1,720,956 research outputs found
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Designer recombinase-mediated reactivation of fetal hemoglobin for the treatment of ß-hemoglobinopathies
No full textHemoglobinopathies are among the most common inherited monogenic disorders worldwide, with sickle cell disease (SCD) and ß-thalassemia (ß-thal) resulting from mutations in the ß-globin gene. These mutations impair or prevent ß-globin production, leading to severe clinical outcomes. Although allogeneic hematopoietic stem cell transplantation (HSCT) is curative, it is limited by low donor availability and the risk of immunological complications. Genetically modified autologous HSCT is an emerging alternative, with reactivation of fetal hemoglobin (HbF) being a major therapeutic strategy. Persistent HbF expression, as seen in hereditary persistence of fetal hemoglobin (HPFH), mitigates disease severity, making it an attractive target for genome editing.
Site-specific recombinases (SSRs) offer a double-strand break-free genome editing platform with high specificity and predictable outcomes between a pair of defined target sites, as they re-ligate DNA independently of host repair pathways. However, engineering SSRs to recognize new genomic targets is technically challenging. To overcome this, directed evolution approaches have been employed to generate recombinases with altered DNA specificity.
In this thesis, I present the successful development of a novel dual recombinase system targeting the BCL11A locus, a key repressor of gamma-globin (HBG) expression. Using substrate-linked directed evolution (SLiDE) in E. coli, I evolved a modular AA-BB heterodimeric recombinase system to recognize loxBCL11A target sites. High-throughput deep-sequencing-based screening identified variants with strong on-target activity. When transiently delivered as mRNA, these recombinases demonstrated activity in HeLa reporter cells, albeit with reduced efficiency compared to E. coli-based assays and a progressive decline in the proportion of edited cells over time.
To enhance functionality, two strategies were explored: fusion with zinc finger DNA-binding domains (ZFDs) and counter-selection-based evolution against known off-target sequences. While ZFD fusion did not enhance performance, counter-selection approach yielded improved recombinases including variant 229, which achieved approximately 70% editing efficiency in HeLa reporter cells. Importantly, it also mediated precise and seamless excision of the ~11.4 kb fragment at the endogenous BCL11A locus.
Variant 229 was further validated in the HUDEP-2 human erythroid progenitor cell line, demonstrating approximately 40% editing efficiency in reporter assays. During erythroid differentiation of wild-type HUDEP-2 cells, variant 229 enabled precise editing at the endogenous BCL11A locus, leading to BCL11A downregulation and robust HBG upregulation at the transcript level and a milder effect at the protein level. Bulk RNA-sequencing of recombinase treated HUDEP-2 cells revealed widespread transcriptional changes, including immune activation and membrane-associated stress responses, indicating cytotoxic effects. Furthermore, specificity profiling of variant 229, using a library of over 6000 computationally predicted human genomic off-target sites in a plasmid-based bacterial assay, showed high fidelity: approximately 97% of sites exhibited little to no recombination. However, a small subset showed higher activity than the intended target, often containing AT-rich motifs with high sequence similarity to the on-target site.
These findings highlight both the therapeutic potential and safety challenges associated with designer recombinases. In this thesis, I present a novel, modular dual-recombinase system capable of mediating precise deletion at the endogenous BCL11A locus, resulting in a therapeutically relevant functional response. Despite this progress, limitations remain, including suboptimal target selection, potential for off-target activity, and cytotoxicity. Overcoming these challenges will require refined evolution strategies in eukaryotic systems, unbiased off-target profiling, and computationally guided protein engineering. Overall, this work establishes SSRs as precise and adaptable genome editing tools and provides a strong foundation for advancing designer recombinase-based gene therapy strategies for the treatment of ß-hemoglobinopathies.:TABLE OF CONTENTS 1
ABBREVIATIONS 5
LIST OF FIGURES 8
LIST OF TABLES 9
1. INTRODUCTION 10
1.1 ß-hemoglobinopathies 10
1.1.1 Description and global prevalence 10
1.1.2 Molecular basis and pathophysiology 11
1.1.3 Treatment options 12
1.1.3.1 Symptomatic management 13
1.1.3.2 Curative therapies 14
1.2 Control of globin gene expression: basis for development of gene therapies 16
1.3 The protective contribution of fetal hemoglobin 17
1.4 BCL11A - an important HbF repressor 18
1.5 Therapeutic genome modifications for ß-hemoglobinopathies 19
1.5.1 Lentivirus-based transgene integration 19
1.5.2 Targeted genome editing approaches 21
