Note: Plasmids for Therapy and Vaccination -- Contents -- Preface -- List of Contributors -- 1 The Biology of Plasmids -- 1 Introduction: What are plasmids? -- 2 General properties of plasmids -- 2.1 Plasmid replication and its control -- 2.2 The molecular basis of incompatibility -- 2.3 Plasmid inheritance -- 2.4 Mechanisms of plasmid spread -- 2.4.1 Conjugation in gram-negative bacteria -- 2.4.2 Conjugation in gram-positive bacteria -- 3 Plasmid-encoded phenotypes -- 3.1 Bacteriocin production and resistance -- 3.2 The Ti plasmids -- 3.3 Heavy metal resistance -- 3.4 Other phenotypical traits -- 4 The clinical importance of plasmids -- 4.1 The spread of antibiotic resistance and the evolution of multiple antibiotic resistance -- 4.2 Transfer of antibiotic resistance genes -- 4.3 Mechanisms of antibiotic resistance -- 4.4 Bacterial virulence genes -- 5 Plasmid cloning vectors -- 6 Perspectives -- References -- 2 Structures of Plasmid DNA -- 1 Introduction -- 2 Topological structures of plasmids -- 3 Supercoiling of DNA -- 4 DNA intercalating dyes -- 5 Analysis of plasmid structures -- 5.1 Electron microscopy (EM) -- 5.2 Agarose gel electrophoresis (AGE) -- 5.3 Capillary gel electrophoresis (CGE) -- 5.4 Analytical chromatography -- 6 Conclusion -- References -- 3 Genetic Vaccination with Plasmid Vectors -- 1 Introduction -- 2 Vector design -- 2.1 Plasmid DNA -- 2.2 Construction of simple transcription units -- 2.3 Construction of complex transcription units -- 3 Strategies for DNA delivery -- 4 Priming humoral and cellular immune responses by DNA vaccines -- 5 Experimental strategies facilitated by DNA vaccination -- 6 Unique advantages of DNA vaccination -- 7 DNA vaccines in preclinical animal models -- 7.1 DNA vaccines to control infectious diseases -- 7.2 Therapeutic tumor vaccines -- 7.3 Autoimmune disease. , 7.4 Treatment of allergy by therapeutic DNA vaccination -- 8 Proposed clinical applications of DNA vaccines -- 9 Risks of nucleic acid vaccination -- 10 Future perspectives -- References -- 4 A Liposomal iNOS-Gene Therapy Approach to Prevent Neointimal Lesion Formation in Porcine Femoral Arteries -- 1 Introduction -- 2 Results and discussion -- 2.1 Therapeutic plasmid -- 2.2 The gene therapy product has a clinically acceptable format -- 2.3 Efficient gene transfer was established in a minipig femoral artery injury model -- 2.4 Transfection efficiency is dose dependent -- 2.5 Non-viral iNOS gene transfer efficiently inhibits neointimal lesion formation -- 3 Summary and perspectives -- References -- 5 lmmunotherapy of Chronic Hepatitis B by pCMV-S2.S DNA Vaccine -- 1 Introduction -- 1.1 Hepatitis B: the disease -- 1.2 Hepatitis B: treatments -- 1.3 Hepatitis B: immune response to infection -- 1.4 What are DNA vaccines? -- 1.5 Which DNA vaccines for hepatitis B? -- 2 DNA vaccines for the prevention of hepatitis B -- 2.1 The mouse model -- 2.1.1 Humoral response -- 2.1.2 Cell-mediated response -- 2.1.3 Mechanisms of DNA-induced immune response to HBsAg -- 2.1.4 The primate model -- 2.1.5 DNA-based vaccination of chimpanzees against HBV -- 2.1.6 Neonatal immunization -- 3 DNA-based vaccination for chronic HBV infections -- 3.1 HBsAg transgenic mice as a model for HBV chronic carriers -- 4 Clinical trials of DNA vaccines -- References -- 6 pSG.MEPfTRAP - A First Generation Malaria DNA Vaccine Vector -- 1 Parasite life cycle and impact of malaria -- 2 Concept of vaccination against malaria -- 3 First-generation plasmid: pSG.MEPfTRAP -- 3.1 Vector backbone -- 3.2 Insert -- 3.3 Production and formulation -- 3.4 Preclinical testing of pSG.MEPfTRAP -- 3.4.1 Toxicity studies -- 3.4.2 Biodistribution -- 3.4.3 Stability