The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.

Thesis advisor: Katie Rapier / Through the years, people began to breed their dogs and cats with the intention for the pets to be useful in a certain skill. However, that has shifted to focus on their looks rather than their skills thanks to kennel clubs imposing standards on each breed. This has led irresponsible breeding practices to occur which in turn caused breeds to evolve negatively as breeds began to suffer from preventable genetic disorders and negative physical changes. Genetically manipulating soon shifted from pets to humans with PGD/IVF and CRISPR-Cas9. At first, there was a focus to use these methods to help cure and prevent genetic disorders. That has since shifted to people wanting to create the perfect child. In this thesis I will argue that designer breeds help lead the way to designer babies, and that any genetic manipulation to embryos should only be done if a medical reason is present. / Thesis (BA) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Philosophy.

Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...

The question of delivery has become a critical part of therapeutic research and development. While nanoparticle formulations are now used in a variety of FDA-approved therapies, these focus on simple oral formulations or systemic lipid nanoparticle administration; therefore, significant research is ongoing in the development of specialized delivery systems for more complex cargos or specific targeting[1]. For therapeutics that have high potential for toxicity, like chemotherapeutics, delivery systems need to transport disproportionately into target organs and further target cells. This is more so true for therapeutics that require delivery to a specific cellular compartment to have significant efficacy, like in the case of gene editing in the nucleus or mitochondria-targeted peptides. To contribute to the development of efficient carriers for gene and drug therapy, this work aims to explore the potential of liposome-coated mesoporous silica nanoparticles and engineering methods that provide functionality to defeat barriers to efficient therapeutic development. Recent advances in CRISPR/Cas9 technology present an attractive toolset to study genes for the development of novel therapeutics. Since traditional delivery methods for gene therapies relied heavily on viruses— requiring biosafety level clearance and eliciting immunogenicity concerns, as well as limitations in multiplex gene editing capabilities in single viral vehicles— recently, nanoparticles have become an attractive nonviral alternative for gene therapy research and development. While lipid-based nanoparticles have been at the forefront of siRNA and mRNA therapies, we looked at increasing the loading capabilitiesof a liposome by using a mesoporous silica nanoparticle (MSN) core. The MSN provides efficient electrostatic loading of the relatively large and non-uniformly charged CRISPR/Cas9 protein and guide RNA ribonucleoprotein complex (RNP) as well as more charge-dense plasmids, while a liposome coating offers the PEGylation and targeting capabilities necessary for selective uptake and in vivo application. After demonstrating gene-editing efficiencies above 20% for both plasmid and RNP modalities of CRISPR/Cas9, we tested its application in multiplex gene editing for the study lipid metabolism pathways. To demonstrate the maintained efficiency of this system, this liposome-coated MSN (lipoMSN) platform was used to deliver a combination of RNPs targeting three genes involved in lipid metabolism in the liver. These genes, Pcsk9, Apoc3, and Angptl3, are derived from research demonstrating that populations with loss-of-function mutations in any one of these genes garner improved cardiovascular health, characterized by lowered blood cholesterol and triglycerides. The lipoMSN system demonstrated that it maintained significant gene editing, above 25% efficiency, at a specific gene target despite reduced dosage of target-specific RNP due to the combination of other target RNP. By leveraging this system to deliver various combinations of targeting RNPs in the same nanoparticle and therefore ensuring a higher probability that any given cell is edited at all targets, synergistic effects on lipid metabolism can be observed in vitro and in vivo. These effects, such as an approximately 50% decrease in serum LDL-cholesterol 4-weeks after treatment with pcsk9 and angptl3- targeted RNPs, have not been observed in previous studies. Continued work with this lipoMSN platform is ongoing, with projects leveraging the system for multiplex gene disruption as well as endosomal delivery of peptide and chemical drugs. We are currently leveraging the comparatively low spread of lipoMSN to provide gene disruption of adra1a, adra1b, and