1.6 Genetic engineering with tyrosine site-specific recombinases 25
1.6.1 Mechanism of the Cre/loxP recombination system 25
1.7 Tyrosine site-specific recombinases as genome editing tools for therapy 27
1.7.1 Principles of directed molecular evolution 28
1.7.2 Directed evolution of tyrosine site-specific recombinases 29
1.7.3 Therapeutic application of designer recombinases 30
2. AIM OF THE THESIS 32
3. MATERIALS AND METHODS 34
3.1 Target site identification and off-target prediction 34
3.2 Molecular cloning methods 34
3.2.1 DNA isolation and purification 35
3.2.2 Polymerase chain reaction (PCR) 35
3.2.3 Restriction enzyme digestion 36
3.2.4 DNA gel electrophoresis 36
3.2.5 Ligation 37
3.2.6 Preparation of electrocompetent E. coli cells 37
3.2.7 E. coli transformation 37
3.2.8 Sanger sequencing 38
3.3 Plasmid construction 38
3.3.1 pEVO plasmids 38
3.3.2 Mammalian expression plasmids 38
3.3.3 Mammalian reporter plasmids 39
3.4 SLiDE-based evolution of designer recombinases 39
3.5 DNA shuffling for recombinase library diversification 40
3.6 Deep-sequencing-based high-throughput screening using DEQSeq 41
3.6.1 Unique molecular identifier (UMI) preparation 41
3.6.2 Barcoding of recombinase library 42
3.6.3 Nanopore sequencing and data analysis 42
3.6.4 Validation of selected recombinase variants 43
3.7 mRNA synthesis using in vitro transcription (IVT) 43
3.8 Cell culture 44
3.8.1 Cell culture maintenance 44
3.8.2 HUDEP-2 cell line differentiation 44
3.8.3 Lentivirus production 45
3.8.4 Generation of reporter cell lines 45
3.8.5 mRNA transfection 46
3.8.6 mRNA electroporation 46
3.8.7 Flow cytometry 47
3.9 PCR-based detection of genomic excision 47
3.9.1 Excision on the integrated reporter cassette 48
3.9.2 Excision on the endogenous BCL11A locus 48
3.10 Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) 48
3.11 Biochemistry 49
3.11.1 SDS-PAGE 49
3.11.2 Western blotting 50
3.12 Endogenous zinc finger domain generation and cloning 50
3.13 RNA sequencing and analysis 51
3.14 Off-target profiling of recombinase variant 229 51
3.14.1 Target site library construction 51
3.14.2 Cloning and preparation for deep sequencing 52
3.14.3 Data analysis 54
4. RESULTS 55
4.1 Recombinase-mediated excision on asymmetric target sites 55
4.1.1 Target site identification for recombinase-mediated knockout of BCL11A 55
4.1.2 Substrate-linked directed evolution of a recombinase heterodimer 58
4.1.3 Substrate-linked directed evolution of a recombinase heterotetramer 61
4.2 Deep-sequencing-based high-throughput screening of heterodimer library 64
4.3 Activity evaluation of heterodimer recombinase variants in human cells 67
4.3.1 Potential causes of recombinase-induced toxicity in mammalian cells 71
4.4 Zinc finger domain-recombinase fusions to increase on-target specificity 73
4.4.1 Evaluation of ZFD-recombinase fusions in bacterial cells 75
4.4.2 Evaluation of ZFD-recombinase fusions in mammalian cells 76
4.5 Counter-selection based directed evolution 79
4.6 Evaluation of counter-selected variants in HeLa reporter cell line 82
4.6.1 Characterization of recombinase variant 229 83
4.7 The HUDEP-2 cell line: a valuable tool in ß-hemoglobinopathy gene therapy development 86
4.7.1 Evaluation of designer recombinase performance in HUDEP-2 reporter cell line 86
4.7.2 Characterization of recombinase variant 229 88
4.8 Functional evaluation of designer recombinase 229 in HUDEP-2 WT cells 91
4.9 Transcriptional profiling of recombinase treated HUDEP-2 WT cells 94
4.10 Off-target profiling of designer recombinase variant 229 97
5. DISCUSSION 100
5.1 Overview of the work 100
5.2 Interpretation of key findings 101
5.2.1 Directed evolution of heteromeric designer recombinases 101
5.2.2 Screening and validation of evolved variants 102
5.2.3 Optimization of recombinase properties 104
5.2.4 Functional evaluation of recombinase-mediated genome editing 107
5.3 Limitations and challenges of the study 109
5.4 Conclusion and outlook 112
5.4.1 Current landscape and future directions 112
5.4.2 Future of gene therapy 114
6. SUMMARY 116
7. ZUSAMMENFASSUNG 118
8. SUPPLEMENTARY INFORMATION 120
8.1 Supplementary figures 120
8.2 Supplementary tables 133
9. REFERENCES 141
10. ACKNOWLEDGMENTS 160
Anlage 1 162
Anlage 2 164Hämoglobinopathien gehören weltweit zu den häufigsten monogenen Erbkrankheiten. Die Sichelzellkrankheit (SCD) und die ß-Thalassämie (ß-thal) entstehen durch Mutationen im ß-Globin-Gen. Diese Mutationen beeinträchtigen oder verhindern die Produktion von ß-Globin, was zu schweren klinischen Folgen führt. Obwohl die allogene hämatopoetische Stammzelltransplantation (HSZT) heilend wirken kann, ist sie durch die geringe Verfügbarkeit von Spendern und das Risiko immunologischer Komplikationen limitiert. Genetisch modifizierte autologe HSZT stellen eine neue Alternative dar, wobei insbesondere die Reaktivierung von fetalem Hämoglobin (HbF) im therapeutsichen Fokus steht. Die anhaltende Expression von HbF, wie sie bei der hereditären Persistenz fetalen Hämoglobins (HPFH) zu beobachten ist, mildert den Schweregrad der Erkrankung und macht HbF damit zu einem attraktiven Ziel für Genome-Editierungen.
