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581. Correcting Exon Skipping Splicing Defects in BTK RNA by Using Bifunctional Oligonucleotides
Oligonucleotide Therapeutics II Oligonucleotide Therapeutics II 580. Prevention of Airway Inflammation by Simultaneous Inhibition of NFkB and STST6 Using Chimeric Decoy Oligonucleotides in a Mouse Model of Asthma Tetsuo Miyake, Takashi Miyake, Hizuki Hamada, Ryuichi Morishita Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Suita, Japan
Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITClabeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
581. Correcting Exon Skipping Splicing Defects in BTK RNA by Using Bifunctional Oligonucleotides
C. I. Edvard Smith1, Burcu Bestas1, Janne J. Turunen1, Jesper Wengel2 1 Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden, 2 Physics, Chemistry and Pharmacy, University of Southern Denmark, Huddinge, Denmark An important percentage of disease causing mutations affects premRNA splicing. The mechanism of pre-mRNA splicing is controlled by sequence elements within the exons and introns. These sequence elements recruit the spliceosome for directing the splicing process. Mutations disrupting the sequence elements commonly result in exon exclusion or cryptic exon inclusion named as pseudoexon. The result is typically a frame-shift in the pre-mRNA causing production of partially functional or a defective protein. A common way to correct splicing mutations is to use antisense oligonucleotides (AONs) that are short complementary sequences that bind to the pre-mRNA and re-direct the splicing. X -linke d a ga mma globuline mia (X LA ) is a pr i m ar y immunodeficiency disease that is caused by the mutations in the gene named Bruton’s Tyrosine Kinase (BTK). BTK has a crucial role in B cell development and the absence of BTK introduces a developmental block at the stage where the transition between pro-B and pre-B cells takes place. A prominent percentage of splicing mutations affect pre-mRNA splicing in XLA [1]. Previously, we have published a proof-of-concept study and shown that AONs can correct a cryptic exon mutation and restore a functional BTK in a humanized transgenic mouse model both in vitro and in vivo [2]. AONs in the previous study were designed to sterically block an intronic mutation and prevent the recruitment of the splicing factors around the cryptic exon. We are here addressing a different scenario, where defective BTK is produced due to exon exclusion. This is typically due to mutations that weaken the splice sites or splicing enhancer sequences in their vicinity, and constitutes the most common type of splicing defect. Here we have designed bifunctional AONs that have a complementary binding site and a free tail region that is able to recruit splicing factors to the site in order to enhance exon inclusion. Initially, we constructed a library of BTK reporters with mutations known to cause XLA, and the bifunctional AONs were then tested in cultures of cells stably transfected with various BTK reporters for intronic mutations affecting the inclusion of BTK exons 16 and 17. These exons were selected owing to the rather small size of the corresponding introns simplifying the generation of relevant reporter cell lines. Screening of different tail sequences show that AONs designed to recruit TIA1 or TDP-43 proteins can correct the splicing defects and give rise to wild-type BTK mRNA. Optimization of the antisense and tail parts of the AONs has also revealed the importance of chemical modifications (2’-O-methyl, phosphorothioate, and/or locked nuclei acid) for enhancing the efficacy of the AONs. We have also studied the influence of combining bifunctional AONs and found that this can profoundly enhance exon inclusion. Thus, depending on the exact type of mutation single or combinatorial approaches provide different outcomes. To the best of our knowledge, this is the first time that exon inclusion has been achieved in a hematopoietic setting and experiments are currently underway to further optimize and explore the design of bifunctional AONs for potential in vivo use. 1. Bestas, B., et al., Splice-correction strategies for treatment of x-linked agammaglobulinemia. Current Allergy and Asthma Reports, 2015. 15(3): p. 510. 2. Bestas, B., et al., Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model. The Journal of Clinical Investigation, 2014. 124(9): p. 4067-81.
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Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITClabeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
581. Correcting Exon Skipping Splicing Defects in BTK RNA by Using Bifunctional Oligonucleotides
C. I. Edvard Smith1, Burcu Bestas1, Janne J. Turunen1, Jesper Wengel2 1 Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden, 2 Physics, Chemistry and Pharmacy, University of Southern Denmark, Huddinge, Denmark An important percentage of disease causing mutations affects premRNA splicing. The mechanism of pre-mRNA splicing is controlled by sequence elements within the exons and introns. These sequence elements recruit the spliceosome for directing the splicing process. Mutations disrupting the sequence elements commonly result in exon exclusion or cryptic exon inclusion named as pseudoexon. The result is typically a frame-shift in the pre-mRNA causing production of partially functional or a defective protein. A common way to correct splicing mutations is to use antisense oligonucleotides (AONs) that are short complementary sequences that bind to the pre-mRNA and re-direct the splicing. X -linke d a ga mma globuline mia (X LA ) is a pr i m ar y immunodeficiency disease that is caused by the mutations in the gene named Bruton’s Tyrosine Kinase (BTK). BTK has a crucial role in B cell development and the absence of BTK introduces a developmental block at the stage where the transition between pro-B and pre-B cells takes place. A prominent percentage of splicing mutations affect pre-mRNA splicing in XLA [1]. Previously, we have published a proof-of-concept study and shown that AONs can correct a cryptic exon mutation and restore a functional BTK in a humanized transgenic mouse model both in vitro and in vivo [2]. AONs in the previous study were designed to sterically block an intronic mutation and prevent the recruitment of the splicing factors around the cryptic exon. We are here addressing a different scenario, where defective BTK is produced due to exon exclusion. This is typically due to mutations that weaken the splice sites or splicing enhancer sequences in their vicinity, and constitutes the most common type of splicing defect. Here we have designed bifunctional AONs that have a complementary binding site and a free tail region that is able to recruit splicing factors to the site in order to enhance exon inclusion. Initially, we constructed a library of BTK reporters with mutations known to cause XLA, and the bifunctional AONs were then tested in cultures of cells stably transfected with various BTK reporters for intronic mutations affecting the inclusion of BTK exons 16 and 17. These exons were selected owing to the rather small size of the corresponding introns simplifying the generation of relevant reporter cell lines. Screening of different tail sequences show that AONs designed to recruit TIA1 or TDP-43 proteins can correct the splicing defects and give rise to wild-type BTK mRNA. Optimization of the antisense and tail parts of the AONs has also revealed the importance of chemical modifications (2’-O-methyl, phosphorothioate, and/or locked nuclei acid) for enhancing the efficacy of the AONs. We have also studied the influence of combining bifunctional AONs and found that this can profoundly enhance exon inclusion. Thus, depending on the exact type of mutation single or combinatorial approaches provide different outcomes. To the best of our knowledge, this is the first time that exon inclusion has been achieved in a hematopoietic setting and experiments are currently underway to further optimize and explore the design of bifunctional AONs for potential in vivo use. 1. Bestas, B., et al., Splice-correction strategies for treatment of x-linked agammaglobulinemia. Current Allergy and Asthma Reports, 2015. 15(3): p. 510. 2. Bestas, B., et al., Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model. The Journal of Clinical Investigation, 2014. 124(9): p. 4067-81.
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