- Email: [email protected]
664. Generation of Epstein Barr Virus Specific Cytotoxic T Lymphocytes (EBV-CTLs) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)
GENE SILENCING that toxicity was diminished when Artemis was regulated by a lower strength PGK promoter ( Molecular Therapy 16(S1) S226, 2008). These results demonstrate the necessity of establishing conditions that provide Artemis expression at a level that is non-toxic but nonetheless sufficient to complement Artemis deficiency. In this study, we recovered the human Artemis promoter region and explored the possibility of employing innate regulation of human Artemis cDNA using its own endogenous promoter sequence. A one kilobase DNA sequence upstream of the human Artemis gene was PCR amplified from genomic DNA to recover the Artemis promoter (APro). The one kb APro sequence conferred significant expression in vitro when luciferase expression constructs were transfected into HEK 293 cells. Deletion constructs were tested to identify the minimal sequence required for human Artemis promoter activity, and the 5’ end of the Artemis message was mapped by 5’-RACE. The effectiveness of the APro sequence in mediating in vivo expression was tested by transduction with a lentiviral vector containing an APro-regulated green fluorescent protein (GFP) sequence. After transplantation with APro-GFP transduced donor marrow, GFP expression was observed in several hematopoietic lineages of recipient mice at reduced mean fluorescence intensity compared to control mice transplanted with EF1α-GFP transduced cells. APro-regulated GFP expression was sustained in secondary transplant recipients. We conclude that the human Artemis promoter provides moderate levels of expression in vitro and in vivo after lentiviral transduction into murine hematopoietic stem cells. This promoter will therefore be useful for the purpose of establishing a clinical vector that provides Artemis expression at a non-toxic level that is nonetheless sufficient to correct the B-T- SCID-A phenotype.
Gene Silencing 663. Effect of siRNA in PEG-Coated siRNALipoplex on the Anti-PEG IgM Production as Induced by the PEG-Coated siRNA-Lipoplex
Tatsuaki Tagami,1 Kazuya Nakamura,1 Taro Shimizu,1 Tatsuhiro Ishida,1 Hiroshi Kiwada.1 1 Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, Tokushima, Japan. Polyethylene glycol (PEG) has been used to prolong the circulation time of drug carriers such as liposome, polymer and nanoparticle. The universal function of PEG in this case is the prevention of PEGcoated substances from recognition by immune system, especially by macrophages in the mononuclear phagocyte system (MPS). PEG is believed to be immunologically inert, but we and other researchers found that repeated injection of PEG-coated liposome with several days interval induced a reduction of blood circulation of second dose. This phenomenon is called the “accelerated blood clearance (ABC) phenomenon”. We recently clarified that anti-PEG IgM production induced by the first injection of PEG-coated liposome is the major cause of the phenomenon. In the present study, we investigated the issue of whether siRNA in PEG-coated siRNA-lipoplex (PSCL) effects on the anti-PEG IgM production as induced by the PSCL following intravenous injection of the lipoplex. We found that PSCL produced anti-PEG IgM production and consequently attenuated the blood circulation time of second dose PEG-coated naked cationic liposome (PCL) (the ABC phenomenon). Anti-PEG IgM responses to PSCL were inversely related to the PSCL dose. Interestingly, anti-PEG IgM responses were significantly lower for PSCL than for PCL. The studies with splenectomized mice and nude mice indicated that anti-PEG IgM response was closely related to an interaction of PSCL and PCL with the spleen in a T cell-independent manner. In addition, PSCL induced apoptosis on IgM-expressing splenic cells more strongly than PCL, suggesting that siRNA in the PSCL rather Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
attenuated anti-PEG IgM production by causing apoptosis in the splenic cells including B cells. In conclusion, our study indicating that PEG-coated cationic liposome (PCL) needs to be used with caution for applications involving siRNA-based therapeutics, increases the importance of evaluating the carrier-mediated effect in siRNA delivery system.