Ortsspezifische Rekombinasen (SSRs) bieten eine doppelstrangbruchfreie Genom-Editierungsplattform mit hoher Spezifität und vorhersehbaren Ergebnissen zwischen zwei definierten Zielorten, da sie die DNA unabhängig von Reparaturwegen der Zelle neu ligieren. Die Entwicklung von SSRs zur Erkennung neuer genomischer Ziele ist jedoch eine technische Herausforderung. Um dies zu überwinden, wurden Ansätze der gerichteten Evolution eingesetzt, um Rekombinasen mit veränderter DNA-Spezifität zu erzeugen.
In dieser Arbeit berichte ich über die Entwicklung eines neuartigen dualen Rekombinasesystems, das auf den BCL11A-Lokus abzielt, einen wichtigen Repressor der gamma-Globin (HBG)-Expression. Unter Verwendung von substratgebundener gerichteter Evolution (SLiDE) in E. coli wurde das modulare AA-BB heterodimere Rekombinasesystem entwickelt, um die loxBCL11A-Zielstellen zu rekombinieren. Durch ein Deep-Sequencing-Screening mit hohem Durchsatz in E. coli wurden Varianten mit starker On-Target-Aktivität identifiziert. Bei transienter Verabreichung als mRNA zeigten ausgewählte Rekombinasen Aktivität in HeLa-Reporterzellen, allerdings mit reduzierter Effizienz im Vergleich zu auf E. coli basierenden Tests und einem fortschreitenden Rückgang des Anteils der editierten Zellen im Laufe der Zeit.
Zur Verbesserung der Funktionalität wurden zwei Strategien verfolgt: die Fusion mit Zinkfinger-DNA-Bindungsdomänen (ZFDs) und die auf Gegenselektion basierende Evolution gegen bekannte Off-Target-Sequenzen. Während die ZFD-Fusion nicht die erhofften Ergebnisse brachte, resultierten aus der Gegenselektion die Identifikation verbesserter Rekombinasen, darunter die Variante 229, die in HeLa-Reporterzellen eine Editierungseffizienz von etwa 70% erreichte und darüber hinaus die präzise und nahtlose Exzision des etwa 11.4 kb großen Fragments am endogenen BCL11A-Lokus ermöglichte.
Die Variante 229 wurde in der menschlichen erythroiden Vorläuferzelllinie HUDEP-2 weiter validiert und zeigte in Reporter-Assays eine Editierungseffizienz von etwa 40%. Während der erythroiden Differenzierung von Wildtyp-HUDEP-2-Zellen ermöglichte die Variante 229 eine präzise Editierung am endogenen BCL11A-Lokus, was zu einer Herunterregulierung von BCL11A und einer robusten Hochregulation von HBG auf der Transkriptionsebene sowie zu einem milderen Effekt auf der Proteinebene führte. Die Bulk-RNA-Sequenzierung rekombinasebehandelter HUDEP-2-Zellen ergab weitreichende transkriptionelle Veränderungen, einschließlich Immunaktivierung und membranassoziierter Stressreaktionen, was auf zytotoxische Wirkungen hindeutet. Darüber hinaus zeigte das Spezifitätsprofils der Variante 229 unter Verwendung einer Bibliothek von über 6000 rechnerisch vorhergesagten genomischen Off-Target-Stellen in einem plasmidbasierten Bakteriensystem eine hohe Präzision: Etwa 97% der Off-Target-Stellen wiesen nur eine geringe bis keine Rekombination auf. Eine kleine Untergruppe zeigte jedoch sogar eine höhere Aktivität im Vergleich zur Zielstelle und enthielt häufig AT-reiche Motive mit hoher Sequenzähnlichkeit zur Zielstelle.
Diese Ergebnisse unterstreichen sowohl das therapeutische Potenzial als auch die Sicherheitsprobleme von Designer-Rekombinasen. In dieser Arbeit präsentiere ich ein neuartiges, modulares duales Rekombinasesystem vorgestellt, das gezielt einen endogenen BCL11A-Lokus entfernen kann und damit eine funktionelle, therapeutisch relevante Reaktion auslöst. Trotz dieses Fortschritts bleiben Einschränkungen bestehen, darunter eine suboptimale Zielstellen-Auswahl, potenzielle Off-Target-Aktivität und Zytotoxizität. Um diese Herausforderungen zu meistern, sind verfeinerte Evolutionsstrategien in eukaryontischen Systemen, eine unvoreingenommene Off-Target-Profilierung und ein computergestütztes Protein-Engineering erforderlich. Insgesamt etabliert diese Arbeit SSRs als präzise und anpassungsfähige Genom-Editierwerkzeuge und bietet eine solide Grundlage für die Weiterentwicklung von Designer-Rekombinase-basierten Gentherapiestrategien zur Behandlung von ß-Hämoglobinopathien.:TABLE OF CONTENTS 1
ABBREVIATIONS 5
LIST OF FIGURES 8
LIST OF TABLES 9
1. INTRODUCTION 10
1.1 ß-hemoglobinopathies 10
1.1.1 Description and global prevalence 10
1.1.2 Molecular basis and pathophysiology 11
1.1.3 Treatment options 12
1.1.3.1 Symptomatic management 13
1.1.3.2 Curative therapies 14
1.2 Control of globin gene expression: basis for development of gene therapies 16
1.3 The protective contribution of fetal hemoglobin 17
1.4 BCL11A - an important HbF repressor 18
1.5 Therapeutic genome modifications for ß-hemoglobinopathies 19
1.5.1 Lentivirus-based transgene integration 19
1.5.2 Targeted genome editing approaches 21
1.6 Genetic engineering with tyrosine site-specific recombinases 25
1.6.1 Mechanism of the Cre/loxP recombination system 25
1.7 Tyrosine site-specific recombinases as genome editing tools for therapy 27
