Problems in Gene Therapy and Transgenesis Prof. Dr. Oliver Kayser Technische Biochemie TU Dortmund 16.03.2015 Chapter 5 - Transgenesis and Gene 1 Therapy Producing transgenic animals Purpose: Production of heterolog proteins in transgenic animals Principle: Integration of heterolog DNA with selected genetic information in fertilised eggs Methods: Mikroinjektion (-) Retrovirus vectors (-) Genetically modified embryonic stem cells (++) 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 2 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 3 Dolly 16.03.2015 Chapter 5 - Transgenesis and Gene 4 Therapy Telomers 16.03.2015 Chapter 5 - Transgenesis and Gene 5 Therapy klausurrelevant 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 6 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 7 Advantage of sheeps and goats as transgenic animals • high milk production capacity (2-3 l/ day) • short gestation period and quick maturation • low capital investment • easy handling and breeding • high expression level of proteins (35g protein/ l milk) • easy downstream processing • ongoing supply of product is guaranteed by breeding 16.03.2015 Chapter 5 - Transgenesis and Gene 8 Therapy Drug safety • Genetic stability from animal to anmial • Variation in produktion und purity during lactation • Microbial, varial, and mykoplasm contamination • TSE, Scrapie • Age and general conditions of animals 16.03.2015 Chapter 5 - Transgenesis and Gene 9 Therapy Atryn • First recombinant therapeutic protein approved in 2009 • Anticoagulant for treatment of antithrombin III (AT alpha) deficiency in newborn • 1 goat replaces 90,000 donators http://16.03.2015www.gtc -bio.comChapter/ 5 - Transgenesis and Gene Therapy 10 Blood Clotting 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 11 Transgenic animals as disease model • Knock out animals with directed genetic defect • Testing of drugs and dosage forms • Hypercholesterinemia • Cancer • Coronar diseases • Onco-Mouse as first example 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 12 Transgenic animals Ageing-Mouse, Defect in DNA-Helcicase 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 13 Lehrbuch Molekulare Zellbiologie, Kapitel 10.5 Kapitel Zellbiologie, Molekulare Lehrbuch Transgenic salmon • „Turbo-salmon“ AquAdvantage, AquaBounty Technologies • Cloning of growth gene for improved growth (growth factors) • Fish grows in 16 to 18 months instead of 3 years • Triploid and not diploid http://www.nature.com/news/transgenic-salmon- (Biosafety) nears-approval-1.12903 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 14 Kuru-Region in Papua-Neuguinea 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 15 Prions 16.03.2015 Chapter 5 - Transgenesis and Gene 16 Therapy Brain autopsy 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 17 Somatic Gene Therapy 16.03.2015 Chapter 5 - Transgenesis and Gene 18 Therapy 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 19 Defective Genes Disease Genetic defect hemophilia A absence of clotting factor VIII cystic fibrosis defective chloride channel protein muscular dystrophy defective muscle protein (dystrophin) sickle-cell disease defective beta globin hemophilia B absence of clotting factor IX severe combined any one of several genes fail to make immunodeficiency a protein essential for T and B cell (SCID) function 16.03.2015 Chapter 5 - Transgenesis and Gene 20 Therapy ADA und purine metabolim IMP AMP ADP ATP dAMP dADP dATP Acumulation Adenosin 2-Deoxyadenosin Adenosin- Deaminase Inosin 2-Deoxyinosin 16.03.2015 Chapter 5 - Transgenesis and Gene 21 Therapy Basics of ADA-Defency dAMP dADP dATP Apoptosis Acumulation Inhibition of Ribonucleotid-Reductase 2-Deoxyadenosin Consequence: No DNA-Replication Adenosin- Deaminase Inhibition of T cell activation 2-Deoxyinosin 16.03.2015 Chapter 5 - Transgenesis and Gene 22 Therapy EX VIVO vs IN VIVO Delivery to target Tumor cells fibroblasts local Hemopathic cells Vector systemic Gene transfer in culture 16.03.2015 Chapter 5 - Transgenesis and Gene 23 Therapy Examples for local gene expression 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 24 Vectors in gene technology Biochemical vectors/Methods: • viral vectors (Retro-, Adenovirus, AAV) • ligands with polylysin • 'Transferinfection' Physical methods: • Hydrodynamic pressure • 'Biolistic