De nombreuses études suggèrent que linhibition de la voie alterne du complément de lhôte est nécessaire aux parasites hématophages pour leur permettre daccomplir leur repas sanguin. Une revue décrivant ces études a été publiée dans Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Plusieurs études suggèrent que linhibition de la voie alterne de lhôte par les protéines salivaires de tiques est importante pour lacquisition du repas sanguin et la transmission subséquente de pathogènes à lhôte infesté. Confortant cette hypothèse, une protéine salivaire capable dinhiber la voie alterne du complément a été clonée chez la tique américaine Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Cette protéine, appelée Isac, ne présente aucune homologie avec les autres molécules anti-complément connues à ce jour, suggérant que cette protéine a été acquise au cours de lévolution par un mécanisme dévolution convergente. En plus de cet aspect fondamental, Isac représente un candidat antigénique prometteur pour le développement dun vaccin anti-tique potentiellement capable dinduire le rejet de la tique et/ou de prévenir la transmission des pathogènes. Le but initial de cette thèse était didentifier le ou les homologue(s) de la protéine Isac de la tique américaine Ixodes scapularis chez la tique européenne Ixodes ricinus. De façon intéressante, deux séquences différentes ont été isolées du transcriptome des glandes salivaires de la tique I. ricinus (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Lexpression de ces séquences a révélé quelles codent pour des protéines secrétées capables dinhiber la voie alterne du complément. Ces protéines ont été appelées IRAC I et II pour « Ixodes ricinus anti-complement protein I and II ». La caractérisation des IRACs à laide danticorps monoclonaux a permis de révéler que ces deux protéines sont exprimées de façon constitutive au sein des glandes salivaires de la tique Ixodes ricinus et sont sur-exprimées au cours du repas sanguin. Létude de lexpression des protéines IRAC I et IRAC II au sein de la population dIxodes ricinus a révélé que ces deux protéines sont des paralogues codés par des gènes différents et non par des allèles dun même locus. Enfin, des analyses phylogéniques portant sur les différentes séquences codant pour les protéines homologues à Isac isolées chez les tiques Ixodes scapularis, Ixodes ricinus et Ixodes pacificus ont révélé que les tiques appartenant au complexe Ixodes ricinus codent pour une famille encore non décrite de molécules anti complément qui se sont diversifiées au cours de lévolution par un processus de sélection darwinienne positive. Les analyses phylogéniques des IRACs suggèrent que ces séquences ont subi une diversification par un processus de pression de sélection darwinienne positive, menant probablement à des molécules possédant des propriétés biologiques différentes. Dans la seconde étude, nous avons testé lhypothèse de travail selon laquelle chaque paralogue pourrait posséder des activités inhibitrices différentes à lencontre du complément de différentes espèces dhôtes naturels, contribuant ainsi à élargir le spectre dhôte des tiques I. ricinus. Les résultats obtenus démontrent que cette hypothèse est correcte (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). Dans la troisième et dernière étude de ce manuscrit, nous avons testé le potentiel des protéines IRACs comme candidat antigénique pour le développement dun vaccin anti tique. Des recombinants de lherpèsvirus bovin 4 (BoHV-4) exprimant IRAC I ou IRAC II ont été produits. De façon intéressante, nous avons observé que bien que les recombinants BoHV-4 expriment de hauts taux de protéines IRACs fonctionnelles in vitro, les lapins immunisés à laide des recombinants BoHV-4 exprimant les IRACs ne développent pas de réponse humorale détectable à lencontre des transgènes. Dans le but daugmenter limmunogénicité des IRACs exprimés comme transgène, une seconde génération de recombinants BoHV-4 a été produite. Ceux-ci induisent lexpression des IRACs sous la forme de protéines de fusion transmembranaires à la surface des cellules infectées. Linoculation de ces recombinants à des lapins a engendré le développement dune forte réponse humorale à lencontre des IRACs. Néanmoins, cette réponse immune na pas engendré deffet majeur sur le repas sanguin de femelles Ixodes ricinus placées sur les lapins immunisés. / An increasing number of studies demonstrate that inhibition of host complement activation is crucial for completion of the blood feeding process of hematophagous parasites. A review of these studies has