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PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization
Hepatology, Mar 1, 2019
RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene e... more RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene expression, that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerases PAP-associated domain-containing protein 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies which phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. Also, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusions: We identify PAPD5 and PAPD7 as novel cellular host factors required for HBV RNA stabilization and as new therapeutic targets for the HBV cure. This article is protected by copyright. All rights reserved.
Alimentary Pharmacology & Therapeutics, Oct 26, 2015
UvA-DARE is a service provided by the library of the University of Amsterdam () UvA-DARE (Digital... more UvA-DARE is a service provided by the library of the University of Amsterdam () UvA-DARE (Digital Academic Repository) MicroRNA's in chronic hepatitis B and C virus infection van der Ree, M.H. Publication date 2017 Document Version Other version License Other Link to publication Citation for published version (APA): van der Ree, M. H. (2017). MicroRNA's in chronic hepatitis B and C virus infection. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website.
observed. In the present study we have evaluated a novel strategy to prevent HCV infection using ... more observed. In the present study we have evaluated a novel strategy to prevent HCV infection using a lectin, Griffithsin (GRFT), that specifically binds N-linked high mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticles (HCVpp) and HCV cell culture (HCVcc) systems. We will demonstrate that pre-incubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA +/+ -SCID mice that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected displayed a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs a... more Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.
VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the vi... more VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection, but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a S375A mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.
Author Reply: Is the HBsAg response to RO7062931 an antisense effect?
Hepatology, Aug 15, 2021
We thank Andrew Vaillant for his correspondence regarding our study of the singlestranded oligonu... more We thank Andrew Vaillant for his correspondence regarding our study of the singlestranded oligonucleotide RO7062931 in chronic hepatitis B[1] and the editor for this opportunity to clarify why we conclude that the results are evidence of target engagement. Firstly, Dr. Vaillant proposes that RO7062931 should have exhibited greater HBsAg reductions, given the efficient RO7062931 liver uptake, rapid turnover of HBsAg, and rapid protein reductions observed with Nacetylgalactosamine antisense oligonucleotides (GalNAcASOs) in other therapeutic indications. We find this reasoning unsatisfactory, primarily as there should be no expectations of similar potency for GalNAcASOs targeting different mRNA species. Numerous factors contribute to an individual agent’s in vivo potency, including target mRNAspecific factors (mRNA prevalence, tissue/cellular distribution, turnover) and underlying oligonucleotide chemical structure (affecting molecular stability, endosomal trafficking). Furthermore, HBsAg turnover itself is not necessarily rapid, given that literaturereported rates vary from 1.3 to 38 days.[2] Secondly, he suggests that preexisting/rapidly evolving escape mutants and offtarget effects (immune activation through tolllike receptor 9) likely account for the modest HBsAg response. We find both of these hypotheses to be unfounded. Presence of rapidly evolving escape mutations would result in ontreatment HBsAg rebound/breakthrough, which is not supported by our data. Preclinical studies with RO7062931 and related GalNAcconjugated molecules have demonstrated a direct correlation between inhibition of viral mRNA and reduction of HBsAg.[3] Here, specific downregulation of target mRNA— not offtarget mRNAs— was observed. While targeting nonannotated transcripts or partially matched targets cannot be ruled out, we have seen no evidence of inhibition of genes that would lead to indirect inhibition of HBsAg expression or increased turnover. Additionally, we have not found evidence in human whole blood ex vivo assays for cytokine or complement responses,[3] and cytokines and complement biomarkers in the clinical study[1] were unremarkable. In summary, the RO7062931 mechanism of action was well characterized in preclinical studies, and we stand behind a logical conclusion that the antiviral effects observed in clinic were through the same proposed RNaseHmediated antisense mechanism, therefore providing evidence of target engagement. We acknowledge that potential offtarget effects cannot be ruled out; however, if present, such effects are likely minimal and certainly not a primary driver of HBsAg response. The modest HBsAg response in clinic showed a suboptimal quantitative translation from the mouse adenoassociated virus model system, which suggests that RO7062931 was not well suited to the demands of potency, stability, and durability at the site of action in humans.
Disclaimer/Complaints regulations If you believe that digital publication of certain material inf... more Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
L'invention concerne des conjugues glucidiques d'oligonucleotides therapeutiques a base d... more L'invention concerne des conjugues glucidiques d'oligonucleotides therapeutiques a base de LNA presentant une puissance considerablement accrue, un index therapeutique plus important et une toxicite reduite.
Compositions et méthodes pour la modulation de l'expression de fgfr3
L'invention concerne des oligonucleotides qui ciblent et s'hybrident a des molecules d... more L'invention concerne des oligonucleotides qui ciblent et s'hybrident a des molecules d'acide nucleique codant pour FGFR3, entrainant une expression reduite de FGFR3. Une reduction de l'expression aberrante de FGFR3 est benefique pour le traitement de certains troubles medicaux, tels que l'achondroplasie.
RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene e... more RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene expression, that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerases PAP-associated domain-containing protein 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies which phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. Al...
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs a... more Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.
VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the vi... more VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection, but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a S375A mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.
Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle prog... more Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 -propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1 C subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1 C , suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G 1 -S transition.
Miravirsen is a β- d -oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide... more Miravirsen is a β- d -oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC 50 ) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the pres...
Proceedings of the National Academy of Sciences, 2008
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansio... more Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.
PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization
Hepatology, Mar 1, 2019
RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene e... more RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene expression, that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerases PAP-associated domain-containing protein 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies which phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. Also, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusions: We identify PAPD5 and PAPD7 as novel cellular host factors required for HBV RNA stabilization and as new therapeutic targets for the HBV cure. This article is protected by copyright. All rights reserved.
Alimentary Pharmacology & Therapeutics, Oct 26, 2015
UvA-DARE is a service provided by the library of the University of Amsterdam () UvA-DARE (Digital... more UvA-DARE is a service provided by the library of the University of Amsterdam () UvA-DARE (Digital Academic Repository) MicroRNA's in chronic hepatitis B and C virus infection van der Ree, M.H. Publication date 2017 Document Version Other version License Other Link to publication Citation for published version (APA): van der Ree, M. H. (2017). MicroRNA's in chronic hepatitis B and C virus infection. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website.
observed. In the present study we have evaluated a novel strategy to prevent HCV infection using ... more observed. In the present study we have evaluated a novel strategy to prevent HCV infection using a lectin, Griffithsin (GRFT), that specifically binds N-linked high mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticles (HCVpp) and HCV cell culture (HCVcc) systems. We will demonstrate that pre-incubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA +/+ -SCID mice that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected displayed a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs a... more Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.
VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the vi... more VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection, but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a S375A mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.
Author Reply: Is the HBsAg response to RO7062931 an antisense effect?
Hepatology, Aug 15, 2021
We thank Andrew Vaillant for his correspondence regarding our study of the singlestranded oligonu... more We thank Andrew Vaillant for his correspondence regarding our study of the singlestranded oligonucleotide RO7062931 in chronic hepatitis B[1] and the editor for this opportunity to clarify why we conclude that the results are evidence of target engagement. Firstly, Dr. Vaillant proposes that RO7062931 should have exhibited greater HBsAg reductions, given the efficient RO7062931 liver uptake, rapid turnover of HBsAg, and rapid protein reductions observed with Nacetylgalactosamine antisense oligonucleotides (GalNAcASOs) in other therapeutic indications. We find this reasoning unsatisfactory, primarily as there should be no expectations of similar potency for GalNAcASOs targeting different mRNA species. Numerous factors contribute to an individual agent’s in vivo potency, including target mRNAspecific factors (mRNA prevalence, tissue/cellular distribution, turnover) and underlying oligonucleotide chemical structure (affecting molecular stability, endosomal trafficking). Furthermore, HBsAg turnover itself is not necessarily rapid, given that literaturereported rates vary from 1.3 to 38 days.[2] Secondly, he suggests that preexisting/rapidly evolving escape mutants and offtarget effects (immune activation through tolllike receptor 9) likely account for the modest HBsAg response. We find both of these hypotheses to be unfounded. Presence of rapidly evolving escape mutations would result in ontreatment HBsAg rebound/breakthrough, which is not supported by our data. Preclinical studies with RO7062931 and related GalNAcconjugated molecules have demonstrated a direct correlation between inhibition of viral mRNA and reduction of HBsAg.[3] Here, specific downregulation of target mRNA— not offtarget mRNAs— was observed. While targeting nonannotated transcripts or partially matched targets cannot be ruled out, we have seen no evidence of inhibition of genes that would lead to indirect inhibition of HBsAg expression or increased turnover. Additionally, we have not found evidence in human whole blood ex vivo assays for cytokine or complement responses,[3] and cytokines and complement biomarkers in the clinical study[1] were unremarkable. In summary, the RO7062931 mechanism of action was well characterized in preclinical studies, and we stand behind a logical conclusion that the antiviral effects observed in clinic were through the same proposed RNaseHmediated antisense mechanism, therefore providing evidence of target engagement. We acknowledge that potential offtarget effects cannot be ruled out; however, if present, such effects are likely minimal and certainly not a primary driver of HBsAg response. The modest HBsAg response in clinic showed a suboptimal quantitative translation from the mouse adenoassociated virus model system, which suggests that RO7062931 was not well suited to the demands of potency, stability, and durability at the site of action in humans.
Disclaimer/Complaints regulations If you believe that digital publication of certain material inf... more Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
L'invention concerne des conjugues glucidiques d'oligonucleotides therapeutiques a base d... more L'invention concerne des conjugues glucidiques d'oligonucleotides therapeutiques a base de LNA presentant une puissance considerablement accrue, un index therapeutique plus important et une toxicite reduite.
Compositions et méthodes pour la modulation de l'expression de fgfr3
L'invention concerne des oligonucleotides qui ciblent et s'hybrident a des molecules d... more L'invention concerne des oligonucleotides qui ciblent et s'hybrident a des molecules d'acide nucleique codant pour FGFR3, entrainant une expression reduite de FGFR3. Une reduction de l'expression aberrante de FGFR3 est benefique pour le traitement de certains troubles medicaux, tels que l'achondroplasie.
RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene e... more RG7834 is a potent orally bioavailable small-molecule inhibitor of Hepatitis B virus (HBV) gene expression, that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerases PAP-associated domain-containing protein 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies which phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. Al...
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs a... more Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.
VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the vi... more VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection, but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a S375A mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.
Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle prog... more Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 -propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1 C subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1 C , suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G 1 -S transition.
Miravirsen is a β- d -oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide... more Miravirsen is a β- d -oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC 50 ) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the pres...
Proceedings of the National Academy of Sciences, 2008
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansio... more Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.
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Papers by Søren Ottosen