664. Generation of Epstein Barr Virus Specific Cytotoxic T Lymphocytes (EBV-CTLs) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)
Biagio De Angelis,1 Gianpietro Dotti,1 Concetta Quintarelli,1 Leslie E. Huye,1 Lan Zhang,1 Ming Zhang,1 Helen E. Heslop,1 Malcolm K. Brenner,1 Cliona M. Rooney,1 Barbara Savoldo.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant and solid organ transplant recipients is safe and effective for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD). However, CTLs expansion, persistence and efficacy can be limited by immunosuppressive drugs, which can often be tapered in patients developing PTLD, but not completely withdrawn due to the risk of graft rejection. One of the most used immunosuppressive agents is FK506 whose effects are highly dependent on binding of FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506. We therefore hypothesize that EBV-CTLs can be rendered resistant to FK506 by knocking down FKBP12, using a small interfering RNA (siRNA) stably expressed from a retroviral vector. After extensive screening of potential target sequences, we identified by Western blotting that siRNA4 knocked down>90% of FKBP12 in CTLs. We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively. These vectors were used to transduce established CTLs generated from 7 EBV-seropositive donors. Transduction efficiency was 46%±22% for siRNA4 and 55%±27% for irrelevant-siRNA. We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures. Using a thymidine uptake assay, we found that the inhibition of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41%±4% inhibition vs 74%±2%, respectively). To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+CTLs were stimulated weekly with autologous LCL, low dose IL-2 (20U/mL) and in the presence or absence of FK506 (5ng/ml). The proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46%±22% to 89%±5% after 5 stimulations) but also numerically (median fold expansion: 39, range 5-111). In contrast, control EBVCTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 55%±27% to 57%±23%) and CTLs ceased to proliferate (median fold expansion: 2, range 0-11). In addition, siRNA4+ CTLs kept their MHC-restricted cytotoxic activity, assessed by Cr release assay (66%±22% killing of autologous LCL S253
GENE SILENCING vs 16%±12% of allogenic LCL at an E:T ratio of 20:1). Finally, in 4 CTL lines, we ensured by that the broad EBV reactivity necessary for effective activity was maintained using tetramer against lytic and latent EBV antigen. We confirmed that EBV responses were functional for both lytic and latent antigens with IFN-g ELISPOT assays. We are currently evaluating the in vivo expansion of siRNA4+CTLs in the presence of FK506. In conclusion, we have developed a strategy that produces EBV-CTLs resistant to FK506. This strategy may be beneficial to improve EBV immune reconstitution in patients at high risk of developing post transplant lymphoma.
665. Multiplexing of snoRNAs for HIV-1 Inhibition
Jane Zhang,1 John J. Rossi.1 Graduate School of Biological Sciences, Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA.
1
Small nucleolar RNAS (snoRNAs) are a group of small RNAs that are shuttled between the nucleus and the nucleolus to regulate RNA gene expression. Early HIV gene expression is known to occur within the nucleolus with the nucleolar trafficking of Tat and other regulatory factors to be critical for HIV-1 viral replication. We have designed various HIV-1 inhibitory molecules that are inserted onto the stem of a C/D box snoRNA. By doing so, we can localize these inhibitory RNAs to the nucleolus to capture the nucleolar trafficking of HIV proteins or with the use of a ribozyme, interfere with the nucleolar trafficking of HIV unspliced RNAs. To minimize viral escape mutants to a single antiviral agent, we have incorporated into a tri-cistronic miRNA expression system 3 snoRNA-chimeric RNA molecules: a Rev decoy, a U5 HIV-1 targeting hammerhead ribozyme, and a TAR decoy. Alternatively, we have also designed a construct that replaces one of the snoRNA molecules with a miRNA mimic targeting the HIV tat/rev transcript. Our approach disrupts the HIV replication cycle by simultaneously targeting HIV proteins and RNAs within the nucleolus. This novel strategy of multiplexing snoRNAs within a single gene construct represents a new approach for inhibiting HIV replication in a gene therapy setting.
666. A Combination of an Effective shRNA and the Weak H1 Promoter Is Required for Efficient and Non-Toxic Antiviral Effects in Hepatitis B Virus Transgenic Mice with Heavy Viral Load
Cheng-Pu Sun,1 Chun-Chi Chen,1 Tze-Hui Wu,1 Pin-Yi Wu,1 MiHua Tao.1 1 Institute of Biomedical Sciences, Taipei, Taiwan, Province of China.
Background & Aims: Using double-stranded adeno-associated virus serotype 8 (dsAAV8) as a delivery vehicle, we previously showed that HBV-S1 is the only shRNA which can achieve profound and sustained HBV suppression in hepatitis B virus (HBV) transgenic mice without causing toxicity (1, 2). However, Grimm et al. reported that shRNAs delivered by a similar dsAAV8 vector caused acute hepatitis and death in the majority of animals (3). This report aims to identify factors which might cause differences in these two studies. Methods: We noted that different promoters (U6 in Grimm’s and H1 in ours) and shRNAs were used in these two studies. For a direct comparison, we constructed dsAAV8 vectors containing U6 or H1 promoters to drive expression of HBV-S1 or two anti-HBV shRNAs used in Grimm’s study, the non-toxic sAg19 and toxic sAg25. These dsAAV8 vectors were compared for their anti-HBV efficacy and toxicity using our HBV transgenic mice, which produce at least 10fold more serum HBV than mice used in the other study. Results: In vitro co-transfection and hydrodynamic assays showed that U6 promoter produced more shRNAs than H1 promoter, and under the S254
weak H1 promoter HBV-S1 is equal to sAg25 but better than sAg19 in HBV suppression. When testing in HBV transgenic mice, all shRNAs expressed by dsAAV8 vectors with U6 promoter were highly toxic, and induced only transient HBV suppression. In contrast, none of the dsAAV8 vectors containing H1 promoter caused apparent liver damage, but only HBVS1, but not sAg19 or sAg25, could achieve profound and persistent HBV suppression under the weaker H1 promoter. Activation of type I interferon and down-regulation of endogenous miRNAs were observed in mice treated with U6-based dsAAV8 vectors which developed severe liver toxicity. However, the fact that toxicity remained in STAT-1-deficient mice, which are not responsive to IFN signaling, argues against the importance of IFN pathway in the RNAi-mediated toxicity. Conclusions: We conclude that a combination of an effective shRNA with the weak H1 promoter in dsAAV8 vectors is required to achieve effective and not-toxic anti-HBV effect in animal models with heavy viral load. 1. Chen et al., Gene Ther. 2007, 14: 11-9. 2. Chen et al., Mol Ther. 2009, 17: 352-9. 3. Grimm et al., Nature. 2006, 441: 537-41.