been published in Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Several observations suggest that inhibition of the host complement alternative pathway by tick salivary proteins is crucial for the achievement of blood feeding and efficient transmission of the pathogens transmitted by the parasite. Strongly supporting this conclusion, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Interestingly, this molecule, termed Isac, has no similarity to any previously reported anti-complement molecules suggesting that it has been acquired through a mechanism of convergent evolution. In addition to this fundamental aspect, Isac is also a promising candidate antigen for the development of an anti-tick vaccine potentially able to induce the reject of the tick and/or to prevent the transmission of the pathogens. The initial goal of the present work was to clone the orthologue of Isac from the European tick Ixodes ricinus. Interestingly, two different sequences were isolated from the transcriptome of I. ricinus salivary glands (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins were called I. ricinus anticomplement (IRAC) protein I and II. Further characterization of IRACs using monoclonal antibodies revealed that both proteins are expressed constitutively in I. ricinus salivary glands and are up-regulated during blood feeding. Analysis of a series of individual ticks revealed that all ticks tested express both IRAC I and IRAC II, demonstrating that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses of the I. ricinus IRAC I and II sequences together with homologues from I. scapularis and I. pacificus demonstrates that ticks belonging to the Ixodes ricinus complex encode a new family of relatively small anti-complement molecules undergoing diversification by positive Darwinian selection. Phylogenetic analyses of IRACs suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the second study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. The data obtained demonstrated that this working hypothesis is correct (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). In the third and last chapter of the present manuscript, we addressed the potential of IRAC I and II as candidate antigens for the development of an anti-tick vaccine. Bovine herpesvirus 4 (BoHV-4) recombinants expressing IRAC I or II were produced. Interestingly, we observed that although both recombinants expressed high levels of functional IRAC proteins in vitro, our attempts to immunize rabbits against IRACs via infection with these viruses invariably failed. In order to improve the immunogenicity of IRACs expressed as transgene, a second generation of BoHV-4 recombinants was produced. The latter expressed IRACs as transmembrane fusion proteins on cell surface. Comparison of the vaccine potential of BoHV-4 recombinant viruses expressing either secreted or transmembrane IRAC proteins revealed that while the former did not induce a detectable immune response against IRACs, the latter led to high titers of anti-IRAC antibodies. However, the immune response induced against IRACs did not lead to the reject of the tick but only slightly increased the duration of the blood feeding process.

Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...

Knowledge on the human genome and specific sequences associated with human diseases is continuously growing. The ability to connect human genetics to cellular mechanisms and physiology raises the need for medicine to get to gene specific therapeutics. In order to achieve gene-specific modification, tools are required to enable sequence-specific DNA cleavage. Not long ago, the RNA-guided endonuclease Cas9 was shown to effectively facilitate gene editing in humans. Cas9 endonuclease, which is naturally part of an adaptable bacterial immune system, can be easily adjusted to recognize and cleave specific DNA sequences in a 20 nt RNA-DNA complementary manner. The easy adjustability and high efficiency of Cas9 gave rise to hopes that this genome engineering tool could pave the way to ‘gene surgery’ in humans. However, to achieve DNA cleavage, the endonuclease and its guiding RNA need to be sufficiently accessible in the nucleus of target cells. Viruses, which evolution has made well adapted to transfer their own genetic information into cells can be exploited for transfer of foreign genetic material. Replication deficient retroviruses therefore represent interesting vehicles for gene delivery. Retroviruses preferentially incorporate their own genetic information in the form of RNA into viral particles. Typically, viral