Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(−) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses

Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments

Segmented RNA viruses are ubiquitous pathogens, which include influenza viruses and rotaviruses. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct RNAs. This process involves complex protein/RNA interactions, which are often obscured by non-specific binding at concentrations approaching in vvo assembly conditions. Here, we present direct experimental evidence of sequence-specific inter-segment interactions in rotaviruses, taking place in a complex RNA- and protein- rich milieu. We show that binding of the rotaviral protein NSP2 to ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. To identify the sites of these interactions, we have developed an RNA-RNA SELEX approach for mapping the sequences involved in inter-segment base-pairing. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for delineating RNA-RNA interactions that govern assembly of other segmented RNA viruse

Identification of resistance mechanism to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer in vivo

Triple-negative breast cancers (TNBCs) (ER/PR/HER2 negative) represent 15% of invasive\nbreast cancers and occur at a higher rate in young and African-American women. Exploration of\nnovel therapeutic approaches is critical, since only 30% of woman with metastatic breast cancer\nwill survive and virtually none with metastatic TNBC. The status quo as it pertains to the\ntreatment of TNBCs can be summarized as: no effective therapies available. In part, the lack of\ntherapeutic success is due to high genetic heterogeneity of TNBCs, challenging single drug\napproaches.\nMany targeted strategies to treat TNBC are being explored, including the inhibition of kinase\npathways (e.g. PI3K/Akt, MEK, VEGFR and PDGFR), the inhibition of DNA repair, of survival\npathways and androgen receptor blockade. In most cases, such single-drug targeted therapy is\ncombined with systemic genotoxic chemotherapy. For example, although about 60% of basallike\nTNBCs over express EGFR, EGFR targeted therapy, including kinase inhibition, has been\ndisappointing due to the development of resistance.\nVarious resistance mechanisms allow cancer cells to evade single-drug targeted therapies:\nmutations in the targeted molecules, extensive crosstalk/pathway redundancy and the upregulation\nof alternate growth or survival pathways. Design of combinatorial approaches of\ntherapeutics for TNBC that overcome resistance is therefore critical. The contribution of the\nproposed research is expected to be the identification of signaling network perturbations that\noccur in response to single targeted therapies, in particular in ADAM17/EGFR axis inhibition,\nand confer resistance. Based on published results, inhibition of the ADAM17/EGFR ligand axis\nin TNBC should provide therapeutic benefit with reduced tumor growth and decreased\nmetastasis, if not possibly cure.\nIn our in vitro studies on PKC? and PPP1R14D gene knockout in MDA-MB-231 cells indeed led\nto decreases in cellular growth and migration. However, to our surprise, when the same cells\nwere injected into mice through orthotropic fat pad transplantation, they produced aggressive,\nmetastatic tumors that showed activation of alternate growth signaling pathways, namely of the\nmitogen-activated protein kinase ERK and of the PI3kinase target Akt, also a mitogen activated\nkinase. This suggested that TNBC cells were developing resistance to EGF ligand regulator\nknockdown by rewiring their growth factor signaling pathways. To determine where these\nadditional growth signals come from, we first considered the tumor cells themselves.