T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

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.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-01-08T14:33:50Z No. of bitstreams: 1 gustavotorresdesouza.pdf: 5085443 bytes, checksum: eada917698a8738ea1947743e940692c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-01-23T11:05:29Z (GMT) No. of bitstreams: 1 gustavotorresdesouza.pdf: 5085443 bytes, checksum: eada917698a8738ea1947743e940692c (MD5) / Made available in DSpace on 2018-01-23T11:05:29Z (GMT). No. of bitstreams: 1 gustavotorresdesouza.pdf: 5085443 bytes, checksum: eada917698a8738ea1947743e940692c (MD5) Previous issue date: 2017-08-08 / 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.

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

[ES] Las leguminosas son un grupo de plantas consideradas de gran importancia por su valor nutricional para la alimentación humana y ganadera. Además, las familias de leguminosas se caracterizan por rasgos distintivos de desarrollo como su inflorescencia compuesta y su compleja ontogenia floral. Para comprender mejor estas características distintivas, es importante estudiar los genes reguladores clave involucrados en el desarrollo de la inflorescencia y la flor. El gen SUPERMAN (SUP) es un factor transcripcional de dedos de zinc (Cys2-Hys2) considerado como un represor activo que controla el número de estambres y carpelos en A. thaliana. Además, SUP está involucrado en la terminación del meristemo floral y el desarrollo de los tejidos derivados del carpelo. El objetivo principal de este trabajo fue la caracterización funcional del ortólogo de SUP en la leguminosa modelo Medicago truncatula (MtSUP). Logramos este objetivo en base a un enfoque de genética reversa, análisis de expresión génica y ensayos de complementación y sobreexpresión. Nuestros resultados muestran que MtSUP es el gen ortólogo de SUP en M. truncatula. MtSUP comparte algunos de los roles ya descritos para SUP con algunas variaciones. Curiosamente, MtSUP controla la determinación del meristemo inflorescente secundario (I2) y de los primordios comunes (CP) a pétalos y estambres. Por tanto, MtSUP controla el número de flores y de pétalos-estambres que producen el meristemo I2 y los primordios comunes, respectivamente. MtSUP muestra funciones novedosas para un gen de tipo SUP, desempeñando papeles clave en los meristemos que confieren complejidad de desarrollo a esta familia de angiospermas. Este trabajo permitió identificar a MtSUP, un gen clave que forma parte de la red reguladora genética que subyace al desarrollo de la inflorescencia compuesta y de las flores en la leguminosa modelo M. truncatula. / [CA] Les lleguminoses són un gran grup de plantes considerades de gran importància pel seu valor nutricional per a l'alimentació humana i ramadera. A més, les famílies de lleguminoses es caracteritzen per trets distintius de desenrotllament com la seua inflorescència composta i la seua complexa ontogènia floral. Per a comprendre millor estes característiques distintives, és important estudiar els gens reguladors clau involucrats en la inflorescència i el desenrotllament floral. El gen SUPERMAN (SUP) és un factor transcripcional de dits de zinc (Cys2-Hys2) considerat com un repressor actiu que controla el nombre d'estams i carpels en A. thaliana. A més, SUP està involucrat en la terminació del meristemo floral i el desenrotllament dels teixits derivats del carpel. "L'objectiu principal d'este treball va ser la caracterització funcional de l'ortòleg de SUP en la lleguminosa model Medicago truncatula (MtSUP) . Aconseguim l'objectiu amb base en un enfocament genètic invers, anàlisi d'expressió gènica i assajos de complementació i sobreexpressió. Els nostres resultats mostren que MtSUP és el gen ortòleg de SUP en M. truncatula. MtSUP compartix alguns dels rols ja descrits per a SUP amb variacions. Curiosament, MtSUP està involucrat en la determinació del meristemo de la inflorescència secundària (I2) i els primordios comuns (CP). Per tant, MtSUP controla el nombre de flors i pètals-estams que produïxen el meristemo I2 i els primordios comuns, respectivament. MtSUP mostra funcions noves per a un gen tipus SUP, exercint papers clau en els meristemos que conferixen complexitat de desenrotllament a esta família d'angiospermes. "Este treball va permetre identificar a MtSUP, un gen clau que forma part de la xarxa reguladora genètica darrere de la inflorescència composta i el desenrotllament de flors en la lleguminosa model M. truncatula. / [EN] Legumes are a large group of plants considered of great importance for their nutritional value in human and livestock nutrition. Besides, legume families are characterized by distinctive developmental traits as their compound inflorescence and complex floral ontogeny. For a better understanding of these distinctive features is important to study key regulatory genes involved in the inflorescence and floral development. The SUPERMAN (SUP) gene is a zinc-finger (Cys2-Hys2) transcriptional factor considered to be an active repressor that controls the number of stamens and carpels in A. thaliana. Moreover, SUP is involved in the floral meristem termination and the development of the carpel marginal derived tissues. The main objective of this work was the functional characterization of the SUP orthologue in the model legume Medicago truncatula (MtSUP). We achieved this objective based on a reverse genetic approach, gene expression analysis, and complementation and overexpression assays. Our results show that MtSUP is the orthologous gene of SUP in M. truncatula. MtSUP shares some of the roles already described for SUP with variations. Interestingly, MtSUP controls the determinacy of the secondary inflorescence (I2) meristem and the common primordia (CP). Thus, MtSUP controls the number of flowers and petal-stamens produced by the I2 meristem and the common primordia respectively. MtSUP displays novel functions for a SUP-like gene, playing key roles in the meristems that confer developmental complexity to this angiosperm family. This work allowed to identify MtSUP, a key gene that participates in the genetic regulatory network underlying compound inflorescence and flower development in the model legume M. truncatula. / I would like to thanks the Spanish Ministry of Economy and Competitiveness for the grant (MINECO; BIO2016-75485-R) that supported this work. Special thanks to the Generalitat Valenciana for funding my doctorate with the Santiago Grisolía predoctoral scholarships / Rodas Méndez, AL. (2021). MtSUPERMAN controls the number of flowers per inflorescence and floral organs in the inner three whorls of Medicago truncatula [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171474 / TESIS

[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31. / [CAT] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31. / [EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch. / Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055 / TESIS

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

In this thesis, I argue that the treatment/enhancement distinction that is central to the ethical debate concerning germline genome editing and CRISPR is too vague to be ethically and normatively guiding. The problem of vagueness is twofold, being both a semantic and epistemic issue. This vagueness creates borderline cases, cases that cannot be properly defined as either treatment or enhancement, I call this The Borderline Cases Argument. These borderline cases enable a slippery slope towards eugenic practices, radical enhancement and dangerous applications of CRISPR. The distinction therefore fails to be action guiding as it cannot distinguish treatment from enhancement as well as failing to correspond to what is genuinely morally problematic with germline genome editing and not, I call this The Argument of Missing the Point. In using the treatment/enhancement distinction we therefore risk losing control over how CRISPR is used and for what purposes.

One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.