adra1d in the discrete area of the thalamus. Further, this system is also well suited for other CRISPR/Cas9 elements, such as deactivated Cas9 (dCas9) fused to epigenetic modifiers, enhancers and repressors. To demonstrate another facet of the lipoMSN delivery platform, we adjusted the formulation for the specific delivery to endosomal compartments of nociceptive neurons. Previous work by our collaborators at the Bunnett Lab provided evidence that signaling in the endosomal compartment is partially responsible for both pain propagation and amelioration. In order to provide highly targeted treatment to reduce the off-target effects linked with opioid-based pain treatment, we wanted to leverage the resonance of these nanoparticles in the endosomes following endocytosis and enhance the drug delivery to the endosomal compartments propagating nociceptive signaling. We did this by first integrating a targeting ligand in the form of a DADLE-peptide, to the outside of the liposomes, providing a 20-40% increase in uptake for delta-opioid receptor (DOR)-expressing cell types. Further, we used an oxidation- and pH-sensitive MSN core to provide increased drug release to the endosomal environment, which is naturally oxidizing and at a lower pH of approximately 5.2. This resulted in an increased therapeutic effect when compared to naked peptide drug in a mouse model of neurogenic pain. We are also looking at leveraging this system for other endosomal signaling pathways and applying our lipoMSN platform to cargos such as glucagon-like peptide-1 (GLP-1) receptor agonists for diabetes and US28 receptor antagonists for reduction of proliferative signaling in cancers. Collectively, these projects provide insight on how to design delivery vehicles for specific gene and drug delivery. This lipoMSN system has the potential to be a versatile platform for the development of combinatorial gene therapeutics in liver-related disease. Further, this platform may inform research and development in the next generation of endosomally-targeted therapeutics for increased efficacy and reduced off-target or side effects.

Tesis por compendio / [ES] El síndrome de Usher (USH) es un trastorno raro autosómico recesivo definido principalmente por sordera neurosensorial (SNHL), y una distrofia retiniana conocida como retinosis pigmentaria (RP). La patología muestra heterogeneidad genética, puesto que se conocen al menos 10 genes responsables. No obstante, las mutaciones en USH2A son la causa más frecuente de la enfermedad, en gran medida por la recurrencia de la variante patogénica c.2299delG. En esta tesis se ha desarrollado un ensayo de edición génica para revertir dicha anomalía genética por medio del sistema CRISPR/Cas9. Se diseñaron y probaron varios complejos CRISPR específicos de locus, y el más eficiente fue usado para la corrección de la mutación c.2299delG en células derivadas de pacientes. La tasa de corrección de la mutación obtenida fue del 2.5%. Otro objetivo de esta tesis ha sido la caracterización genética de pacientes USH aún sin diagnóstico molecular. Una primera fase implicó la secuenciación masiva dirigida de las regiones codificantes de todos los genes asociados a la enfermedad. Este estudio, cuya cohorte incluyó 58 pacientes no escrutados previamente, permitió la identificación de 42 nuevas mutaciones presuntamente patológicas, y una tasa general de detección de alelos responsables de la enfermedad de prácticamente el 83%. Sorprendentemente, uno de los sujetos presentaba mutaciones en CEP250, uno de los últimos genes correlacionados con la enfermedad. Una exhaustiva revisión clínica reveló que la degeneración retiniana se trataba en realidad de una distrofia de conos y bastones en lugar de RP clásica, lo cual permitió consolidación del gen CEP250 como responsable de un fenotipo similar al USH. El resto de casos sin resolver induce a sospechar de la existencia de otros genes vinculados con USH. Así pues, se analizó el exoma íntegro de dichos casos negativos del panel por medio de secuenciación de exoma completo, lo cual proporcionó resultados relevantes en seis de las muestras estudiadas. Uno de tales sujetos resultó ser un claro caso de fenocopia de USH, al albergar mutaciones patogénicas en dos genes independientes, TECTA y REEP6, siendo el primero responsable de la SNHL y el segundo de la RP. De forma parecida, en otro paciente se detectaron variantes patológicas para RP en el gen EYS, pero no se identificó paralelamente ningún cambio genético que explicara la SNHL. Tres individuos adicionales resultaron haber sido erróneamente diagnosticados como USH, dada la conclusiva inexistencia o ambigüedad de la sordera. Uno de ellos fue definido como homocigoto de una mutación en CNGB1, ya reconocido como responsable de RP. En el segundo de dichos sujetos se identificó una mutación