testing -- 3.4.4 Potency testing. , 4 Regulatory aspects -- 5 Future perspectives -- References -- 7 Polyvalent Vectors for Coexpression of Multiple Genes -- 1 Introduction -- 2 Polycistronic expression vectors -- 2.1 Mechanisms of translation initiation -- 2.2 Characteristics of IRES elements -- 2.3 Application of IRES elements in cells and animals -- 2.4 Polycistronic vector systems -- 2.5 Expression properties of IRES vectors -- 3 Bidirectional promoters -- 3.1 Natural bidirectional promoters -- 3.2 Artificial bidirectional promoters -- 3.3 Combining polycistronic and bidirectional expression -- 4 Perspectives -- References -- 8 Form Follows Function: The Design of Minimalistic lmmunogenically Defined Gene Expression (MIDGE®) Constructs -- 1 The problem -- 2 The solution -- 2.1 MIDGE -the concept -- 2.2 Simple MIDGE -- 2.3 Smart MIDGE -- 2.4 Applications -- 2.5 Practical aspects of vector sequence design -- References -- 9 Synthetic Genes for Prevention and Therapy: Implications on Safety and Efficacy of DNA Vaccines and Lentiviral Vectors -- 1 Introduction -- 2 Paradoxon: HIV-derived vaccines and gene delivery systems -- 3 Synthetic genes: Novel tools contributing to the understanding of HIV replication -- 3.1 Construction of a synthetic, HIV-1 derived gag gene -- 3.2 Codon usage modification in the gag gene abolishes Rev dependency and increases expression yields -- 3.3 Codon usage modification in the gag gene increases nuclear RNA stability and promotes constitutive nuclear translocation -- 3.4 Codon usage modification in the gag gene alters the nuclear export pathway of otherwise CRM1 dependent RNAs -- 3.5 Codon usage modification increases RNA stability, modulates nuclear RNA export and increases translational efficiency -- 4 Synthetic genes: Implications on the development of safe and effective DNA vaccines -- 4.1 Safety issues to be considered for DNA vaccine development. , 4.2 Codon optimization of a gag-specific candidate vaccines results in increased antibody responses -- 4.3 Enhanced in vitro cytokine release of splenocytes from mice immunized with synthetic gag plasmid DNA -- 4.4 Induction of CTL responses in mice immunized with the modified Gag expression plasmids -- 5 Synthetic genes: Implications on the development of safe lentiviral vectors for gene delivery into quiescent cells -- 5.1 Safety issues to be considered for lentiviral vector development -- 5.2 Construction and characterization of synthetic gagpol expression plasmids -- 5.3 Production of lentiviral vectors using synthetic gagpol genes -- 5.4 Transduction of non-dividing cells -- 5.5 Absence of replication-competent recombinants (RCRs) -- 6 Future perspectives -- References -- 10 Plasmids in Fish Vaccination -- 1 Introduction -- 2 Fish -- 3 Fish immunology -- 3.1 Innate defence mechanisms -- 3.2 Adaptive defence mechanisms -- 4 Vaccination of fish -- 5 Nucleic acid vaccination of fish -- 6 Plasmid constructs used in fish studies -- 7 Routes of plasmid administration -- 7.1 Intramuscular injection of plasmid DNA -- 7.2 Other routes of plasmid administration -- 8 Fate of injected plasmid DNA -- 9 Magnitude, distribution and longevity of expressed antigen -- 10 Responses of fish to injection with plasmid DNA -- 10.1 Inflammatory responses -- 10.2 Avirulent antigens -- 10.3 Virulent antigens -- 11 Regulatory issues and future directions -- References -- 11 Plasmid Manufacturing - An Overview -- 1 Introduction -- 2 Structure of nucleic acids -- 2.1 Brief structural description of DNA and RNA structures -- 2.2 DNA supercoiling -- 3 Plasmid DNA Manufacturing -- 3.1 Major impurities and main product specifications -- 3.1.1 Host nucleic acids -- 3.1.2 