Gene therapy is advancing rapidly, with Recombinant Adeno-associated virus (rAAV) being investigated for potential use in treating cancer and neurological disorders. Plasmid DNA transfection and viral infection are standard methods for producing large-scale rAAV vectors. However, improving yield production requires careful monitoring and control of process state variables, which can be expensive and time-consuming. This thesis proposes a model predictive control (MPC) model that can efficiently monitor, predict, and optimize the final product by controlling state variables like DOT and temperature. The model relies on an unstructured mechanistic kinetic model designed explicitly based on rAAV upstream production. Monitoring viral vector production based on substrate or biomass concentration enhances bioprocess production efficiency. However, other state variables like dissolved oxygen (DO), pH, and temperature should also be considered. The objective of this thesis is to enhance cell growth in bioreactors by regulating dissolved oxygen and temperature levels using a Model Predictive Control (MPC) system. This model can be employed in different processes to enhance cell growth and examine the impact of control measures. The goal is to achieve a high cell density, increase productivity, and lower costs in a shorter duration. Simulink, a software tool developed by MATLAB, seamlessly integrates Ordinary Differential Equations (ODEs) to optimize bioprocesses in bioreactors. The Model Predictive Control (MPC) controller expertly regulates Dissolved Oxygen Tension (DOT) and temperature, thereby increasing cell growth concentrations. This sophisticated controller efficiently manages multiple variables simultaneously and exceeds the Proportional Integral Derivative (PID) controller. The model is straightforward to comprehend and promptly responds to anomaly data. To evaluate the suggested resolution, we conducted tests on both PID and MPC controllers by introducing measurement noise to the DOT. Our analysis indicated that MPC demonstrated superior performance based on the ISE (Integral of Squared Error), IAE (Integral of Absolute Error), and ITAE (Integral of Time-weighted Absolute Error), all of which were substantially higher for the PID controller. Regardless of changing conditions, MPC adeptly tracks the setpoint and optimizes the variable to enhance production efficiency.

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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

In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels. / Ph. D.

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.

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