en homocigosis en el gen GRN, cuyos defectos en estado heterocigoto están asociados a demencia frontotemporal y más raramente combinada con RP si ambos alelos se encuentran alterados. Por otro lado, el tercer paciente fue resuelto como heterocigoto compuesto de variantes en WDR19, un gen asociado en mayor medida a una distrofia retiniana acompañada de trastornos renales y, más raramente, a la forma aislada del síntoma. En el último de los seis casos resaltados de este objetivo se detectó una mutación homocigota sin sentido en el gen ASIC5, cuyo papel en el organismo todavía se desconoce. Sin embargo, se han correlacionado funciones visuales y auditivas para miembros de la misma familia proteica. En conjunto, los hallazgos obtenidos en este trabajo avalan la importancia del uso de las más novedosas tecnologías en la búsqueda de soluciones para enfermedades raras, las cuales presentan por ahora un pronóstico terapéutico bastante desamparado. Asimismo, otras consecuencias positivas en cuanto a la caracterización genética de los pacientes son la corroboración (o rectificación) del diagnóstico inicial, así como la contribución a la estimación demográfica y correlaciones de genotipo-fenotipo, que en definit / [CA] La síndrome d'Usher (USH) és una malaltia rara autosòmic recessiu definit principalment per sordera neurosensorial (SNHL) i una distròfia retiniana coneguda com a retinosi pigmentària (RP). La patologia mostra heterogeneïtat genètica, ja que es coneixen almenys 10 gens responsables. No obstant això, les mutacions en USH2A són la causa més freqüent de la malaltia, a causa de la recurrència de la variant patogènica c.2299delG. En aquesta tesi s'ha desenvolupat un assaig d'edició gènica per a revertir la dita anomalia genètica per mitjà del sistema CRISPR/Cas9. Es van dissenyar i van probar diversos complexos CRISPR específics de locus, i el més eficient va ser usat per a la correcció de la mutació en cèl·lules derivades de pacients. La taxa de correcció de la mutació obtinguda va ser del 2.5%. Un altre objectiu d'aquesta tesi ha sigut la caracterització genètica de pacients USH encara sense diagnòstic molecular. Una primera fase va implicar la seqüenciació massiva dirigida de les regions codificants de tots els gens associats a la malaltia. Aquest estudi, la cohort de la qual va incloure 58 pacients no escrutats prèviament, va permetre la identificació de 42 noves mutacions presumptament patològiques, i una taxa general de detecció d'al·lels responsables de la malaltia de pràcticament el 83%. Sorprenentment, un dels subjectes presentava mutacions en CEP250, un dels últims gens correlacionats amb la malaltia. Una exhaustiva revisió clínica va revelar que la degeneració retiniana es tractava en realitat d'una distròfia de cons i bastons en lloc de RP clàssica. Aquestes troballes han permés la consolidació del gen CEP250 com a responsable d'un fenotip similar al USH. La resta de casos sense resoldre induïx a sospitar de l'existència d'altres gens vinculats amb USH. Així, doncs, es va analitzar l'exoma íntegre dels casos negatius del panell a través de seqüenciació d'exoma complet, cosa que va proporcionar resultats rellevants en sis de les mostres estudiades. Un de tals subjectes va resultar ser un clar cas de fenocopia d'USH, a l'albergar mutacions patogèniques en dos gens independents, TECTA i REEP6, sent el primer responsable de la SNHL i el segon de la RP. De forma semblant, en un altre pacient es van detectar variants patològiques per a RP al gen EYS, però no es va identificar paral·lelament cap canvi genètic que explicara la SNHL. Tres individus addicionals van resultar haver sigut erròniament diagnosticats com USH, donada la final inexistència o ambigüitat de la sordera. Un d'ells va ser definit com a homozigot d'una mutació en CNGB1, ja reconegut com a responsable de RP. En el segon d'aquestes subjectes es va identificar una mutació en homozigosi en el gen GRN, els defectes del qual estan associats a demència frontotemporal en estat heterozigot, i més rarament en combinació amb RP si ambdós al·lels es troben alterats. D'altra banda, el tercer pacient va ser resolt com a heterozigot compost de variants en WDR19, un gen associat en major grau a una distròfia retiniana acompanyada de trastorns renals i, més rarament, a la forma aïllada del símptoma. En l'últim dels sis casos ressaltats d'aquest objectiu es va detectar una mutació homozigota sense sentit en el gen ASIC5, el paper en l'organisme del qual encara es desconeix. Amb tot, s'han correlacionat funcions visuals i auditives per a membres de la mateixa família proteica. En conjunt, les troballes obtingudes en aquest treball avalen la importància de l'ús de les més noves tecnologies en la recerca de solucions per a malalties rares, les quals presenten per ara un pronòstic terapèutic prou desemparat. Així