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Preclinical Assessment of a Gene Editing Approach for MNGIE Marta

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Preclinical Assessment of a Gene Editing Approach for MNGIE Marta ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Doctoral thesis Preclinical assessment of a gene editing approach for MNGIE Marta Parés Casellas Thesis from Departament de Bioquímica i Biologia Molecular of Universitat Autònoma de Barcelona: PhD degree in Biochemistry, Molecular Biology and Biomedicine This work was carried out at Gene and Cell Therapy group of Institut de Recerca Hospital Universitari Vall d’Hebron Director Jordi Barquinero Máñez Tutor Assumpció Bosch Merino Universitat Autònoma de Barcelona 2020 The doctoral thesis titled “Preclinical assessment of a gene editing approach for MNGIE” was carried out by Marta Parés Casellas under the direction of Dr. Jordi Barquinero in the Gene and Cell Therapy group of Institut de Recerca Hospital Universitari Vall d’Hebron. The director certifies that this memory is suitable to be presented for the PhD degree in Biochemistry, Molecular Biology and Biomedicine of Universitat Autònoma de Barcelona. This research was funded by Fondo de Investigaciones Sanitarias (FIS) of Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación del Gobierno Español) through the project “Nuevas estrategias para mejorar la eficacia y reducir la inmunogenicidad en terapia génica de enfermedades hereditarias” (PI15/00172). The Fundació “la Caixa” provided the scholarship for the PhD student. Barcelona, 16th of September of 2020. Director: Dr. Jordi Barquinero PhD student: Marta Parés Agraïments En primer lloc vull agrair a en Jordi, el meu director de tesi, el seu suport, orientació constant i interès al llarg de la realització de la meva tesi. Gràcies per haver confiat en mi per tal de dur a terme aquesta investigació. Els teus valuosos comentaris, revisions i suport al llarg de tot aquest temps m'han ajudat a posar en ordre les idees i estructurar el meu treball. Treballant amb tu he pogut comprovar que la teràpia gènica és un camp molt apassionant en el qual m'agradaria poder seguir vinculada. També m'agradaria agrair-te la teva proximitat, i haver estat disponible sempre que ho he necessitat. Així mateix, vull agrair a la Sílvia la formació rebuda durant les pràctiques del màster. Has estat un dels meus referents a seguir. Això va fer que em decidís a seguir treballant en el camp d'investigació i m'animés a fer el doctorat. Durant tots aquests anys he compartit laboratori amb molta gent diferent, alguns més temps i d'altres menys, però tots han tingut un paper rellevant en la meva vida. Molts d'aquests companys i amics no han intervingut directament en la tesi, però sí que han participat del meu dia a dia durant aquests anys. Andrea, Èlia, Paula i Bet, tot i que ja fa un temps que vau marxar, encara recordo les nostres escapades per fer un cafè i les nostres xerrades. I molta altra gent, com en Lluís, la Kelly, en Rafa, l’Eva… Tots heu contribuït a fer que sempre hi hagi hagut molt bon ambient al laboratori; ha estat un plaer compartir aquesta etapa de la meva vida amb tots vosaltres. No em puc oblidar de mencionar a totes les estudiants de màster que he supervisat al llarg de tot aquest temps. Laura M, Laura P, Lídia, i Carla, m'ha agradat molt poder-vos ajudar durant la vostra estada. Algunes de vosaltres ja heu començat el vostre propi doctorat, i d’altres encara esteu meditant el vostre futur, però espero que totes us emporteu bons records d’aquestes pràctiques. Us desitjo molta sort en el que decidiu fer a partir d’ara. Penélope, encara que no compartíssim laboratori sí que hem compartit tutor. Gràcies a tu el congrés de Barcelona va ser més entretingut durant els descansos. Molta sort en la teva tesi, segur que et va molt bé. No em puc deixar de mencionar a en Jesús, l'última incorporació al laboratori. Els dinars són molt més animats des que hi ets i amb tu sempre hi ha temes de conversa. Tot i que ha sigut curt, m'ho he passat molt bé. També vull agrair la col·laboració de la Cristina i en Salvador del IQS. M’han ajudat en l'elaboració de les nanopartícules i m'han assessorat en tot moment. I a en Ferran, ja que sense ell una part important de la meva tesi no hauria estat possible. Et desitjo molta sort en la teva tesi també, ja que la presentarem gairebé al mateix temps. Així mateix també li vull agrair a en Ramon el seu interés i assessorament. A més, vull agrair a tots els treballadors d’Acuitas Therapeutics® que ens han ajudat en la col·laboració científica d’elaboració de nanopartícules. També