Many biopharmaceuticals, already approved for sale or currently under development, are post-translationally modified proteins, such as recombinant monoclonal antibodies or recombinant hormones. These are generally expressed in continuous (stable) mammalian cell lines, which are capable of long-term, commercial-scale production of recombinant proteins of the highest complexity. Yet, the development of a stable cell line capable of expressing heterologous proteins is very costly and can take up to 9–15 months. Therefore, transient gene expression (TGE) in animal cells has become the method of choice for many researchers who wish to obtain small to moderate quantities (1-500 mg) of novel complex recombinant proteins for further functional and structural characterisation within weeks of cDNA discovery. TGE is more cost-effective than the time-consuming establishment of stable cell clones, but a key factor in ensuring that these transient systems have practical application is the availability of efficient and robust transfection agents/methods. While chemical transfection methods currently dominate transient systems, the underlying fundamentals such as the formation of DNA complexes or their mode of function are not fully understood and the characteristics of the complexes and their subsequent ability to transfect cells are variable. This often renders the development of a successful transfection protocol for a new cell line random and researchers frequently have to resort to a trial-and-error approach, testing different media and/or conditions during DNA complex formation, as well as having to fine-tune the cell culture regime pre-, during, and post-transfection. This thesis aimed to explore novel transfection agents and develop DNA complex structure/property—transfection efficiency relationships for these reagents. Two different chemical approaches to transient transfection were investigated: i) a recently suggested inorganic nanoparticle based transfection system which utilises the anion exchange capacity of nanoparticles of a particular family of anionic clays, layered double hydroxides (LDHs), and ii) a modified polyethyleneimine (PEI)-based system, which aimed to reduce the inherent cytotoxicity of high molecular weight (MW) PEI, which is a very effective transfection agent, by constructing high MW mimics from low MW building blocks that are linked to each other via biodegradable linkers such as azomethine groups. While the LDH nanoparticles failed to give satisfactory transfection results for plasmid DNA, they were able to functionally deliver smaller nucleic acids such as siRNA. A mechanism different to that currently accepted for the transfection of mammalian cells with plasmid DNA using LDH nanoparticles as carriers is proposed. The modified polymeric transfection agents were shown to result in significantly less cell death, while maintaining the ability to transfect mammalian cells with almost similar efficiency to that obtained with high MW polyethyleneimine. Generic DNA complex structure/property—transfection efficiency relationships were developed by systematically studying the influence of particle size and zeta potential on transfection results.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...

BACKGROUND: DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). METHODS: Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. RESULTS: Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. CONCLUSIONS: For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.

Nosso grupo tem estudado uma estratégia alternativa de tratamento para a deficiência de hormônio de crescimento (DGH), utilizando a administração in vivo do plasmídeo pUC-UBI-hGH, seguida de eletroporação, no músculo quadríceps de camundongos anões e imunodeficientes (lit/scid), que proporcionou níveis sustentáveis de hGH na circulação durante 60 dias e um aumento significativo de peso dos animais tratados. Visando investigar os efeitos autócrinos/parácrinos e endócrinos derivados deste tipo de tratamento, e compará-los aos da administração diária por via parenteral do hormônio de crescimento humano recombinante (r-hGH) neste modelo animal, os seguintes parâmetros de crescimento foram avaliados no presente trabalho: peso corpóreo, comprimento total do corpo e da cauda, pesos de órgãos (fígado, rins, coração, baço, músculos quadríceps e gastrocnêmio) e os níveis plasmáticos de mIGF-I (fator de crescimento semelhante à insulina I de camundongo) e de glicose. Em um primeiro experimento, foram observados aumentos altamente significativos (P<0,001) do peso corpóreo e do comprimento total do corpo e da cauda, após 50 dias de tratamento. Todos os órgãos analisados para o grupo tratado, exceto os músculos gastrocnêmios