\nIn this context we discovered that when kept in culture, MDA-MB-231 cells expressing sh-\nRNAs targeting either PKCa or PPP1R14D maintained knockdown of the target for up to 35days\ntested. At the same time EGFR and ERK showed low activity as expected due to a decrease in\nEGF ligand cleavage; Akt activity was undetectable. Since we observed strong reactivation of\nERK and new activation of Akt in tumors in vivo, we considered possible up-regulation of other\ngrowth factor receptors on the cell surface that would be engaged by factors released from the\ntumor stroma once cells are inserted in vivo. Indeed, we found that FGFR2 and Erbb4 were\nupregulated. It is therefore likely that reactivation of ERK and new activation of Akt was due to\nFGFR2 and Erbb4. This would suggest that combination therapy of EGF ligand release regulator\ninhibition and FGFR inhibition would decrease growth of these tumors in vivo.Fil: Sharma, Neha. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaEl cáncer de mama triple negativo (TNCB) es aquel que no expresa el receptor de estrógenos\n(ER), ni el de progesterona (PR) o el HER2. Esta patología representa el 15% de los tumores de\nmama invasivos y tiene una alta incidencia en mujeres jóvenes Afro-Americanas. Es responsable\nde una alta tasa de mortalidad por cáncer de mama ya que generalmente el TNCB causa\nmetástasis; además, responde pobremente a las terapias con quimioterápicos a largo plazo y\ngeneralmente desarrolla resistencia a las terapias dirigidas, incluyendo las que implican al EGFR.\nPor todo ello, es fundamental el desarrollo de terapias alternativas, dado que solo el 30% de las\nmujeres con cáncer de mama metastásico sobrevive pero ninguna de las que presentan TNBC\nmetastásico.\nActualmente, no existe una terapia adecuada y efectiva para el TNBC. En parte, esto se debe a la\nalta heterogeneidad genética que presentan estos tumores, lo cual redunda en la inefectividad de\nterapias basadas en una única droga. Terapias basadas en blancos terapéuticos específicos están\nen investigación y desarrollo, como aquellas basadas en la inhibición de quinasas implicadas en\nseñalización (ejemplo: /Akt, MEK, VEGFR, PDGFR), reparación del DNA, supervivencia\ncelular o acciones androgénicas. Mayormente, estas terapias específicas son combinadas con\nquimioterapia sistémica. Sin embargo, hasta el momento, los beneficios de tales propuestas\nterapéuticas no son claros. Aproximadamente el 60% de los TNBC de tipo basal sobreexpresan\nEGFR; sin embargo, las terapias que implican la inhibición del receptor son mayormente\ninefectivas debido al desarrollo de resistencia. Distintos mecanismos están involucrados en el\ndesarrollo de resistencia a las terapias dirigidas, como ser mutaciones en la proteína blanco o la\nredundancia y sobreactivación de vías de señalización compartidas con otros factores de\ncrecimiento.\nPor lo tanto, es fundamental diseñar terapias combinadas para TNBC que contemplen el posible\ndesarrollo de resistencia. El trabajo de investigación propuesto intenta identificar alteraciones de\nvías de señalización intracelular ocasionadas por las terapias dirigidas, particularmente en lo que\nrespecta al eje ADAM17/EGFR, con el fin de establecer su posible implicancia en el desarrollo\nde resistencia.\nDado que se desconoce como es regulada la actividad y selectividad de ADAM17, se realizó un\namplio estudio mediante shRNA para dilucidar como se regula el clivaje de and PPP1R14D\nregulan el clivaje de TGFa, AREG y HB-EGF sin afectar la actividad proteasa de ADAM17. La\ninhibición del eje ADAM17/EGFR sería beneficioso para el tratamiento del TNBC. Nuestros\nestudios in vitro revelaron que células MDA MB 231 knockout para PKC? and PPP1R14D no\npresentan sobreactivación de RTKs, sugiriendo que en estos modelos podría verse potenciada la\neficacia terapéutica de la inhibición del eje ADAM17/EGFR.\nSin embargo, cuando las mismas células fueron inyectadas a ratones, produjeron un fenotipo de\ntumor agresivo y metastásico, asociado a la reactivación de vías de señalización intracelular\ncomo las mediadas por ERK y PI3K/Akt. Ello se asoció a un aumento de la expresión y\nactivación de distintas RTKs, incluido el EGFR como así también de Akt.\nEstos resultados sugieren la activación alternativa de vías de señalización que permiten que las\ncélulas tumorales proliferen y produzcan metástasis

