No description available.

No description available.

No description available.

Sirtuin-1 (SirT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase expressed ubiquitously in the body. In the vasculature, SirT1 is present in endothelial and vascular smooth muscle cells (VSMCs), where it has been shown to promote anti-inflammatory and anti-oxidant effects. As a result, SirT1 is known to play a protective role in the vasculature wall from pathologies such as atherosclerosis, arterial stiffness, and aortic aneurysm. Hence, SirT1 is considered an attractive therapeutic target for vascular diseases and potentially, aging-related and metabolic diseases. However, currently available SirT1 activators have failed to reach the clinic. Thus, novel approaches to activate SirT1 are needed. In this study, we first sought to optimize a novel fluorescence-based SirT1 activity assay, with which to reliably assess intracellular SirT1 activity and the efficacy of SirT1 activators and inhibitors. We next sought to use the SirT1 activity assay to screen novel compounds identified by an in silico docking analysis and hypothesized to activate SirT1. Lastly, we generated adeno-associated viruses (AAV) overexpressing wildtype (WT) or a redox-resistant (3M) SirT1 to analyze the effects of overexpressing SirT1 in VSMCs, in normal and oxidative stress conditions. For the activity assay, our results showed that an optimal standard curve range was between 0 ng and 12 ng of substrate (acetylated-p53 peptide). After testing different commercially available human recombinant SirT1s, the Anaspec SirT1 of the highest concentration showed a decrease in measured fluorescence for acetylated-p53 peptide with higher SirT1 (ng), indicating the enzyme and the assay were functional. However, when novel small molecules (A4, B4, and G3) hypothesized to activate SirT1 were added to reactions, the total p53 peptide fluorescence values increased compared to the control, suggesting some interference of the molecules with the assay detection. After AAV infection in VSMCs, SirT1 expression, measured by HA-tag, increased for AAV WT (n=3, p=0.04) and similarly for AAV 3M SirT1, indicating that the AAVs efficiently infect VSMCs. SirT1 activity, measured by Western Blot as decreased acetylated-histone (H3), also appeared to increase for both AAV WT and AAV 3M. A similar trend was shown for VSMCs under oxidant stress conditions (n=2). In conclusion, we successfully established a standard curve range for a novel SirT1 activity assay. Further trials are needed to ensure activity assay reproducibility before testing the efficacy of SirT1 activators and inhibitors. Infection of AAV WT and 3M SirT1 led to an increase in the expression and activity of SirT1 in VSMCs. The expression of SirT1 by AAV may be a promising therapeutic option for in vivo prevention and treatment of vascular diseases. / 2026-03-01T00:00:00Z

Efficient targeting of genes to either inhibit or increase their expression in specific tissues in vivo remains a challenge. Adeno-Associated Virus (AAV) has emerged as an efficacious delivery method in both humans and murine model systems. AAV is a non-enveloped, single-stranded DNA virus that is non-integrating with long-term expression. Due to its low immunogenicity and various serotypes with specific tissue tropisms, AAV is a preferred choice for organ specific-gene delivery in many experimental settings. This project focused on protocol optimization for high-volume production of AAV plasmids, improved transfection efficiency, and increased viral yield and purity to specifically target noncoding RNAs (ncRNAs) expressed from the imprinted Dlk1-Dio3 locus. Five AAV9 viruses were produced, each containing one of the following transgenes: 1) human Meg3 cDNA for overexpression of this long noncoding RNA, 2) Meg3-specific short hairpin RNA for knockdown analysis, 3) eGFP cDNA to demonstrate AAV9 tissue tropism, 4) Cas9 cDNA, and 5) gene-specific guide RNAs to target the Meg3 proximal promoter. The AAV9 virus production protocol optimized in this project expands the tools available for in vivo study of the Dlk1-Dio3 ncRNA locus.

