Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Insertion of the viral genome into host cell chromatin is a hallmark ofHuman Immunodeficiency Virus type 1 (HIV-1) replication. Catalyzed by the viral integrase (IN) enzyme, this step irreversibly links the fate ofthe provirus with that of the cell. IN however, has proved to be a tough nut for researchers to crack. Nevertheless, a number of milestone discoveries and insights accumulating over the last decade have propelled the field forward. In this thesis are bundled five research manuscripts reflecting distinct lines of research on HIV-1 IN, yet all focusing on itsstructure, function and inhibition.Since the approval of raltegravir in 2007, IN strand transfer inhibitors (INSTIs) have become an integral part of antiretroviral therapy. Three are in clinical use today, potently inhibiting viral replication with a dramatic drop in viral load.However, the emergence of resistance to these drugs underscores the need to develop next-generation IN catalytic site inhibitors with improved resistance profiles. In chapter 3, we present a 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivative (MB-76) which potently blocks integration of wild type as well as raltegravir-resistant viruses. A crystal structure of MB-76 bound to the Prototype Foamy Virus (PFV) intasome reveals an overall binding mode identical to that of INSTIs but with a number of unique features. Its characterization highlights MB-76 as a promising candidate for further development.Chapters 4 and 5 describe an entirelydifferent approach to target IN. HIV-1 is capable of infecting non-dividing cells and therefore needs to be imported into the nucleus prior to integration. Transportin-SR2 (TRN-SR2) is a cellular β-karyopherin thought to import the viral preintegration complex (PIC) into the nucleus through a direct interaction with IN. As for most nuclear import cargoes, the driving force behind PIC import is likely a gradient of theGDP- and GTP-bound forms of Ran, a small GTPase. In chapter 4 we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. We demonstrate stable complex formation of TRN-SR2 and RanGTP in solution.Consistent with the behavior of normal nuclearimport cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. While in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2–RanGTP complex was significantly morecompact. We present a homology model of the TRN-SR2–RanGTP complex, which is in excellent agreement with the experimental small-angle X-ray scattering data and represents a stepping stone towards modulation of TRN-SR2 function for therapeutic purposes. In chapter 5 we describe the development and use of a high-throughput screening pipeline for small molecule inhibitors of the HIV-1 IN–TRN-SR2 interaction that block viral nuclear import and replication. We identify and confirm 5 active compound families from a 25,608-compound library. Modest antiviral activities are detected across all 5 classes. Two representative compounds significantly reducethe percentage of nuclear PICs in a fluorescence-based HIV-1 nuclear import assay. These compounds represent the first small molecule inhibitors of HIV-1 nuclear import and corroborate the role of the IN–TRN-SR2interaction for HIV-1 nuclear import.As IN requires a dynamic equilibrium between at least dimers and tetramers for its catalytic activities, another candidate drug target is IN oligomerization. In chapter 6, we develop, characterize and validate an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits proved to act as allosteric IN inhibitors, are devoid of cross-resistance with INSTIs and may become true next-generationIN inhibitors. Additionally, the assay offers a platform to study IN dimerization and unravel the full mechanism of action of LEDGINs, allosteric inhibitors recognizing the Lens epithelium-derived growth factor/p75 (LEDGF/p75) site on HIV-1 IN.Finally, in chapter 7 we identify the amino acids in HIV-1 IN that directly contact target DNA bases and affect local integration site sequence selection during viral replication.These residues also determine the propensity of the virus to integrate into flexible sequences. Remarkably, natural polymorphisms INS119G and INR231G redirect viral integration away from gene dense regions. Precisely these variants are associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Cystic fibrosis (CF) is the most common monogenic life-threatening disease in the Caucasian population, caused by mutations in CFTR, a chloride/bicarbonate channel that regulates fluid transport across epithelium of different organs (airways, pancreas, intestine, sweat glands and vas deferens). CF affects multiple organs, but lung pathology is the major clinical manifestation. Mutations in the CFTR gene lead to an imbalance in ion and water transport followed by the formation of viscous mucus which lines the lung epithelium and contributes to vicious cycle of airway obstruction, persistent infection and inflammation resulting in irreversible decline of lung function. For the majority of CF patients the treatment is symptomatic and there is no cure available.Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recently, rAAV-based gene therapy made remarkable success showing efficacy for several hereditarynbsp;and progressing to the market with a first gene-based product approved by the European Medicines Agency. This encouraged us to re-explore rAAV-based gene therapeutic approach for cystic fibrosis. Furthermore, the conceptual advantage of an early treatment gains more and more support in the gene therapy field since several studies showed beneficial effect of an early treatment on the clinical outcome. In that light, early gene therapy, prior to disease onset, may even prevent disease, rather than having to cure it.In parallel with the immune system disorders, CF was at the forefront of the gene therapy field since its inception in early 1990`s. Since then, more than 20 clinical trials for CF have been conducted, but none have led to a persistent clinical benefit. Hence the question on how to further improve pulmonary gene transfer remains unanswered. In that perspective, the work presented in this thesis aimed at developing a preclinical strategy for viral vector-based gene therapy, as a first step towards a cure (curative treatment) for CF airway disease.In a first part of my thesis, I developed and characterized a model for pulmonary gene transfer using adeno-associated viral vector with airway tropism (rAAV2/5) carrying reporter genes in fetal, neonatal and adult mice to answer generic questions on stability and efficacy of gene transfer. Combining non-invasive bioluminescent imaging and histological analysis, efficient transduction of both the upper (nose) and the lower (lung) mouse airways was observed. In order to cure an inherited disease, like CF, lifelong expression of the therapeutic gene is required. rAAV2/5-mediated gene transfer in the fetal or neonatal mouse airways, followed by a single re-administration later in adult animals resulted in sustained gene expression up to 7 months, without a marked decrease. In addition, we demonstrated that a single dose of rAAV2/5 administered to adult mice also resulted in reporter gene expression at least up to 15 months, which corresponds to the expected life-span of a mouse (1.5-2 years).nbsp;generic model highlights the clinical potential of rAAV2/5 vector for treatment of inherited and acquired pulmonary disorders for which long-term gene correction is required and no effective therapy is available.In a last part of this thesis, I translated the above-mentioned generic rAAV2/5-based technology fornbsp;gene transfer to a therapeutic model for CF gene therapy. Compared to previous unsuccessful rAAV-based clinical trials, we designed an improved vector based on an airway-tropic serotype (rAAV2/5) and carrying a truncated, but functional transgene (CFTRΔR) that allows incorporation of an external promoter for optimal gene expression. Combining different functional tests (iodide efflux assay, patch-clamp and forskolin-induced swelling), we demonstrated that CFTRΔR, a CFTR version that lacks a portion of the regulatory R-domain, retains ion channel activity and it is regulated by cAMP/PKA pathway in cultured cells. Finally we assessed the therapeutic potential of rAAV-CFTRΔR vector in two complementary models: intestinal organoids derived from CF patients and in vivo across the nasal mucosa of CF mice homozygous for ΔF508 mutation. A single dose of the therapeutic vector rAAV-CFTRΔR restored chloride secretion in both models proffering a rAAV-based gene therapy option for CF, a small butnbsp;step forward opening avenues towards a curative treatment for CF.