DEVELOPMENT OF AN AAV-BASED GENE EDITING SYSTEM FOR NEURONAL HTT INACTIVATION IN THE MOUSE BRAIN

Huntington's disease (HD) is a fatal neurodegenerative disorder, caused by a dominant genetic mutation within the first exon of the huntingtin gene (HTT). HD patients go through a progressive and severe motor and cognitive decline, associated with the selective degeneration of striatal and cortical neurons in the brain. Currently, no curative treatment is available, and the pathology management is mainly focused on symptoms reduction. Among the therapeutic strategies under development, CRISPR/Cas9-mediated inactivation of the HTT gene represents a promising approach. However, most studies to date have focused exclusively on the treatment of the striatum with less regard to the involvement of cortical neurons in the HD pathogenesis. ln the present project, we aim to improve this approach, by developing an innovative and powerful gene editing tool, to simultaneously target cortical and striatal projection neurons in the mouse brain, leading to consistent HTT inactivation in both cell types. In order to simplify the assessment of technical readouts in vivo, we took advantage of wild-type mice and a sgRNA targeting the mouse HTT to establish and characterize the system. We used CRISPR/Cas9 to selectively inactivate the mouse HTT by targeting a region close the translation start site of the gene, using either the spCas9 or the saCas9 nuclease variant. The latter, because of its small size, was used to develop a single AAV vector driving the expression of both the nuclease and the sgRNA, in order to potentially increase the efficiency of our approach, with ail transduced cells expressing both elements. A first, in vitro screening, demonstrated promising results using both systems. However, the dual vector spCas9 showed higher efficiency in vivo compared to the single vector saCas9 and was therefore selected for further studies. We combined the efficiency of the spCas9 gene editing system with the retrograde transport feature of the newly characterized AAV2-retro variant, and the broad local diffusion promoted by the AAV2/10 serotype, reaching consistent and simultaneous HTT inactivation in both striatal and cortical projection neurons within the mouse brain, following a single injection procedure. The efficiency of our system in vivo was confirmed by a substantial reduction of the HTT protein expression observed in the mouse striatum two month after the surgery. Finally, we improved the biosafety of our approach by developing an optimized AAV version of the self-inactivating KamiCas9 system previously described, showing transient Cas9 expression while maintaining high on-target efficiency both in vitro and in vivo. Based on this promising results, our system represents an innovative and powerful tool for the future development of new and efficient genome editing therapeutic approaches for HD, potentially maximizing their therapeutic effect by promoting the simultaneous HTT inactivation in both striatal and cortical projection neurons within the brain. -- La maladie de Huntington (MH) est une maladie neurodégénérative mortelle, causée par une mutation génétique dominante dans le premier exon du gène huntingtin (HTT). Les patients atteints de la MH connaissent un déclin moteur et cognitif progressif et sévère, associé à la dégénérescence sélective des neurones striataux et corticaux du cerveau. Actuellement, aucun traitement curatif n'est disponible, et la prise en charge de la pathologie est principalement axée sur la réduction des symptômes. Parmi les stratégies thérapeutiques en cours de développement, l'inactivation du gène HTT par CRISPR/Cas9 représente une approche prometteuse. Cependant, la plupart des études menées jusqu'à présent se sont concentrées exclusivement sur le traitement du striatum, sans tenir compte de l'implication des neurones corticaux dans la pathogenèse de la MH. Dans le présent projet, nous visons à améliorer cette approche, en développant un outil d'édition génétique innovant et puissant, pour cibler simultanément les neurones de projection corticaux et striataux dans le cerveau de la souris, conduisant à une inactivation HTT cohérente dans les deux types de cellules. Afin de simplifier l'évaluation des résultats techniques in vivo, nous avons utilisé des souris de type sauvage et un ARNg ciblant la HTT de la souris pour établir et caractériser le système. Nous avons utilisé CRISPR/Cas9 pour inactiver sélectivement la HTT de la souris en ciblant une région proche du site de début de traduction du gène, en utilisant la variante de nucléase spCas9 ou saCas9. Cette dernière, en raison de sa petite taille, a été utilisée pour développer un vecteur AAV unique permettant l'expression à la fois de la nucléase et du sgRNA, afin d'augmenter potentiellement l'efficacité de notre approche, toutes les cellules transduites exprimant les deux éléments. Un premier criblage in vitro a montré des résultats prometteurs en utilisant les deux systèmes. Cependant, le vecteur double spCas9 a montré une plus grande efficacité in vivo par rapport au vecteur simple saCas9 et a donc été sélectionné pour des études supplémentaires. Nous avons combiné l'efficacité du système d'édition de gènes spCas9 avec la caractéristique de transport rétrograde de la variante AAV2-retro nouvellement caractérisée, et la large diffusion locale favorisée par le sérotype AAV2/l 0, pour atteindre une inactivation cohérente et simultanée des HTT dans les neurones de projection striataux et corticaux du cerveau de la souris, après une seule procédure d'injection. L'efficacité de notre système in vivo a été confirmée par une réduction substantielle de l'expression de la protéine HTT observée dans le striatum de la souris deux mois après la chirurgie. Enfin, nous avons amélioré la biosécurité de notre approche en développant une version AAV optimisée du système KamiCas9 auto-inactivant précédemment décrit, montrant une expression transitoire de Cas9 tout en maintenant une efficacité élevée sur la cible à la fois in vitro et in vivo. Sur la base de ces résultats prometteurs, notre système représente un outil innovant et puissant pour le développement futur de nouvelles approches thérapeutiques efficaces d'édition du génome pour la MH, maximisant potentiellement leur effet thérapeutique en favorisant l'inactivation simultanée de la HTT dans les neurones de projection striataux et corticaux du cerveau