Les vecteurs viraux dérivés du virus adéno-associé (AAV) apparaissent depuis deux décennies, comme des outils efficaces pour le transfert de gène in vivo. Cependant, malgré une faible immunogénicité et une absence de toxicité in vivo, leur optimisation requiert encore un effort important vers une meilleure compréhension de leur biologie et, en particulier, de leur interaction avec le système immunitaire. Au cours de ce travail de thèse, nous avons utilisé une méthode de sélection dirigée in vitro dans le but d’obtenir un variant de capside capable de transduire efficacement un type cellulaire non-permissif aux vecteurs AAV : les cellules dendritiques (DC). En effet, ces cellules jouent un rôle primordial dans l’établissement de la réponse immunitaire et, par conséquent, dans la persistance de l’expression du transgène in vivo. Cette technologie, très répandue dans la communauté AAV, a permis de sélectionner un variant de capside aux propriétés très intéressantes. La mutation sélectionnée, caractérisée in vitro comme induisant une instabilité de la capside, a permis d’identifier et de surmonter un point de blocage majeur dans le processus de transduction des DC par les vecteurs AAV consistant dans l’étape de décapsidation du génome du vecteur dans le noyau cellulaire. De manière intéressante, le variant obtenu exhibe un avantage en terme de transduction non seulement dans les DC mais aussi dans différents modèles de cellules primaires humaines (e.g. HUVEC) ou animales (OBC), peu ou pas permissive à l’AAV. De plus, des expériences de transfert de gène in vivo réalisées dans un modèle murin, indiquent que le variant sélectionné conduit à une meilleure expression du transgène, possiblement due à la mise en place d’un processus de tolérisation. Les propriétés remarquables de ce variant de capside, font de lui un candidat intéressant pour des applications médicales. / Vectors derived from the Adeno-associated virus (AAV) have emerged as an efficient system for in vivo gene transfer. However, despite their low immunogenicity and good tolerance in vivo, a better characterization of the host-AAV interaction is required to be able to fully exploit AAV’s potential fora gene therapy or gene vaccination. In this PhD project, we have used an in vitro directed evolution strategy to select an AAV capsid variant able to transduce human dendritic cell (DC), a non-permissive cell type which plays a critical role in the initiation of immune responses and, consequently, on the persistence of the expression of transgene in vivo. This procedure allowed us to identify an AAV variant characterized by a decreased stability of the capsid in vitro. The use of this mutant as a vector to transduce human DC resulted in an improved uncoating of the vector genome in the cell nucleus, thus identifying this step as major barrier toward DC transduction. Interestingly, the selected variant also displayed an increased transduction efficiency not only in DC but also in different primary human and animal cell types, poorly or non-permissive to AAV. Finally, when injected in mice, this AAV variant resulted in a higher expression of the transgene, associated to a low level of immune responses, suggesting the induction of tolerant state. The remarkable features suggest that our selected variant capsid is a promising candidate for medical applications.

No description available.

No description available.

Photoreceptors are of prime importance for humans, since vision is one of the most important senses for us. In our daily life, where nearly every action is dependent on visual input, an impairment or a loss of eyesight leads to severe disability. With a non-syndromic prevalence of 1:4000, retinitis pigmentosa, a collective term for a group of inherited retinal eye diseases, represents, together with age-related macula degeneration, one of the main causes for visual impairment and blindness in industrialized countries. The dominant reason for vision loss is, in both cases, the irreversible loss of photoreceptor cells located in the outer nuclear layer of the retina. To date, no effective treatment is available to preserve or regain visual function in affected patients. Recent promising strategies for new retinal therapeutical approaches focus on one hand on the development of gene therapies, where an introduced wild-type allele compensates a mutated gene, and on the other hand on cell therapies, where stem or photoreceptor precursor cells (PPCs) are transplanted to the sub-retinal space to replace degenerated host photoreceptors. The current study is subdivided into three parts, addressing the issue of non-reversible photoreceptor cell loss due to retinal degenerative diseases by investigating in the first two parts new qualitative as well as quantitative approaches in the field of retinal cell therapy, while in the third part an ocular gene therapeutical approach targeting prominin-1, a gene involved in retinal degenerative disorders, was investigated. Briefly, this study shows in the first part, a significant enhancement of the integration rate of PPCs in wild-type host retinas, achieved by pre-transplantational sorting, using the recently discovered PPC - specific cell surface marker CD73. This sets another step further towards retinal cell therapy by increasing the effectiveness of such treatment. Next to this quantitative approach, it is also shown that the quality of transplanted photoreceptor precursor cells is comparable to native photoreceptors by demonstrating, that an indispensable prerequisite of every photoreceptor cell, the outer segment, is developed by transplanted PPCs after proper integration. Importantly, transplanted PPCs develop native outer segments even when not integrated in the host tissue but located in the sub-retinal space, as it is predominantly observed after transplantation into severely degenerated retinas. These results substantiate the feasibility of cell therapeutical treatment of severely degenerated retinas. At the end of this part, it is demonstrated, that outer segments are not formed properly by PPCs transplanted to the vitreal side of the retina. This suggests an influence of signaling molecules, presumably secreted by retinal pigment epithelial cells into the sub-retinal space, on transplanted PPC final differentiation. Since intensive research is done to differentiate stem cells into PPCs for cell therapeutical transplantation, these results may contribute significantly to this research by demonstrating, that factors secreted by the retinal pigment epithelium might play a crucial role for successful stem cell to PPC differentiation. The last part of my work investigates a gene therapeutical approach to cure inherited retinal degenerative diseases. One gene, where reported mutations cause retinal degeneration in humans is prominin-1, a protein expressed at cell membrane evaginations in a variety of cell types. Interestingly, the prominin-1 knock-out mouse is characterized exclusively by disorganized photoreceptor outer segment formation and progressive retinal degeneration. Successful delivery of a wild-type form of mouse prominin-1 using adeno-associated viral vector transfer, into the photoreceptors of prominin-1 - deficient mice is demonstrated. The divergent results show on one hand a rescue of the thickness of the photoreceptor outer nuclear layer on a short time period (3 weeks post treatment), and on the other hand long-term data (8-10 weeks post treatment) suggests histologically as well as functionally a negative effect on treated photoreceptors. This might be due to effects caused by an over-expression of prominin-1 and will be investigated in future studies. In conclusion, distinct and important investigations were made which contribute significant puzzle pieces to new cell- as well as gene therapeutical approaches for the treatment of retinal degenerative disorders.