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context

KSHV Rta Promoter Specification and Viral Reactivation

Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi’s sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation

Dynamics of DNA methylation in differentiating hematopoietic cells

The methylation of CpG dinucleotides represents an epigenetic mark that is crucial for regulating the normal progression of numerous biological processes including development and cell differentiation. During the last decade, it became increasingly clear that methylation patterns are not static but may adapt to various cellular requirements. Regarding normal somatic cells, the dynamic of DNA methylation including its extent throughout the genome as well as its implication in cellular differentiation is largely unknown. In the context of the present thesis, it was demonstrated that several cell type or cell lineage specific genes harboured a specific methylation profile. Interestingly, those differences in DNA methylation were mostly confined to regions upstream or downstream of the core promoter and preferentially affected CpG poor DNA regions. The gene regulatory relevance of DNA sequences affected by dynamical alterations in the methylation pattern, may be studied by means of transient transfection assays. For this purpose, a novel CpG free luciferase reporter vector was designed that provides a simple and robust tool for analysing effects of DNA methylation within CpG poor as well as CpG rich DNA stretches on gene expression. As particularly the regulated and active removal of methyl CpG marks still remains controversial, the major aim of the present work was the characterization of this epigenetic phenomenon in a natural setting of post mitotic cells: the proliferation independent differentiation of human peripheral blood monocytes into dendritic cells or macrophages, respectively. Using a global, comparative CpG methylation profiling approach that was directed to detect differentially methylated regions in CpG rich as well as CpG poor DNA stretches, 45 examples for active demethylation were identified. The validation by a bisulfite conversion based technique and the characterization of a selected subset revealed that DNA demethylation was not restricted to promoter regions and that the time course varied for individual CpGs. Irrespective of their location, the removal of methylated cytosines strictly coincided with the appearance of activating histone marks indicating the presence of cis acting elements. Since demethylation events were highly reproducible between monocyte derived dendritic cells from distinct donors, the present data suggest that active demethylation is a precisely targeted process. The comparison of the global methylation data with the genome wide mRNA expression profiles demonstrated that active DNA demethylation is not always directly followed by transcriptional activation. Probably, gene activation is a multilevel process that is dependent on various genetic and epigenetic factors. Thereby, CpG demethylation seems to be a necessary prerequisite for priming the chromatin structure for transcription factor binding

Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells

Numerous mutations contribute to tumorigenesis of cancer cells. For most of them it remains unclear whether they are driver or passenger mutations. A classic knock-out to study their function in cancer cells used to take a lot of effort. The CRISPR/Cas-system can be used as a programmable “genome editing” tool. In this work, oncogenes have been inactivated with the CRISPR/Cas-system. Considering off-targets, Streptococcus pyogenes sgRNAs can be designed for 88% of the known cancer mutations. The activity of 15 sgRNAs, targeting 13 mutations in proto-oncogenes (deletions, insertions and point mutations), has been tested with a RFP-GFP-reporter plasmid. For 13 sgRNAs, activity prediction scores correlated with measured activity. Furthermore, sgRNAs have shown preferential binding to mutated versions of targeted proto-oncogene sequence and did not induce double strand breaks in the wild type sequence. For 10 sgRNAs, the activity against their target sequence has been more than 4 times higher than against the wild type sequence. Most of those sgRNAs target insertions or deletions and fewer target point mutations. Permanent knock-out of three mutated proto-oncogenes NPM1, BRAF and PIK3CA has been achieved with a lentiviral expression of CRISPR/Cas. Accordingly, effects on proliferation and phenotype have been studied. Knock-out of NPM1 c.863_864insTCTG mutation has been studied in heterozygous mutated OCI AML3 cell line. Proliferation was strongly inhibited by the corresponding sgRNA. Cells arrested in G0/1-phase of cell cycle (77%) compared to control cells (56%), although no difference was observed for sub-G1 phase, indicating no induction of apoptosis. Cells treated with NPM1 sgRNA had 88% reduced expression of NPM1 c.863_864insTCTG mRNA as well as less cytoplasmic localization of nucleophosmin as assessed by immunostaining. The activity of sgRNA has been confirmed by deep sequencing, showing a shift of wild type to mutated allele ratio from 51:49 to 68:32. This effect was enhanced by the additional treatment with the NHEJ inhibitor SCR7. A BRAFV600E sgRNA was tested in homozygously mutated melanoma cell lines A-375 and SK MEL-28. No differences were detected in comparison to controls. However, in the CRC cell line RKO, heterozygous for BRAFV600E and PIK3CAH1047R, proliferation was inhibited through sgRNAs against either BRAF or PIK3CA. A combination of both had no synergistic effect on proliferation. Activity and specificity of the sgRNA targeting BRAF were confirmed by deep sequencing, while the PIK3CA sgRNA showed a moderate induction of double strand breaks also in the wild type allele. The relation of wild type to mutated allele of BRAF was changed from 32:68 before treatment to 51:49 afterwards. This effect can be explained by a “re mutation” to the wild type after DSB via HDR with wild type sister chromatid as template. This effect was observed for PIK3CA sgRNA to a lesser extent. In conclusion, these results show the applicability of the CRISPR/Cas-system for the inactivation of mutated proto-oncogenes.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIIIIn Krebszellen tritt eine Vielzahl von Mutationen auf. Für den Großteil der Mutationen ist ungeklärt, ob es sich um krebsverursachende oder passagere Mutationen handelt. Ein gezieltes Ausschalten (Knock-out) dieser Gene zur Untersuchung ihrer Funktion in Krebszellen war bisher mit großem Aufwand verbunden. Das CRISPR/Cas-System lässt sich als programmierbares „Genome-editing“ Werkzeug einsetzen und wurde in der vorliegenden Arbeit verwendet, um gezielt mutierte Protoonkogene zu inaktivieren. Für 88% der bekannten, in Krebszellen auftretenden Mutationen lassen sich, unter Berücksichtigung von off-targets, Streptococcus pyogenes sgRNAs entwerfen. Mit Hilfe eines RFP-GFP-Reporter-Plasmides wurde die Aktivität von 15 sgRNAs gegen 13 Mutationen (Deletionen, Insertionen und Punktmutationen) in Protoonkogenen überprüft. Für 13 der sgRNAs zeigte sich eine Aktivität, die mit der Vorhersage durch den Algorithmus korrelierte. Außerdem wurde gezeigt, dass die sgRNAs spezifisch genug binden, um zwar bei der mutierten Sequenz eines Protoonkogens, jedoch nicht bei der Wildtyp-Sequenz Doppelstrangbrüche zu erzeugen. Unter den sgRNAs waren 10 mit mehr als 4-fach höherer Aktivität bei komplett übereinstimmender Zielsequenz gegenüber der Wildtyp-Sequenz. Diese spezifischen sgRNAs waren vor allem gegen Insertions- oder Deletionsmutationen gerichtet, einige auch gegen Punktmutationen. Durch permanente, lentivirale Expression von CRISPR/Cas wurden die Effekte eines Knock-out von drei mutierten Protoonkogenen, NPM1, BRAF und PIK3CA, auf das Wachstum und phänotypische Aspekte humaner Krebszelllinien untersucht. Ein Knock-out der NPM1 c.863_864insTCTG Mutation wurde in heterozygot mutierten OCI AML3 Zellen untersucht, es zeigte sich eine starke Proliferationshemmung. In der Zellzyklusanalyse trat ein G0/1-Arrest dieser Zellen (77%) im Vergleich mit Kontroll-Zellen (56%) auf, jedoch keine Unterschiede in der sub-G1-Analyse, sodass nicht von einer vermehrten Apoptose auszugehen ist. Die mit sgRNA behandelten OCI-AML3 Zellen zeigten sowohl eine um 88% verminderte NPM1 c.863_864insTCTG mRNA-Expression als auch verminderte zytoplasmatische Sublokalisation des Nucleophosmins in der Immunfärbung. Die hohe Aktivität der gRNA gegen mutiertes NPM1 wurde durch Deep Sequencing bestätigt, außerdem hat sich das Verhältnis vom Wildtyp- zu mutiertem Allel von 51:49 zu 68:32 verschoben. Dieser Effekt wurde durch Zugabe des NHEJ-Hemmstoffes SCR7 noch verstärkt. Die sgRNA gegen BRAFV600E wurde in den homozygot mutierten Melanom-Zelllinien A-375 und SK-MEL-28 getestet. Bei Proliferationsversuchen zeigten sich keine Unterschiede im Vergleich zu Kontrollzellen. In der kolorektalen Krebszelllinie RKO, die heterozygot BRAFV600E und PIK3CAH1047R ist, zeigte sich bei der Testung von sgRNAs gegen BRAF, PIK3CA und Kombination beider sgRNAs eine Wachstumshemmung. Jedoch lag kein synergistischer Effekt bei sgRNA-Kombination vor. Zudem bestätigten sich Aktivität und Spezifität der sgRNA gegen BRAF im Deep Sequencing, während die sgRNA gegen PIK3CA in mäßigem Umfang Doppelstrangbrüche im Wildtyp-Allel verursachte. Das Verhältnis vom Wildtyp- zu mutiertem BRAF Allel verschob sich von 32:68 ohne sgRNA zu 51:49 nach sgRNA-Behandlung. Eine mögliche Erklärung dieser Beobachtung ist die Rückmutation zum Wildtyp-Allel nach Doppelstrangbruch mit Hilfe homologer Rekombination durch das Wildtyp-Schwesterchromatid. Für PIK3CA konnte dieser Effekt in schwächerem Ausmaß ebenfalls beobachtet werden. Zusammengefasst zeigen diese Ergebnisse, dass das CRISPR/Cas-System zur Inaktivierung mutierter Protoonkogene genutzt werden kann.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VII