1.7.1 Principles of directed molecular evolution 28
1.7.2 Directed evolution of tyrosine site-specific recombinases 29
1.7.3 Therapeutic application of designer recombinases 30
2. AIM OF THE THESIS 32
3. MATERIALS AND METHODS 34
3.1 Target site identification and off-target prediction 34
3.2 Molecular cloning methods 34
3.2.1 DNA isolation and purification 35
3.2.2 Polymerase chain reaction (PCR) 35
3.2.3 Restriction enzyme digestion 36
3.2.4 DNA gel electrophoresis 36
3.2.5 Ligation 37
3.2.6 Preparation of electrocompetent E. coli cells 37
3.2.7 E. coli transformation 37
3.2.8 Sanger sequencing 38
3.3 Plasmid construction 38
3.3.1 pEVO plasmids 38
3.3.2 Mammalian expression plasmids 38
3.3.3 Mammalian reporter plasmids 39
3.4 SLiDE-based evolution of designer recombinases 39
3.5 DNA shuffling for recombinase library diversification 40
3.6 Deep-sequencing-based high-throughput screening using DEQSeq 41
3.6.1 Unique molecular identifier (UMI) preparation 41
3.6.2 Barcoding of recombinase library 42
3.6.3 Nanopore sequencing and data analysis 42
3.6.4 Validation of selected recombinase variants 43
3.7 mRNA synthesis using in vitro transcription (IVT) 43
3.8 Cell culture 44
3.8.1 Cell culture maintenance 44
3.8.2 HUDEP-2 cell line differentiation 44
3.8.3 Lentivirus production 45
3.8.4 Generation of reporter cell lines 45
3.8.5 mRNA transfection 46
3.8.6 mRNA electroporation 46
3.8.7 Flow cytometry 47
3.9 PCR-based detection of genomic excision 47
3.9.1 Excision on the integrated reporter cassette 48
3.9.2 Excision on the endogenous BCL11A locus 48
3.10 Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) 48
3.11 Biochemistry 49
3.11.1 SDS-PAGE 49
3.11.2 Western blotting 50
3.12 Endogenous zinc finger domain generation and cloning 50
3.13 RNA sequencing and analysis 51
3.14 Off-target profiling of recombinase variant 229 51
3.14.1 Target site library construction 51
3.14.2 Cloning and preparation for deep sequencing 52
3.14.3 Data analysis 54
4. RESULTS 55
4.1 Recombinase-mediated excision on asymmetric target sites 55
4.1.1 Target site identification for recombinase-mediated knockout of BCL11A 55
4.1.2 Substrate-linked directed evolution of a recombinase heterodimer 58
4.1.3 Substrate-linked directed evolution of a recombinase heterotetramer 61
4.2 Deep-sequencing-based high-throughput screening of heterodimer library 64
4.3 Activity evaluation of heterodimer recombinase variants in human cells 67
4.3.1 Potential causes of recombinase-induced toxicity in mammalian cells 71
4.4 Zinc finger domain-recombinase fusions to increase on-target specificity 73
4.4.1 Evaluation of ZFD-recombinase fusions in bacterial cells 75
4.4.2 Evaluation of ZFD-recombinase fusions in mammalian cells 76
4.5 Counter-selection based directed evolution 79
4.6 Evaluation of counter-selected variants in HeLa reporter cell line 82
4.6.1 Characterization of recombinase variant 229 83
4.7 The HUDEP-2 cell line: a valuable tool in ß-hemoglobinopathy gene therapy development 86
4.7.1 Evaluation of designer recombinase performance in HUDEP-2 reporter cell line 86
4.7.2 Characterization of recombinase variant 229 88
4.8 Functional evaluation of designer recombinase 229 in HUDEP-2 WT cells 91
4.9 Transcriptional profiling of recombinase treated HUDEP-2 WT cells 94
4.10 Off-target profiling of designer recombinase variant 229 97
5. DISCUSSION 100
5.1 Overview of the work 100
5.2 Interpretation of key findings 101
5.2.1 Directed evolution of heteromeric designer recombinases 101
5.2.2 Screening and validation of evolved variants 102
5.2.3 Optimization of recombinase properties 104
5.2.4 Functional evaluation of recombinase-mediated genome editing 107
5.3 Limitations and challenges of the study 109
5.4 Conclusion and outlook 112
5.4.1 Current landscape and future directions 112
5.4.2 Future of gene therapy 114
6. SUMMARY 116
7. ZUSAMMENFASSUNG 118
8. SUPPLEMENTARY INFORMATION 120
8.1 Supplementary figures 120
8.2 Supplementary tables 133
9. REFERENCES 141
10. ACKNOWLEDGMENTS 160
Anlage 1 162
Anlage 2 16
Designer recombinase-mediated reactivation of fetal hemoglobin for the treatment of ß-hemoglobinopathies
No full textHemoglobinopathies are among the most common inherited monogenic disorders worldwide, with sickle cell disease (SCD) and ß-thalassemia (ß-thal) resulting from mutations in the ß-globin gene. These mutations impair or prevent ß-globin production, leading to severe clinical outcomes. Although allogeneic hematopoietic stem cell transplantation (HSCT) is curative, it is limited by low donor availability and the risk of immunological complications. Genetically modified autologous HSCT is an emerging alternative, with reactivation of fetal hemoglobin (HbF) being a major therapeutic strategy. Persistent HbF expression, as seen in hereditary persistence of fetal hemoglobin (HPFH), mitigates disease severity, making it an attractive target for genome editing.