Photoreceptors are of prime importance for humans, since vision is one of the most important senses for us. In our daily life, where nearly every action is dependent on visual input, an impairment or a loss of eyesight leads to severe disability. With a non-syndromic prevalence of 1:4000, retinitis pigmentosa, a collective term for a group of inherited retinal eye diseases, represents, together with age-related macula degeneration, one of the main causes for visual impairment and blindness in industrialized countries. The dominant reason for vision loss is, in both cases, the irreversible loss of photoreceptor cells located in the outer nuclear layer of the retina. To date, no effective treatment is available to preserve or regain visual function in affected patients. Recent promising strategies for new retinal therapeutical approaches focus on one hand on the development of gene therapies, where an introduced wild-type allele compensates a mutated gene, and on the other hand on cell therapies, where stem or photoreceptor precursor cells (PPCs) are transplanted to the sub-retinal space to replace degenerated host photoreceptors. The current study is subdivided into three parts, addressing the issue of non-reversible photoreceptor cell loss due to retinal degenerative diseases by investigating in the first two parts new qualitative as well as quantitative approaches in the field of retinal cell therapy, while in the third part an ocular gene therapeutical approach targeting prominin-1, a gene involved in retinal degenerative disorders, was investigated. Briefly, this study shows in the first part, a significant enhancement of the integration rate of PPCs in wild-type host retinas, achieved by pre-transplantational sorting, using the recently discovered PPC - specific cell surface marker CD73. This sets another step further towards retinal cell therapy by increasing the effectiveness of such treatment. Next to this quantitative approach, it is also shown that the quality of transplanted photoreceptor precursor cells is comparable to native photoreceptors by demonstrating, that an indispensable prerequisite of every photoreceptor cell, the outer segment, is developed by transplanted PPCs after proper integration. Importantly, transplanted PPCs develop native outer segments even when not integrated in the host tissue but located in the sub-retinal space, as it is predominantly observed after transplantation into severely degenerated retinas. These results substantiate the feasibility of cell therapeutical treatment of severely degenerated retinas. At the end of this part, it is demonstrated, that outer segments are not formed properly by PPCs transplanted to the vitreal side of the retina. This suggests an influence of signaling molecules, presumably secreted by retinal pigment epithelial cells into the sub-retinal space, on transplanted PPC final differentiation. Since intensive research is done to differentiate stem cells into PPCs for cell therapeutical transplantation, these results may contribute significantly to this research by demonstrating, that factors secreted by the retinal pigment epithelium might play a crucial role for successful stem cell to PPC differentiation. The last part of my work investigates a gene therapeutical approach to cure inherited retinal degenerative diseases. One gene, where reported mutations cause retinal degeneration in humans is prominin-1, a protein expressed at cell membrane evaginations in a variety of cell types. Interestingly, the prominin-1 knock-out mouse is characterized exclusively by disorganized photoreceptor outer segment formation and progressive retinal degeneration. Successful delivery of a wild-type form of mouse prominin-1 using adeno-associated viral vector transfer, into the photoreceptors of prominin-1 - deficient mice is demonstrated. The divergent results show on one hand a rescue of the thickness of the photoreceptor outer nuclear layer on a short time period (3 weeks post treatment), and on the other hand long-term data (8-10 weeks post treatment) suggests histologically as well as functionally a negative effect on treated photoreceptors. This might be due to effects caused by an over-expression of prominin-1 and will be investigated in future studies. In conclusion, distinct and important investigations were made which contribute significant puzzle pieces to new cell- as well as gene therapeutical approaches for the treatment of retinal degenerative disorders.