How to Apply the Templates for Sustainable Product Development : Support for Sustainability Practitioners

The Templates for Sustainable Product Development (TSPD) is a tool for sustainability practitioners to assist product development teams with bringing sustainability considerations to the early phases of product development in a strategic, quick and resource-efficient way. This thesis project builds on the earlier TSPD work by investigating the ways in which sustainability practitioners could apply the TSPD in order to improve the outcomes. The factors influencing the quality of the outcomes of a TSPD application were investigated. Then, support was developed for use by sustainability practitioners to address these factors. This support was field tested and refined in four iterations. It was discovered that achieving high quality outcomes from a TSPD application is dependent on having both high quality strategic sustainable product development content and high quality facilitation of the people considering this content. The quality of both content and facilitation influences the participants’ level of engagement, which influences the quality of the outcomes of the TSPD application. The support was developed such that it assists sustainability practitioners in addressing both content and facilitation through a participatory approach. There is some evidence that use of the developed support contributed to both high level of participants’ engagement and high quality outcomes of the TSPD applications.Blog at http://sustainableproductdevelopment.blogspot.se/</p

Semi-automated workflows to quantify AAV transduction in various brain areas and predict gene editing outcome for neurological disorders

One obstacle to the development of gene therapies for the central nervous system is the lack of workflows for quantifying transduction efficiency in affected neural networks and ultimately predicting therapeutic potential. We integrated data from a brain cell atlas with 3D or 2D semi-automated quantification of transduced cells in segmented images to predict AAV transduction efficiency in multiple brain regions. We used this workflow to estimate the transduction efficiency of AAV2/rh.10 and AAV2.retro co-injection in the corticostriatal network affected in Huntington’s disease. We then validated our pipeline in gene editing experiments targeting both human and mouse huntingtin genes in transgenic and wild-type mice, respectively. Our analysis predicted that 54% of striatal cells and 7% of cortical cells would be edited in highly transduced areas. Remarkably, in the treated animals, huntingtin gene inactivation reached 54.5% and 9.6%, respectively. These results demonstrate the power of this workflow to predict transduction efficiency and the therapeutic potential of gene therapies in the central nervous system

Maximizing lentiviral vector gene transfer in the CNS

AbstractGene transfer is a widely developed technique for studying and treating genetic diseases. However, the development of therapeutic strategies is challenging, due to the cellular and functional complexity of the central nervous system (CNS), its large size and restricted access. We explored two parameters for improving gene transfer efficacy and capacity for the selective targeting of subpopulations of cells with lentiviral vectors (LVs). We first developed a second-generation LV specifically targeting astrocytes for the efficient expression or silencing of genes of interest, and to better study the importance of cell subpopulations in neurological disorders. We then made use of the retrograde transport properties of a chimeric envelope to target brain circuits affected in CNS diseases and achieve a broad distribution. The combination of retrograde transport and specific tropism displayed by this LV provides opportunities for delivering therapeutic genes to specific cell populations and ensuring high levels of transduction in interconnected brain areas following local administration. This new LV and delivery strategy should be of greater therapeutic benefit and opens up new possibilities for the preclinical development of gene therapy for neurodegenerative diseases.</jats:p

IL CONSOLE(The Consul) Dramma musicale in tre atti - Libretto e Musica di Gian Carlo Menotti -Versione in lingua inglese.

Dramma musicale in tre atti, rappresentato al Teatro Regio di Torino in un nuovo allestimento l’11 aprile 2006. Stagione lirica 2005-2006. […] Le Moli, nel rimettere in scena l'opera di Menotti cosa l'ha colpita di più […]? «Sicuramente il fatto che in quegli anni l'opera è stata vista come espressione contro le dittature dell'Est, contro il razzismo, sentimento ancor oggi comune in gran parte del mondo. […] La mia idea è che si tratta di una società malata. Per questo ho pensato di fare una regia non dal punto di vista della vittima, ma della cecità del carnefice. Per combattere il quale bisogna essere altrettanto forti». Nell'opera di Menotti il carnefice è il capo della polizia segreta. «Esattamente. Il quale dice ai suoi sgherri: ''Dovete agire sul cuore delle vittime con crudeltà, fino a portarle al suicidio. Estremo sacrificio che la vittima, moglie del clandestino, compie, schiacciata da una brutalità inesorabile».[...] «La vittima che si trova in queste condizioni è anche ingenua, si convince che il Paese che la ospita sia un Paese amico. Ma il Paese ospitante non sempre può dichiarare amicizia verso coloro che chiedono asilo. La storia non cambia, è attualissima». […] (Armando Caruso a colloquio con il regista Walter Le Moli, La Stampa 09-04-2006