Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souches. / Lentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols.

Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Re oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität. / In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to cell lines known to produce HERV-K virus particles, such as the teratocarcinoma cell line GH and breast cancer cell lines, recently melanoma cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma cell lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma cell lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem cells was investigated. The investigated stem cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem cells, particularly for a tumourigenic potential.

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

Chromatin insulators are short DNA sequences that, together with enhancers and silencers, orchestrate gene transcription through DNA-protein interactions in eukaryotic genomes. It has been proposed that insulators operate at the chromatin level by generating functionally independent higher-order chromatin domains. Insulators may maintain the integrity of such domains using two properties: blocking enhancer-promoter interactions and blocking heterochromatin spreading. The gypsy insulator of Drosophila was identified as a region of the gypsy retrovirus responsible for the production of tissue-specific mutations in many genes. The Suppressor of Hairy wing [Su(Hw)] protein contains 12 zinc fingers that specifically bind the gypsy insulator. Upon DNA binding, Su(Hw) recruits a second protein, Modifier of Mdg4 67.2 [Mod(mdg4) 67.2], and the interaction of both proteins is required for insulator function in vivo. We have found that three different arrays of gypsy retrovirus insertions in a yellow transgene result in unique yellow phenotypes, showing that the enhancer-blocking activity of the Drosophila gypsy insulators depends on the relative orientation of the gypsy retroviruses on the chromosome. We also observed from transgenic lines with gypsy retrovirus or insulator insertions that interaction of insulators may be regulated by active enhancers according to the relative positions of the insulators flanking the enhancers. Moreover, we show that gypsy insulators can positively modulate yellow activation and result in wild-type levels of expression when placed upstream of enhancers in yellow transgenes in which enhancers are placed out of context by &#;-DNA spacers and fail to reproduce the expression levels of yellow in wings and body cuticle. Our results provide evidence indicating that this phenomenon is independent of the boundary activity. Genetic analysis using mod(mdg4)67.2 mutant lines containing gypsy retrovirus insertions revealed that the gypsy insulator may be placed close to the yellow promoter region and be intimately involved in transcriptional activation and repression. Therefore, we suggest that insulators may also function by mediating long range interactions between enhancers and promoters.

Long terminal repeats (LTRs) are non-coding repeats flanking the protein-coding genes of LTR retrotransposons. The variability of LTRs poses a challenge in studying them. Hidden Markov models (HMMs), probabilistic models widely used in pattern recognition, are useful in dealing with this variability. The aim of this work was mainly to study LTRs of retroviruses and LTR retrotransposons using HMMs. Paper I describes the methodology of HMM modelling applied to different groups of LTRs from exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs). The detection capabilities of HMMs were assessed and were found to be high for homogeneous groups of LTRs. The alignments generated by the HMMs displayed conserved motifs some of which could be related to known functions of XRVs. The common features of the different groups of retroviral LTRs were investigated by combining them into a single alignment. They were the short inverted terminal repeats TG and CA and three AT-rich stretches which provide retroviruses with TATA boxes and AATAAA polyadenylation signals. In Paper II, phylogenetic trees of three groups of retroviral LTRs were constructed by using HMM-based alignments. The LTR trees were consistent with trees based on other retroviral genes suggesting co-evolution between LTRs and these genes. In Paper III, the methods in Paper I and II were extended to LTRs from other retrotransposon groups, covering much of the diversity of all known LTRs. For the first time an LTR phylogeny could be achieved. There were no major disagreement between the LTR tree and trees based on three different domains of the Pol gene. The conserved LTR structure of paper I was found to apply to all LTRs. Putative Integrase recognition motifs extended up to 12 bp beyond the short inverted repeats TG/CA. Paper IV is a review article describing the use of sequence similarity and structural markers for the taxonomy of ERVs. ERVs were originally classified into three classes according to the length of the target site duplication. While this classification is useful it does not include all ERVs. A naming convention based on previous ERV and XRV nomenclature but taking into account newer information is advocated in order to provide a practical yet coherent scheme in dealing with new unclassified ERV sequences. Paper V gives an overview of bioinformatics tools for studies of ERVs and of retroviral evolution before and after endogenization. It gives some examples of recent integrations in vertebrate genomes and discusses pathogenicity of human ERVs including their possible relation to cancers. In conclusion, HMMs were able to successfully detect and align LTRs. Progress was made in understanding their conserved structure and phylogeny. The methods developed in this thesis could be applied to different kinds of non-coding DNA sequence element.

