The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

The discovery and development of clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have revolutionized targeted genomic medicine. In my thesis, we discuss our efforts to improve and optimize various CRISPR-Cas systems for therapeutic genome editing applications. In part one, we propose that aberrant splice site disruption could be a simple and efficient strategy for treating some mutations associated with β-thalassemia. Specifically, we show that disruption of common mutant alleles in the HBB gene by Cas9 and Cas12a results in restoration of normal β-globin splicing, functional expression of HBB, and improved quality of erythroid maturation in edited β-thalassemia patient CD34+ hematopoietic stem and progenitor cells. In part two, we demonstrate that optimization of the nuclear localization signal (NLS) sequence framework is an effective method to improve the mutagenesis frequencies of Cas12a nuclease. In particular, the 3xNLS-NLP-cMyc-cMyc framework improves genome editing in mammalian and primary cells, relative to previous Cas12a NLS frameworks. We show that our NLS optimization approach can be applied to various Cas12a orthologs resulting in high editing activity without sacrificing the high intrinsic specificity of Cas12a nucleases. Furthermore, we demonstrate that NLS-optimized enAspCas12a can efficiently disrupt the ATF4-binding motif at the +55 enhancer of BCL11A, which may serve as an alternative therapeutic strategy for β-hemoglobinopathies. In part three, we develop and characterize fusion enhanced base editor (feBE) systems, which are fusions of base editors to our Cas9-programmable DNA binding domain (pDBD) and Cas9-Cas9 platforms. We report that our feBEs are more active than previously described base editor platforms at canonical and noncanonical PAMs. Furthermore, we show that feBEs can selectively edit a therapeutically relevant target site, CCR5, with minimal editing at a highly homologous off-target site. Taken together, my thesis research aimed to engineer CRISPR-Cas tools to improve their efficiency and specificity for therapeutic genome editing applications.

Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20930号 / 理博第4382号 / 新制||理||1630(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24878号 / 医博第5012号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24673号 / 農博第2556号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5454(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 矢﨑 一史, 教授 梅澤 俊明, 教授 伊福 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Advances in genome editing, human induced pluripotent stem cells (iPSC), and cardiac tissue engineering have significantly improved the ability of in vitro models to model cardiac disease. The objective of this dissertation is to leverage cardiac tissue engineering to generate meaningful biological insights into human genetic cardiomyopathies. First, we studied a novel, de novo mutation in the filamin C (FLNC) gene which causes restrictive cardiomyopathy in a young patient. Using engineered cardiac tissues, we showed that this mutation causes a restrictive phenotype marked by increased passive tension and slowed contraction velocities. Complementing our engineered tissues, we used high-throughput calcium imaging to identify compounds which improved myocardial relaxation in mutant cardiomyocytes. These compounds improved function of mutant cardiac tissues, suggesting a potentially targetable pathway in the patient’s mutation. In another study, engineered cardiac tissues and stem cells were used to study BAG3, a dilated cardiomyopathy- related gene, in cardiac fibroblasts. BAG3-/- and wild-type iPSCs were differentiated to cardiac fibroblasts and cardiomyocytes. By generating fully isogenic cardiac tissues and altering cellular genotypes, we determined that the loss of BAG3 in cardiac fibroblasts was deleterious to cardiac tissue function despite genetically normal cardiomyocytes. Further work studying cardiac fibroblasts revealed a mechanistic function of BAG3 in regulating cardiac fibroblast extracellular matrix synthesis. Together, this work highlights the ability of cardiac tissues and stem cells to unravel the complexities of genetic heart disease.

Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency affecting approximately 1 in 100,000 live XY births in North America and is caused by a mutation to the WAS gene which is expressed across hematopoietic lineages. The WAS protein (WASp) plays a role in regulating actin polymerization. On a cellular level, there are a variety of effects of a lack of WASp or expression of a dysfunctional WASp protein for patients including issues with migration, adhesion, chemotactic response, phagocytosis, activation, and proliferation across different cell types in addition to reduced platelet size and output. This can lead to several systematic effects for the patients however because mutations to the WAS gene are not limited to one location or type there is a great amount of variability between patient symptoms making it challenging to diagnose. Major symptoms include frequent and recurrent infections, uncontrolled bleeding episodes, issues associated with autoimmunity, and malignancy, the most common form being lymphoma. Without treatment, the life expectancy of an individual diagnosed with WAS is 14 years of age, and the only curative treatment strategy available is hematopoietic stem and progenitor cell transfer (HSPCT). If not performed with an HLA-identical donor, which is available to less than 10% of patients, and within the first two years of life, the risk of graft versus host disease (GvHD) increases drastically for the patient. A gene therapy using autologous and genetically corrected CD34+ cells would be advantageous to the patients due to a reduction in preparative conditioning, immunosuppressive aftercare, and the risk of GvHD. CSL Behring is currently in the development of a lentiviral gene therapy to fulfill this gap in care, however, to develop the assays required to assess and characterize the drug substance usually an uncorrected patient sample is compared with a gene-edited sample. The limitation here is that due to the risk of infection and bleeding patient sample is very limited and therefore the development of a mock patient sample is necessary for early development. The goal of the project is to develop a WAS-KO protocol utilizing CRISPR/Cas9 and its characterization.

In the growing landscape of innovative non-viral delivery vehicles, polymeric and lipid nanoparticles remain at the forefront for their versatility in encapsulating a variety of therapeutic payloads. This thesis investigates their potential for facilitating the transport of nucleic acid components into cells, with a focus on targeted delivery to the liver and brain. To achieve this, we address key considerations including the composition of the delivery vector, the nature of the therapeutic cargo, and the chosen delivery route. The challenge of targeted delivery to specific organs or cell types, i.e. hepatocytes or neurons, is addressed through rational design and development of libraries of nanoparticulate systems tailored for nucleic acid therapeutics. Although liver gene editing using non-viral systems has been extensively studied, oral delivery for liver targeting remains challenging due to the mucosal barrier. To that end, we explore intraduodenal delivery as a strategy to bypass the mucosal barrier and target the liver. Furthermore, insights from collaborative research with the Mao lab at Johns Hopkins University reveal that tuning the composition of lipid nanoparticles (LNPs) can influence their preferential targeting of specific cell types. Leveraging this, we employed an in vitro library screening and machine learning approach to identify populations of LNPs capable of preferentially transfecting hepatocytes. The efficacy of these LNPs in liver gene editing is then evaluated through “cluster-mode” screening in vivo, and therapeutic efficacy was demonstrated using a proof-of-concept in vivo model for PCSK9 and ANGPTL3 knockdown, resulting in 27% serum cholesterol knockdown. In addition to liver-targeted gene delivery, this thesis also investigates the potential of polymeric and lipid nanoparticles for delivering nucleic acid therapeutics to the brain. However, overcoming the blood-brain barrier (BBB) is crucial for systemic delivery to the brain. To circumvent the BBB, we explored two methods: intracranial injection and theranostic ultrasound (THUS)-mediated temporary opening of the BBB. While intracranial injection achieves localized gene editing, THUS offers a non-invasive approach for transient and widespread BBB opening. Utilizing the previously validated in vitro screening and machine learning approaches for chitosan-grafted bPEI (CS-PEI) and lipid nanoparticle (LNP) carriers with tunable compositions, we assessed their efficacy in systemic gene delivery to the brain, and specifically their capability in preferentially transfecting neuronal cells over hepatocytes. Subsequently, we validated their efficiency via intracranial administration using the Ai14 reporter mouse model and observed up to 20% gene editing of the targeted cross-sectional area of the brain hemisphere using the top-performing cluster. Through comprehensive investigations into both brain and liver gene delivery, this thesis aims to contribute to the advancement of non-viral nanoparticle-based gene therapy strategies for treating a range of cholestatic liver diseases and hereditary neurodegenerative diseases.