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1
BONOMELLI, SARA. « L'EDITING GENETICO GERMINALE UMANO, TRA PROBLEMI ETICI E QUESTIONI DI GOVERNANCE ». Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/922688.
Texte intégralRésumé :
The dissertation aims at identifying and analyzing the scientific, legal, and ethical issues raised by the perspective of intentional modification of human germline by the potential future use of gene editing techniques in the context of human reproduction.
Such a study makes it possible to formulate some critical considerations about human germline gene editing governance. The dissertation claims that the best option to regulate the use of this biotechnological innovation for reproductive purposes consists of a regulation on a state-by-state-basis, which should however be developed within an international governance framework. Several arguments are suggested to underpin this thesis, and some recent initiatives adhering to such governance pattern are examined.
The research is organized in three chapters.
The first chapter, which is introductive to the real research, focuses on the scientific and technical aspects of the thesis topic. More specifically, this chapter aims at laying the foundations for the subsequent discussion, by defining and explaining the notions of i) DNA, gene, chromosome; ii) genetic mutation and genetic disease; and iii) gene therapy and gene editing. Special attention is paid to this latter technology and especially to its potential use on the human germline. Such use is highly controversial, mainly – but not exclusively – since, unlike modifications made by somatic gene editing, those affecting germinal cells – namely, gametes and zygotes – are transmitted to descendants, and thus to next generations.
The second chapter is divided into two sections. The first section reconstructs and analyses the existing regulations in the field of human germline gene editing at international, supranational and national level, stressing their vagueness, fragmentation and lack of specificity. Given the impossibility of extensively examining all relevant domestic laws, guidelines and policies, those of four countries only – the USA, the UK, China and Italy – have been considered in detail. This choice is motivated by the geographical and cultural representativeness of their respective regulations, as well as by the fact that, except for Italy, those countries conducted nearly all the experiments carried out so far in the field of human germline gene editing. The second section of the chapter precisely focuses on these experiments – both for research and reproductive purposes. Jiankui He’s experiment – which resulted in the birth of the world’s first gene-edited babies in 2018 – and Denis Rebrikov’s germline gene editing clinical trial project are thoroughly described and analyzed.
The third and last chapter deals with the ethical issues raised by the perspective of the potential future implementation of germline gene editing interventions in the context of human reproduction. This chapter too is articulated into two sections. The first section provides the theoretical bases for the subsequent ethical analysis, by dividing the possible future uses of germline gene editing techniques into three categories: i) therapeutic interventions; ii) medical enhancement interventions; and iii) non-medical enhancement interventions. Such categorization is paramount, since the various ethical issues related to human germline gene editing do not always involve all three of these categories, and, even when they do, they tend to carry different connotations according to each category. This becomes clear in the second section of the chapter, which critically explores six main ethically problematic areas related to this biotechnological innovation and their numerous articulations.
Finally, the dissertation argues that the scientific, legal and ethical issues identified and examined throughout the research must be taken into account by proper germline gene editing governance mechanisms, which should be the result of parallel and complementary regulatory initiatives promoted both at national and international level.
2
JACOB, AURELIEN MARC FLORENT. « IMPROVING TARGETED GENE EDITING IN HEMATOPOIETIC STEM CELLS FOR CLINICAL TRANSLATION ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/304800.
Texte intégralRésumé :
Negli ultimi anni, l’editing genetico nelle cellule staminali/progenitrici ematopoietiche umane (HSPC) per il trattamento di malattie genetiche del sangue è migliorato drasticamente trasformando inserzioni genetiche casuali in precise e mirate modificazioni del genoma. La modifica mirata dei geni mutati ereditati consente la correzione in situ e la ricostituzione funzionale con il mantenimento del controllo endogeno dell'espressione. Recentemente abbiamo dimostrato che sia le rotture del DNA a doppio filamento indotte dall’editing che il genoma stesso dell’Adeno-Associated Virus 6 (AAV) innescano una risposta dipendente da p53 nell'HSPC che risulta in un ritardo della proliferazione con conseguente diminuzione della ricostituzione ematopoietica dopo il trapianto delle cellule editate in animali immuno-compromessi. Per cui, abbiamo quindi dimostrato come la soppressione di questa risposta mediante l’espressione transitoria della forma negativa dominante di p53 preservi la ricostituzione del lineage ematopoietico. Tuttavia, la biologia sottostante è rimasta sconosciuta, così come l'impatto dell'editing genetico sulle dinamiche clonali dell'HSPC modificate con riparo diretto per omologia (Homology Directed Repair, HDR) al momento del trapianto. Inoltre, lo stato quiescente delle HSC primitive costituisce un limite per l’editing genetico mediato da HDR, riducendo le sue possibili applicazioni cliniche. In questo lavoro, abbiamo prima superato tale limite esprimendo transitoriamente la proteina dell'adenovirus 5 E4orf6/7, che regola il principale controllore del ciclo cellulare, E2F, insieme alla nucleasi. Mediante un'analisi dell'espressione genica globale e mirata, abbiamo dimostrato come E4orf6/7 spinga le cellule in fase S/G2 con concomitante sovra-regolazione di tutti i principali componenti del macchinario HDR, aumentando così l'efficienza dell'inserimento del transgene in cellule precedentemente quiescenti. Nel contesto dello xenotrapianto, l'espressione combinata di E4orf6/7 e l'inibizione di p53 hanno migliorato l'efficienza del HDR (>50%) all'interno dell'innesto umano totale, superando i livelli riportati fino ad ora in letteratura. Tale risultato è stato riprodotto in diversi donatori da diverse fonti di HSPC e sono stati modificati più loci genomici, dimostrando la maggior versatilità di questa piattaforma se paragonata ad altre strategie di editing. In parallelo, abbiamo ideato una nuova tecnologia (BAR-seq) che consente il monitoraggio clonale di singole HSC modificate con HDR. Questo approccio prevede l’introduzione di un codice a barre ereditabile univoco (BAR) nel templato AAV6 necessario al HDR. Il sequenziamento ad alta copertura di tali sequenze negli xenotrapianti ha mostrato come l’editing genetico risulti in un attecchimento di pochi cloni dominanti. Mentre l'inibizione transitoria di p53 durante l’editing ha consentito un aumento sostanziale della composizione clonale dell'innesto senza alterare la capacità ripopolante delle HSC. Inoltre, questi dati suggeriscono come la risposta mediata da p53 sia responsabile di un'ematopoiesi oligoclonale. È importante sottolineare che il BAR-seq ha fornito la prima prova diretta che le HSC umane modificate con HDR mantengono un potenziale multilineage e subiscono più cicli di divisioni simmetriche e asimmetriche nei trapianti primari e secondari. In conclusione, auspichiamo che i miglioramenti messi a punto nel nostro protocollo di editing possano ampliare le possibili applicazioni cliniche dell’editing genetico.The scope of genome engineering in hematopoietic stem/progenitor cells (HSPCs) has broadened from random to precise genome insertions for treating genetic diseases of the blood lineages. Targeted editing of inherited mutant genes allows in situ correction and functional reconstitution with preserved expression control. We recently showed that both the induced double-strand DNA breaks and the AAV6 genome trigger a p53-dependent DNA damage response in HSPC delaying proliferation and decreasing hematopoietic reconstitution after xenotransplantation. Suppression of this response by transient expression of a dominant negative p53 released cell-cycle block and rescued hematopoietic reconstitution. Yet, the underlying biology remained unknown as well as the impact of gene editing on clonal dynamics of HDR-edited HSPC upon transplantation. Moreover, it has long been contended that the quiescence of primitive HSC constrains HDR-mediated gene editing, thus limiting its perspective clinical applications in several diseases. Here, we first overcame such constraints by transiently expressing the adenovirus 5 protein E4orf6/7, which operates the major cell cycle controller E2F, together with the nuclease. By global and targeted gene expression analysis we showed engagement of targeted cells in S/G2 phases with concomitant upregulation of all major components of the HDR machinery, thus increasing the efficiency of targeted transgene insertion. Combined E4orf6/7 expression and p53 inhibition enhanced >50% HDR efficiency within human graft surpassing the levels reported until now in the literature. Such outcome was reproducible across several HSPC donors and sources, genomic loci and conceivably portable to most types of editing platforms. In parallel, we devised a novel technology (BAR-seq) which enables clonal tracking of individual HDR-edited HSC by introducing a unique heritable barcode in the AAV6 template. Deep sequencing of integrated BARs in human hematochimeric mice showed that only few (5-10) dominant clones of edited HSC robustly contributed to the hematopoietic graft long-term after transplant. Transient p53 inhibition during editing enabled substantial increase in polyclonal graft composition without altering individual HSC output, thus explaining the improved engraftment and highlighting the p53-mediated response as culprit of an otherwise oligoclonal hematopoiesis. Importantly, BAR-seq provided the first direct evidence that human HDR-edited HSC maintain multilineage potential and undergo multiple rounds of symmetric and asymmetric divisions in primary and secondary xenogeneic hosts. Altogether, we expect that the substantial gains obtained in HDR efficiency and polyclonal repopulation by our improved editing protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.
3
INSANGUINE, MINGARRO Ferdinando Achille. « MODIFICAZIONI GERMINALI DEL PATRIMONIO GENETICO E BIODIRITTO. I paradossi della de-differenziazione tra bioetica e biodiritto ». Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/507066.
Texte intégralRésumé :
Il presente lavoro, incominciato nel novembre del 2017, è partito con l'ambizione di ricostruire la risposta che il sistema giuridico fornisce innanzi alle nuove tecniche di ingegneria genetica che, a fronte della loro applicabilità sugli esseri umani, hanno prodotto, negli ultimi anni, il sorgere di nuovi stakeholders e, ancor prima, di nuovi interessi meritevoli di tutela.
