Academic literature on the topic 'Base editors'

APOBEC-1 is a cytosine-to-uracil deaminase that exerts its primary physiological role in the editing of the Apolipoprotein B mRNA at C6666. Recent studies outlined the ability of APOBEC1 to edit also DNA, thus linking its mutational property to the its overexpression in cancer. Several reports suggest that APOBEC1 can alter the cellular state by targeting RNA molecules. Our hypothesis portrays APOBEC1 as the trigger for a dual path towards cancer through its DNA- and RNA- targeting abilities, and its activity as central to the onset of tumorigenic features. Aim of my PhD project is to understand whether APOBEC1 oncogenic potential is mainly related to its ability to edit RNA or DNA. To this aim, I assessed the tumorigenic potential of a set of APOBEC1 mutants, selected for a dissociation in their RNA and DNA editing ability: they are DNA editing proficient but unable to edit RNA. I characterized these mutants and I tested them in mice to assess their ability to trigger liver tumors. Even if unable to edit RNA, the APOBEC1 mutants are still able to induce tumor formation. This means that RNA editing does not play a central role in the oncogenic potential of APOBEC1. Moreover, considering the recent developments in genome editing, I have exploited these mutants as base editors -fusions of the DNA targeting Cas9 with a DNA deaminase- that allow the correction of single bases: the most common mutator moiety in cytosine-targeting base editors is based on APOBEC1, but overexpression of the APOBEC1-Cas9 chimera results in a substantial amount of RNA and DNA off-target alterations. Once fused with Cas9, our RNA-editing deficient mutants were able to mutate the target DNA. On the other hand, I demonstrated that their off-target activity on RNA is orders of magnitude lower than that of wild type APOBEC1, at the same levels of the negative controls. Exploitation of these mutants could thus provide tools for safer base editing both in vitro and in vivo.

Identification of small molecules against APOBEC3B The APOBECs are deaminases that act on DNA and RNA to restrict exogenous nucleic acids. Yet, the signature of their mutagenic activity –especially that of APOBEC3A and APOBEC3B- has been observed in the cancer genomes and their ability to increase the genetic heterogeneity of tumours has been linked to the onset of drug resistance in cancer. As such inhibition of their enzymatic activity represents a potential target for anticancer therapies. During my PhD I worked at the identification of APOBEC3B small-molecule inhibitors. To this aim, I used a computational approach to perform a virtual screening on large library of molecules to block APOBEC3B enzymatic activity. I then tested selected molecules from the virtual screening using biochemical assays to quantify their effect on APOBEC3B activity and their capacity to interfere with APOBEC3B binding to DNA. Through this, I was able to identify two small molecules that reduce the activity of this protein, which could provide basis for the development of the first drug for anti-APOBEC activity. Engineering ADAR2 to act on DNA Genome editing technologies have revolutionized our ability to target and modify the genomes of living cells and organisms. The fusion of AID/APOBECs to genome editing tools such as Cas9 allowed the development the first base editor, molecules that can be targeted to mutate specific cytosines. The pool of available Base Editors is in constant expansion as new molecules are developed to target DNA with more specificity and efficiency. As the only adenine-targeting Base Editor is based on TadA- an RNA deaminase-, I focused on the development of a A•T base editor based on the catalytic domain of ADAR2. Adenosine Deaminases Acting on RNA (ADARs), are editing enzymes that catalyse the C6 deamination of adenosine (A) to produce inosine (I) in double-stranded RNA. As human ADAR2 is able to target DNA/RNA hybrids, I first tried -without success- to use chimeras of n/dCas9 and the deaminase domain of ADAR2 to induce mutations in a fluorescent reporter. I then used a bacterial screen to select for mutants of ADAR2 that act on DNA. I selected a mutant that induces a mutator phenotype in bacteria and DNA damage in mammalian cells. I am currently working to engineer this mutant into a Base Editor suitable for biotechnological applications such as gene therapy, antiviral treatment and cancer therapy.