Site-specific recombinases (SSRs) offer a double-strand break-free genome editing platform with high specificity and predictable outcomes between a pair of defined target sites, as they re-ligate DNA independently of host repair pathways. However, engineering SSRs to recognize new genomic targets is technically challenging. To overcome this, directed evolution approaches have been employed to generate recombinases with altered DNA specificity.
In this thesis, I present the successful development of a novel dual recombinase system targeting the BCL11A locus, a key repressor of gamma-globin (HBG) expression. Using substrate-linked directed evolution (SLiDE) in E. coli, I evolved a modular AA-BB heterodimeric recombinase system to recognize loxBCL11A target sites. High-throughput deep-sequencing-based screening identified variants with strong on-target activity. When transiently delivered as mRNA, these recombinases demonstrated activity in HeLa reporter cells, albeit with reduced efficiency compared to E. coli-based assays and a progressive decline in the proportion of edited cells over time.
To enhance functionality, two strategies were explored: fusion with zinc finger DNA-binding domains (ZFDs) and counter-selection-based evolution against known off-target sequences. While ZFD fusion did not enhance performance, counter-selection approach yielded improved recombinases including variant 229, which achieved approximately 70% editing efficiency in HeLa reporter cells. Importantly, it also mediated precise and seamless excision of the ~11.4 kb fragment at the endogenous BCL11A locus.
Variant 229 was further validated in the HUDEP-2 human erythroid progenitor cell line, demonstrating approximately 40% editing efficiency in reporter assays. During erythroid differentiation of wild-type HUDEP-2 cells, variant 229 enabled precise editing at the endogenous BCL11A locus, leading to BCL11A downregulation and robust HBG upregulation at the transcript level and a milder effect at the protein level. Bulk RNA-sequencing of recombinase treated HUDEP-2 cells revealed widespread transcriptional changes, including immune activation and membrane-associated stress responses, indicating cytotoxic effects. Furthermore, specificity profiling of variant 229, using a library of over 6000 computationally predicted human genomic off-target sites in a plasmid-based bacterial assay, showed high fidelity: approximately 97% of sites exhibited little to no recombination. However, a small subset showed higher activity than the intended target, often containing AT-rich motifs with high sequence similarity to the on-target site.
These findings highlight both the therapeutic potential and safety challenges associated with designer recombinases. In this thesis, I present a novel, modular dual-recombinase system capable of mediating precise deletion at the endogenous BCL11A locus, resulting in a therapeutically relevant functional response. Despite this progress, limitations remain, including suboptimal target selection, potential for off-target activity, and cytotoxicity. Overcoming these challenges will require refined evolution strategies in eukaryotic systems, unbiased off-target profiling, and computationally guided protein engineering. Overall, this work establishes SSRs as precise and adaptable genome editing tools and provides a strong foundation for advancing designer recombinase-based gene therapy strategies for the treatment of ß-hemoglobinopathies.:TABLE OF CONTENTS 1
ABBREVIATIONS 5
LIST OF FIGURES 8
LIST OF TABLES 9
1. INTRODUCTION 10
1.1 ß-hemoglobinopathies 10
1.1.1 Description and global prevalence 10
1.1.2 Molecular basis and pathophysiology 11
1.1.3 Treatment options 12
1.1.3.1 Symptomatic management 13
1.1.3.2 Curative therapies 14
1.2 Control of globin gene expression: basis for development of gene therapies 16
1.3 The protective contribution of fetal hemoglobin 17
1.4 BCL11A - an important HbF repressor 18
1.5 Therapeutic genome modifications for ß-hemoglobinopathies 19
1.5.1 Lentivirus-based transgene integration 19
1.5.2 Targeted genome editing approaches 21
1.6 Genetic engineering with tyrosine site-specific recombinases 25
1.6.1 Mechanism of the Cre/loxP recombination system 25
1.7 Tyrosine site-specific recombinases as genome editing tools for therapy 27
1.7.1 Principles of directed molecular evolution 28
1.7.2 Directed evolution of tyrosine site-specific recombinases 29
1.7.3 Therapeutic application of designer recombinases 30
2. AIM OF THE THESIS 32
3. MATERIALS AND METHODS 34
3.1 Target site identification and off-target prediction 34
3.2 Molecular cloning methods 34