apresentaram aumentos de peso altamente significativos. O quadríceps direito, o que recebeu a injeção do DNA plasmidial, apresentou um aumento de peso de 48,1% em comparação com o controle injetado com salina (P<0,001), enquanto o aumento de peso dos quadríceps esquerdo, não injetados, dos animais tratados foi de 31% (P<0,005). Estes dados sugerem efeitos locais (autócrino e / ou parácrinos) e sistêmicos (endócrinos) resultantes da injeção do plasmídeo contendo o gene do hGH e da consequente expressão e secreção do hGH. Em outro experimento, a comparação da administração única de DNA plasmidial com injeções diárias de r-hGH proporcionou aumentos significativos dos pesos corpóreos, em relação ao grupo controle, de 23,1% (P<0,01) e de 35,5% (P<0,001) para os grupos tratados com DNA e proteína, respectivamente, enquanto as inclinações das curvas de crescimento foram praticamente idênticas: 0,094 g / animal / dia para a injeção de DNA e 0,095 g / animal / dia para a proteína. Entre os dois grupos tratados, não houve diferenças significativas entre o peso dos seguintes órgãos: rins direito e esquerdo, baço e músculo quadríceps direito. Os níveis de glicose no plasma dos grupos tratados e controle não apresentaram diferenças significativas ao final dos tratamentos de 28 e 50 dias. As diferenças das concentrações de mIGF-I entre os grupos tratados foram significativas apenas após 7 dias de tratamento. Os valores foram sempre maiores para o grupo tratado com DNA plasmidial em relação ao grupo do r-hGH, e os níveis de ambos os grupos tratados foram significativamente superiores ao do grupo controle. Apesar da necessidade de outros estudos para confirmar a segurança e a viabilidade deste tratamento, este tipo de terapia gênica para a DGH pode vir a ser uma alternativa viável ao tratamento padrão desta doença, baseado em repetidas injeções de r-hGH altamente purificado. / Our group has studied an alternative strategy for the treatment of growth hormone deficiency (GHD), using the in vivo administration of the plasmid pUC-UBI-hGH, followed by electroporation, into the quadriceps muscle of immunodeficient dwarf mice (lit/scid), that provided sustainable levels of hGH in the circulation during 60 days and a significant increase of body weight in the treated animals. In order to investigate the autocrine / paracrine and endocrine effects derived from this type of treatment, and compare them to the parentheral daily administration of the recombinant human growth hormone (r-hGH) in this animal model, the following growth parameters were evaluated in this study: body weight, total body length, tail length, organ weights (liver, kidneys, heart, spleen, quadriceps and gastrocnemius muscles) and the plasmatic levels of mIGF-I (mouse insulin-like growth factor I) and glucose. In a first experiment, higly significant increases of body weight, total body length and tail length were observed, after 50 days of treatment. All the analised organs of the treated group, except the two gastrocnemius muscles, presented a highly significant weight increase. The right quadriceps, that received the DNA injection, presented a 48.1% weight increase versus saline injected control (P<0.001), whereas the weight increase of left quadriceps, non injected, of treated animals was 31.0% (P<0.005). These data suggest local (autocrine and / or paracrine) and systemic (endocrine effects as a result of hGH DNA injection and the consequent hGH expression and secretion. In a second experiment, the comparison between single plasmid DNA administration with daily r-hGH injections provided significant body weight increases (P<0.001) of both treated groups, compared to control group, of 23.1% (P<0.01) and 35.5% for DNA and protein treated groups respectively, while the slopes of the growth curves were practically identical: 0.094 g / mouse / day for DNA and 0.095 g / mouse / day for protein injection. In both treated groups, the weights of the following organs did not present significant differences: right and left kidneys, spleen and right quadriceps muscle. The levels of glucose in the plasma of treated and control groups showed no significant differences after 28 and 50 days. The differences in mIGF-I concentration, among the treated groups, were significant only 7 days after treatment. The values were always higher in the group treated with plasmid DNA in relation to the r-hGH group, and the levels of both treated groups were significantly higher than the control group. Despite the need for further studies to confirm the safety and feasibility of this treatment, this type of gene therapy for DGH can be a viable alternative to the standard treatment for this disease, based on repetitive injections of highly purified r-hGH.