Se fino a qualche anno fa pareva impensabile modificare il genoma umano e, men che meno, farlo in maniera precisa, efficiente ed economica, oggi grazie al sistema di modificazione genetica CRISPR/Cas9 è possibile, intervenendo sulla linea germinale degli embrioni umani, prevenire la contrazione di odiose malattie genetiche e, addirittura, a medio termine sradicarle dalla nostra società. Le enormi potenzialità terapeutiche di questa tecnica hanno addirittura attirato l’attenzione dell’Accademia Reale Svedese delle Scienze che, proprio mentre si stanno scrivendo queste righe, ha attribuito alle sue inventrici, Jennifer Doudna ed Emmanuelle Charpentier, il Premio Nobel per la Chimica 2020, definendo CRISPR/Cas9 come “un rivoluzionario metodo di editing genetico che contribuisce allo sviluppo di nuove terapie contro il cancro e può realizzare il sogno di curare malattie ereditarie” (The Royal Swedish Academy of Sciences 2020a). Al fianco di queste prospettive, che dal 2017 ad oggi si sono fatte sempre più evidenti, si annidano però rischi e pericoli derivanti dall’uso delle tecniche d’ingegneria genetica che il diritto deve tenere in adeguata considerazione al momento della loro regolamentazione.
Nei primi mesi di lavoro, dedicati proprio alla ricostruzione delle fonti giuridiche applicabili, ci ha subito colpito che nonostante le tecniche in parola costituiscano, ancora oggi, un’assoluta novità in continuo cambiamento, le norme giuridiche, sia sovranazionali che nazionali, siano relativamente risalenti nel tempo: la legge 40 che, in Italia, si propone di regolare la procreazione medicalmente assistita e alla lett. b) del co. 3 del suo art. 13 si occupa delle manipolazioni genetiche è del 2004, mentre la norma più rilevante sul punto a livello internazionale, l’art. 13 della Convenzione di Oviedo, è addirittura datata aprile 1997. Insomma, in questo campo il diritto anziché presentarsi in fisiologico ritardo, ha enucleato delle regolamentazioni in sospetto anticipo. Questa constatazione, combinata con gli esiti della ricostruzione del dibattito dottrinale, dove anche autorevolissimi autori combinano continuamente argomentazioni etiche ed argomentazioni giuridiche, spesso senza neppure differenziarle, ci ha condotto ad appurare come prima di affrontare il tema della regolamentazione specifica del genome editing fosse necessario riflettere su come il diritto s’interfacci innanzi al bios come oggetto normativo e, soprattutto, in quale relazione si ponga con la bioetica nell’espletare siffatta funzione.
Pertanto, abbiamo deciso di dedicare la Parte Prima dell’opera proprio ad un’indagine sulla relazione tra la bioetica ed il biodiritto, che costituiscono la proiezione applicativa di etica e diritto al bios, finalizzata a dotare di un adeguato fondamento epistemologico l’intuizione della deriva di de-differenziazione tra essi. Per raggiungere tale obiettivo abbiamo ritenuto necessario partire, nel Capitolo I, da una breve genealogia della bioetica in cui ci siamo interrogati sulla nascita di questa disciplina e sulle sue successive svolte metodologiche. Il Capitolo II, invece, è stato dedicato alle origini di quello specifico ambito della comunicazione giuridica, comunemente identificato ormai come biodiritto, mettendo in evidenza i contributi interni che la scienza giuridica ha fornito per lo sviluppo dello stesso e riflettendo, in particolare, sul ruolo che ha giocato in tal senso l’istituzione giuridica dei diritti umani. Al contrario, il Capitolo III parte dai contributi esterni alla nascita del biodiritto e specificatamente quelli forniti dalla bioetica per proseguire, poi, con una riflessione sul rapporto tra questi. Mediante una ricostruzione delle posizioni dominanti in dottrina e soprattutto attraverso uno sguardo fisso alla prassi, si è posto in evidenza come, ad oggi, via sia un problema di de-differenziazione tra bioetica e biodiritto che ha portato quest’ultimo a trasformarsi in una scienza ancillare alla prima al punto da essere definito come “diritto della bioetica”. Lungi dal fermarci su posizioni unicamente critiche, abbiamo dotato l’ultima parte del Capitolo di una pars construens in cui abbiamo evidenziato i vantaggi di una relazione funzionalmente differenziata tra bioetica e biodiritto, senza però trascurare anche i problemi ad essa sottesi.
Con il chiaro intento di testare i nostri approdi teorici nell’esperienza empirica e, allo stesso tempo, per assolvere all’intento originario della nostra opera, abbiamo deciso di dedicare la Parte II interamente alle implicazioni etiche, sociologiche e giuridiche derivanti dalle tecniche di manipolazione genetica germinale. Per farlo si è reso necessario, anzi tutto, dedicare il Capitolo IV a comprendere, tecnicamente, cosa sia una modificazione genetica germinale e quali siano le posizioni rinvenibili all’interno della comunità scientifica. Il Capitolo V, invece, è stato dedicato ad affrontare i problemi, i rischi, le promesse e le speranze che si annidano intorno alla nostra tecnica: dal timore per una deriva eugenetica alla compatibilità delle modificazioni con l’autocomprensione e la dignità del genere umano, passando per le preoccupazioni delle comunità delle persone diversamente abili e dei genitori, che rischiano di restare schiacciati dalle pressioni sociali, giungendo a prendere in seria considerazione però anche le possibilità di sradicare odiose malattie genetiche una volta per tutte, liberando l’umanità di alcune atroci sofferenze. Con un quadro chiaro dei diversi valori che mette in gioco ed in potenziale conflitto tra loro la tecnica germinale, abbiamo finalmente affrontato il problema della regolamentazione delle nostre tecniche. Abbiamo cercato di farlo non con l’animo di produrre una mera attività compilativa sulle regolamentazioni esistenti e neanche con il solo intento di mostrare lacune e paradossi che in esse si annidano, ma con la finalità più ambiziosa di verificare se le nostre conclusioni teoriche della Parte Prima fossero fondate: se effettivamente il diritto si propone come un mero trasformatore permanente di principi bioetici in precetti coercitivi e se l’approccio regolativo vigente sia adeguato per cogliere i benefici che una tecnica premiata con il Nobel per la Chimica può dare alla società, senza rinunciare a tutelare i diritti fondamentali delle persone.
4
INSANGUINE, MINGARRO Ferdinando Achille. « Modificazioni germinali del patrimonio genetico e biodiritto. I paradossi della de-differenziazione tra bioetica e biodiritto ». Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/499144.
Texte intégralRésumé :
Il presente lavoro, incominciato nel novembre del 2017, è partito con l'ambizione di ricostruire la risposta che il sistema giuridico fornisce innanzi alle nuove tecniche di ingegneria genetica che, a fronte della loro applicabilità sugli esseri umani, hanno prodotto, negli ultimi anni, il sorgere di nuovi stakeholders e, ancor prima, di nuovi interessi meritevoli di tutela.
Se fino a qualche anno fa pareva impensabile modificare il genoma umano e, men che meno, farlo in maniera precisa, efficiente ed economica, oggi grazie al sistema di modificazione genetica CRISPR/Cas9 è possibile, intervenendo sulla linea germinale degli embrioni umani, prevenire la contrazione di odiose malattie genetiche e, addirittura, a medio termine sradicarle dalla nostra società. Le enormi potenzialità terapeutiche di questa tecnica hanno addirittura attirato l’attenzione dell’Accademia Reale Svedese delle Scienze che, proprio mentre si stanno scrivendo queste righe, ha attribuito alle sue inventrici, Jennifer Doudna ed Emmanuelle Charpentier, il Premio Nobel per la Chimica 2020, definendo CRISPR/Cas9 come “un rivoluzionario metodo di editing genetico che contribuisce allo sviluppo di nuove terapie contro il cancro e può realizzare il sogno di curare malattie ereditarie” (The Royal Swedish Academy of Sciences 2020a). Al fianco di queste prospettive, che dal 2017 ad oggi si sono fatte sempre più evidenti, si annidano però rischi e pericoli derivanti dall’uso delle tecniche d’ingegneria genetica che il diritto deve tenere in adeguata considerazione al momento della loro regolamentazione.
Nei primi mesi di lavoro, dedicati proprio alla ricostruzione delle fonti giuridiche applicabili, ci ha subito colpito che nonostante le tecniche in parola costituiscano, ancora oggi, un’assoluta novità in continuo cambiamento, le norme giuridiche, sia sovranazionali che nazionali, siano relativamente risalenti nel tempo: la legge 40 che, in Italia, si propone di regolare la procreazione medicalmente assistita e alla lett. b) del co. 3 del suo art. 13 si occupa delle manipolazioni genetiche è del 2004, mentre la norma più rilevante sul punto a livello internazionale, l’art. 13 della Convenzione di Oviedo, è addirittura datata aprile 1997. Insomma, in questo campo il diritto anziché presentarsi in fisiologico ritardo, ha enucleato delle regolamentazioni in sospetto anticipo. Questa constatazione, combinata con gli esiti della ricostruzione del dibattito dottrinale, dove anche autorevolissimi autori combinano continuamente argomentazioni etiche ed argomentazioni giuridiche, spesso senza neppure differenziarle, ci ha condotto ad appurare come prima di affrontare il tema della regolamentazione specifica del genome editing fosse necessario riflettere su come il diritto s’interfacci innanzi al bios come oggetto normativo e, soprattutto, in quale relazione si ponga con la bioetica nell’espletare siffatta funzione.
Pertanto, abbiamo deciso di dedicare la Parte Prima dell’opera proprio ad un’indagine sulla relazione tra la bioetica ed il biodiritto, che costituiscono la proiezione applicativa di etica e diritto al bios, finalizzata a dotare di un adeguato fondamento epistemologico l’intuizione della deriva di de-differenziazione tra essi. Per raggiungere tale obiettivo abbiamo ritenuto necessario partire, nel Capitolo I, da una breve genealogia della bioetica in cui ci siamo interrogati sulla nascita di questa disciplina e sulle sue successive svolte metodologiche. Il Capitolo II, invece, è stato dedicato alle origini di quello specifico ambito della comunicazione giuridica, comunemente identificato ormai come biodiritto, mettendo in evidenza i contributi interni che la scienza giuridica ha fornito per lo sviluppo dello stesso e riflettendo, in particolare, sul ruolo che ha giocato in tal senso l’istituzione giuridica dei diritti umani. Al contrario, il Capitolo III parte dai contributi esterni alla nascita del biodiritto e specificatamente quelli forniti dalla bioetica per proseguire, poi, con una riflessione sul rapporto tra questi. Mediante una ricostruzione delle posizioni dominanti in dottrina e soprattutto attraverso uno sguardo fisso alla prassi, si è posto in evidenza come, ad oggi, via sia un problema di de-differenziazione tra bioetica e biodiritto che ha portato quest’ultimo a trasformarsi in una scienza ancillare alla prima al punto da essere definito come “diritto della bioetica”. Lungi dal fermarci su posizioni unicamente critiche, abbiamo dotato l’ultima parte del Capitolo di una pars construens in cui abbiamo evidenziato i vantaggi di una relazione funzionalmente differenziata tra bioetica e biodiritto, senza però trascurare anche i problemi ad essa sottesi.