3.2.1 DNA isolation and purification 35
3.2.2 Polymerase chain reaction (PCR) 35
3.2.3 Restriction enzyme digestion 36
3.2.4 DNA gel electrophoresis 36
3.2.5 Ligation 37
3.2.6 Preparation of electrocompetent E. coli cells 37
3.2.7 E. coli transformation 37
3.2.8 Sanger sequencing 38
3.3 Plasmid construction 38
3.3.1 pEVO plasmids 38
3.3.2 Mammalian expression plasmids 38
3.3.3 Mammalian reporter plasmids 39
3.4 SLiDE-based evolution of designer recombinases 39
3.5 DNA shuffling for recombinase library diversification 40
3.6 Deep-sequencing-based high-throughput screening using DEQSeq 41
3.6.1 Unique molecular identifier (UMI) preparation 41
3.6.2 Barcoding of recombinase library 42
3.6.3 Nanopore sequencing and data analysis 42
3.6.4 Validation of selected recombinase variants 43
3.7 mRNA synthesis using in vitro transcription (IVT) 43
3.8 Cell culture 44
3.8.1 Cell culture maintenance 44
3.8.2 HUDEP-2 cell line differentiation 44
3.8.3 Lentivirus production 45
3.8.4 Generation of reporter cell lines 45
3.8.5 mRNA transfection 46
3.8.6 mRNA electroporation 46
3.8.7 Flow cytometry 47
3.9 PCR-based detection of genomic excision 47
3.9.1 Excision on the integrated reporter cassette 48
3.9.2 Excision on the endogenous BCL11A locus 48
3.10 Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) 48
3.11 Biochemistry 49
3.11.1 SDS-PAGE 49
3.11.2 Western blotting 50
3.12 Endogenous zinc finger domain generation and cloning 50
3.13 RNA sequencing and analysis 51
3.14 Off-target profiling of recombinase variant 229 51
3.14.1 Target site library construction 51
3.14.2 Cloning and preparation for deep sequencing 52
3.14.3 Data analysis 54
4. RESULTS 55
4.1 Recombinase-mediated excision on asymmetric target sites 55
4.1.1 Target site identification for recombinase-mediated knockout of BCL11A 55
4.1.2 Substrate-linked directed evolution of a recombinase heterodimer 58
4.1.3 Substrate-linked directed evolution of a recombinase heterotetramer 61
4.2 Deep-sequencing-based high-throughput screening of heterodimer library 64
4.3 Activity evaluation of heterodimer recombinase variants in human cells 67
4.3.1 Potential causes of recombinase-induced toxicity in mammalian cells 71
4.4 Zinc finger domain-recombinase fusions to increase on-target specificity 73
4.4.1 Evaluation of ZFD-recombinase fusions in bacterial cells 75
4.4.2 Evaluation of ZFD-recombinase fusions in mammalian cells 76
4.5 Counter-selection based directed evolution 79
4.6 Evaluation of counter-selected variants in HeLa reporter cell line 82
4.6.1 Characterization of recombinase variant 229 83
4.7 The HUDEP-2 cell line: a valuable tool in ß-hemoglobinopathy gene therapy development 86
4.7.1 Evaluation of designer recombinase performance in HUDEP-2 reporter cell line 86
4.7.2 Characterization of recombinase variant 229 88
4.8 Functional evaluation of designer recombinase 229 in HUDEP-2 WT cells 91
4.9 Transcriptional profiling of recombinase treated HUDEP-2 WT cells 94
4.10 Off-target profiling of designer recombinase variant 229 97
5. DISCUSSION 100
5.1 Overview of the work 100
5.2 Interpretation of key findings 101
5.2.1 Directed evolution of heteromeric designer recombinases 101
5.2.2 Screening and validation of evolved variants 102
5.2.3 Optimization of recombinase properties 104
5.2.4 Functional evaluation of recombinase-mediated genome editing 107
5.3 Limitations and challenges of the study 109
5.4 Conclusion and outlook 112
5.4.1 Current landscape and future directions 112
5.4.2 Future of gene therapy 114
6. SUMMARY 116
7. ZUSAMMENFASSUNG 118
8. SUPPLEMENTARY INFORMATION 120
8.1 Supplementary figures 120
8.2 Supplementary tables 133
9. REFERENCES 141
10. ACKNOWLEDGMENTS 160
Anlage 1 162
Anlage 2 164Hämoglobinopathien gehören weltweit zu den häufigsten monogenen Erbkrankheiten. Die Sichelzellkrankheit (SCD) und die ß-Thalassämie (ß-thal) entstehen durch Mutationen im ß-Globin-Gen. Diese Mutationen beeinträchtigen oder verhindern die Produktion von ß-Globin, was zu schweren klinischen Folgen führt. Obwohl die allogene hämatopoetische Stammzelltransplantation (HSZT) heilend wirken kann, ist sie durch die geringe Verfügbarkeit von Spendern und das Risiko immunologischer Komplikationen limitiert. Genetisch modifizierte autologe HSZT stellen eine neue Alternative dar, wobei insbesondere die Reaktivierung von fetalem Hämoglobin (HbF) im therapeutsichen Fokus steht. Die anhaltende Expression von HbF, wie sie bei der hereditären Persistenz fetalen Hämoglobins (HPFH) zu beobachten ist, mildert den Schweregrad der Erkrankung und macht HbF damit zu einem attraktiven Ziel für Genome-Editierungen.
