitor System (PMIS), and it is the only miR inhibitor that can be used to generate transgenic mice to study the role of miRs during embryonic development and as disease models. The PMIS is very stable, efficient, has high specificity and affinity for specific miRs and is not toxic to cells or animals. We focused on miR-210 as it is increased in human colon cancer. The PMIS-miR-210 lentivirus construct and plasmid both express GFP. We made PMIS-miR-210 and PMIS-vector only stably transduced human SW620 colon cancer cell lines to determine the effect of inhibiting miR-210 on cell growth and phenotypes. The cells were transduced with the lentivirus and GFP expressing cells were FACS sorted and analyzed for growth. The PMIS-miR-210 cells proliferated very slowly (25% of the rate of PMIS-vector only cells). RNA-sequencing experiments and bioinformatics analyses revealed several new genes targeted by miR-210 and new pathways in colon tumor progression. We have identified a miR regulatory loop controlling the expression of other miRs. Nude mice were injected with PMIS-miR-210 or PMIS-vector stably expressing human colon cancer cells and 5 out of 8 mice with colon cancer cells expressing PMIS-miR-210 did not form tumors compared to the controls (PMIS-vector, 8 of 8 had tumors). To demonstrate the effectiveness of PMIS-miR-210 as a therapeutic, nude mice were injected with colon cancer cells and tumors were allowed to form (3 weeks), mice were then injected with naked plasmid DNA PMIS-miR-210 or vector control into and surrounding the solid tumor mass at several concentrations. No tumors were observed in 6 of the 10 mice injected with PMIS-miR-210 and the other 4 mice had small tumors compared to controls after 2 weeks of further growth (PMIS-vector, 10 of 10 had no effect on tumor growth). This is the first time that direct injection of a naked plasmid DNA expressing the PMIS microRNA inhibitor reduced or completely inhibited tumor growth, without using toxic delivery systems. The PMIS has great potential as a new therapeutic biological drug for cancer treatments. Support or Funding Information This work was supported by National Institutes of Health grant DE026433. A) Transduced colon cancer cells with either PMIS empty vector or PMIS-miR-210 were injected into nude mice and after 4 weeks the tumors were analyzed. B,C) Untreated colon cancer cells were put into nude mice and after 3 weeks, tumors were injected with PMIS empty vector or PMIS-miR-210, 2 weeks later the tumors were harvested and analyzed. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Amine-Functionalized Silica Nanoparticles with Drug and Gene Co-Delivery for Anti-Angiogenesis Therapy of Breast Cancer.

In our study, we report on the design and characterization of a combined angiogenesis therapy for breast cancer based on well-formed amine-functionalized silica nanoparticles (SLNs). The aminefunctionalized SLNs was employed to simultaneously deliver angiostatin (ANG) plasmid and candesartan (CD) to the same cancer cell. The well-formed ANG/CD/SLNs exhibited small particle size, reasonable positive charges, excellent loading of drug and gene in vitro. Moreover, aminefunctionalized SLNs were almost no cytotoxicity. ANG/CD/SLNs resulted in enhanced gene transfection compared to naked plasmid. More importantly, ANG/CD/SLNs as a co-delivery system achieved a stronger inhibitory effect on angiogenesis in vitro, possibly resulting from significant downregulation of vascular endothelial growth factor (VEGF) expression via different pathways. In particular, in vivo investigation on nude mice bearing MCF-7 xenografts confirmed that ANG/CD/SLNs codelivery system exerted strong anti-tumor efficacy by synergistic antiangiogenic mechanism.

Gene transfer of a naked plasmid (pUDK-HGF) encoding human hepatocyte growth factor attenuates skin/muscle incision and retraction-induced chronic post-surgical pain in rats.

Chronic post-surgical pain (CPSP) remains a major clinical problem and is often refractory to current treatments. New analgesic medications and strategies for pain relief are needed. Hepatocyte growth factor (HGF) is known to be a multi-functional growth factor and regulates various biological activities. We investigated the analgesic effect and underlying mechanism of plasmid pUDK-HGF encoding human HGF gene on CPSP induced by skin/muscle incision and retraction (SMIR) in rats. The possible changes of inflammatory factors, glial cell activation and pain sensitivity after pUDK-HGF administration were investigated by ELISA, western blot and Von Frey tests, respectively. In behavioural assays, we found that a single intramuscular or intrathecal injection of pUDK-HGF significantly attenuated mechanical hypersensitivity to von Frey stimulation of plantar ipsilateral hind paw after SMIR. Intramuscular injection of pUDK-HGF promoted blood flow and proliferation of satellite cells and inhibited inflammatory cells recruitment, collagen accumulation and expression of pronociceptive factors. Intrathecal injection of pUDK-HGF inhibited activation of spinal glial cells and production of inflammatory mediators induced by SMIR. pUDK-HGF has a strong analgesic potency and efficacy in CPSP induced by SMIR in rats. This study highlights a new strategy for the treatment of CPSP. The CPSP occurs following various surgical procedures and remains a major clinical problem due to the lack of study on the mechanisms of CPSP. Our findings provide the first evidence that pUDK-HGF attenuates SMIR-induced pain behaviuors through peripheral or central mechanisms. The peripheral analgesic effect of pUDK-HGF is associated with promoting tissue repair and inhibiting inflammatory response; furthermore, pUDK-HGF inhibits activation of spinal glial cells and overexpression of inflammatory mediators in spinal cord. Therefore, naked pUDK-HGF may be a potential therapeutic strategy for treatment of CPSP in clinic.

