Myosin II, das Motorprotein des kardialen Muskels, besteht aus zwei schweren und vier leichten Ketten. Der Hebelarmbereich der schweren Myosinkette (MyHC) enthält das IQ-Konsensus-Motiv für die Bindung der essentiellen leichten Myosinkette (ELC), welche wesentlich für eine normale Kraftentwicklung des Myosinmoleküls ist. Im Rahmen dieser Arbeit wurden fünf, mit hypertropher Kardiomyopathie assoziierte, Mutationen im humanen essentiellen ventrikulären leichten Myosinketten (hVLC1)-Gen (MYL3) untersucht (E56G, A57G, E143K, M149V, R154H). Von keiner dieser Mutationen war der Pathomechanismus bekannt. Ziel der Arbeit war es, die Effekte der Mutationen im MYL3-Gen auf Proteinstruktur und Funktion zu untersuchen und daraufhin einen möglichen Pathomechanismus zu formulieren. Dazu erfolgten Strukturanalysen (CD-Spektren, Schmelzkurven, FLIM), Versuche auf Protein- und Zellebene (Protein-Protein-Interaktionsstudien, Sorting Assay) sowie Untersuchungen in vitro (Zell-Verkürzungsmessungen, isoliert perfundierte Herzen nach Langendorff) und in vivo (Echokardiographie) im transgenen Mausmodell. / Myosin II, the motor protein of cardiac muscle, is composed of two heavy chains (MyHC) and four non-covalently linked light chains (MLC). The lever arm of the MyHC contains the IQ motif that binds the essential myosin light chain (ELC), which is necessary for the normal force production of the myosin molecule. Five with HCM associated mutations in the human ventricular essential myosin light chain (hVLC1) -gen (MYL3) were investigated in this study (E56G, A57G, E143K, M149V, R154H). The pathomechanisms of the mutations were not known. Aim of the study was i) to test the hypothesis that mutations in the ventricular essential myosin light chain affect the protein structure, the binding to the IQ motif of MyHC and the force production of the myosin molecule as well as ii) to postulate an accompanying pathomechanism. Structural analyses (circular dichroism, melting curves, fluorescence lifetime imaging microscopy), functional investigations (surface plasmon resonance spectroscopy, sorting assay) and in vivo (echocardiography) and in vitro studies in a transgenic mouse model were performed.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.

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