Con il chiaro intento di testare i nostri approdi teorici nell’esperienza empirica e, allo stesso tempo, per assolvere all’intento originario della nostra opera, abbiamo deciso di dedicare la Parte II interamente alle implicazioni etiche, sociologiche e giuridiche derivanti dalle tecniche di manipolazione genetica germinale. Per farlo si è reso necessario, anzi tutto, dedicare il Capitolo IV a comprendere, tecnicamente, cosa sia una modificazione genetica germinale e quali siano le posizioni rinvenibili all’interno della comunità scientifica. Il Capitolo V, invece, è stato dedicato ad affrontare i problemi, i rischi, le promesse e le speranze che si annidano intorno alla nostra tecnica: dal timore per una deriva eugenetica alla compatibilità delle modificazioni con l’autocomprensione e la dignità del genere umano, passando per le preoccupazioni delle comunità delle persone diversamente abili e dei genitori, che rischiano di restare schiacciati dalle pressioni sociali, giungendo a prendere in seria considerazione però anche le possibilità di sradicare odiose malattie genetiche una volta per tutte, liberando l’umanità di alcune atroci sofferenze. Con un quadro chiaro dei diversi valori che mette in gioco ed in potenziale conflitto tra loro la tecnica germinale, abbiamo finalmente affrontato il problema della regolamentazione delle nostre tecniche. Abbiamo cercato di farlo non con l’animo di produrre una mera attività compilativa sulle regolamentazioni esistenti e neanche con il solo intento di mostrare lacune e paradossi che in esse si annidano, ma con la finalità più ambiziosa di verificare se le nostre conclusioni teoriche della Parte Prima fossero fondate: se effettivamente il diritto si propone come un mero trasformatore permanente di principi bioetici in precetti coercitivi e se l’approccio regolativo vigente sia adeguato per cogliere i benefici che una tecnica premiata con il Nobel per la Chimica può dare alla società, senza rinunciare a tutelare i diritti fondamentali delle persone.
5
Pérez, Álvarez Lucía. « Metabolic engineering and genome editing in rice ». Doctoral thesis, Universitat de Lleida, 2018. http://hdl.handle.net/10803/665272.
Texte intégralRésumé :
El meu programa d’investigació s’ha basat en la utilització de l’arròs com a model experimental per a estudiar mecanismes i colls d’ampolla que limiten la transició de l’enginyeria metabòlica a la biologia sintètica en les plantes. Em vaig concentrar en la caracterització a nivell molecular i bioquímica de plantes que vaig generar en dos conjunts de línies d’investigació diferents però interrelacionades. En el primer conjunt d’experiments vaig abordar la hipòtesi que en eliminar gens específics en una ruta metabòlica primària, concretament la biosíntesi de midó, les plantes mutants resultants podrien mostrar fenotips propicis per a aplicacions de biologia sintètica en termes de redireccionar el flux i limitar els precursors biosintètics a vies metabòliques secundàries específiques. En aquest context, vaig utilitzar CRISPR/Cas9 per crear dos mutants heterozigots, un amb una glucosa-1-fosfat adenil transferasa (AGPasa) citosòlica severament truncada i no funcional i l’altre amb una modificació estructural C-terminal causant una pèrdua parcial d’activitat. Inesperadament, vaig observar una reducció del midó en les fulles de tots dos mutants i un augment concomitant en el nivell de sucres solubles. Això es va reflectir en l’expressió no prevista d'OsAPL2 i OsAPS2b en les fulles, generant una AGPasa ectòpica completa en el citosol de la fulla, i una disminució en l’expressió de la subunitat petita plastidial OsAPS2a que es va complementar només parcialment amb un augment en l’expressió de OsAPS1
En un conjunt posterior d’experiments amb base similar, vaig investigar els efectes més amplis de les mutacions en un gen de la biosíntesi de midó, la midó sintasa unida a grànuls (GBBS, waxy). Vaig utilitzar CRISPR / Cas9 per introduir un rang de mutacions amb diferents efectes en aquest gen específic. Vaig trobar que les mutacions produïdes van reduir, però no van abolir totalment, l’activitat de GBSS en les llavors a causa d’una compensació parcial causada per la regulació ectòpica de GBSSII. L’activitat de GBSS en els mutants va ser de 61 a 71% dels nivells dels controls, però el contingut d'amilosa, va disminuir del 8 al 12% en llavors heterozigotes i va ser tan baix com nomes un 5% en llavors homozigotes, acompanyat per una organització cel•lular anormal en la capa d'aleurona i amb estructures del gra de midó amorfes. Gairebé tots els gens de la via del midó es van veure afectats a diferents nivells en les fulles i llavors. Aquests canvis en l’expressió gènica van donar com a resultat canvis en l’activitat de la AGPasa i de la sacarosa sintasa que coincidien amb els nivells corresponents de midó i sucres solubles.
La segona línia del meu programa d’investigació es va centrar en l’enginyeria d'una via ectòpica de MVA en plastidis d’arròs per investigar la hipòtesi que al reconstituir la via ectòpica, la regulació estricta de la via de MVA nativa podria relaxar-se en cert grau a mesura que augmentés el conjunt de precursors terpenoides essencials. Els resultats van indicar un augment molt elevat dels nivells d’àcids grassos, luteïna i tocoferol, i una reducció en els nivells d'esqualè i d’esterols.
Els meus resultats són el fonament per a futurs experiments encaminats a determinar si el germoplasma que he creat i caracteritzat pot servir de base per a intervencions d’enginyeria metabòlica i biologia sintètica més complexes.Mi programa de investigación se ha basado en la utilización del arroz como modelo experimental para estudiar mecanismos y cuellos de botella que limitan la transición de la ingeniería metabólica a la biología sintética en las plantas. Me concentré en la caracterización a nivel molecular y bioquímica de plantas que generé en dos conjuntos de líneas de investigación distintas pero interrelacionadas. En el primer conjunto de experimentos abordé la hipótesis de que al eliminar genes específicos en una ruta metabólica primaria, concretamente la biosíntesis de almidón, las plantas mutantes resultantes podrían mostrar fenotipos propicios para aplicaciones de biología sintética en términos de redireccionar el flujo y limitar los precursores biosintéticos a vías metabólicas secundarias específicas. En este contexto, utilicé CRISPR/Cas9 para crear dos mutantes heterocigotos, uno con una glucosa-1-fosfato adenil transferasa (AGPasa) citosólica severamente truncada y no funcional y el otro con una modificación estructural C-terminal causando una pérdida parcial de actividad. Inesperadamente, observamos una reducción del almidón en las hojas de ambos mutantes y un aumento concomitante en el nivel de azúcares solubles. Esto reflejó la expresión no prevista de OsAPL2 y OsAPS2b en las hojas, generando una AGPasa ectópica completa en el citosol de la hoja, y una disminución en la expresión de la subunidad pequeña plastidial OsAPS2a que se complementó solo parcialmente con un aumento en la expresión de OsAPS1 En un conjunto posterior de experimentos, con similar base, investigué los efectos más amplios de las mutaciones en un gen de la biosintésis de almidón, la almidon sintasa unida a gránulos (GBBS, waxy). Utilicé CRISPR/Cas9 para introducir un rango de mutaciones con diferentes efectos en este gen específico. Encontre que las mutaciones producidas redujeron, pero no abolieron la actividad de GBSS en las semillas, debido a una compensación parcial causada por la regulación ectópica de GBSSII. La actividad de GBSS en los mutantes fue de 61 a 71% de los niveles de los controles, pero el contenido de amilosa, sin embargo, disminuyó a 8 a 12% en semillas heterocigotas y fue tan bajo como 5% en semillas homocigotas, acompañado por una organización celular anormal en la capa de aleurona y con estructuras del grano de almidón amorfas. Casi todos los genes de la vía del almidón se vieron afectados a diferentes niveles en las hojas y semillas. Estos cambios en la expresión génica dieron como resultado cambios en la actividad de la AGPasa y de la sacarosa sintasa que coincidían con las alteraciones en los niveles de almidón y azúcares solubles. La segunda línea de mi programa de investigación se centró en la ingeniería de una vía ectópica de MVA en plastidios de arroz para investigar la hipótesis de que al reconstituir la vía ectópica, la regulación estricta de la vía de MVA nativa podría relajarse en cierto grado a medida que aumentase el conjunto de precursores terpenoides esenciales. Los resultados indicaron un aumento en los niveles de ácidos grasos, luteína y tocoferol, una reducción en los niveles de escualeno y niveles similares de esteroles. Mis resultados son el fundamento para futuros experimentos encaminados a determinar si el germoplasma que he creado y caracterizado puede servir de base para intervenciones de ingeniería metabólica y biología sintética más complejas.