mateix, altres conseqüències positives quant a la caracterització genètica dels pacients són la corroboració (o rectificació) del diagnòstic inicial, així com la contribució a l'estimació demogràfica i correlacions de genotip-fenotip, que en definitiva ajuden en la compressió d'US / [EN] Usher syndrome (USH) is a rare autosomal recessive disorder defined essentially by sensorineural hearing loss (SNHL) and a retinal dystrophy known as retinitis pigmentosa (RP). The condition shows a genetic heterogeneity, since there are at least 10 genes known to be causative of the syndrome. However, mutations in USH2A are the most frequent cause of the disease, due in a large measure to the recurrence of the c.2299delG pathogenic variant. A gene editing assay to reverse this specific genetic anomaly was developed in this thesis by means of the groundbreaking CRISPR/Cas9 system. Several locus-specific CRISPR complexes were designed and tested, and the most efficient was used to proceed with the c.2299delG mutation correction on patient-derived cells. The trial resulted in a mutation correction rate of 2.5%. Another goal of this thesis was the genetic characterization of molecularly undiagnosed USH patients. Given the genetic diversity of the disease, the procedure required the implementation of high-throughput sequencing, a technology that enables in bulk sequencing of any number of selected loci (or the indiscriminate totality) of the genome. The first phase implied the targeted sequencing of the coding-relevant regions of all known causative or disease-associated genes at the moment. The study, comprising a cohort of 58 previously unscreened patients, enabled the identification of 42 novel putative pathogenic mutations, and an etiologic-allele detection ratio shy of 83%. Remarkably, one of the subjects harbored nonsense mutations in CEP250, which is one of the latest USH-associated genes. However, an exhaustive review of the clinical features unmasked the retinal degeneration as a cone-rod dystrophy rather than RP, which reinforced the linkage of the gene to an USH-like phenotype. The remaining portion of unresolved cases lead to suspicion of the existence of other genes accountable for USH. Hence, the complete exome of such panel-negative cases was screened through whole exome sequencing. This venture provided relevant findings in six of the surveyed samples. One subject was plainly exposed as an USH phenocopy by harboring pathogenic splice-site mutations in two independent genes, TECTA and REEP6, the former responsible for the SNHL and the latter for the RP. Similarly, RP-causative variants in EYS were detected in another patient, yet no pathogenic changes explaining the HL were discovered. Three additional individuals were ultimately unveiled as USH misdiagnosed cases, being the HL actually absent or ambiguous. One of the patients in this set was homozygous for a mutation in CNGB1, already known to be accountable for RP. The other two cases showed a more peculiar outcome being compound heterozygous for putatively pathogenic variants in genes generally associated to other disorders. One presented a homozygous mutation in GRN, a gene associated to frontotemporal dementia under heterozygous condition and less commonly to combined RP for homozygous alterations. The third subject was found to be a carrier of mutations in WDR19, a gene best associated with retinal disorders accompanied by renal signs and rarely with the isolated visual symptom. The last case presented a homozygous nonsense variant in the ASIC5 gene, whose role has yet to be learned. However, some correlations to visual and hearing functions have been reported for members of the same protein family. Altogether, the results obtained from this work attest to the importance of applying the most up-to-date technologies in the search of solutions for rare diseases that realistically pose a despairing therapeutic prognosis. In addition, the positive consequences of the genetic characterization of the patients are the corroboration (or else correction) of the initial diagnosis, and the contribution to the appraisal of demographic and genotype-phenotype correlations, which ultimately aid in the understanding USH and other related diseases. / This work was financially supported by the Institute of Health Carlos III and FEDER funds (ISCIII; grants PI13/00638, PI16/00425, PI16/00539, and PIE13/00046), Fundación ONCE (grant 2015/0398), XVIII Fundaluce-FARPE, and “Telemaratón: Todos Somos Raros, Todos Somos Únicos” (grant IP58). C.F.-G. is a recipient of a fellowship (grant IFI14/00021) from the ISCIII. R.P.V.-M. is a Miguel Servet researcher (grant CP11/00090 funded by ISCIII, Madrid, Spain). The funds from the ISCIII are partially supported by the European Regional Development Fund. R.-P.V.M. is also a Marie Curie fellow (grant CIG322034 from the European Commission). / Fuster García, C. (2020). Therapeutic approaches and development of genomic diagnostic tools for Usher syndrome [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137034 / TESIS / Compendio