RNA of retroviruses is reverse transcribed into DNA during viral infection and integrated into host cell chromosomes. In this respect, integration-competent or integration-deficient lentiviral (HIV-derived) vectors (ICLV/IDLV) were reported to be efficient ‘gene shuttles’ for Cas9 delivery. In contrast, up to now Foamy viruses (FV), which represent a distinct subfamily in the family of retroviruses have not previously been tested for their efficiency to transduce CRISPR/Cas9 components. FV show several unique characteristics some of which make them interesting candidates for gene therapy, such as high transduction efficiency on a wide variety of human cell lines or a special capability to efficiently transfer and provide non-viral RNA in target cells. In this thesis the unique characteristic of FVs, which allow for the efficient transduction of non-viral RNAs, was exploited for transient FV mediated (TraFo) Cas9 expression. It is shown in this thesis that gene knock-out (KO) achieved with TraFo Cas9 particles appears to have several advantages over ICLV or IDLV mediated Cas9 transduction. In this work, it could be demonstrated that a single application of TraFo Cas9 supernatant results in high efficiency of GFP KO in osteosarcoma cells (U2OS). The efficiency of gene KO with TraFo Cas9 particles exceeded gene KO frequencies achieved with similar volumes of ICLV or IDLV supernatant for Cas9 transduction. In addition, transient Cas9 delivery achieved with TraFo particle supernatant resulted in remarkably reduced Cas9 off-target cleavage compared to corresponding infections with ICLV or IDLV particles. The results show, that TraFo Cas9 represents an interesting addition to the currently utilized methods for transient Cas9 delivery. One particular feature of TraFo particle transduction is especially noteworthy – TraFo mediated transduction does not depend on any particular adjustment on the encapsidated non-viral RNA sequence (such RNA only needs to be present in sufficient amounts during virus assembly) nor does it depend on any modification of viral proteins. The easy adaptability of TraFo mediated non-viral RNA transfer is an especially remarkable feature, since science continues to both developing new variants of Cas9 and continues to find new and interesting members of the pool of CRISPR enzymes. In this regard TraFo particles represent interesting vehicles to transiently provide mRNA transcripts of such new protein candidates in cells. The ability of TraFo particles to provide the RNA sequence needed to guide Cas9 (termed sgRNA) to its target DNA sequence in cells was additionally investigated. It was assumed that typically engaged RNA polymerase (RNAP) III transcription of sgRNAs hampers transduction with TraFo particles, since RNAP III-derived transcripts are not actively exported into the cytoplasm and show low stability. An additional CRISPR enzyme Csy4 was used, which is able to specifically cleave RNA. This enabled TraFo mediated transfer of RNAP II transcripts (with active nuclear export and higher stability than RNAP III transcripts) with embedded sgRNA sequences. It was demonstrated that a simultaneous infection of cells with TraFo particles providing bicistronic transcripts of Cas9 and Csy4 on the one side and RNAP II-derived transcripts with embedded sgRNA sequences on the other, enabled reasonable GFP gene inactivation in U2OS cells. Gene KO with RNAP II transcripts as a result significantly exceeds TraFo transduction of RNAP III-derived sgRNA. Interestingly, with regard to gene KO, it was found that de novo transcription of sgRNAs from viral DNA (by integration-competent or integration-deficient retroviral vector [ICRV/IDRV] transduction) when combined with TraFo Cas9 transduction was superior to a TraFo transduction of sgRNA transcripts. IDRV mediated transduction was optimized in order to minimize the risk of unfavorable genome modification of cells by viral DNA integration. By adding the coding sequence of a fluorescent marker to the viral vector, it was demonstrated that a smaller number of viral particles helps to significantly lower the frequency of viral DNA integration. In addition, the expression of a fluorescent marker opened up the opportunity to further reduce the cell fraction with continuous marker gene expression by flow cytometric cell sorting. The IDRV/ICRV sgRNA and TraFo Cas9 delivery system was then challenged for use on immortalized and primary T cells. Primary T cells represent interesting targets for genetic engineering since modified T cells can be utilized as ‘living drugs’ (by expression of chimeric antigen receptors – CARs) against cancer cells. Efficient gene inactivation was observed on the immortalized T cell line – Jurkat. Transduction of primary T cells pointed to certain restrictions of the split two-virus delivery system for sgRNA and Cas9 transduction. However, despite certain limitations, it was possible to demonstrate that this FV-derived Cas9 delivery system is also feasible on primary tissue, and further optimization could make it an interesting alternative delivery method for CAR therapy. The ability of IDRV vector genomes to provide repair template donor DNA to induce homologous recombination (HR) was additionally investigated. DNA double-strand breaks in eukaryotic cells are typically repaired by the error prone non-homologous end joining pathway (often leading to frame-shift mutations by small insertions or deletions) or HR. Delivery of a homologous DNA sequence during DNA cleavage enables site-specific integration of exogenous DNA sequences. The work of this thesis showed that IDRV vector genomes providing repair template donor DNA allow for HR in a homology length dependent manner. Besides the length of homology, it was also observed, that the length of sequence which should be integrated (KI) remarkably influences the frequency of HR. HR is therefore engaged significantly more frequently if single nucleotides, rather than a whole gene, are provided as sequences within a repair template. In addition, viral vectors were augmented with additional fluorescent marker sequences. It could subsequently be demonstrated that the majority of cells showed accurate sequence-specific DNA integration. Furthermore, several indications were found, which lead to the assumption that the ratio of KI to homologous sequence markedly influences the accuracy of HR. Using the previously obtained knowledge it was further possible to tag an essential human protein by FV vector mediated transient Cas9 and repair template transduction. It was found that the large packaging capacity of FV vectors can be exploited to enable selection and flow cytometric sorting of cells with correct site-specific DNA integration. In summary, the results of this thesis demonstrate for the first time that FV mediated non-viral mRNA Cas9 transduction in combination with retroviral delivery of sgRNA (and repair template sequence) are a promising basis for several different interesting applications with relevance for not only basic research, but also for gene therapy.:1. Introduction 1 1.1 Gene therapy 1 1.2 Viral vectors for gene therapy 2 1.3 History of retroviral research 2 1.4 Taxonomy of Retroviruses 3 1.5 Foamy Viruses 4 1.5.1 Morphology of Foamy Virus 6 1.5.2 Foamy Virus replication 7 1.5.3 Foamy virus proteins, as part of a viral vector system 10 1.6 Genetic engineering 14 1.6.1 ‘DNA scissors’ – Zinc-finger and Transcription-activator like effector nucleases 15 1.6.2 History of CRISPR/Cas9 as a tool for genetic engineering 16 1.7 CRISPR/Cas immunity in prokaryotes 18 1.8 CRISPR/Cas9 functioning 21 1.9 Double-strand break repair in eukaryotic cells 21 1.9.1 Classical NHEJ 23 1.9.2 Homologous recombination 24 1.9.3 DSB repair in vertebrates 26 1.10 DSBs in context of CRISPR/Cas9 cleavage 27 1.11 Thesis Aim: CRISPR/Cas9 transduction with FV particles 28 2. Materials and Methods 30 2.1 Materials 30 2.1.1 Chemicals 30 2.1.2 Buffers and Solutions 30 2.1.3 Bacterial Growth Media 33 2.1.4 Cell Culture Media 34 2.1.5 Antibodies 34 2.1.6 Enzymes 35 2.1.7 Commercial Kits and additional reagents 36 2.1.8 Size Markers 36 2.1.9 Antibiotics 36 2.1.10 Bacterial strains 37 2.1.11 Cell lines 37 2.1.12 Devices and Software 37 2.1.13 Oligonucleotides 38 2.1.14 Plasmids 46 2.1.15 sgRNA sequences 56 2.1.16 Consumable material 57 2.2 Molecular Biology Methods 58 2.2.1 Restriction of DNA 58 2.2.2 Polymerase chain reaction 59 2.2.3 Gibson assembly 60 2.2.4 Agarose gel electrophoresis 60 2.2.5 Ligation 61 2.2.6 Cultivation of bacteria 62 2.2.7 Transformation 62 2.2.8 Plasmid Preparation 63 2.2.9 Sequencing 65 2.3 Cell culture methods 66 2.3.1 Passaging of cells 66 2.3.2 Cell counting 66 2.3.3 Freezing and thawing of cells 66 2.3.4 Seeding and fixation of cells for microscopy 67 2.4 Virological Methods 67 2.4.1 Polyethyleneimine transfection 67 2.4.2 Integration-competent, integration-deficient and ‘transient’ retroviral vectors 68 2.4.3 Infection of adherent cells 70 2.4.4 Infection of suspension cells 71 2.4.5 Flow cytometry 72 2.4.6 Multiplicity of infection (MOI) 72 2.4.7 Particle preparation 73 2.5 Nucleic acid composition in viral particles and culture cells 73 2.5.1 Isolation of total RNA from viral particles 73 2.5.2 RNA isolation from culture cells 73 2.5.3 Reverse transcription of viral or cellular RNA 73 2.5.4 DNA isolation from culture cells 74 2.5.5 Quantitative PCR (qPCR) analysis 74 2.5.6 T7 endonuclease assay 75 2.6 Protein biochemistry methods 76 2.6.1 Cell lysates 76 2.6.