vull agrair a en Fran i la Irene la seva col·laboració en la seqüenciació de mostres i el seu assessorament en diferents aspectes de la tesi. Agraïments A més a més, vull agrair la col·laboració de totes aquelles persones i institucions que ha fet possible la realització d'aquesta tesi. En especial, agraeixo als membres de la comissió de seguiment i a la meva tutora l'interès mostrat en l'evolució de la meva tesi. També a la Fundació “la Caixa” per haver-me atorgat la beca que m’ha permès realitzar aquest treball. També agrair tots aquells que d'una manera o altra m'han ajudat encara que el seu nom no figuri de forma explícita en aquestes línies; sense ells tampoc hauria estat possible. Tots vosaltres heu fet de la meva estada al VHIR una experiència única. Per últim, vull agrair a la meva família, en especial a la meva mare, tot el suport i ànims rebuts. Tots han estat al meu costat durant la realització d'aquest treball. Abstract ABSTRACT MNGIE is a rare metabolic disease caused by recessive mutations in the TYMP gene, which encodes the enzyme TP. This causes a systemic accumulation of nucleosides which results in mitochondrial toxicity that is usually lethal during the first decades of life. The goal of the present work was to achieve an efficient integration of the human TYMP cDNA on introns of the Tymp and Alb genes of murine hepatocytes by the coordinated action of two elements: CRISPR/Cas9 system and TYMP cDNA templates. These templates were designed to be integrated in a region downstream a genomic promoter, so that the TYMP cDNA expression will be under its control. In the case of the Alb locus the successful gene editing produced a hybrid Alb-hTP protein with a secretory ability. With this approach, hepatocytes would be permanently edited and the TYMP cDNA would be expressed long-term, thus overcoming the problem of loss of transgene expression often detected in conventional gene therapies. Two distinct strategies were tested. In the first one, CRISPR/Cas9 RNAs were delivered by nanoparticles (NPs). This allowed a high but transient expression. We tested NPs of two different sources: polymeric NPs (PNPs) of GEMAT-IQS, and lipid NPs (LNPs) of Acuitas Therapeutics®. In the second approach, CRISPR/Cas9 was delivered as DNA inside AAV vectors. Both in vitro and in vivo results corroborated that the TYMP cDNA could be successfully inserted in both loci. We also assessed the presence of TYMP mRNA and functional TP protein in the edited cells. In vivo experimentation was performed with the mice model of MNGIE. The Thd and dUrd plasma levels of treated animals were monitored to assess the effectivity of the different genome editing approaches. The best results were obtained in animals treated with LNPs carrying the CRISPR/Cas9 RNAs and rAAV2/8 vectors carrying the DNA templates. These mice showed a consistent and stable biochemical correction that correlated with the presence of TYMP mRNA and functional TP enzyme in liver cells. Moreover, mice of the Alb locus gene editing group presented significant TP activity in plasma, which confirms that the hybrid enzyme can be successfully secreted and maintains its activity in plasma. However, the reduction of plasma nucleoside levels was not enough to reach the levels observed in WT mice, so there is room for optimization. Unexpectedly, in some cases genome edition was observed in the absence of CRISPR/Cas9, which suggests that the DNA templates alone could trigger the correct insertion through homologous recombination. All these results confirm that our genome editing approach is viable, although we need to increase the overall efficiency of the procedure to achieve a full biochemical correction in the murine model of MNGIE. Index INDEX 1. ABBREVIATIONS ........................................................................................................................ 13 2. INTRODUCTION .......................................................................................................................... 21 2.1. MITOCHONDRIAL NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE) ................... 23 2.1.1. TYMP gene and mRNA ........................................................................................................................... 23 2.1.2. TP enzyme .................................................................................................................................................. 23 2.1.3. Nucleoside and nucleotide metabolism......................................................................................... 24 2.1.4. Molecular
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