Nosso grupo demonstrou que a captura de baixas doses de DNA plasmideal pode inibir a apresentação de antígeno e induzir um padrão de resposta anti-inflamatória, e que após a captação do DNA plasmideal por macrófagos, estas moléculas podiam prevenir a acidificação de vesículas endossomais, um passo essencial para a apresentação de antígenos para células T CD4+. Além disso, em modelos in vivo, a baixa dose de DNA foi suficiente para amenizar o quadro inflamatório e diminuir a produção de citocinas inflamatórias. Estes resultados estão em contraste com os modelos de vacinas gênicas comumente utilizadas. A baixa dose de DNA plasmideal, no contexto da indução da resposta imune, pode levar à modificação na apresentação do antígeno e ativação celular levando ao controle da resposta imune exacerbada desencadeada por outro antígeno, tornando o tratamento por DNA um importante foco de estudo para o combate de doenças autoimunes e inflamações. O objetivo deste trabalho foi identificar os microRNAs expressos em macrófagos tratados com alta ou baixa dose de DNA plasmideal e avaliar o papel destas moléculas na modulação da cascata de sinalização intracelular visando esclarecer o mecanismo imunomodulador observado. Macrófagos da linhagem J774 foram estimulados por 2 horas com o vetor plasmideal pcDNA3, nas concentrações de 10 g ou 100 g de pcDNA3/mL de RPMI. O RNA total foi extraído e o ensaio de microarray para microRNAs foi realizado. Os miRNAs diferencialmente expressos foram confirmados pela RT-qPCR, e o programa de bioinformática miRDB foi utilizado para predição de genes alvos. Os miRNAs e genes selecionados também foram dosados em macrófagos estimulados com LPS e tratados com pcDNA3. Foram detectados 6 miRNAs (miR-494-3p, miR-21-3p, miR-1897-5p, miR-1894-3p, miR-294-3p e miR-483-5p) diferencialmente expressos em macrófagos estimulados somente com pcDNA3. A expressão do miR-494-3p foi aumentado somente em células tratadas com DNA em baixa concentração tanto na ausência quanto presença de LPS. O gene IBk (IKK-) foi identificado como alvo do miR-494-3p, dosado e teve sua função detectada por western blot, mostrando que seu papel biológico foi alterado na presença deste miRNA. Além disso, os dados do tratamento com pcDNA3 em camundongos knockout para o receptor TLR9, sugerem que a expressão do miR-494-3p é independente deste tipo de receptor, porém é inibida na presença do mesmo quando no tratamento com alta dose de DNA. Os dados sugerem que quando macrófagos são tratados com baixa dose de DNA plasmideal (10 g/mL), o miR-494-3p age como regulador negativo do fator de transcrição NF-B, por meio da inibição da expressão e função da proteína IKK-. / Our group demonstrated that the capture of low doses of plasmid DNA can inhibit antigen presentation and induce an anti-inflammatory response pattern together with the decrease of pro-inflammatory cytokines expression, as observed in in vivo experimental models. Moreover, we observed that after the uptake of plasmid DNA by macrophages, these molecules could prevent endosomal vesicles acidification, an essential step for antigen presentation to CD4+ T cell. These results are in contrast with the usual DNA vaccines models commonly used. The low dose of plasmid DNA, in the context of immune response induction, can take to a modification in antigen presentation leading to the control of the response already established, what can be a potential treatment in auto-immune diseases and inflammation. The objective of this work is to identify expressed microRNAs on J774 macrophages triggered by different concentrations of plasmid DNA stimuli in order to evaluate the observed immunomodulatory mechanism. J774 macrophages were treated with low (10 µg/mL) and high (100 µg/mL) doses of pcDNA3 for 2 hours. Total RNA was extracted and microarray (Agilent plataform) was performed for detection of differentially expressed microRNAs. All obtained data was normalized in Genespring GX11.5 software. The microRNAs expression was confirmed by RT-qPCR and their mRNA targets were predicted by miRDB software. The miRNAs and their respective targets were also evaluated on DNA and LPS-treated macrophages. We detected 6 differentially expressed microRNAs (miR-494-3p, miR-21-3p, miR-1897-5p, miR-1894-3p, miR-294-3p e miR-483-5p) between the two conditions (fold change 2, p-value 0,05). The miR-494-3p expression increased only in cells treated with low dose of pcDNA3, on either previously LPS-stimulated cells or not. IBk (IKK-) was one of the predicted targets for miR-494-3p. RT-qPCR was performed to calculate its expression and its biological function was assessed by western blot. Besides, results from pcDNA3-treated cells from TLR9 knockout mice suggest that miR-494-3p expression is TLR9 independent although the receptor presence impaired miR-494-3p expression on high dose of pcDNA3-treated cells. Together, the data indicate that when macrophages are treated with low dose of plasmid DNA, miR-494-3p expression is increased, acting as negative regulator of the transcription factor NF-B, through IKK- inhibition.