Proteins -- 3.1.3 Endotoxins. , 3.2 Factors influencing the production of plasmid DNA: Some considerations on the upstream processing and fermentation stages -- 3.2.1 The plasmid vector -- 3.2.2 The bacterial host strain -- 3.2.3 Plasmid fermentation -- 3.3 Downstream processing of plasmid DNA -- 3.3.1 Cell lysis -- 3.3.2 Pre-chromatography processing: clarification and concentration -- 3.3.3 Chromatographic processing: purification of supercoiled plasmid DNA -- 3.4 Purification Strategies -- 4 Concluding remarks -- References -- 12 Quality Control of pDNA -- 1 Introduction -- 2 Characterization and quality control of pDNA -- 3 Validation of test procedures -- 4 GLP -- 5 Detailed description of the characterization of pDNA (final product) -- 5.1 Sterility -- 5.2 Purity -- 5.2.1 Content -- 5.2.2 Homogeneity -- 5.2.3 Host DNA -- 5.2.4 Host cell protein impurities -- 5.2.5 Endotoxins -- 5.3 Identity -- 5.3.1 Restriction analysis -- 5.3.2 Determination of the DNA sequence -- 6 Conclusion -- References -- 13 From Research Data to Clinical Trials -- 1 Introduction -- 2 Approaching regulators -- 3 Vaccine manufacture -- 4 Predinical safety testing -- 5 Clinical trials -- 6 Approval for clinical trials -- 7 Clinical trial applications in Germany -- References -- 14 Market Potential for DNA Therapeutics -- 1 Definition of biotechnology -- 2 History -- 3 Process of pharmaceutical development -- 4 Human society and technical revolution -- 5 From sequence to product: Applications of biotechnology -- 5.1 Milestones in biotechnology: The Human Genome Project -- 5.2 The future is now: Examples for existing therapeutic approaches using gene products -- 5.2.1 Gene therapy in cardiovascular diseases -- 6 Legal aspects of gene technology and pDNA derived products -- 6.1 The extension of patent law to living creatures and their components -- 6.2 Impacts of biomedical patents. , 6.3 Resisting corporate ownership of life forms.

Note: Intro -- DNA Pharmaceuticals -- Preface -- Contents -- List of Contributors -- Abbreviations -- 1 DNA Vaccines - An Overview -- 1.1 Rationale for DNA Vaccines -- 1.2 Preclinical Proof of Concept -- 1.3 Clinical Trials -- 1.4 Second-Generation Vaccines -- 1.5 Conclusions -- References -- 2 DNA as a Pharmaceutical - Regulatory Aspects -- 2.1 Introduction -- 2.2 Quality Requirements for DNA used as a Gene Therapy Product -- 2.2.1 Introduction -- 2.2.2 Production and Purification -- 2.2.2.1 Raw Materials -- 2.2.2.2 Antibiotics -- 2.2.2.3 Solvents -- 2.2.2.4 Fermentation -- 2.2.2.5 Purification -- 2.2.3 Cell Banking System Procedures -- 2.2.3.1 Generation and Characterization of Master and Working Cell Banks -- 2.2.4 Product Characterization and Quality Criteria -- 2.2.4.1 Identity -- 2.2.4.2 Purity -- 2.2.4.3 Adventitious Agents -- 2.2.4.4 Potency -- 2.3 Safety Studies for Clinical Trials -- 2.3.1 General Considerations -- 2.3.2 Conduct of Preclinical Safety Studies -- 2.3.2.1 Regulations -- 2.3.2.2 Design of an Appropriate Toxicology Program -- 2.3.2.3 Single- and Repeat-Dose Toxicity Studies -- 2.3.2.4 Safety of the Formulated Plasmid DNA -- 2.3.2.5 Specific Safety Considerations -- 2.3.2.6 Choice of Animal Model -- 2.4 Special Issues -- 2.4.1 Comparability of Plasmid Gene Therapy Products -- 2.4.2 Mixed Plasmid Preparations -- 2.4.3 Plasmid Molecular Structure -- 2.5 Biosafety Issues and Environmental Risk Assessment -- References -- 3 From Bulk to Delivery: Plasmid Manufacturing and Storage -- 3.1 Introduction -- 3.1.1 Gene Therapy -- 3.1.2 DNA Vaccination -- 3.2 Manufacturing of Plasmid DNA -- 3.2.1 Bacterial Cultivation -- 3.2.2 Plasmid DNA Purification -- 3.2.3 Innovative Aspects in Plasmid Manufacturing -- 3.3 Quality Control of Plasmid DNA Vectors -- 3.3.1 Proteins, Ribonucleic Acid, and Lipopolysaccharides -- 3.3.2 Chromosomal DNA. , 3.3.3 Plasmid Identity -- 3.3.4 Plasmid Topology (Structural Homogeneity) -- 3.4 Plasmid Stability during Storage and Application -- 3.4.1 Long-Term Stability of Plasmid DNA -- 3.4.2 Lyophilization for Long-Term Storage -- 3.4.3 Stability during Application -- 3.5 Future Developments -- References -- 4 Minimized, CpG-Depleted, and Methylated DNA Vectors: Towards Perfection in Nonviral Gene Therapy -- 4.1 Introduction -- 4.2 The Mammalian Immune System as a Barrier to Nonviral Gene Delivery -- 4.3 Strategies to Minimize DNA Vectors -- 4.3.1 Excision of a DNA Fragment Containing a Transgene Expression Cassette from Plasmid DNA -- 4.3.2 Intramolecular Site-Specific Recombination Within a Bacterial Plasmid -- 4.3.3 Synthesis of Minimized DNA Vectors by PCR -- 4.3.4 Improvement of Minimized DNA Vector Yield and Purity -- 4.4 Depletion of CpG Dinucleotides in the Bacterial Vector Backbone -- 4.5 Methylation of CpG Dinucleotides in Plasmid DNA -- 4.6 Towards an Ideal Nonviral Vector -- 4.7 Conclusion -- References -- 5 Localized Nucleic Acid Delivery: A Discussion of Selected Methods -- 5.1 Foreword -- 5.2 Nucleic Acid Delivery - What For? -- 5.3 Nucleic Acid Delivery - How? -- 5.3.1 Nucleic Acid Compaction -- 5.3.2 Receptor-Ligand Interactions -- 5.3.3 Endocytosis and Endosomal Escape -- 5.3.4 Nuclear Transport -- 5.3.5 Genome Organization -- 5.3.6 Biocompatibility -- 5.4 Why is Localization of Drug and Nucleic Acid Delivery Important? -- 5.5 Hierarchies of Localization (Targeting) -- 5.5.1 Methods of Localization and of Local Control -- 5.5.2 Nuclear Transport of Macromolecules in Living Cells -- 5.5.3 Nuclear Localization Signals and Gene Transfer -- 5.5.4 Localization Hierarchies I and II - Establishing Target Cell Contact -- 5.5.5 Vector Localization by Magnetic Force (Magnetofection) -- 5.5.6 Hydrodynamic Methods of Nucleic Acid Delivery. , 5.5.7 Local Vector Implantation. Carrier-Mediated Nucleic Acid Delivery -- 5.5.8 Injectable Implants for Localized Nucleic Acid Delivery -- 5.5.9 Aerosol Application of Nucleic Acids -- 5.5.10 Use of Ultrasound to Trigger Localized Delivery -- 5.6 Concluding Remarks -- References -- 6 DNA Needle Injection -- 6.1 From Mouse to Human -- 6.1.1 DNA Vaccines -- 6.1.2 Successful Strategy for Vaccination -- 6.2 Intramuscular Injection -- 6.2.1 Biology of Muscle Fibers -- 6.2.1.1 Resting Stem Cells -- 6.2.2 Uptake of Plasmid DNA -- 6.2.3 Activation of the Immune System -- 6.2.3.1 Receptors and other Signals -- 6.2.3.2 Antigen Presentation -- 6.2.4 Cross-Priming -- 6.2.5 Safety Aspects -- 6.2.5.1 Uptake of the DNA by Muscle Cells -- 6.2.5.2 Antigen Processing -- 6.2.5.3 Antigen Presentation -- 6.2.6 DNA Vaccination of Horses against Infection with Equine Arteritis Virus I -- 6.3 Intradermal Injection -- 6.3.1 Skin-Associated Lymphoid Tissue (SALT) -- 6.3.2 DNA Vaccination of Horses Against Infection with Equine Arteritis Virus II -- 6.4 Concluding Remarks -- References -- 7 Needleless Jet Injection of Naked DNA for Nonviral in vivo Gene Transfer -- 7.1 Introduction -- 7.2 In vivo Application of Jet Injection -- 7.2.1 Intratumoral Jet Injection of Naked Plasmid DNA -- 7.2.2 Analysis of Reporter Gene Expression in Jet-Injected Tumors -- 7.2.3 Analysis of the Stability of Jet-Injected Naked DNA -- 7.3 Conclusions -- References -- 8 Plasmid Inhalation: Delivery to the Airways -- 8.1 Introduction -- 8.2 Delivery Methods -- 8.2.1 Lung Delivery by Instillation -- 8.2.2 Delivery by Aerosol -- 8.2.3 Aerosol Deposition -- 8.2.4 Aerosolization Devices -- 8.2.4.1 Metered