Lentivirus efficiently transduce stem cells, however are notably silenced in embryonic stem cells (ESC). Provirus can be silent, expressing, or variegated when clonal single copy ESCs spawn daughters that revert expression despite containing identical integration sites (IS) indicating epigenetic regulation. In the silent state, variegated provirus are bound by H1 and MeCP2, where H1 compensates for MeCP2 binding in DNA methylation null ESCs, consistent with a model of heterochromatin formation dependent on concentrations of its constituent components. ESC Variegation was hypothesized to result from spreading of nearby heterochromatin. Global IS analysis indicates Variegated IS favour gene deserts, repeat clusters, and LINEs while Expressers prefer gene density with stable modest expression and SINEs. Chromatin data does not support a role for the spread of heterochromatin possibly a consequence of the dynamic/dispersed nature of ESC heterochromatin. Variegation thus may depend on stochastic chromatin regulation by pluripotency factors at proximal genome organizing repeats.

Lentivirus efficiently transduce stem cells, however are notably silenced in embryonic stem cells (ESC). Provirus can be silent, expressing, or variegated when clonal single copy ESCs spawn daughters that revert expression despite containing identical integration sites (IS) indicating epigenetic regulation. In the silent state, variegated provirus are bound by H1 and MeCP2, where H1 compensates for MeCP2 binding in DNA methylation null ESCs, consistent with a model of heterochromatin formation dependent on concentrations of its constituent components. ESC Variegation was hypothesized to result from spreading of nearby heterochromatin. Global IS analysis indicates Variegated IS favour gene deserts, repeat clusters, and LINEs while Expressers prefer gene density with stable modest expression and SINEs. Chromatin data does not support a role for the spread of heterochromatin possibly a consequence of the dynamic/dispersed nature of ESC heterochromatin. Variegation thus may depend on stochastic chromatin regulation by pluripotency factors at proximal genome organizing repeats.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-147).

Die Familie der Retrovirinae wird in zwei Unterfamilien untergliedert, die Orthoretrovirinae und die Spumaretrovirinae. Foamyviren stellen aufgrund einiger besonderer Eigenschaften die einzigen Vertreter dieser Unterfamilie, die sie als Bindeglied zwischen den Retroviren und den Hepadnaviren erscheinen lassen. So erfolgt beispielsweise die reverse Transkription des viralen Genoms nicht erst nach Eintritt in die Zielzelle, sondern, anders als bei Orthoretroviren, bereits in der Produzentenzelle noch während oder kurz nach der Morphogenese. Diese Eigenschaft teilen Foamyviren mit den Hepadnaviren ebenso wie die obligate Koexpression der Kapsidproteine mit den viralen Hüllproteinen für die Freisetzung von Viruspartikeln. Im Gegensatz zu Orthoretroviren sind Foamyviren folglich nicht in der Lage virusähnliche Partikel (VLP) zu sekretieren und die spezifische Funktion des PFV Env Proteins kann nicht durch heterologe Hüllproteine übernommen werden. Die Synthese des PFV Env Vorläuferproteins erfolgt am rER, wobei es eine Typ III Membrantopologie erhält, mit sowohl dem N- als auch dem C-Terminus im Zytoplasma. Während des Transports des Proteins zum Ort der Partikelknospung, wird es posttranslational im Golgi-Apparat, oder dem trans-Golgi Netzwerk, durch Furin oder eine Furin-ähnliche Protease in drei partikelassoziierte Untereinheiten prozessiert. Eine Partikelassoziation retroviraler Signalpeptide ist bislang nur für Foamyviren nachgewiesen worden, genauso wie eine essentielle Rolle dieses Proteins bei der Interaktion zwischen dem

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