My research program used rice as an experimental model to address fundamental bottlenecks and mechanisms limiting the transition from metabolic engineering to synthetic biology in plants. I concentrated on an in depth molecular and biochemical characterization of plants I generated in two distinct, yet interrelated sets of research lines. In the first set of experiments I addressed the hypothesis that by knocking out specific genes in a primary metabolic pathway, starch biosynthesis, resulting mutant plants might exhibit phenotypes conducive to synthetic biology applications in terms of redirecting flux and limiting biosynthetic precursors to specific secondary metabolic pathways. In this context I used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and non-functional cytosolic glucose-1-phosphate adenylyl transferase (AGPase) and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, both mutants exhibited depletion of starch in the leaves and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. In a subsequent set of experiments along similar lines I investigated the broader effects of mutations in an additional starch biosynthetic gene, granule bound starch synthase (GBBS, waxy). I used CRISPR/Cas9 to introduce a range of mutations with different effects in this specific gene. All mutations I recovered reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the ectopic upregulation of GBSSII. The GBSS activity in the mutants was 61–71% of wild-type levels, but the amylose content nevertheless declined to 8–12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Almost every starch pathway gene was impacted at different degrees in leaves and seeds. These gene expression changes resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars. The second line of my program focused on the engineering of an ectopic MVA pathway in rice plastids in order to investigate the hypothesis that by reconstituting such an ectopic pathway the strict regulation of the native MVA pathway might be relieved to a certain degree in turns increasing the pool of essential terpenoid precursors. Results indicated a profound increase in the levels of fatty acids, lutein and tocopherol, a decrease in squalene levels and similar levels of sterols. My results set the stage for further experiments to ascertain whether germplasm I created and characterized, can serve as a basis for more complex metabolic engineering and synthetic biology interventions.
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McGurk, Leeane. « Drosophila lacking RNA editing ». Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/2695.
Texte intégralRésumé :
ADAR is an adenosine deaminase that acts on dsRNA. Once bound to dsRNA, ADAR deaminates specific adenosines into inosines. If this occurs within the coding region of a transcript the inosine will be read as a guanosine. This can lead to a change in the amino acid at this position and increase protein diversity. In mammals there are three ADAR genes: ADAR1, ADAR2 and ADAR3. However, only ADAR1 and ADAR2 have been shown to be enzymatically active. ADAR1 is widely expressed and can edit both coding RNA and non-coding RNA. ADAR2 is restricted to the CNS and the key transcript that it edits encodes the GluR-B subunit of the glutamate-gated ion channel receptor. Editing of the Q/R site in the GluR-B transcript occurs with an efficiency of more than 99.9% and changes the genomically encoded glutamine into an arginine. This results in an ion channel that is impermeable to calcium. The ADAR2 knock-out mice are viable, but suffer from epileptic seizures and die by day 20. This phenotype can be rescued by expressing the edited R isoform of GluR-B, suggesting that this site is the most important target for ADAR2. Drosophila has only one Adar gene and its product has been reported to edit more than one hundred adenosines in different transcripts. Many of these transcripts encode subunits of ion channels, and it has been hypothesised that lack of ion channel editing causes the behavioural defects and age-related neurodegeneration observed in Adar deletion mutants. In this thesis I investigate the function of ADAR in an uncharacterised Adar mutant, Adar5G1. To characterise the Adar5G1mutant I not only used standard histology but a 3D imaging technique, optical projection tomography (OPT), that had not been reported to be used with Drosophila before this work. OPT allows the internal organs to be imaged without any manual sectioning or dissecting. I used OPT to identify neurodegenerative vacuoles from within the intact head and present the data both in 2D and in 3D. In addition to this, I demonstrate that this technique can be used to image global expression patterns in the Drosophila adult and I relate the TAU-β galactosidase expression pattern to the Drosophila anatomy. The neurodegeneration observed by OPT was confirmed by detailed analysis of stained wax sections. Complete loss of Adar, in the Adar5G1 mutant revealed age-dependent vacuolisation of the retina and mushroom body calyces. The vacuolisation observed in the Adar5G1 mutant was rescued by expression of Drosophila Adar and human ADAR1 p110, and ADAR2. However the cytoplasmic form of ADAR1, ADAR1 p150, did not rescue the vacuolisation of the Adar5G1 mutant. ADAR3, a catalytically inactive ADAR, rescued the vacuolisation phenotype of the Adar5G1 mutant, suggesting that ADAR may have an additional function independent of editing activity. The vacuolisation of the Adar5G1mutant was found not to be associated with type I programmed cell death. However, it was associated with swollen nerve fibres and degrading ommatidia containing multilamellar whorls. Neurodegeneration in various Drosophila mutant models and human neuropathies has been associated with similar cellular structures, suggesting that loss of ADAR results in neurodegeneration common to many of the known neuropathies. Finally, I found that expression of edited isoforms of the nicotinic receptor channel 34E subunit (Nic 34E) failed to rescue the locomotion phenotype of the Adar mutant. However, I found preliminary evidence that one of the lines generated for an edited isoform of Rdl, a subunit of the GABA receptor ion channel, gave a partial rescue of both locomotion and neurodegeneration of the Adar1F4 and Adar5G1 mutant.
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Chew, Wei Leong. « Postnatal Genome Editing With CRISPR ». Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493352.
Texte intégralRésumé :
Targeted genome editing holds tremendous promise for permanent correction of many genetic diseases. The recently developed CRISPR/Cas9 genome-editing tool exhibits facile programmability and robust gene-editing efficiency, and has been applied in cell cultures and animal tissues. However, multi-organ gene-editing in live mammals has not been examined or achieved. This study demonstrates genetic modification in multiple organs of postnatal mice by systemic delivery of CRISPR with adeno-associated viruses (AAVs). I resolved the AAV payload limitation by splitting Cas9 and reconstituting the native protein in vivo using scarless split-intein protein trans-splicing, which preserves full activity of Cas9. I determined that the delivery efficiency of AAV-CRISPR dictates gene-targeting rates in vivo, with the preferential gene-editing in liver and heart, and more modest editing efficiencies in skeletal muscle, brain and gonads, directly reflecting the infection profile of the virus serotype. To track CRISPR biodistribution, I established two reporter systems that apply in situ fluorescence activation to demarcate CRISPR-targeting events at single-cell resolution, identifying rare gene-edited cells that normally evade detection by sequencing. This exquisite detection sensitivity further allows evaluation of inter-generational transmission of gene-editing viruses. Finally, although Cas9 elicits host immune responses, these can be ameliorated by immunosuppression. I also identified a public Cas9-responsive T-cell clonotype and mapped the B-cell epitopes on Cas9 and AAV. Engineering tolerance to immunodominant epitopes may provide an avenue for avoiding immune rejection of AAV-CRISPR. The ability to create programmable genetic modifications in multiple organs of postnatal mammals provides a powerful tool for biological research, and foretells that the genomes of whole mammals may be rewritten at will.Medical Sciences
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Neadeau, Joseph Francis. « Comparing Genetic Modification and Genetic Editing Technolgies : Minimal Required Acreage ». Thesis, North Dakota State University, 2018. https://hdl.handle.net/10365/29878.
Texte intégralRésumé :
There are many technologies being developed for crop breeding. Two interesting technologies are genetic modification and genetic editing. Competitive pressures and changing consumer preferences are forcing organizations to invest heavily in these two technologies. Organizations must decide which traits they want to target and must commit significant time a money to the project. Traditionally, firms would decide which project to embark on if the project is net present value positive. Throughout the research and development process managers have flexibility to abandon the project once new information is received. That flexibility has value and real option analysis must be performed to value that flexibility. Once the value of a GM and GE project is determined, how might an organization decide which project to do? The concept of minimum required acreage (MRA) is developed in this study, allowing organizations to compare GM and GE technologies and decide which project to invest it.
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Robinson, Jason M. « Functional Significance of mtDNA Cytosine Modification Tested by Genome Editing ». VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4561.
Texte intégralRésumé :
The field of epigenetics is gaining popularity and speed, due in part to its capability to answer lingering questions about the root cause of certain diseases. Epigenetics plays a crucial role in regulation of the cell and cell survival, particularly by cytosine methylation. It remains controversial if DNMT’s which facilitate methylation are present in mammalian mitochondria and what the functional significance they may have on modification of mitochondrial DNA. CRISPR-Cas9 technology enabled genome editing to remove the MTS (mitochondrial targeting sequence) from DNMT1 of HCT116 cells, purposefully minimizing effects on nuclear cytosine methylation, while exclusively impacting mitochondrial modification. Removal of the DNMT1 MTS did not completely prevent the localization of this enzyme to the mitochondria according to immunoblot analysis. As well, deletion of the MTS in DNMT1 revealed only a small decline in transcription; not until removal of DNMT3B did we see a two-fold decrease in transcription from mitochondrial protein coding genes. No significant decline in transcription occurred when a DNMT3B knockout also lost the MTS of DNMT1; this study is evidencing that DNMT3B is possibly the more significant methyltransferase in the mitochondria. Our aim from this study and future research is to clearly characterize which enzymes in the mitochondria are controlling cytosine modifications and to understand the mechanistic complexities that accompany cause and consequence of epigenetic modifications.
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Rodríguez, José A. « Genetic editing with CRISPR/Cas9 : A scientific, ethical, and pastoral approach ». Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108890.
Texte intégralRésumé :
Thesis advisor: Andrea ViciniThesis advisor: Colleen M. Griffith
Thesis (STL) — Boston College, 2019
Submitted to: Boston College. School of Theology and Ministry
Discipline: Sacred Theology
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McVey, David Graham. « Investigating genetic risk factors of coronary artery disease using genome editing ». Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/36614.
Texte intégralRésumé :
Genome-wide association studies (GWAS) have identified the genetic loci associated with many complex diseases including coronary artery disease (CAD). The challenge now is to elucidate the biological and cellular pathways affected by disease-associated loci. In order to fully understand the functional mechanisms, the causal genetic variants need to be identified. The majority of GWAS loci lack candidate genes, and may instead be located in regulatory regions, making the functional effects of specific variants difficult to appreciate. Recently, genome editing techniques have become available that allow targeted alteration of the genome, producing isogenic cell lines that differ only at the site of interest. In this study, recombinant adeno-associated virus (rAAV) genome editing was established and used to investigate potentially functional disease-associated variants in the 1p13 and 9p21 CAD loci. Evidence from previous work suggests that 1p13 (rs12740374) and 9p21 (rs10811656 and rs10757278) single nucleotide polymorphisms (SNPs) affect transcription factor binding, leading to dysregulation of local genes. Specific alteration of these SNPs using this technique enabled the examination of these hypotheses directly. The 1p13 study has provided evidence to support the hypothesis that rs12740374 is the causal SNP at this locus. We observed genotype-dependent effects upon C/EBPα binding and the expression of four 1p13 genes (SORT1, CELSR2, PSRC1 and MYBPHL). Our examination of the 9p21 SNPs showed that these variants are capable of influencing STAT1 binding, but local gene expression was not affected. This suggests that variation of just rs10811656 and rs10757278 is insufficient to affect gene expression, and that other pathways may be involved. The first study to utilise the rAAV technique to examine non-coding, regulatory SNPs, this work demonstrates that isogenic cell lines produced by rAAV genome editing allow for the quantification of subtle, genotype-specific effects. This work suggests that this adaptable technology may be beneficial for other studies examining the genetics of complex diseases.