Gene therapy for neuropathic pain induced by spared nerve injury with naked plasmid encoding hepatocyte growth factor.

Neuropathic pain (NP) is a refractory disease in the clinic with a tremendous impact on the quality of life of patients. Gene therapy is a potential strategy for the management of NP. In the present study, we examined the analgesic effect and mechanism of hepatocyte growth factor (HGF) in vitro and in vivo. We examined the proinflammatroy gene changes in lipopolysaccharide (LPS)-induced microglia BV2 cells with a quantitative real-time polymerase chain reaction of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Mechanical stimulation tests were performed five times at 5-min intervals to assess pain thresholds using Von Frey Hair in mice following spared nerve injury (SNI). The glial cell activation of spinal cord was examined by western blotting. Statistical significance was determined by a Tukey's test and a paired t-test. We found that recombinant human HGF protein suppressed LPS-induced BV2 cell activation in vitro, marked by the down-regulation of IL-1β, IL-6, TNF-α and iNOS expression, as well as decrease of nitric oxide production. Moreover, intrathecal injection of naked plasmid encoding HGF gene (pUDK-HGF) significantly attenuated SNI-induced pain behaviors in mice by direct inhibition of spinal cord microglia and astrocyte activation. The results of the present study indicate that pUDK-HGF can reduce cytotoxicity products released from activated glial cells, which may provide a promising therapeutic strategy for treating NP.

Bacterial exposure to ZnO nanoparticles facilitates horizontal transfer of antibiotic resistance genes

Bacterial exposure to ZnO nanoparticles (nZnO) at sublethal concentrations (1 to 10mg/l) for 24h significantly increased the conjugative frequency of antibiotic resistance plasmid RP4. A 24.3-fold increase was observed in E. coli pure cultures, and an 8.3-fold increase occurred in a mixed culture of indigenous aquatic microbiota. In addition, nZnO increased by three fold the transformation efficiency of E. coli via the uptake of naked plasmid pGEX4T-1, which was confirmed by confocal fluorescence microscopy. Enhanced horizontal transfer of resistance plasmids was a nanoparticle-specific effect, since 5.3mg/l Zn2+ (representing the maximum release of Zn2+ from 10mg/l nZnO) did not enhance RP4 transfer or pGEX4T-1 uptake frequency. This phenomenon was attributed to increased bacterial cell permeability after exposure to nZnO (but not to Zn2+), which was confirmed by flow cytometry with propidium iodide staining. Overall, this study forewarns that accidental or incidental release of nZnO may facilitate environmental dissemination and propagation of antibiotic resistance genes through horizontal gene transfer.

Functionalizing titanium surface with PAMAM dendrimer and human BMP2 gene via layer-by-layer assembly for enhanced osteogenesis.

The study reports on the gene functionalization of titanium substrates with multilayered functional human BMP2 (hBMP2) gene plasmid and its effects on osteogenesis in vitro and in vivo. The multilayers comprising cationic poly(amidoamine) (PAMAM) dendrimer/EGFP-hBMP2 plasmid DNA complex (d-DNA) and anionic naked plasmid were deposited on titanium substrates via layer-by-layer (LbL) assembly technique, which was revealed by atomic force microscopy (AFM), water contact angle measurement, and release profiles. The expression of marker gene EGFP and functional gene hBMP2 were observed in osteoblasts. The assays of alkaline phosphatase activity, collagen secretion, ECM mineralization, and osteogenesis-related genes expression indicated that the multilayered structure improved the osteogenic differentiation in vitro. Moreover, the femur and subcutaneous transplantation of multilayered titanium implants were also investigated to reveal osteogenesis peri-implant by using histological examination and X-ray imaging. The in vivo histologic results showed that the BMP2 group (containing functional gene hBMP2) resulted in improved osteogenic proteins expression in subcutaneous and femur tissue, as well as high level of bone formation and % bone-implant contact (%BIC) peri-implant. The study offers an effective dendrimer and hBMP2 based strategy for surface functionalization of titanium implants in potential orthopedic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 706-717, 2018.

Effects of spinal non-viral interleukin-10 gene therapy formulated with d-mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-β1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.