Drépanocytose et bêta-thalassémie sont dues à des mutations affectant la production de la chaîne de globine β. La sévérité clinique est atténuée par des mutations augmentant la quantité d'hémoglobine fœtale (HbF), une condition appelée persistance héréditaire d'HbF à l'âge adulte. La transplantation autologue de cellules souches/progénitrices hématopoïétiques (CSPH) génétiquement corrigées est prometteuse. Une approche CRISPR/Cas9 pour réprimer BCL11A, un répresseur de la globine γ, a été approuvée mais comporte des risques de génotoxicité dues aux cassures double brin (CDB). Nous avons ciblé les sites de liaison (SL) des activateurs transcriptionnels GATA1 et ATF4, présents dans les régions +58-kb et +55-kb, respectivement, des enhancers érythroïdes de BCL11A, avec des éditeurs de base (BEs) pour introduire des mutations ponctuelles précises sans créer de CDBs. Des ARN guides (ARNg) ont été testés dans des cellules souches hématopoïétiques (CSH) de patients drépanocytaires en combinaison avec des BEs, générant différents nucléotides dans les motifs de liaison avec une efficacité d'édition atteignant 90 %, avec peu ou pas d'indels induits par les CDB. La réactivation d'HbF a été observée dans tous les échantillons édités, mesurée par HPLC, mais pas suffisante pour un sauvetage complet du phénotype dans les cellules érythroïdes issues des CSPHs drépanocytaires éditées. Nous avons donc ciblé simultanément les SL de GATA1 et ATF4 pour augmenter les niveaux d'HbF. Les cellules éditées au niveau des enhancers +58 et +55 ont montré une augmentation de l'expression d'HbF par rapport aux cellules recevant des ARNg individuels, dépassant les niveaux atteints avec la stratégie CRISPR/Cas9. Enfin, la réactivation de l'HbF était suffisante pour permettre un sauvetage substantiel du phénotype malade, diminuant le nombre de cellules drépanocytaires à 16,4%. Pour évaluer les effets hors cibles, nous avons utilisé le GUIDE-seq couplé au séquençage ciblé, le séquençage de l'exome entier et le RNA-seq, tandis que les effets indésirables présent dans les sites cibles ont été évalués par séquençage Long read. L'efficacité du BE pour repeupler les CSH a été démontrée en transplantant des CSH éditées dans des souris immunodéficientes, prouvant l'efficacité de l'édition simultanée des éléments transrégulateurs de la globine γ pour la réactivation de l'HbF. Cette preuve de concept permettra le développement préclinique et clinique de CSH modifiées pour le traitement des β-hémoglobinopathies. Cependant, des travaux récents montrent que les BEs peuvent également générer de larges délétions ou indels (Antoniou et al. 2022). Ainsi, une nouvelle stratégie basée sur l'édition de l'épigénome a été développée pour moduler l'expression des gènes sans modifier la séquence d'ADN sous-jacente. Nous avons analysé les marques épigénétiques dans deux régions cis-régulatrices clés, les promoteurs de HBG et les enhancers de BCL11A, dans des cellules érythroïdes dérivées de CSPH. Des marques épigénétiques répressives, telles que la méthylation de l'ADN, ont été détectées au niveau des promoteurs de HBG dans des cellules érythroïdes adultes n'exprimant pas la globine γ. En revanche, la déméthylation de l'ADN et les marques épigénétiques activatrices telles que l'acétylation de la lysine 27 de l'histone 3 (H3K27ac) et la triméthylation de la lysine 4 (H3K4me3) ont été détectées au niveau des promoteurs de HBG dans les cellules érythroïdes exprimant la globine γ. La forte expression de BCL11A dans les cellules érythroïdes adultes est associée à de faibles niveaux de méthylation de l'ADN au niveau des enhancers de BCL11A. L'inactivation des enhancers de BCL11A est associée à une augmentation de la méthylation de l'ADN. Ces données nous ont permis de concevoir des modificateurs de l'épigénome pour manipuler l'architecture épigénétique des promoteurs de HBG et des enhancers de BCL11A afin d'atteindre des niveaux