Nosso grupo tem estudado uma estratégia alternativa de tratamento para a deficiência de hormônio de crescimento (DGH), utilizando a administração in vivo do plasmídeo pUC-UBI-hGH, seguida de eletroporação, no músculo quadríceps de camundongos anões e imunodeficientes (lit/scid), que proporcionou níveis sustentáveis de hGH na circulação durante 60 dias e um aumento significativo de peso dos animais tratados. Visando investigar os efeitos autócrinos/parácrinos e endócrinos derivados deste tipo de tratamento, e compará-los aos da administração diária por via parenteral do hormônio de crescimento humano recombinante (r-hGH) neste modelo animal, os seguintes parâmetros de crescimento foram avaliados no presente trabalho: peso corpóreo, comprimento total do corpo e da cauda, pesos de órgãos (fígado, rins, coração, baço, músculos quadríceps e gastrocnêmio) e os níveis plasmáticos de mIGF-I (fator de crescimento semelhante à insulina I de camundongo) e de glicose. Em um primeiro experimento, foram observados aumentos altamente significativos (P<0,001) do peso corpóreo e do comprimento total do corpo e da cauda, após 50 dias de tratamento. Todos os órgãos analisados para o grupo tratado, exceto os músculos gastrocnêmios apresentaram aumentos de peso altamente significativos. O quadríceps direito, o que recebeu a injeção do DNA plasmidial, apresentou um aumento de peso de 48,1% em comparação com o controle injetado com salina (P<0,001), enquanto o aumento de peso dos quadríceps esquerdo, não injetados, dos animais tratados foi de 31% (P<0,005). Estes dados sugerem efeitos locais (autócrino e / ou parácrinos) e sistêmicos (endócrinos) resultantes da injeção do plasmídeo contendo o gene do hGH e da consequente expressão e secreção do hGH. Em outro experimento, a comparação da administração única de DNA plasmidial com injeções diárias de r-hGH proporcionou aumentos significativos dos pesos corpóreos, em relação ao grupo controle, de 23,1% (P<0,01) e de 35,5% (P<0,001) para os grupos tratados com DNA e proteína, respectivamente, enquanto as inclinações das curvas de crescimento foram praticamente idênticas: 0,094 g / animal / dia para a injeção de DNA e 0,095 g / animal / dia para a proteína. Entre os dois grupos tratados, não houve diferenças significativas entre o peso dos seguintes órgãos: rins direito e esquerdo, baço e músculo quadríceps direito. Os níveis de glicose no plasma dos grupos tratados e controle não apresentaram diferenças significativas ao final dos tratamentos de 28 e 50 dias. As diferenças das concentrações de mIGF-I entre os grupos tratados foram significativas apenas após 7 dias de tratamento. Os valores foram sempre maiores para o grupo tratado com DNA plasmidial em relação ao grupo do r-hGH, e os níveis de ambos os grupos tratados foram significativamente superiores ao do grupo controle. Apesar da necessidade de outros estudos para confirmar a segurança e a viabilidade deste tratamento, este tipo de terapia gênica para a DGH pode vir a ser uma alternativa viável ao tratamento padrão desta doença, baseado em repetidas injeções de r-hGH altamente purificado. / Our group has studied an alternative strategy for the treatment of growth hormone deficiency (GHD), using the in vivo administration of the plasmid pUC-UBI-hGH, followed by electroporation, into the quadriceps muscle of immunodeficient dwarf mice (lit/scid), that provided sustainable levels of hGH in the circulation during 60 days and a significant increase of body weight in the treated animals. In order to investigate the autocrine / paracrine and endocrine effects derived from this type of treatment, and compare them to the parentheral daily administration of the recombinant human growth hormone (r-hGH) in this animal model, the following growth parameters were evaluated in this study: body weight, total body length, tail length, organ weights (liver, kidneys, heart, spleen, quadriceps and gastrocnemius muscles) and the plasmatic levels of mIGF-I (mouse insulin-like growth factor I) and glucose. In a first experiment, higly significant increases of body weight, total body length and tail length were observed, after 50 days of