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Kentner, Jeffrey Louis. « Engineering the zinc finger recombinase for use in targeted genomic editing ». Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6910/.
Texte intégral
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Muller, Emily A. « Developing a Gene Editing System to Study Haplodiploidy in the Jewel Wasp, Nasonia Vitripennis ». Scholarship @ Claremont, 2015. http://scholarship.claremont.edu/scripps_theses/576.
Texte intégralRésumé :
Hymenopteran insects, which include all ants, bees and wasps, reproduce through a poorly understood form of reproduction known as haplodiploidy. A promising experimental system for understanding this developmental process is the jewel wasp, Nasonia vitripennis. A critical aspect of using Nasonia as a model is establishing an effective means for editing specific genes of interest so that their functions can be studied through genetic means. For my thesis research, I performed a pilot study of the gene editing method known as CRISPR in Nasonia. I targeted the single heterochromatin protein 1 (HP1) gene present in the Nasonia genome in order to assess the feasibility of this gene editing approach. Targeting HP1 would provide a clear phenotype when this gene is mutated due to its essential functions in early development known from studies in other eukaryotes. Additionally, creating a mutant of this gene will provide a means for studying the role of HP1 in wasp spermatogenesis, an aim that interlinks with the broader chromatin-based goals of our laboratory. Through this study I worked out a streamlined procedure for injecting CRISPR molecules into young wasp embryos, conducting genetic crosses with injected wasps, and screening through their progeny for potential mutants. I observed no mutant phenotypes in injected wasps, but instead, I isolated four potential mutants in F1 progeny. My work has helped to create a solid framework for improving this procedure in Nasonia, and they allow for a better overall understanding of the limitations of producing mutants through CRISPR gene editing in non-model organisms such as Nasonia.
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Canver, Matthew. « Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing ». Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493407.
Texte intégralRésumé :
Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders.Medical Sciences
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Gupta, Ankit. « Getting a Tight Grip on DNA : Optimizing Zinc Fingers for Efficient ZFN-Mediated Gene Editing : A Dissertation ». eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/637.
Texte intégralRésumé :
The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity.
Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics.
In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.
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Kaahui, Soncy. « The Future of Mosquito Control : Wolbachia and Genome Editing ». Scholarship @ Claremont, 2019. https://scholarship.claremont.edu/scripps_theses/1234.
Texte intégralRésumé :
The impact that mosquitoes and vector-borne diseases have on humans is vast and continues to grow with our expanding global interactions, such as international travel and shipping, so the need for effective vector controls is imperative. Aedes aegypti is a species of mosquito that spreads some of the most common vector-borne diseases, including zika virus, dengue fever, chikungunya, and yellow fever. A. aegypti have yet to be successfully contained, so they are favorable targets for implementing these new vector-control techniques. A review of scientific literature was performed from 1965 to present, timeline was constructed of studies on A. aegypti and their diseases, with inclusion criteria of techniques like bacterial controls and genome editing. Bacterial controls, such as using an endosymbiont like Wolbachia, can result in sterilization of mosquitoes as well as inhibiting the ability for mosquitoes to be infected by pathogens. Genome editing techniques involve CRISPR and gene drives, allowing the manipulation of certain genes to decrease fitness or susceptibility of pathogens. Combining newly discovered genes that play a role in sterilization with the introduction of sterilizing Wolbachia bacteria could result in a more effective method for controlling A. aegypti. Neither technique is known to be entirely effective on its own, but research indicates that highly effective vector-controls could be developed by combining aspects from both fields.
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Pigini, Paolo <1991>. « Neuroblastoma targeted therapy : employment of CRISPR gene-editing to explore relevant markers and potential targets in aggressive tumours ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8752/1/Pigini_Paolo_tesi.pdf.
Texte intégralRésumé :
Neuroblastoma is a tumour originating from the sympathetic nervous system, and represents the most common extracranial solid cancer in childhood. Despite the malignancy is extremely heterogeneous, about 25% of all cases is characterized by MYCN-gene amplification, aggressive tumour and poor survival. The network of genes that are deregulated in this group of patients represents a focal point for targeted-therapy discovery. Along this research line, the first objective of the present project was to investigate the prognostic significance of a single nucleotide polymorphism (SNP) located in the promoter of ODC1, a neuroblastoma prognostic marker involved in polyamine biosynthesis. The SNP genotype was first associated with survival of a large cohort of patients with aggressive neuroblastoma. Then, CRISPR-editing revealed that the SNP genotype affects ODC1 expression and proliferation of neuroblastoma cells. At last, the SNP was found to influence cell sensibility to DFMO, an ODC1 inhibitor that is currently under trial for treatment of aggressive neuroblastoma. The second objective was to investigate the role in neuroblastoma development and progression of RUNX1T1, a poorly studied transcription repressor involved in distinct development events and cancers. Survival analysis of a cohort of neuroblastoma patients revealed that RUNX1T1 is a potential oncosuppressor. In apparent contrast, RUNX1T1 knockout by CRISPR-editing demonstrated that the gene promotes aggressiveness of neuroblastoma cells. Transcriptome analysis of the mutant cells then evidenced deregulation of a significant number of genes and pathways that are prognostic markers in neuroblastoma, therefore depicting a multifunctional regulation network that could be exploited for new therapies. The third and last objective was to test a novel therapeutic approach based on MYCN-amplification targeting via CRISPR-cleavage. In vitro experiments demonstrated that the system efficiently and specifically impairs the survival of aggressive neuroblastoma cells, thus providing a proof of principle for the development of an innovative therapy.
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Moshiri, Houta. « Fluorescence-based reporter substrate for monitoring RNA editing in Trypanosomatid pathogens ». Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116117.
Texte intégralRésumé :
Mitochondrial gene expression in trypanosomatid pathogens requires extensive post transcriptional modification called RNA editing. This unique molecular mechanism, catalyzed by a multiprotein complex (the editosome), generates translatable transcripts for essential components of parasite respiratory complex. How editosome proteins are assembled and perform RNA editing is not fully understood. Moreover, previous studies have shown that editosome proteins are essential for parasite survival, which makes editosome as a suitable target for drug discovery. Currently, researchers use radio-labeled based assays to monitor RNA editing process. However, these assays are not suitable for high throughput screening of editosome inhibitors, have low detection limits, and cannot monitor RNA editing in real time.Therefore, to develop a sensitive high throughput RNA editing assay, we have designed a sensitive hammerhead ribozyme-based fluorescence assay. Ribozyme structure was remodeled by adding or removing uridylate in its conserved catalytic core to make an inactive ribozyme. In the presence of the editosome, inactive ribozyme is edited to an active ribozyme. Consequently, hammerhead ribozyme activity can be measured by cleaving its fluorescently labeled substrate. We have shown that higher sensitivity is achieved using fluorescent based assay than conventional radio-labeled assay. Moreover, we can use this assay for rapid identification and characterization of the editosome inhibitors against RNA editing activities in trypanosomatids.
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MINGOIA, MAURA. « Terapia genica della β Talassemia mediante editing del DNA ». Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266632.
Texte intégralRésumé :
β (HBB) gene, resulting in absence (β0) or deficiency (β+) of β globin chain synthesis. This genetic disorder occurs most frequently in people from Mediterranean countries, such as Italy. In particular, the data indicates that about 12.6% of the Sardinian subjects are carriers of β thalassemia and these are among the highest frequencies of thalassemia genes found in a Caucasian population. In Sardinia, the disease is generally determined by a nonsense mutation at codon 39 (E39X) of exon 2 causing the interruption of β globin synthesis. Patients homozygous with E39X mutation have a severe anemia and require frequent transfusions and iron chelation. The only definitive cure today possible for β chain hemoglobinopathies is the hematopoietic stem cells transplantation, but it is limited by availability of HLA matched donors. However, in the last few years new therapeutic approaches for this genetic disease are taking place. The correction of disease-causing mutation through the technique of Genome-Editing in patient-specific stem cells and subsequent autologous transplantation would be the ideal approach for the treatment of monogenic diseases such as β thalassemia. However, due to difficulties in obtaining sufficient homologous recombination percentages for therapeutic purposes, the aim of my PhD project is to reproduce artificially the HPFH mutations identified in non-coding regions of the β globin cluster, using the system CRISPR/Cas9 associated with NHEJ pathway. In this way, we hope to restore at therapeutic levels the expression of HBG genes and consequently the synthesis of a functional HbF in order to ameliorate the phenotype of β thalassemia.
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Chan, Robin F. « Epigenetic editing to validate findings from methylome-wide association studies of neuropsychiatric disorders ». VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5003.
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DNA methylation is necessary for learning, memory consolidation and has been implicated in a number of neuropsychiatric disorders. Obtaining high quality and comprehensive data for the three common forms of methylation in brain is challenging for methylome-wide association studies (MWAS). To address this we optimized a panel of enrichment methods for screening the brain methylome. Results show that these enrichment techniques approach the coverage and fidelity of the current gold standard bisulfite based techniques. Our MBD-based method can also be used with low amounts of genomic material from limited human biomaterials. Psychiatric disorders have high prevalence and are often chronic making them a leading contributor to disability. Major depressive disorder (MDD) has a lifetime prevalence of ~15% and high recurrence leading to substantial morbidity and costs to society. The underlying biological processes that contribute to MDD are poorly understood. Noting the importance of DNA methylation in neurobiology, we conducted the largest MWAS in human post-mortem brain uncover novel candidate genes and biomarkers associated with MDD. The top result of this MDD MWAS was within the gene ANKS1B. This gene has been implicated in many past genetic studies of psychiatric disorders and has experimental support as a regulator of neurotransmission. Targeted epigenetic editing technologies allow for precise modification of DNA methylation in living cells. However, an appropriate model system is critical to properly interpreting such experiments. An accelerated protocol for differentiating Ntera2 cells into human neurons was developed for this purpose. Ntera2-derived neurons express key neuronal markers and are well suited to use in epigenetic editing experiments. Concurrently, the generation of the reagents necessary for recapitulating the aberrant methylation at ANKS1B linked to MDD was undertaken. Using a modified CRISPR/Cas9 approach demethylating enzyme was directed to target sites to attempt perform editing of DNA methylation. Results indicate that significant but biologically irrelevant changes to methylation at ANSK1B were achieved. The novelty of the technology employed presented challenges to the success of the current work. However, the field of epigenetic editing is advancing rapidly and will remain an attractive method for functional characterization of future MWAS findings and basic neuroscience research.