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, featuring motor signs such as tremors, bradykinesia, and impaired gait that are often preceded by nonmotor symptoms such as anxiety/depression and olfactory dysfunction. Interestingly, significant olfactory loss was found to be manifested in the majority of PD patients and may precede motor symptoms by years, and thus can be used for the risk assessment of developing PD in asymptomatic individuals. The main pathological feature of PD is the progressive and irreversible loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain. Although the detailed etiology of PD remains unclear, most PD cases were found to be sporadic and can be associated with environmental factors. Only 5–10% of patients result from familial PD. With considerable effort in the past two decades, a number of genes associated with familial PD have been identified and interestingly, many of these genes are involved in regulating and maintaining mitochondrial function. The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we show novel information concerning the role of PARL in zebrafish by establishing a loss-of-function mutation in parla and parlb via CRISPR/Cas9- mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function, as well as loss of both parla and parlb function result in loss of DA neurons in the olfactory bulb and telencephalon of adult zebrafish brain. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants and in fish with loss of parla and parlb function. Furthermore, we showed that loss of parla and/or parlb function translates into altered locomotion parameters and that loss of parla but not parlb function results in impaired olfaction. Finally, increased susceptibility to neurotoxin exposure was identified in mutants with loss of both parla and parlb function but not with loss of parla or parlb function. These results suggest an evident role for parla in the development and/or maintenance of DA neuron function in zebrafish and confirm the existence of redundant and non-redundant functions for the two paralogs, parla and parlb.

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.

The development of a drug able to distinguish between tumor and host cells has been long sought, but the solid tumor microenvironment (TME) confounds many current therapeutics. Solid tumors present several challenges for oncotherapeutics, primarily, (1) aberrant vascularization, resulting in hypoxia, necrosis, abnormally high pH, and (2) tumor immune suppression. Oncolytic microbes are drawn to this microenvironment by an innate ability to selectively penetrate, colonize, and eradicate solid tumors as well as reactivate tumor associated immune components. To consider oncolytic bacteria deployment into this microenvironment, Chapter 1 dives into the background of oncolytic microbes. A discussion of the oncolytic bacterial field state, identifying Clostridium novyi? as a promising species, and details genetic engineering techniques to develop customized bacteria. Despite the promise of C.novyi in preclinical/clinical trials when administered intratumorally, the genetic and biochemical uniqueness of C.novyi necessitated the development of new methodologies to facilitate more widespread acceptance. Chapter 2 reports the development of methods that facilitate experimental work and therapeutic translation of C.novyi, including the ability to work with this obligate micro-anaerobe aerobically on the benchtop. While methods development is a necessary step in the clinical translation of C.novyi so too is choosing the correct model of the TME within which to test a potential anti-cancer therapy. While the typical solid TME includes both phenotypic and genotypic heterogeneity, the methods used to model this disease state often do not reflect this complexity. This simplistic approach may have contributed to stagnant five-year survival rates over the past four decades. Nevertheless, simplistic models are a necessary first step in clinical translation. Chapter 3 explores the impact of cancer cell lines co-cultured with C. novyi to establish the efficacy of this oncolytic bacteria in a monolayer culture. Chapter 4 extends this analysis adding not only a level of complexity by using an in vivo model, but also using CRISPR/Cas9 to modify the genome of C.novyi to encode a tumor targeting peptide, RGD, for expression within the spore coat. The combination of these studies indicates that C. novyi is uniquely poised to accomplish the long sought after selective tumor localization via intravenous delivery.

