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<p>Glycogen storage disease type Ia is an autosomal recessive disorder caused by a mutation in the glucose-6-phosphatase (G6Pase) catalytic subunit, encoded in humans by G6PC. G6Pase dephosphorylates glucose-6-phosphate (G6P) in the liver to generate glucose that can be shuttled to the bloodstream to maintain normoglycemia. Patients with GSD Ia typically present at 6 months of age with sever hypoglycemia, which is lethal if untreated. The current treatment is a strict dietary regimen in which children must be fed every 2 hours overnight or given nasogastric tube feeding, and adults must consume uncooked cornstarch around the clock to maintain normal blood sugar levels. This treatment maintains survival but fails to prevent other symptoms related to metabolism of the excess G6P, and patients develop hepatic adenomas that may become hepatocellular carcinoma later in life, in addition to progressive renal complications.</p><p>To overcome the problems persisting during dietary therapy, the Koeberl lab has sought to develop gene therapy approaches that use adeno-associated virus (AAV) vectors to replace the G6pase activity, restoring normoglycemia and normal metabolic processes. However, the vast majority of AAV-delivered genetic material exists as episomes that do not replicate as cells divide, so the effects of AAV gene therapy on GSD Ia mouse and dog models have proven temporary. We hypothesized that driving integration of therapeutic vector genomes into an affected individual's genome would improve beneficial effects' longevity.</p><p>We tested several approaches to accomplish this, and have found positive effects using a zinc finger nuclease (ZFN) that targets the mouse safe harbor ROSA26 locus to induce homologous recombination of the G6PC donor vector into the mouse genome. We were able to see an improvement in mouse survival to 8 months of age, an increase in G6Pase activity at 3 months of age, and a decrease in glycogen accumulation at 3 months of age, when the ZFN vector is administered alongside the G6PC vector, compared with mice that received the G6PC vector alone.</p><p>We have also taken an alternative approach to overcoming the long-term complications of the current dietary treatment, which would augment rather than replace the current treatment. We have examined several drugs known to induce autophagy in other disease models or cell culture systems, to determine if we could manipulate autophagic activity in G6PC knockdown hepatocytes or GSD Ia mice. We have found positive results using rapamycin, a well-studied MTOR inhibitor, in mice and cells, and have screened several other drugs as well, finding positive effects for bezafibrate, mifepristone, carbamazepin, and lithium chloride, in terms of lipid reduction (which accumulates as a symptom of GSD Ia) and/or LC3-II enhancement, which is reduced in GSD Ia due to downregulation of autophagy during G6P accumulation.</p> / Dissertation

Zinc finger nucleases (ZFNs) induce double-strand DNA breaks at specific recognition sites. ZFNs can dramatically increase the efficiency of incorporating desired insertions, deletions, or substitutions in living cells. These tools have revolutionized the field of genome engineering in several model organisms and cell types including zebrafish, rats, and human pluripotent stem cells. There have been numerous advances in ZFN engineering and characterization strategies, some of which are detailed in this work. The central theme of this dissertation is improving the activity and specificity of engineered zinc finger nucleases with the ultimate goal of increasing the safety and efficacy of these tools for human therapy. As a first step, I undertook a large-scale effort to demonstrate that the modular assembly method of ZFN synthesis has a significantly higher failure rate than previously reported in the literature. This strongly suggested that engineering of ZFNs should better account for context-dependent effects among zinc fingers. The second advance reported in this dissertation is a method for biasing repair of zinc finger protein-induced DNA breaks toward homology-driven rather than error-prone repair in the presence of a donor template. Catalytically inactivating one monomer of a ZFN dimer results in a zinc finger nickase (ZFNickase) whose cleavage preference is directed at only one DNA strand. In human cell reporter assays, these ZFNickases exhibit a higher likelihood of repair by homology-driven processes, albeit with reduced absolute rates of correction. With further optimization, zinc finger nickases could provide a safer alternative to ZFNs in the context of gene correction therapies. Third, realizing there was no robust method for determining off-target cleavage sites of ZFNs in a genome-wide manner, I validated a collaborator’s novel in vitro selection system in human cells by identifying eight new potential off-target cleavage sites for a ZFN pair currently being used in clinical trials. Although it is unlikely these low-frequency mutations would be deleterious to patients, these results demonstrated that ZFNs induced more off-target effects than had been appreciated by previous work in the field. Collectively, the findings of this dissertation have contributed to more robust strategies for designing and evaluating ZFNs.

This thesis describes the establishment of a precise gene targeting methodology in the silkworm Bombyx mori by technologies based on engineered endonucleases. Two classes of engineered endonucleases, ZFNs and full length TALENs were used for creating DSBs at specified sites in the colour marker genes (BmBlos2 and Bmwh3). Direct embryo microinjection of engineered nucleases mRNA were performed and let the nuclease proteins to disrupt the functions of these marker genes by creating DSBs and inducing error prone NHEJ mechanism. These experiments showed that both ZFNs and TALENs could be used for targeted gene disruption in silkworms.

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen. / Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.