thérapeutiques d'HbF<br>B-thalassemia and sickle cell disease (SCD) result from mutations that affect the synthesis or structure of adult hemoglobin. Historically, allogeneic hematopoietic stem cell (HSC) transplantation from a compatible donor was the only curative treatment. Transplantation of autologous, genetically modified HSCs offers a promising therapeutic alternative for patients lacking a suitable donor. The clinical severity in b-hemoglobinopathies is mitigated by co-inheritance of hereditary persistence of fetal hemoglobin (HPFH), a benign condition characterized by mutations occurring in the genes encoding the fetal y-globin chains, which lead to increased fetal hemoglobin (HbF, a2y2) expression, which can rescue the b-thalassemic and SCD phenotypes. HbF reactivation can be achieved by down-regulating BCL11A, encoding a key repressor of HbF. A CRISPR/Cas9 strategy targeting the GATA1 binding site (BS) within the +58-kb erythroid-specific enhancer of BCL11A has recently been approved as the first gene-editing therapy for b-thalassemia and SCD. Indeed, the targeting of the BCL11A erythroid-specific enhancer led to an efficient reduction of BCL11A in the erythroid cells, without impacting the differentiation of HSPCs in the other cell lineages. However, site-specific nucleases induce double strand breaks (DSBs), posing significant risks, such apoptosis and generation of large genomic rearrangements. In addition, to obtain an adequate number of corrected cells to transplant, several collections of HSCs are necessary to compensate for the cell loss due to DSB-induced apoptosis. Finally, the clinical study showed variability in the extent of HbF reactivation, still high HbS levels and modest correction of ineffective erythropoiesis. Novel CRISPR/Cas9 derived tools are currently available and can be used to develop therapeutic strategies associated with a low risk of DSB generation and increased HbF expression. In this project, we intend to develop universal, safe and efficacious therapeutic strategies for b-hemoglobinopathies aimed at modifying HSCs using base editors (BEs) and epigenome editors to reactivate HbF expression in their erythroid progeny. BEs are a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations with little DSB generation. In this work we used this technology to inactivate the GATA1 or the ATF4 transcriptional activator BS in the +58-kb and +55-kb BCL11A erythroid-specific enhancers through the insertion of point mutations. In particular, to reach levels of HbF sufficient to rescue the sickling phenotype, we performed simultaneous targeting of the two BS, achieving similar HbF levels compared to CRISPR/Cas9 nuclease-based approach. Additionally, we showed that BEs generated fewer DSBs and genomic rearrangements compared to the CRISPR/Cas9 nuclease approach. In parallel, we developed a novel epigenome-editing strategy aimed at modulating gene expression without altering the DNA sequence (e.g. without generating DSBs). We designed two approaches to upregulate HbF expression: a first strategy targeting and activating the y-globin promoters and a second approach downregulating BCL11A by targeting its erythroid-specific enhancers. We first identified the epigenetic marks in these trans- and cis-regulatory regions that are associated with active or inactive transcription in adult versus fetal erythroid cells. Then we used epigenome editors to deposit active histone modifications at the y-globin promoters and remove inactive marks such as DNA methylation. In parallel, we decorated the BCL11A enhancers with inactive epigenetic marks. Preliminary results demonstrated y-globin reactivation using both strategies, though the effects diminished over time, indicating the need for further optimization. In conclusion, we proposed two different editing approaches that allow to reduce DSB-associate issues as strategies to treat b-hemoglobinopathies