POLYCISTRONIC HSV VECTORS FOR THE DIFFERENTIATION OF EMBRYONIC STEM CELLS TOWARD A CARDIAC LINEAGE

Cardiovascular disease is the leading cause of death in developed countries, but we lack the ability to regenerate cardiac tissue. Cell-based therapy holds promise to repopulate a damaged heart with functional cardiomyocytes. Developing technologies to produce cells for transplantation is key to the success of this approach. Pluripotent stem cells (PSC) are an ideal starting material for cell-based therapies because they can be expanded indefinitely in culture and their plasticity gives them the potential to regenerate any tissue or organ. The issue of teratoma formation in the host may be avoided by devising methods to differentiate PSC toward a desired lineage before transplantation. Because the heart is vital for life, and the biggest source of human morbidity and mortality, in vitro differentiation of PSC into cardiomyocytes for cell-based treatment of heart disease is an area of intense research. Exogenous expression of vital cardiogenic genes in PSC can be a powerful tool. Transduction of PSC with recombinant viral vectors can deliver genes to activate cardiac programming and drive differentiation toward a cardiac lineage. Replication defective HSV vectors efficiently transduce PSC and can be engineered to express genes that alter the cellular differentiation program. The goal of this research was to develop highly defective HSV vectors to express multiple cardiac transcription factors in embryonic stem cells to increase their cardiogenic potential. Vectors vββG4Nk, vG4Nk, and vGTM were engineered to express GATA4 and NKX2.5 or GATA4, TBX5, and MEF2c in PSC with high efficiency and low toxicity. Transduction of mESC with these vectors induced the expression of endogenous genes that are vital for cardiogenesis. Differentiation of mESC transduced with cardiogenic HSV vectors had a positive impact on terminal cardiomyocyte differentiation, producing many more embryoid bodies with beating cardiomyocytes than those transduced with control vectors. In addition, we found that delaying the drastic dilution of viral genomes that occurs over the time interval to terminal differentiation could enhance the outcome. Our results indicate that infection of mESC with cardiogenic HSV vectors has long reaching effects on mESC differentiation, supporting the suggestion that HSV vectors can be a useful tool for producing lineage related changes in differentiating PSC to generate specialized cell types

Identification of neuronal caspases and involvement of death domain proteins in neuronal apoptosis

Apoptosis is implicated in both developmental and disease related neuronal loss. The characteristic morphological changes that define apoptosis appear to result from the activation of members of a class of cysteine proteases known as caspases. While identification of the relevant caspases and their means of regulation have been investigated in many cell types, the corresponding pathways in neurones are still unclear. One activating mechanism in non neuronal cell types depends upon caspase-8 autocatalytic activation upon aggregation induced by interactions with protein domains referred to as death domains (DD) and death effector domains (DED). The aim of this study was the identification of neuronal caspases and potential DD/DED upstream regulators. These were studied in a model of developmental neuronal apoptosis, rat P1 superior cervical ganglion (SCG) neurones which are dependent upon nerve growth factor (NGF) for survival. The screening of a rat brain library for DD/DED encoding cDNAs resulted in the cloning of the rat homologue of FADD. Wild type (WT) FADD and a dominant negative (DN) FADD mutant were overexpressed by microinjection in SCG neurones. Overexpression of FADD WT did not induce cell death in neurones in the presence of NGF, whereas both the WT and DN rescued neurones from NGF withdrawal induced apoptosis, suggesting that DDs are involved in neuronal apoptosis. The expression of several caspases and DD-related proteins was assessed in SCG neurones with an optimised RT-PCR protocol. While caspases-2, -3, -6, -7 and -9 were found to be normally expressed, caspase-8, a known partner of FADD in non neuronal systems, was absent. Furthermore, the expression of caspase-8 and DD-related mRNAs such as FADD, Fas and Fas ligand were not induced after NGF withdrawal. These findings suggest a regulatory role for DD proteins in SCG neuronal apoptosis, which is different from the Fas-FADD-caspase-8 pathway described for non neuronal cells