Ortsspezifische Rekombinasen (SSRs) bieten eine doppelstrangbruchfreie Genom-Editierungsplattform mit hoher Spezifität und vorhersehbaren Ergebnissen zwischen zwei definierten Zielorten, da sie die DNA unabhängig von Reparaturwegen der Zelle neu ligieren. Die Entwicklung von SSRs zur Erkennung neuer genomischer Ziele ist jedoch eine technische Herausforderung. Um dies zu überwinden, wurden Ansätze der gerichteten Evolution eingesetzt, um Rekombinasen mit veränderter DNA-Spezifität zu erzeugen.
In dieser Arbeit berichte ich über die Entwicklung eines neuartigen dualen Rekombinasesystems, das auf den BCL11A-Lokus abzielt, einen wichtigen Repressor der gamma-Globin (HBG)-Expression. Unter Verwendung von substratgebundener gerichteter Evolution (SLiDE) in E. coli wurde das modulare AA-BB heterodimere Rekombinasesystem entwickelt, um die loxBCL11A-Zielstellen zu rekombinieren. Durch ein Deep-Sequencing-Screening mit hohem Durchsatz in E. coli wurden Varianten mit starker On-Target-Aktivität identifiziert. Bei transienter Verabreichung als mRNA zeigten ausgewählte Rekombinasen Aktivität in HeLa-Reporterzellen, allerdings mit reduzierter Effizienz im Vergleich zu auf E. coli basierenden Tests und einem fortschreitenden Rückgang des Anteils der editierten Zellen im Laufe der Zeit.
Zur Verbesserung der Funktionalität wurden zwei Strategien verfolgt: die Fusion mit Zinkfinger-DNA-Bindungsdomänen (ZFDs) und die auf Gegenselektion basierende Evolution gegen bekannte Off-Target-Sequenzen. Während die ZFD-Fusion nicht die erhofften Ergebnisse brachte, resultierten aus der Gegenselektion die Identifikation verbesserter Rekombinasen, darunter die Variante 229, die in HeLa-Reporterzellen eine Editierungseffizienz von etwa 70% erreichte und darüber hinaus die präzise und nahtlose Exzision des etwa 11.4 kb großen Fragments am endogenen BCL11A-Lokus ermöglichte.
Die Variante 229 wurde in der menschlichen erythroiden Vorläuferzelllinie HUDEP-2 weiter validiert und zeigte in Reporter-Assays eine Editierungseffizienz von etwa 40%. Während der erythroiden Differenzierung von Wildtyp-HUDEP-2-Zellen ermöglichte die Variante 229 eine präzise Editierung am endogenen BCL11A-Lokus, was zu einer Herunterregulierung von BCL11A und einer robusten Hochregulation von HBG auf der Transkriptionsebene sowie zu einem milderen Effekt auf der Proteinebene führte. Die Bulk-RNA-Sequenzierung rekombinasebehandelter HUDEP-2-Zellen ergab weitreichende transkriptionelle Veränderungen, einschließlich Immunaktivierung und membranassoziierter Stressreaktionen, was auf zytotoxische Wirkungen hindeutet. Darüber hinaus zeigte das Spezifitätsprofils der Variante 229 unter Verwendung einer Bibliothek von über 6000 rechnerisch vorhergesagten genomischen Off-Target-Stellen in einem plasmidbasierten Bakteriensystem eine hohe Präzision: Etwa 97% der Off-Target-Stellen wiesen nur eine geringe bis keine Rekombination auf. Eine kleine Untergruppe zeigte jedoch sogar eine höhere Aktivität im Vergleich zur Zielstelle und enthielt häufig AT-reiche Motive mit hoher Sequenzähnlichkeit zur Zielstelle.
Diese Ergebnisse unterstreichen sowohl das therapeutische Potenzial als auch die Sicherheitsprobleme von Designer-Rekombinasen. In dieser Arbeit präsentiere ich ein neuartiges, modulares duales Rekombinasesystem vorgestellt, das gezielt einen endogenen BCL11A-Lokus entfernen kann und damit eine funktionelle, therapeutisch relevante Reaktion auslöst. Trotz dieses Fortschritts bleiben Einschränkungen bestehen, darunter eine suboptimale Zielstellen-Auswahl, potenzielle Off-Target-Aktivität und Zytotoxizität. Um diese Herausforderungen zu meistern, sind verfeinerte Evolutionsstrategien in eukaryontischen Systemen, eine unvoreingenommene Off-Target-Profilierung und ein computergestütztes Protein-Engineering erforderlich. Insgesamt etabliert diese Arbeit SSRs als präzise und anpassungsfähige Genom-Editierwerkzeuge und bietet eine solide Grundlage für die Weiterentwicklung von Designer-Rekombinase-basierten Gentherapiestrategien zur Behandlung von ß-Hämoglobinopathien.:TABLE OF CONTENTS 1
ABBREVIATIONS 5
LIST OF FIGURES 8
LIST OF TABLES 9
1. INTRODUCTION 10
1.1 ß-hemoglobinopathies 10
1.1.1 Description and global prevalence 10
1.1.2 Molecular basis and pathophysiology 11
1.1.3 Treatment options 12
1.1.3.1 Symptomatic management 13
1.1.3.2 Curative therapies 14
1.2 Control of globin gene expression: basis for development of gene therapies 16
1.3 The protective contribution of fetal hemoglobin 17
1.4 BCL11A - an important HbF repressor 18