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with a poor prognosis. Currently, prognosis for HCC patients remains poor as few therapies are available. The clinical need for more effective HCC treatments remains unmet partially because HCC is genetically heterogeneous and HCC driver genes amenable to targeted therapy are largely unknown. Mutations in the TP53 gene are found in ~30% of HCC patients and confer poor prognosis to patients. Identifying genes whose depletion can inhibit HCC growth, and determining the mechanisms involved, will aid the development of targeted therapies for HCC patients. Therefore, the first half of this thesis focuses on identifying genes that are required for cell growth in HCC independent of p53 status. We performed a kinome-wide CRISPR screen to identify genes required for cell growth in three HCC cell lines: HepG2 (p53 wild-type), Huh7 (p53-mutant) and Hep3B (p53-null) cells. The kinome screen identified 31 genes that were required for cell growth in 3 HCC cell lines independent of TP53 status. Among the 31 genes, 8 genes were highly expressed in HCC compared to normal tissue and increased expression was associated with poor survival in HCC patients. We focused on TRRAP, a co-factor for histone acetyltransferases. TRRAP function has not been previously characterized in HCC. CRISPR/Cas9 mediated depletion of TRRAP reduced cell growth and colony formation in all three cell lines. Moreover, depletion of TRRAP reduced its histone acetyltransferase co-factors KAT2A and KAT5 at the protein level with no change at the mRNA level. I found that depletion of KAT5, but not KAT2A, reduced cell growth. Notably, inhibition of proteasome- and lysosome-mediated degradation failed to rescue protein levels of KAT2A and KAT5 in the absence of TRRAP. Moreover, tumor initiation in an HCC mouse model failed after CRISPR/Cas9 depletion of TRRAP due to clearance via macrophages and HCC cells depleted of TRRAP and KAT5 failed to grow as subcutaneous xenografts in vivo. RNA-seq and bioinformatic analysis of HCC patient samples revealed that TRRAP positively regulates expression of genes that are involved in mitotic progression. In HCC, this subset of genes is clinically relevant as they are overexpressed compared to normal tissue and high expression confers poor survival to patients. I identified TOP2A as one of the mitotic gene targets of the TRRAP/KAT5 complex whose inhibition greatly reduces proliferation of HCC cells. Given that this was the first time the TRRAP/KAT5 complex has been identified as a therapeutic target in HCC, the second half of this thesis focuses on identifying the mechanism via which depletion of this complex inhibits proliferation of HCC cells. I discovered that depletion of TRRAP, KAT5 and TOP2A reduced proliferation of HCC cells by inducing senescence. Typically, senescence is an irreversible state of cell cycle arrest at G1 that is due to activation of p53/p21 expression, phosphorylation of RB, and DNA damage. Surprisingly, induction of senescence after loss of TRRAP, KAT5 and TOP2A arrested cells during G2/M and senescence was independent of p53, p21, RB and DNA damage. In summary, this thesis identifies TRRAP as a potential oncogene in HCC. I identified a network of genes regulated by TRRAP and its-cofactor KAT5 that promote mitotic progression. Moreover, I demonstrated that disruption of TRRAP/KAT5 and its downstream target gene TOP2A result in senescence of HCC cells independent of p53 status. Taken together, this work suggests that targeting the TRRAP/KAT5 complex and its network of target genes is a potential therapeutic strategy for HCC patients.

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.