La surdité est le déficit sensoriel le plus fréquent chez l'Homme et touche plus de 360 millions de personnes dans le monde. En France, un enfant sur 700 naît avec une surdité sévère ou profonde, et un enfant sur 1000 deviendra malentendant avant l'âge adulte. Environ 80% des cas de surdité neurosensorielle ont une cause génétique. La surdité peut être associée à des troubles de l'équilibre rendant difficile l'exécution de simples taches quotidiennes. La cause la plus fréquente de déficience auditive et vestibulaire, sont l'atteinte de, respectivement, la cochlée, organe sensoriel de l'audition, et du vestibule, organe sensoriel de l'équilibre, localisés dans l'oreille interne.Face à l'inexistence de traitement curatif, la thérapie génique semble être une alternative afin de traiter les patients atteints de surdités et/ou de troubles vestibulaires héréditaires. L'objectif de mon projet de thèse est de restaurer l'audition et l'équilibre dans des souris modèles des surdités et troubles vestibulaires humains (DFNB9, DFNB59, syndrome de Usher de type 1G et 3A), en utilisant la thérapie génique virale.Les résultats obtenus ont apporté une preuve de principe que le transfert intracochléaire de gènes thérapeutiques contenus dans un adénovirus associé in vivo, permet de restaurer la structure et la fonction des cellules ciliées sensorielles de l'oreille interne, au niveau de l'appareil mécano-sensitif et de la synapse. Ainsi, nous avons restauré de manière significative l'audition, et corriger complètement le trouble vestibulaire. Ce projet ouvre la voie à de nouvelles approches thérapeutiques pour des patients atteints de formes génétiques d'atteinte de l'oreille interne. / Hearing loss is one of the most common human sensory deficits affecting over 360 millions people worldwide. In France, over one child out of 700 suffers from profound deafness at birth, and 1/1000 will be affected by hearing impairment prior to adulthood. The early-onset forms of severe, nonsyndromic deafness are mostly genetic in origin. Deafness can be associated with vestibular impairments which can complicate daily simple tasks. In most cases, hearing and vestibular impairments are due to defects in, respectively, the cochlea, the hearing organ, and the vestibule, the balance organ.In front of the non-existence of curative treatment, gene transfer technology is an alternative therapeutic approach to rescue hereditary deafness and vestibular impairments. The aim of my project is the use of viral gene therapy to restore hearing and balance in mice established as model for human deafness (DFNB9, DFNB59, Usher syndrome type IG and 3A). Our results provide a proof-of-principle that in vivo intracochlear delivery of therapeutic genes using adeno-associated virus can restore the structure and the function of inner ear sensory hair cells, at the mecano-sensitive apparatus and at the synapse. Thus, we restore significantly the hearing, and completely the vestibular impairment. This project open the way to new methods for restoring hearing in patients with genetic forms of deafness.

abstract: The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2020

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Simple Summary Gene therapy is a novel approach to treat diseases by introducing corrective genetic information into target cells. Adeno-associated virus vectors are the most frequently applied gene delivery tools for in vivo gene therapy and are also studied as part of innovative anticancer strategies. Here, we report on the natural preference of AAV2 vectors for hepatocellular carcinoma (HCC) compared to nonmalignant liver cells in mice and human tissue. This preference in transduction is due to the improved intracellular processing of AAV2 vectors in HCC, resulting in significantly more vector genomes serving as templates for transcription in the cell nucleus. Based on this natural tropism for HCC, novel therapeutic strategies can be designed or existing therapeutic approaches can be strengthened as they currently result in only a minor improvement of the poor prognosis for most liver cancer patients. Abstract Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

CRISPR-Cas is the state-of-the-art biological tool that allows precise and fast manipulation of the genetic information of cellular genomes. The translation of the CRISPR-Cas technology from in vitro studies into clinical applications highlighted a variety of limitations: the currently available systems are limited by their off-target activity, the availability of a Cas-specific PAM sequence next to the target and the size of the Cas protein. In particular, despite high levels of activity, the size of the CRISPR-SpCas9 editing machinery is not compatible with an all-in-one AAV delivery system and the genomic sequences that can be targeted are limited by the 3-NGG PAM-dependency of the SpCas9 protein. To further expand the CRISPR tools repertoire we turned to metagenomic data of the human microbiome to search for uncharacterized CRISPR-Cas9 systems and we identified a set of novel small Cas9 orthologs derived from the analysis of reconstructed bacterial metagenomes. In this thesis study, ten candidates were chosen according to their size (less than 1100aa). The PAM preference of all the ten orthologs was established exploiting a bacterial-based and an in vitro platform. We demonstrated that three of them are active nucleases in human cells and two out of the three showed robust editing levels at endogenous loci, outperforming SpCas9 at particular targets. We expect these new variants to be very useful in expanding the available genome editing tools both in vitro and in vivo. Knock-out-based Cas9 applications are very efficient but many times a precise control of the repair outcome through HDR-mediated gene targeting is required. To address this issue, we also developed an MS2-based reporter platform to measure the frequency of HDR events and evaluate novel HDR-modulating factors. The platform was validated and could allow the screening of libraries of proteins to assess their influence on the HDR pathway.

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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