1.5 Therapeutic genome modifications for ß-hemoglobinopathies 19
1.5.1 Lentivirus-based transgene integration 19
1.5.2 Targeted genome editing approaches 21
1.6 Genetic engineering with tyrosine site-specific recombinases 25
1.6.1 Mechanism of the Cre/loxP recombination system 25
1.7 Tyrosine site-specific recombinases as genome editing tools for therapy 27
1.7.1 Principles of directed molecular evolution 28
1.7.2 Directed evolution of tyrosine site-specific recombinases 29
1.7.3 Therapeutic application of designer recombinases 30
2. AIM OF THE THESIS 32
3. MATERIALS AND METHODS 34
3.1 Target site identification and off-target prediction 34
3.2 Molecular cloning methods 34
3.2.1 DNA isolation and purification 35
3.2.2 Polymerase chain reaction (PCR) 35
3.2.3 Restriction enzyme digestion 36
3.2.4 DNA gel electrophoresis 36
3.2.5 Ligation 37
3.2.6 Preparation of electrocompetent E. coli cells 37
3.2.7 E. coli transformation 37
3.2.8 Sanger sequencing 38
3.3 Plasmid construction 38
3.3.1 pEVO plasmids 38
3.3.2 Mammalian expression plasmids 38
3.3.3 Mammalian reporter plasmids 39
3.4 SLiDE-based evolution of designer recombinases 39
3.5 DNA shuffling for recombinase library diversification 40
3.6 Deep-sequencing-based high-throughput screening using DEQSeq 41
3.6.1 Unique molecular identifier (UMI) preparation 41
3.6.2 Barcoding of recombinase library 42
3.6.3 Nanopore sequencing and data analysis 42
3.6.4 Validation of selected recombinase variants 43
3.7 mRNA synthesis using in vitro transcription (IVT) 43
3.8 Cell culture 44
3.8.1 Cell culture maintenance 44
3.8.2 HUDEP-2 cell line differentiation 44
3.8.3 Lentivirus production 45
3.8.4 Generation of reporter cell lines 45
3.8.5 mRNA transfection 46
3.8.6 mRNA electroporation 46
3.8.7 Flow cytometry 47
3.9 PCR-based detection of genomic excision 47
3.9.1 Excision on the integrated reporter cassette 48
3.9.2 Excision on the endogenous BCL11A locus 48
3.10 Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) 48
3.11 Biochemistry 49
3.11.1 SDS-PAGE 49
3.11.2 Western blotting 50
3.12 Endogenous zinc finger domain generation and cloning 50
3.13 RNA sequencing and analysis 51
3.14 Off-target profiling of recombinase variant 229 51
3.14.1 Target site library construction 51
3.14.2 Cloning and preparation for deep sequencing 52
3.14.3 Data analysis 54
4. RESULTS 55
4.1 Recombinase-mediated excision on asymmetric target sites 55
4.1.1 Target site identification for recombinase-mediated knockout of BCL11A 55
4.1.2 Substrate-linked directed evolution of a recombinase heterodimer 58
4.1.3 Substrate-linked directed evolution of a recombinase heterotetramer 61
4.2 Deep-sequencing-based high-throughput screening of heterodimer library 64
4.3 Activity evaluation of heterodimer recombinase variants in human cells 67
4.3.1 Potential causes of recombinase-induced toxicity in mammalian cells 71
4.4 Zinc finger domain-recombinase fusions to increase on-target specificity 73
4.4.1 Evaluation of ZFD-recombinase fusions in bacterial cells 75
4.4.2 Evaluation of ZFD-recombinase fusions in mammalian cells 76
4.5 Counter-selection based directed evolution 79
4.6 Evaluation of counter-selected variants in HeLa reporter cell line 82
4.6.1 Characterization of recombinase variant 229 83
4.7 The HUDEP-2 cell line: a valuable tool in ß-hemoglobinopathy gene therapy development 86
4.7.1 Evaluation of designer recombinase performance in HUDEP-2 reporter cell line 86
4.7.2 Characterization of recombinase variant 229 88
4.8 Functional evaluation of designer recombinase 229 in HUDEP-2 WT cells 91
4.9 Transcriptional profiling of recombinase treated HUDEP-2 WT cells 94
4.10 Off-target profiling of designer recombinase variant 229 97
5. DISCUSSION 100
5.1 Overview of the work 100
5.2 Interpretation of key findings 101
5.2.1 Directed evolution of heteromeric designer recombinases 101
5.2.2 Screening and validation of evolved variants 102
5.2.3 Optimization of recombinase properties 104
5.2.4 Functional evaluation of recombinase-mediated genome editing 107
5.3 Limitations and challenges of the study 109
5.4 Conclusion and outlook 112
5.4.1 Current landscape and future directions 112
5.4.2 Future of gene therapy 114
6. SUMMARY 116
7. ZUSAMMENFASSUNG 118
8. SUPPLEMENTARY INFORMATION 120
8.1 Supplementary figures 120
8.2 Supplementary tables 133
9. REFERENCES 141
10. ACKNOWLEDGMENTS 160
Anlage 1 162
Anlage 2 16
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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