treatment. All the analised organs of the treated group, except the two gastrocnemius muscles, presented a highly significant weight increase. The right quadriceps, that received the DNA injection, presented a 48.1% weight increase versus saline injected control (P<0.001), whereas the weight increase of left quadriceps, non injected, of treated animals was 31.0% (P<0.005). These data suggest local (autocrine and / or paracrine) and systemic (endocrine effects as a result of hGH DNA injection and the consequent hGH expression and secretion. In a second experiment, the comparison between single plasmid DNA administration with daily r-hGH injections provided significant body weight increases (P<0.001) of both treated groups, compared to control group, of 23.1% (P<0.01) and 35.5% for DNA and protein treated groups respectively, while the slopes of the growth curves were practically identical: 0.094 g / mouse / day for DNA and 0.095 g / mouse / day for protein injection. In both treated groups, the weights of the following organs did not present significant differences: right and left kidneys, spleen and right quadriceps muscle. The levels of glucose in the plasma of treated and control groups showed no significant differences after 28 and 50 days. The differences in mIGF-I concentration, among the treated groups, were significant only 7 days after treatment. The values were always higher in the group treated with plasmid DNA in relation to the r-hGH group, and the levels of both treated groups were significantly higher than the control group. Despite the need for further studies to confirm the safety and feasibility of this treatment, this type of gene therapy for DGH can be a viable alternative to the standard treatment for this disease, based on repetitive injections of highly purified r-hGH.

Gene therapy provides an opportunity to ameliorate several genetic disorders and treat numerous diseases by using nucleic acid-based materials to modulate gene activity. However, the greatest challenge for successful gene therapy applications remains delivery. Two general approaches are currently under investigation to improve gene delivery efficiencies. The first is by encapsulating therapeutic genes into modified viruses that are effective at transfecting cells but that have also caused serious side effects during clinical evaluations in 1999 and 2003. In contrast, non-viral gene therapy provides the safety of conventional pharmaceutical products, but possesses inadequate transfection efficiencies for clinical use. Successful non-viral gene delivery systems require evasion of the reticuloendothelial system (RES) while in circulation, a targeting ligand for efficient cellular uptake, and perhaps several additional components for efficient cellular disposition once the carrier has been internalized. Engineering sophisticated gene delivery systems requires modular designs that are well characterized and optimized to circumvent each limiting barrier associated with gene delivery. The following thesis is focused on developing stabilized DNA polyplexes for in vivo applications and coupling their administration with current physical methods of non-viral gene delivery. The aim behind this approach is to systematically prepare gene carriers and evaluate their ability to maintain DNA transfection competent in order to determine which bioconjugate is the most successful for ultimately creating gene carriers that do not require physical interventions for gene expression. The non-viral gene delivery systems presented in the thesis are based on PEGylated polyacridine peptides that bind to DNA predominantly by intercalation rather than by ionic interactions with DNA. The initial experimental chapters deal with the discovery of these novel DNA polyplexes, and the latter chapters focus on the optimization of their design for targeted in vivo gene delivery. The results demonstrate that PEGylated polyacridine DNA polyplexes possess improved compatibility for in vivo administration and that their flexible design is beneficial for preparing multi-component gene delivery systems.

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.