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Maeder, Morgan Lee. « Engineered DNA-Binding Proteins for Targeted Genome Editing and Gene Regulation ». Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10770.
Texte intégralRésumé :
Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.
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LLADO, SANTAEULARIA MANEL. « THERAPEUTIC GENOME EDITING IN RETINA AND LIVER ». Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/696628.
Texte intégralRésumé :
In vivo gene therapy with adeno-associated viral (AAV) vectors has been successful at treating several inherited diseases, specifically those caused by loss of function mutations which require transfer of a correct copy of a gene. This would not benefit dominant diseases due to gain of function mutations which produce toxic protein products. In addition, since AAV genomes persist as episomes in target cells, AAV mediated transgene expression might be short lived in tissues where cell proliferation occurs when newborn or after damage, like for example the liver. To overcome these challenges, I have developed AAV-based therapeutic approaches which use genome editing to introduce stable modifications at specific genomic loci. First, an allele-specific approach which targets the Rhodopsin P347S dominant mutation was developed and tested both in vitro and in vivo. I achieved allele-specific targeting of human P347S rhodopsin, which reduced mRNA levels and improved retinal electrical function in a mouse model of autosomal dominant retinitis pigmentosa. Second, I developed a mutation- and homology-independent targeted integration (HITI) approach for gene correction in photoreceptors. I demonstrated feasibility of this approach in mouse and pig photoreceptors using a reporter gene and characterized on-target precision of HITI in the murine rhodopsin locus. I then tested the therapeutic potential of this approach in a mouse model of autosomal dominant retinitis pigmentosa and observed mild and transient improvement of retinal function in treated eyes, which suggests that the levels of editing obtained need optimization. Third, I developed a HITI approach for expressing therapeutic genes from the liver by targeting the albumin locus, which is highly transcribed in hepatocytes. I demonstrated feasibility and efficiency of this approach using a reporter gene, and characterized on-target precision of HITI, as well as off-target integration due to Cas9 cleavage. I then tested the therapeutic potential of the integration of a copy of the human arylsulfatase B (ARSB) gene, which is mutated in a rare lysosomal storage disease, mucopolysaccharidosis type VI (MPS VI), in the albumin locus in the liver of newborn MPSVI mice. I demonstrated that this approach achieves stable expression of ARSB at levels that reduce glucosaminoglycan (GAG) urinary secretion, one of the main readouts of MPSVI phenotype. This stable expression of ARSB is contrary to the decrease of transgene expression observed in neonatal MPSVI mice injected with the same dose of a conventional gene therapy vector, thus overcoming the potential loss of transgene expression caused by hepatocyte proliferation. Overall, I have developed different genome editing approaches for conditions that are inherited as either dominant or recessive. I have tested these approaches in two relevant tissues for gene therapy like retina and liver and shown the potential to provide AAV with persistent transgene expression in proliferating tissues like the newborn liver.
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Edraki, Alireza. « Compact Cas9s and Their Natural Inhibitors for Genome Editing ». eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1052.
Texte intégralRésumé :
Recent advances with the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as genome editing tools have opened a new avenue for targeting disease-causing mutations. The programmability of the Cas9 endonuclease by RNA makes it a potentially powerful therapeutic tool to correct such mutations. The CRISPR-Cas9 system consists of a Cas9 endonuclease that is guided by RNA (sgRNA) to create double-stranded breaks in a target DNA segment complementary to the guide. This process is dependent on a 2-8 nucleotide sequence (called PAM) that is adjacent to the target and functions as a Cas9 binding signal. Each Cas9 ortholog recognizes a unique PAM.
However, factors such as the size of Cas9 or the frequency of its PAM sequence in the genome have hindered its clinical use. The Cas9 from Streptococcus pyogenes (SpyCas9) is commonly used in research because its PAM (NGG, where “N” symbolizes any nucleotide) is present every ~8 bp in the genome, providing robust targeting potential. However, it is too large to fit into typical viral vectors used for in vivo delivery, namely adeno-associated vectors (AAV). While several Cas9 orthologs have been characterized, none satisfied the need for a compact, accurate Cas9 with a short PAM.
In this thesis, we use two approaches to identify new compact Cas9 orthologs with small PAMs, one using anti-CRISPR proteins and one by searching through closely related Cas9s. First, we use the presence of anti-CRISPRs (naturally occurring, phage-encoded peptides that inhibit CRISPR-Cas9 described in chapter 2) in a genome as indicators of Cas9s that may be highly active. These orthologs come with the added advantage of having inhibitors that can be used as off-switches. We characterize four Cas9s that are targeted by anti-CRISPR proteins and show that they recognize diverse PAMs in vitro. One of the four Cas9’s, namely HpaCas9 from Haemophilus parainfluenzae, induces efficient genome editing in mammalian cells. However, its long N4GATTT PAM does not satisfy the short PAM criterion.
For our second approach, we asked whether closely related Cas9 orthologs with drastically different PAM-interacting domains (PIDs, the domain responsible for PAM recognition) recognize different PAMs, and if so, can be used for genome editing. To this end, we exploited natural variation in the PID of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis (Nme2Cas9). Nme2Cas9 recognizes a simple dinucleotide PAM (N4CC) that provides a high target site density. All-in-one
AAV delivery of Nme2Cas9 with a guide RNA into adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Finally, we show preliminary data on how CRISPR-Cas9 can be used for therapeutic genome editing for Amytrophic Lateral Sclerosis. Our new findings promise to accelerate the development of genome editing tools for biomedical and therapeutic applications.
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Yang, Luhan. « Development of Human Genome Editing Tools for the Study of Genetic Variations and Gene Therapies ». Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11117.
Texte intégralRésumé :
The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.
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Sandhu, Mandeep. « CRISPR Genetic Editing : Paths for Christian Acceptance and Analysis of In Vivo and In Vitro Efficiency ». Scholarship @ Claremont, 2018. https://scholarship.claremont.edu/scripps_theses/1363.
Texte intégralRésumé :
With advancements in CRISPR-cas9 broadening the potential paths for clinical usage of genetic editing, conversations about genetic editing have grown to outside simply scientific communities and into mainstream conversations. This study focuses specifically on Christian discourse of genetic editing and locates four major tensions for many Christians when they think about genetic editing: beginning of life, Creator-human relationship, imago Dei, and stewardship. With these major concerns in mind, I identify epigenetics, somatic cell genetic editing, and in vivo genetic editing research as important research paths to pursue as they can potentially produce techniques that more Christian individuals would feel comfortable using. I pursue one of these paths and conclude with an experimental proposal for an analysis of in vivo and in vitro CIRSPR-Cas9 efficiency in regards to on- and off-target rates.
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Brazel, Ailbhe Jane. « A genetic and epigenetic editing approach to characterise the nature and function of bivalent histone modifications ». Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/29603.
Texte intégralRésumé :
In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.
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Amaya, Colina Anais Karime. « Towards the Treatment of Human Genetic Liver Disease by AAV-Mediated Genome Editing and Selective Expansion of Repaired Hepatocytes ». Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21893.
Texte intégralRésumé :
Gene repair involves the correction of the genetic mutation directly at the defective locus with retention of physiological expression. The biggest challenge of this approach, however, is that gene repair by homologous recombination occurs at levels that are unlikely to be sufficient to confer therapeutic benefit in the majority of cell-autonomous liver disease phenotypes, such as OTC deficiency, the most common urea cycle disorder. To overcome this challenge, gene correction can be complemented by selective expansion strategies designed to expand repaired hepatocytes to frequencies required for therapeutic benefit. In vivo expansion can be achieved, for instance, by conferring a selective advantage to gene-corrected cells. In this study, human-specific genetic inhibitors were designed to exploit a selective expansion strategy based on the modulation of the tyrosine catabolism pathway and were successfully validated in humanised (Fah-/-, Rag2-/-, IL2rg-/-) FRG mice. Another way to increase the frequency of gene repair is to use nucleases to create DNA breaks at the target site to promote homology-directed repair (HDR). Recombinant AAV vectors carrying human-specific reagents for CRISPR/Cas9-mediated genome editing were developed in order to correct a single nucleotide mutation in exon 9 of the OTC gene. Initially, the editing reagents were evaluated in OTC-deficient mice with a transposed engineered “minigene” version of the OTC gene. Editing reagents functionally validated in this model were then evaluated in vivo on the native OTC locus in primary human hepatocytes, including patient-derived hepatocytes, xenografted into FRG mice. Availability of novel synthetic AAV capsids, such as NP59, facilitated high targeting efficiency of human hepatocytes which in turn resulted in up to 29% OTC alleles being corrected by HDR. The studies described in this thesis show for the first time precise gene repair of a disease-causing mutation in primary human hepatocytes in vivo.
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Alotiby, Amna. « Exploring gene editing using site-specific endonucleases as an approach to improve in vitro models of TNF Receptor-Associated Periodic Syndrome (TRAPS) ». Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/39955/.