Dose Inhalers -- 8.2.4.2 Dry Powder Inhalers -- 8.2.4.3 Nebulizers -- 8.2.5 Aerosolization of Plasmid DNA -- 8.2.6 Plasmid DNA/Lipid Complexes -- 8.2.6.1 Optimization of Aerosol Formulation. , 8.2.6.2 Aerosol Delivery of Lipid/pDNA to Human Lung -- 8.2.7 Plasmid Delivery with Cationic Polymers -- 8.3 Future Directions -- 8.4 Conclusions -- References -- 9 Hydrodynamic Gene Delivery -- 9.1 Definition -- 9.2 Initial Discovery of the Technique -- 9.3 The Systemic Hydrodynamic Approach -- 9.4 The Regional Hydrodynamic Approach to the Liver -- 9.5 Gene Delivery to the Liver in Large Animals -- 9.6 Hydrodynamic Gene Delivery to Tissues other than Liver -- 9.6.1 Skeletal Muscle -- 9.6.2 Kidney -- 9.7 Mechanisms of Gene Delivery -- 9.8 Safety and Clinical Applicability -- References -- 10 DNA Pharmaceuticals for Skin Diseases -- 10.1 Introduction -- 10.2 Recombinant DNA-Based Skin Gene Therapy -- 10.2.1 Correction of Genetic Disorders -- 10.2.2 "Suicide" Gene Therapy -- 10.2.3 Genetic Pharmacology -- 10.3 DNA Vaccines -- 10.3.1 DNA Vaccination Through Skin -- 10.3.2 DNA Vaccines Against Skin Cancers -- 10.4 Physical Methods of DNA Delivery -- 10.4.1 Delivery of DNA to the Skin by Particle Bombardment -- 10.4.2 Microparticles for DNA Delivery -- 10.4.3 Genetic Immunization by Jet Injection -- 10.4.4 Epidermal Powder Immunization -- References -- 11 Electrotransfection - An Overview -- 11.1 Theory and Mechanisms -- 11.1.1 History -- 11.1.2 Mechanism of in vitro Electrotransfection at the Scale of a Single Cell -- 11.1.2.1 Permeabilization -- 11.1.2.2 Uptake of DNA -- 11.1.3 Mechanism of in vivo DNA Electrotransfer -- 11.2 In vivo DNA Electrotransfer in Practice -- 11.2.1 Device and Electrical Parameters -- 11.2.2 DNA Electrotransfer and Toxicity -- 11.2.3 Plasmid Biodistribution -- 11.3 Targeted Tissues -- 11.3.1 Skeletal Muscle -- 11.3.2 Tumor Tissue -- 11.3.3 Skin -- 11.3.4 Liver -- 11.3.5 Lung -- 11.3.6 Vasculature -- 11.3.7 Eye -- 11.3.8 Embryos -- 11.3.9 Cartilage -- 11.3.10 Gonads -- 11.4 Therapeutic Applications. , 11.4.1 Intramuscular Electrotransfer -- 11.4.1.1 Ectopic Secretion of Proteins -- 11.4.1.2 Muscle Disease Therapy -- 11.4.2 Vaccination -- 11.4.3 Cancer Gene Therapy -- 11.4.3.1 Strengthening Antitumor Response -- 11.4.3.2 Suicide Genes -- 11.4.3.3 Apoptosis-Inducing Genes -- 11.4.3.4 Inhibition of Tumor Angiogenesis -- 11.4.3.5 Other Strategies -- 11.4.4 Electrotransfer as a Tool -- 11.5 Conclusion -- References -- 12 Electrogenetransfer in Clinical Applications -- 12.1 Summary of the Basis of Electrogenetherapy -- 12.1.1 Tissue Electropermeabilization -- 12.1.2 DNA Electrophoresis -- 12.1.3 The Interest of Electrogenetherapy -- 12.2 The Road to Clinical Electrogenetherapy -- 12.2.1 Basic Difficulties and Requirements -- 12.2.1.1 Electrogenetherapy is a Local Treatment -- 12.2.1.2 DNA Injection -- 12.2.1.3 Need for Appropriate Electrodes -- 12.2.1.4 Need for Appropriate Electrical Pulse Generators -- 12.2.1.5 Electrogenetherapy and Public and Professional Perceptions of the Biomedical Use of Electricity -- 12.2.2 The CLINIPORATOR Project -- 12.2.3 The ESOPE Project -- 12.2.4 Future Perspectives -- References -- 13 Cancer Inhibition in Mice After Systemic Application of Plasmid-Driven Expression of Small Interfering RNAs -- 13.1 Introduction -- 13.2 Plasmid-Expressed siRNA -- 13.2.1 PLK1 shRNA-Mediated Inhibition of PLK1 Expression -- 13.2.2 Nuclease Inhibitor ATA and Stability of Plasmid DNA in Mammalian Blood -- 13.2.3 Antitumor Activity of PLK1 shRNA in vivo -- 13.2.4 Vector-Induced Decreased Expression of PLK1 and Antitumor Activity -- 13.3 Conclusion and Future Directions -- References -- Subject Index.