The constant improvements in device integration, the development of new technologies and the emergence of new design techniques call for flexible, maintainable and robust software tools. The generic nature of compiler-compiler systems, with their semi-formal specifications, can help in the construction of those tools. This thesis describes the Wright editor generator which is used in the synthesis of language-based graphical editors (LBGEs). An LBGE is a programming environment where the programs being manipulated denote pictures. Editing actions can be specified through both textual and graphical interfaces. Editors generated by the Wright system are specified using the formalism of attribute grammars. The major example editor in this thesis, Stick-Wright, is a design entry system for the construction of VLSI circuits. Stick-Wright is a hierarchical symbolic layout editor which exploits a combination of text and graphics in an interactive environment to provide the circuit designer with a tool for experimenting with circuit topologies. A simpler system, Pict-Wright: a picture drawing system, is also used to illustrate the attribute grammar specification process. This thesis aims to demonstrate the efficacy of formal specification in the generation of software-tools. The generated system Stick-Wright shows that a text/graphic programming environment can form the basis of a powerful VLSI design tool, especially with regard to providing the designer with immediate graphical feedback. Further applications of the LBGE generator approach to system design are given for a range of VLSI design activities.

Vid utveckling av tyngre fordon inför man allt fler avancerade funktione. Mycket av denna funktionalitet handlar om att maskiner automatiskt ska utföra uppgifter för att assistera föraren. Detta leder till att nya risker uppstår. Och till följd av detta har man börjat skapa nya funktionella säkerhetsstandarder. ISO 26262 är en ny funktionell säkerhetsstandard som finns för vanliga personbilar men som ännu inte trätt i kraft för lastbilar. I ISO-26262 standarden ska krav kunna mappas till andra krav samt till systemarkitektur. I nuläget finns det vissa verktyg på marknaden som stödjer användaren när den skriver kravspecifikationer. Men undersökningar av verktyg ledde till att vi kommit fram till att alla hade någon brist. Och ingen hade bra stöd för mappning mellan krav och systemarkitektur. I detta examensarbete har arbetet varit att testa implementera funktionalitet för ett verktyg som assisterar användaren på olika sätt när den skriver kravspecifikationer. Baserat på kontraktteori och konceptet om portar som hjälp för att koppla samman krav med systemarkitektur ska applikationen se till att det finns en formell koppling mellan dessa. För att testa och validera att portar går att använda för att testa intressant funktionalitet har också en applikation utvecklats där mycket funktionalitet implementerats. Resultatet har varit lyckat då vi baserat på kontraktteori lyckats implementera och validera att det är möjligt att använda portar för att skapa koppling mellan krav och systemarkitektur, samt mellan krav och krav. Validering av att det valda lagringsformatet JSON också förser implementeraren med nog starkt stöd för att kunna spara dessa krav så att data i filerna kan brytas ner och lagras i temporära databasen Neo4J och på så sätt skapa ett fungerande kretslopp.<br>When developing new heavy vehicles today demands for increasingly more advanced features are asked for. A lot of the new functionality is about machines performing tasks automatically to assist the driver when driving. This leads to new risks, and as a result a new functional safety standard has been created. ISO 26262 is a functional safety standard that today exists for ordinary cars, but has not yet became a standard for trucks. According to the ISO 26262-standard requirements can be mapped to other requirements as well as to the system architecture. At present there are several tools on the market that supports the user when writing specifications. However, our research of the tools has led us to conclude that all lacked something. For example neither of the tools had good support for mapping between requirements and system architecture. In this thesis work, functionality for a tool which is supposed to support the user in various ways when writing requirements specifications was to be examined. Based on contract theory and the concept of ports that links requirements together with system architecture, an application can ensure that there is a formal link between the two. To test the suggested functionality a prototype is being developed. The result has been a successful as we based on contract theory could validate that using ports to create links between different requirements as well as between requirements and system architecture works through the implementation of the tool. Validation that the selected storage format JSON also provides the implementer with enough support to save the requirements in a way so that the data files can be decomposed and stored in the Neo4J database.

Collaborative editing systems (or group editors) allow a geographically dispersed group of human users to view and modify shared multimedia documents, such as research papers, design diagrams, web pages and source code together over a computer network. In addition to being useful tools, group editors are a classic research vehicle and model of interactive groupware applications, based on which a variety of social and technical issues have been investigated. Consistency maintenance as a fundamental problem in group editors has attracted constant research attention. Operational transformation (OT) is an optimistic consistency maintenance method that supports unconstrained collaboration among human users. Although significant progress has been achieved over the past decade, there is still a large space for improvement on the theoretical part of OT. In this dissertation, we are concerned with three problems: (1) How to evaluate the correctness of OT-based consistency maintenance protocols; (2) How to design and prove correct OT-based protocols; (3) What are the consistency correctness conditions for group editing systems in general. This dissertation addresses the above three problems and makes the following contributions: (1) propose a total order based framework including a new consistency model and the associated design methodology. This framework reduces the complexities of the OT design; (2) improve the total order based framework by introducing a natural order based framework. In contrast, this framework removes the requirement of defining a total order that is not necessary to the OT design; (3) establish a generic consistency model and propose the first set of practical design guidelines in OT based on this model.

The Web Based Audio Editor thesis deals with the requirements specification, selection of technologies and with the implementation itself. The thesis uses modern approaches of HTML5 standards and is divided into the client and the server parts. Both the web application and the server are implemented in JavaScript. The Web based audio editor allows basic editing features like cut, shifting, deleting. All this in multiple audio tracks. The application runs in recent versions of the most widely used web browsers.