Texte intégralRésumé :
Tumour necrosis factor receptor-associated periodic syndrome is an autoinflammatory disorder caused by mutations in the tumour necrosis factor receptor-type 1 (TNFR1) gene, leading to misfolding of the TNFR1 protein and a resultant chronic inflammatory condition. This study aimed to explore the whether a more physiologically relevant model of TRAPS could be made by gene editing the cell’s endogenous TNFR1 genes. We utilized two site-specific nucleases, TALEN and CRISPR-Cas9 as gene editing tools. These nucleases are able to introduce double-strand DNA breakage at a pre-determined DNA sequence, resulting in gene modification, by two mechanisms: non-homologous end joining (NHEJ) or homology directed repair (HDR). TALEN pairs were designed and constructed for targeting six sites on exon 2 of TNFR1 in the SK-Hep-1 Cell line. However, despite evidence of correct TALEN construction, gene editing at the target sites in TNFR1 was undetectable by the methods employed (Surveyor mutagenesis assay, DNA sequencing, Flow cytometry). This suggested that our TALEN pairs might be of very low efficiency or be non-functional in vivo. Consequently, the relatively simpler CRISPR-Cas9 system was used as an alternative gene editing tool to target the TNFR1 gene. Two plasmids expressing guide RNAs and Cas9 enzyme were designed to target exon 2 of TNFR1, and were functionally verified in vitro. Both were applied separately to induce gene editing in vivo, firstly by NHEJ, and subsequently by HDR. Analysis of DNA from pooled clones of transfected cells for NHEJ yielded little evidence of successful gene editing by Surveyor assay or pooled DNA sequencing. In silico analysis of exon 2 sequences using TIDE suggested a maximal efficiency of ≤3%. Cells co-transfected with the cas9-guideRNA plasmids and a HDR template, designed to insert the FLAG epitope sequence into TNFR1, was however more successful. PCR detection of FLAG sequence insertion into exon 2 of TNFR1 indicated successful editing had occurred. However, examination of transfected cells by flow cytometry staining for FLAG epitope expression indicated that gene editing was still very low efficiency, ≤ 1.37%, despite high efficiency of transfection. In conclusion, the CRISPR-Cas9 system, in our hands, shows evidence which supports it’s use in gene editing of TNFR1. However, the very poor efficiency of editing detected, suggests much further reagent and process/detection optimization is needed, or more fundamental issues, including accessibility of the TNFR1 gene within the SK-Hep1 cell line need to be addressed, before it’s application to generate disease specific mutations.
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Ghanta, Krishna S. « An Exploration of the Properties of Repair Template DNA that Promote Precision Genome Editing ». eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1150.
Texte intégralRésumé :
CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms.
In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing.
Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.
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Ling, Jiqiang. « Role of phenylalanyl-tRNA synthetase in translation quality control ». Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1212111223.
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Toffessi, Tcheuyap Vanina. « Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing ». Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc984227/.
Texte intégralRésumé :
von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.
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Fleming, Ian Murray Cameron. « Studies on RNA Modification and Editing in Trypanosoma brucei ». The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1452245560.
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Der, Joshua P. « Genomic Perspectives on Evolution in Bracken Fern ». DigitalCommons@USU, 2010. https://digitalcommons.usu.edu/etd/663.
Texte intégralRésumé :
The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.
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Wei, Yulei. « Genetic Knowledge-based Artificial Control over Neurogenesis in Human Cells Using Synthetic Transcription Factor Mimics ». Kyoto University, 2018. http://hdl.handle.net/2433/232265.
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Tennant, Peter Andrew. « Genome editing using site-specific nucleases : targeting highly expressed genomic regions for robust transgene expression and genetic analysis ». Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22857.
Texte intégralRésumé :
Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.
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Stens, Cassandra, Isabella Enoksson et Sara Berggren. « The CRISPR-Cas system ». Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-171997.
Texte intégralRésumé :
Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.
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Hedberg, Rickard. « Preimplantation genetic diagnosis and therapy in humans- Opportunities and risks ». Thesis, Örebro universitet, Institutionen för medicinska vetenskaper, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-81532.
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IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.
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Calatayud, Aristoy Carles. « Investigating the genetic component of Parkinson’s disease through the use of human induced pluripotent stem cells and gene editing ». Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457667.
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Despite the advances in the identification of genes and proteins involved in Parkinson’s disease (PD), there are still appreciable gaps in our understanding of the mechanisms underlying the chronic neurodegenerative process in PD. In the lab, it has been demonstrated that iPSC technology can be used to observe phenotypes relevant to neurodegeneration in PD, and also provided first proof-of-principle evidence that neurons with the genome of a sporadic PD patient exhibited similar phenotypes as seen in iPSC derived from patients with monogenic LRRK2 (G2019S) PD. In the present study we generated a complementary set of iPSC lines from asymptomatic individuals carrying pathogenic LRRK2 mutations, whose gene pool may have a prevailing protective effect. We then corrected the LRRK2 mutation by using TALEN-mediated genetic engineering in the symptomatic LRRK2-iPSC lines, as well as well as introduced it in our already established control-iPSC lines. Dopaminergic neurons differentiated in parallel from this subset of iPSC lines have been cultured over a long time span and monitored for the appearance of neurodegeneration phenotypes (including reduced numbers of neurites and neurite arborization and α-synuclein accumulation) after 75 days in culture. Interestingly we found that while PD iPSC-derived DA neurons showed altered morphology and shorter/fewer neurites, DAn derived from NMC show mature morphology and long neurites with complex arborization, similar to those differentiated from Ctrl-iPSC. We have also identified mutation-linked phenotypes such as α-synuclein accumulation whose appearance was delayed in NMC neurons compared to LRRK2-PD neurons. Complementarily, we have sequenced the exome of our cohort in order to identify the genetic modifiers of LRRK2 mutation penetrance. Importantly, the availability of a refined set of PD patient-specific iPSC lines representing symptomatic and asymptomatic cases of familial PD sharing the same pathogenic mutation in LRRK2, as well as isogenic iPSC lines in which the mutation has been edited out, will open a new window for the early diagnosis and individualized treatment of the prodromic period of the disease.
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Foster, Robert Graham. « Development of a modular in vivo reporter system for CRISPR-mediated genome editing and its therapeutic applications for rare genetic respiratory diseases ». Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33040.
Texte intégralRésumé :
Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.
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Chai, Shin Luen Chai. « Novel Genetic Modifiers in a Monogenic Cardiac Arrhythmia ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1516618028568975.
Texte intégral
41
Waghulde, Harshal B. « Mapping and CRISPR/Cas9 Gene Editing for Identifying Novel Genomic Factors Influencing Blood Pressure ». University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1470402637.
Texte intégral
42
Iacobucci, Ilaria <1980>. « Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all) : from genetic alterations to impaired RNA editing ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/804/1/Tesi_Iacobucci_Ilaria.pdf.
Texte intégralRésumé :
The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it
represents the single most significant adverse prognostic marker. Despite imatinib has led to
significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases
resistance developed quickly and disease progressed. Some mechanisms of resistance have been
widely described but the full knowledge of contributing factors, driving both the disease and
resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia
in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a
zinc finger transcription factor required for normal hemopoietic differentiation and proliferation,
particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several
proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding
isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer
partners to bind DNA.
The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to
determine if molecular abnormalities involving the Ik gene could associate with resistance to
imatinib and dasatinib.
Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with
Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were
collected. We set up a fast, high-throughput method based on capillary electrophoresis technology
to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non
DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display
abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6
failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA
binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and
at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and
Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a
60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the
end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the
insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The
molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6
expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration
could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative
Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically
demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase
inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line
(SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6
strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a
previously unknown link between specific molecular defects that involve the Ikaros gene and the
resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon
splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs):
rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice
junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of
patients was also found. Other two different single nucleotide substitutions not recognized as SNP
were observed. Some mutations were predicted by computational analyses (RESCUE approach) to
alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional
regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients
treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to
resistance opening a time frame, during which leukaemia cells acquire secondary transforming
events that confer definitive resistance to imatinib and dasatinib.
43
Iacobucci, Ilaria <1980>. « Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all) : from genetic alterations to impaired RNA editing ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/804/.
Texte intégralRésumé :
The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it
represents the single most significant adverse prognostic marker. Despite imatinib has led to
significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases
resistance developed quickly and disease progressed. Some mechanisms of resistance have been
widely described but the full knowledge of contributing factors, driving both the disease and
resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia
in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a
zinc finger transcription factor required for normal hemopoietic differentiation and proliferation,
particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several
proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding
isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer
partners to bind DNA.
The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to
determine if molecular abnormalities involving the Ik gene could associate with resistance to
imatinib and dasatinib.
Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with
Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were
collected. We set up a fast, high-throughput method based on capillary electrophoresis technology
to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non
DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display
abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6
failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA
binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and
at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and
Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a
60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the
end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the
insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The
molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6
expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration
could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative
Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically
demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase
inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line
(SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6
strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a
previously unknown link between specific molecular defects that involve the Ikaros gene and the
resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon
splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs):
rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice
junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of
patients was also found. Other two different single nucleotide substitutions not recognized as SNP
were observed. Some mutations were predicted by computational analyses (RESCUE approach) to
alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional
regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients
treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to
resistance opening a time frame, during which leukaemia cells acquire secondary transforming
events that confer definitive resistance to imatinib and dasatinib.
44
Zhang, Yingxiao. « Genetic Engineering of Rubber Producing Dandelions ». The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480626773100647.
Texte intégral
45
Woodfint, Rachel M. woodfint. « Identification of the LB-FABP promoter as a liver specific promoter via the generation of transgenic quail expressing eGFP within their liver cells ». The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523880800285644.
Texte intégral
46
TORCHIO, SILVIA. « WOLFRAM SYNDROME : FROM POINT MUTATION TO CELL DYSFUNCTION ». Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/133066.
Texte intégralRésumé :
Wolfram Syndrome 1 (WS1) is a devastating genetic disease manifesting with diabetes mellitus, diabetes insipidus, optic nerve atrophy and deafness. It is caused by dominant or recessive mutations in the WFS1 gene, coding for Wolframin, a transmembrane protein implicated in ER stress response, autophagy, Ca++ handling and insulin secretion.
In the present, study, we sought to investigate the case of a patient carrying novel WFS1 heterozygous mutations, aiming to characterize the genetic, molecular and functional components that determine WS1 manifestation. To do so, we employed iPSC technology, reprogramming mononucleated blood cells into pluripotent progenitors.