Bombardment' • electroporation • direct tissue inclusion Chemical vectors: • cationic Liposomes • polycations like DEAE-Dextran, Polyethylen-Imin 16.03.2015• poly -Lysin-polymersChapter 5 - Transgenesis and Gene 25 Therapy Retroviral vector system 16.03.2015 Chapter 5 - Transgenesis and Gene 26 Therapy Summary 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 27 16.03.2015 Chapter 5 - Transgenesis and Gene 28 Therapy Non-viral delivery • The non-viral approaches provide a safety (no virus) to gene therapy. However, the route to nucleus for stable expression is not completely understood 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 29 Vectors in gene technology Biochemical vectors/Methods: • viral vectors (Retro-, Adenovirus, AAV) • ligands with polylysin • 'Transferinfection' Physical methods: • Hydrodynamic pressure • 'Biolistic Bombardment' • electroporation • direct tissue inclusion Chemical vectors: • cationic Liposomes • polycations like DEAE-Dextran, Polyethylen-Imin 16.03.2015• poly -Lysin-polymersChapter 5 - Transgenesis and Gene 30 Therapy Electroporation Physical Principle CHO-cells, 20 ms, 1 kV cm-1 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 31 Gehl J (2003) Acta Physiol Scand 177:437 Electroporation Examples Pros and Cons + High safety + High compliance + No risk of infection – low transfection – no tissue specifity – transient expression K562 cells with phGFPS65T transfection Target cells / organs • liver • skin • muscel Mainly: Beta-Galactosidase transfection Transdermal applications 16.03.2015in vivo in muscel cells Chapter 5 - Transgenesis and Gene Therapy 32 Mir et al. (1999) Proc Natl Acad Sci USA 96:4262 Biobalistics Principle Gene Gun Gold partikel Skin Expression 1-3 µm Examples Pros and Cons • Application of vaccines + easy to use ® ® • Arilvax , Hepagene + using already existing technology ® • Powderject Inc: Accell ® • Biorad Inc: Helios - high costs • Transdermal Application - not only transdermal Application 16.03.2015 Chapter 5 - Transgenesis- low efficancy and Gene Therapy 33 Powderject Inc.: Accell® 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 34 Maa et al. (2000) Curr Pharm Biotechnol 3:283 Yang et al. (1999) in Nonviral vectors for gene therapy, Academic Press, 171 Liposomes • This technique relies upon the negative charge of DNA-phosphate, cationic lipids, and cell surfaces (negative charge, sialic acid). • Two classes of cationic lipids have been used: – two alkyl chain in each cationic lipid molecules (DOTMA) – cholesterol as a backbone. • Dioleoylphosphoatidylethanolamine, a helper lipid is usually included to improve efficiency. • Expression is transient for several days only. 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 35 Complexing DNA (+/- -ratio) O O O + N O BASE O O H H H H O O H O + N - O P O O O BASE O H H H H O H - O P O O 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 36 DNA Condensation 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 37 Cellular uptake and migration pH 4,5-6 Long persistence DNA Hydrolysis DNA degradation by nucleases active uptake into nucleus 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 38 Wattiaux et al. (2000) ADDR 41:201 Polyethylenimine (PEI) Gene transfer complex Uptake Migration / Transfection High stability High stability transient Half life time: 30-60 min „Sponge effect“ expression High toxicity (40-100 µg in mice) 50-100 nm Linear structure high transfection High branching low transfection Loading up to 800 kB Coupling with ligands Nuclease degradation 16.03.2015 (Transferrin,Chapter Monoclonal 5 - Transgenesis and Gene Therapy 39 antibodies, sugar, folate) Wightman et al. (2001) J. Gene Med 3:362 Proton sponge + + + Cl- + -+ + + H+ + - +- + H2O -+ +- + - -+ + - -+ -+ - -- - + - H+ + + + + + + + + + + - +- - + + -+ + + + + + - +- + +- + - + + - - - +- - Cl + + - --+ + -+ + - -+ +- - - - - -+ - -- + - + - + + + + Endocytosis + -+ + + H2O + - +- + + + + + + +- - + + + + + - + + + +- - + + + - -+ + - + -+ - -- + + + + + + - + - + - - - + + -+ + - -+ - -+ - -- - + - + + + PEI-Gene transfer complex + - + - - + + + - - + Nucleus Inner tertiar amines neutralise low pH in endosome swelling breaking 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 40 „Proton sponge“ Behr (1997) Chimia 51:34 Summary 16.03.2015 Chapter 5 - Transgenesis and Gene Therapy 41 .