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 76 2.6.3 Semi-dry Western Blot 77 2.6.4 Immunodetection 78 2.6.5 Stripping of Western blot membranes 78 2.6.6 Immunostaining of cells for FACS analysis 78 2.7 Microscopy methods 79 2.7.1 Fluorescence microscopy 79 2.7.2 Confocal Laser scanning Microscopy (CLSM) 79 2.7.3 Live-cell imaging 79 3. Results 80 3.1 Transient foamy virus transduction of non-viral mRNA transcripts 80 3.2 Transient foamy virus transduction of Cas9-encoding mRNA transcripts 81 3.3 Cas9-encoding nucleic acids and their ‘effects’ in cells after retroviral transduction 84 3.4 Off-target analysis after TraFo Cas9 delivery 87 3.5 Transient fomy virus transduction of Cas9 and sgRNAs 89 3.6 Retroviral vectors providing sgRNAs and a fluorescent protein 92 3.6.1 Gene knock-out with retroviral vectors under saturated conditions 92 3.6.2 MOI adjusted ID sgRNA vector supernatants for comparison of residual vector integration 94 3.6.3 Gene knock-out in murine embryonic fibroblasts 95 3.7 Influence of Cas9 expression on IDRV vector genome integration 96 3.8 TraFo Cas9 mediated T cell receptor knock-out in immortalized and primary human T cells 97 3.9 Homology-directed repair after FV CRISPR/Cas9 mediated double-strand breaks 99 3.9.1 Length of homologous donor DNA and its influence on HDR 100 3.9.2 Effect of freezing viral supernatants on the frequency of HDR 102 3.9.3 Effect of donor DNA mismatches on the frequency of HDR 104 3.10 Investigation on donor DNA integration with additional fluorescent markers 105 3.11 Lentiviral and foamyviral transduction of HDR donor DNA 107 3.12 HDR mediated single nucleotide substitutions after TraFo CRISPR/Cas9 delivery 109 3.13 Tagging of an endogenous protein after TraFo CRISPR/Cas9 delivery 111 3.13.1 Specific CRISPR/Cas9 mediated cleavage of endogenous hPLK1 gene 111 3.13.2 Homology-directed repair of the hPLK1 gene for endogenous gene tagging 113 3.13.3 Confocal fluorescence microscopy analysis of GFP-Plk1 HeLa cell populations 118 4. Discussion 120 4.1 Genetic engineering – potential and risks 120 Chapter I Transient FV vectors – mRNA delivery vehicles for CRISPR/Cas9 mediated gene editing 122 4.2 Non-viral Cas9-encoding mRNA transfer in foamy virus particles 122 4.2.1 Fate of Cas9-encoding nucleic acids in cells after TraFo Cas9 transduction 124 4.2.2 Potential adjustments to further improve TraFo Cas9 transduction 125 4.2.3 Lentiviral in contrast to TraFo transduction of Cas9-encoding nucleic acids 126 4.3 Efficiency of Cas9-mediated gene knock-out with different retroviral vectors 127 4.4 Type of retroviral Cas transduction and its influence on the specificity of Cas9 cleavage 127 4.5 Alternative approaches to deliver Cas9-encoding mRNA in human cells 129 4.6 Transient sgRNA transduction with TraFo particles 131 Chapter II Delivery of foreign DNA with FV-derived vectors – enabling gene knock-out and homology-directed repair 133 4.7 Gene inactivation by TraFo Cas9 transduction and sgRNA expression from retroviral vector genomes 133 4.7.1 Gene editing in immortalized and primary T cells 135 4.8 Homology-directed repair with IDRV genomes 137 4.8.1 The influence of the length of sequence homology on HR 138 4.8.2 The influence of freezing viral supernatants on HR 139 4.8.3 Widening the applicability of TraFo vector particles for improved HR 140 4.8.4 The influence of mismatching nucleotides on HR 140 4.8.5 Visualization of inaccurate HR or additional dsDNA integration 141 4.8.6 The influence of the ratio of knock-in and homologous sequence on the accuracy of HR 142 4.8.7 Alternatives to double-stranded donor DNA 143 4.9 Endogenous gene tagging with IDPV donor DNA transduction 145 4.9.1 Alternative approaches for endogenous protein tagging 146 5. Conclusion 148 6. Summary 150 6.1 Summary 150 6.2 Zusammenfassung 153 7. Supplementary 157 8. References 159 9. Appendices 182 9.1 Indices 182 9.1.1 Abbreviations 182 9.1.2 Index of Figures 185 9.1.3 Index of Tables 187 9.2 Curriculum Vitae 188 9.3 Publication Record 189 9.4 Congress Contributions 189 9.5 Patent Applications 189 10. Statement of Authorship 190

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène. / Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.