Concerning genetics, we determined that one of the two mutations, falling at an acceptor splice site, causes the appearance of multiple alternative isoforms lacking variable portions of the original mRNA; some of them retain the reading frame and code for internally truncated isoforms of the protein. In light of this, we genetically corrected with CRISPR/Cas9 said allele and obtained a syngeneic counterpart.
We demonstrated that WS1-derived iPSCs differentiate in the endocrine lineage, but they show anomalies in the composition of the endocrine subpopulations and β cell subtypes.
We investigated molecular alterations both in iPSCs and iPSC-derived β cells: we found that β cells have higher basal levels of ER stress response, and stress induction further exacerbates their anomalous response. Additionally, both WS1 iPSCs and iPSC-derived β cells have abnormal activation of the autophagic flux.
Functional studies were performed on β cells, highlighting irregularities in Ca++ fluxes and insulin secretion in response to glucose stimulation. Lastly, all the individuated mechanisms concur to predispose WS1 cells to undergo apoptosis more than controls in response to ER stress and inflammatory stimuli.
Of interest, we show that Liraglutide administration, a GLP-1 receptor agonist that proved effective in clinics for the patient, can ameliorate molecular and functional parameters in WS1-derived cells.
In conclusion, this study provides a novel perspective on the molecular basis of a peculiar case of WS1, connecting the genetic mutations with a unique molecular signature and with downstream functional alterations.La sindrome di Wolfram è una grave malattia genetica che si manifesta con diabete mellito, diabete insipido, atrofia del nervo ottico e sordità. È causato da mutazioni dominanti o recessive nel gene WFS1, che codifica per Wolframina, una proteina transmembrana implicata nella risposta allo stress del RE, nell'autofagia, nella gestione del calcio e nella secrezione insulinica. In questo studio, abbiamo indagato il caso di una paziente portatrice di nuove mutazioni in eterozigosi nel gene WFS1, con l'obiettivo di caratterizzare le componenti genetiche, molecolari e funzionali che determinano la manifestazione della patologia. Per fare ciò, abbiamo utilizzato la tecnologia delle iPSCs, riprogrammando cellule mononucleate del sangue in progenitori pluripotenti. Per quanto riguarda l’aspetto genetico, abbiamo determinato che una delle due mutazioni, situata in un sito accettore di splicing, provoca la comparsa di più isoforme alternative che risultano prive di alcune porzioni dell'mRNA originale; alcune di esse conservano il frame di lettura e generano isoforme della proteina troncate internamente. Alla luce di ciò, abbiamo corretto geneticamente tramite tecnologia CRISPR/Cas9 questo allele e ottenuto una controparte singenica. Abbiamo dimostrato che le iPSCs derivate da paziente Wolfram si differenziano nel comparto endocrino pancreatico, ma mostrano anomalie nella composizione delle sottopopolazioni endocrine e dei sottotipi di β cellule. Abbiamo studiato le alterazioni molecolari sia nelle iPSCs che nelle β cellule derivate da iPSCs: abbiamo scoperto che le β cellule hanno livelli basali alterati di risposta allo stress del RE e l'induzione dello stress esacerba ulteriormente la loro risposta anomala. Inoltre, sia le iPSCs Wolfram che le β cellule derivate da iPSCs hanno un'attivazione anormale del flusso autofagico. Sono stati condotti studi funzionali sulle β cellule, evidenziando irregolarità nei flussi di calcio e nella secrezione di insulina in risposta alla stimolazione con glucosio. Infine, tutti i meccanismi individuati concorrono a predisporre le cellule Wolfram ad andare incontro ad apoptosi in risposta a stress del RE e stimolo infiammatorio. Abbiamo dimostrato inoltre che la somministrazione di Liraglutide, un agonista del recettore GLP-1 che si è dimostrato efficace in clinica per la paziente, migliora i parametri molecolari e funzionali nelle β cellule Wolfram. In conclusione, questo studio fornisce una nuova prospettiva sulle basi molecolari di un caso particolare di sindrome di Wolfram, mettendo in connessione le mutazioni genetiche con uno schema molecolare unico e con conseguenti alterazioni funzionali.
47
Schneider, Sara Jane. « Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish : Potential for Genome Editing in Somatic Cells ». Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1011754/.
Texte intégralRésumé :
Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.
48
Iyengar, Preethi Ranganathan. « MYSTERIES OF THE TRYPANOSOMATID MAXICIRCLES : CHARACTERIZATION OF THE MAXICIRCLE GENOMES AND THE EVOLUTION OF RNA EDITING IN THE ORDER KINETOPLASTIDA ». VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4010.
Texte intégralRésumé :
The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.
This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 new trypanosomatids and compared them with 8 previously published maxicircle genomes of other trypanosomatids. Transcripts of ~13 of the 20 total genes in these maxicircles undergo post-transcriptional modifications involving the insertion and deletion of U residues at precise sites, to yield the final, fully-edited, translatable mRNA. We have deciphered the diverse patterns and extents of RNA editing of each edited gene in the maxicircle of each organism, and inferred the sequences of the putative fully edited mitochondrial transcripts and proteins. Using a binary value - based strategy (1/0), we quantified the RNA editing in all these trypanosomatids and estimated the evolution of RNA editing in the group. Additionally, we conducted phylogenetic analyses using a subset of unedited maxicircle genes to predict the relationships between the various trypanosomatids in this project, and compared them to the previously published nuclear gene-based phylogenies.
For convenience of analysis, the 28 total trypanosomatids in this work were divided into two groups: the first group consisting of the endosymbiont-bearing and related insect trypanosomatids, which constitute the first half of the project, and the second group consisting of trypanosomatids of the Trypanosoma genus, including T. cruzi-related and unrelated parasites, constituting the latter half of the project.
In summary, most of the trypanosomatid maxicircles showed a syntenic panel of 20 protein-coding genes (excluding any guide RNA genes), beginning with the mitochondrial ribosomal genes and ending with the gene encoding NADH dehydrogenase-5. Although some genes were partially or completely absent in the maxcircles of some species, the remaining genes were completely syntenic. The total number of genes edited and their editing patterns varied considerably among the first group of insect trypanosomatids, but were remarkably similar in the second group of the Trypanosoma genus. On a broad scale, the mitochondrial phylogeny reflects the nuclear phylogeny for these trypanosomatids, except within the T. cruzi population. Similarly, RNA editing appears to have evolved in parallel with the nuclear genes, although subtle differences are again noticeable within the T. cruzi family.
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Rieblinger, Beate [Verfasser], Angelika [Akademischer Betreuer] Schnieke, Wolfgang [Gutachter] Wurst, Reinhard [Gutachter] Schwinzer et Angelika [Gutachter] Schnieke. « Genetic porcine models for in vivo genome editing and for xenotransplantation / Beate Rieblinger ; Gutachter : Wolfgang Wurst, Reinhard Schwinzer, Angelika Schnieke ; Betreuer : Angelika Schnieke ». München : Universitätsbibliothek der TU München, 2020. http://d-nb.info/1226287425/34.
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Souza, Gustavo Torres de. « Produção de células MDBK expressando a enzima CAS9 e edição do gene da beta-lactoglobulina pelo sistema CRISPR/Cas9 ». Universidade Federal de Juiz de Fora (UFJF), 2017. https://repositorio.ufjf.br/jspui/handle/ufjf/6049.
Texte intégralRésumé :
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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O advento sistema CRISPR/Cas9 tornou o processo de edição gênica consideravelmente mais fácil e direto, uma vez que retirou empecilhos técnicos relacionados aos sistemas já disponíveis. Desta forma, foram permitidos diversos avanços no entendimento da função de elementos genômicos, assim como a produção de embriões geneticamente modificados com diversas finalidades. O atual trabalho objetivou a edição gênica no gene da beta-lactoglobulina em células somáticas bovinas objetivando a produção futura de embriões da espécie geneticamente modificados. Considerando-se que a hipersensibilidade a essa proteína responde pela maior parte das alergias ao leite bovino, a produção de animais cujo leite não contenha essa molécula é de grande interesse para a indústria de laticínios. Durante os experimentos, foi possível obter uma linhagem de células bovinas MDBK expressando a enzima Cas9 (MDBK-Cas). Usando células MDBK e as células MBDK-Cas foi possível se obter com sucesso edições gênicas no locus beta-lactoglobulina utilizando-se os componentes do sistema CRISPR/Cas9 na forma de mRNA da proteína Cas9 e sgRNAs. Conclui-se que o sistema CRISPR/Cas9 pode ser usado com os sgRNA desenhados neste estudo para editar o gene da betalactoglobulina em células MDBK. Assim, células MDBK podem ser utilizadas como alvo o locus em estudo. Modelos de estudos para edição do genoma bovino. Em vista da escassa literatura constando de trabalhos em que tenha sido feita a edição gênica em embriões bovinos, os dados gerados por esse trabalho colaborarão para o avanço do estado da arte no que diz respeito a engenharia gênica de bovinos e no conhecimento do funcionamento do sistema CRISPR/Cas9.
The advent of the CRISPR / Cas9 system made the process of gene editing considerably easier and more straightforward, since it removed technical impediments related to the systems already available. In this way, several advances were made in the understanding of the function of genomic elements, as well as the production of genetically modified embryos for various purposes. The present work aimed at the genetic editing of the beta-lactoglobulin gene in bovine somatic cells aiming at the future production of genetically modified embryos of the species. Considering that hypersensitivity to this protein accounts for most of the allergies to bovine milk, the production of animals whose milk does not contain this molecule is of great interest to the dairy industry. During the experiments, it was possible to obtain a lineage of bovine MDBK cells expressing the Cas9 enzyme (MDBK-Cas). Using MDBK cells and MBDKCas cells it was possible to successfully obtain gene editions at the beta-lactoglobulin locus using the components of the CRISPR / Cas9 system as mRNA of the Cas9 protein and sgRNAs. It is concluded that the CRISPR / Cas9 system can be used with the sgRNAs designed in this study to edit the beta-lactoglobulin gene in MDBK cells. Thus, MDBK cells can be targeted as the locus under study. Models of studies for editing the bovine genome. In view of the scarce literature consisting of studies in which bovine embryos have been genetically engineered, the data generated by this work will contribute to the advancement of the state of the art regarding the genetic engineering of cattle and the knowledge of the functioning of the system CRISPR / Cas9.