667. Therapeutic Effect of Ribbon Type NFkB Decoy Oligonucleotides on Inflammatory Bowel Disease Model Rats
Kazunari Ozaki,1,2 Hirofumi Makino,1 Yasushi Takeya,1 Takashi Miyake,1 Hiromi Rakugi,2 Ryuichi Morishita.1 1 Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Background: The pathogenesis of inflammatory bowel disease (IBD) involves with local expression of inflammatory cytokines. Nuclear factor-kB (NFkB), a key transcriptional factor, plays an important role in the coordinated transactivation of the inflammatory cytokines. We have developed decoy oligonucleotide (ODN) targeting NF-kB which offers a novel gene therapy approach for prevention and treatment for inflammatory diseases. In addition, we developed a novel Ribbon-type NFkB decoy ODN (RODN) with loop ends that increase the stability. RODN is resistant to digestion by endonucleases, whereas double-stranded decoy ODN ( dsODN) is easily digested. Aim: To investigate the therapeutic potential of a locally administered RODN in dextran sulphate sodium (DSS)-induced colitis, experimental model of IBD. Methods: We administered NFkB decoys (RODN, dsODN) in rats with DSS-induced IBD. Scrambled decoy was used as a control. Mixture of decoy ODNs and echo contrast microbubble (Optison) was administrated intrarectally with the use of ultrasound on day 0. The therapeutic effects of these ODNs were evaluated on day 3,7,10 with the survival rate, length of large intestine, and disease activity index (DAI), representing clinical symptoms such as body weight loss, bleeding in stool, and stool consistency. Furthermore, expression of inflammatory cytokines was assessed using real-time RT PCR. Results: The survival rate, the length of large intestine and DAI significantly improved in the rat treated with dsODN or RODN than in the control group. Although expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the IBD group significantly increased, treatment with dsODN or RODN significantly reduced expression of these cytokines. In addition, RODN was more effective than dsODN. Conclusions: These data suggest that intracolonic administration of RODN may be effective in DSS-induced colitis, experimental model of IBD, and support the potential use of RODN that increase the stability as a future therapeutics in human IBD.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Gene Silencing 663. Effect of siRNA in PEG-Coated siRNALipoplex on the Anti-PEG IgM Production as Induced by the PEG-Coated siRNA-Lipoplex
Tatsuaki Tagami,1 Kazuya Nakamura,1 Taro Shimizu,1 Tatsuhiro Ishida,1 Hiroshi Kiwada.1 1 Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, Tokushima, Japan. Polyethylene glycol (PEG) has been used to prolong the circulation time of drug carriers such as liposome, polymer and nanoparticle. The universal function of PEG in this case is the prevention of PEGcoated substances from recognition by immune system, especially by macrophages in the mononuclear phagocyte system (MPS). PEG is believed to be immunologically inert, but we and other researchers found that repeated injection of PEG-coated liposome with several days interval induced a reduction of blood circulation of second dose. This phenomenon is called the “accelerated blood clearance (ABC) phenomenon”. We recently clarified that anti-PEG IgM production induced by the first injection of PEG-coated liposome is the major cause of the phenomenon. In the present study, we investigated the issue of whether siRNA in PEG-coated siRNA-lipoplex (PSCL) effects on the anti-PEG IgM production as induced by the PSCL following intravenous injection of the lipoplex. We found that PSCL produced anti-PEG IgM production and consequently attenuated the blood circulation time of second dose PEG-coated naked cationic liposome (PCL) (the ABC phenomenon). Anti-PEG IgM responses to PSCL were inversely related to the PSCL dose. Interestingly, anti-PEG IgM responses were significantly lower for PSCL than for PCL. The studies with splenectomized mice and nude mice indicated that anti-PEG IgM response was closely related to an interaction of PSCL and PCL with the spleen in a T cell-independent manner. In addition, PSCL induced apoptosis on IgM-expressing splenic cells more strongly than PCL, suggesting that siRNA in the PSCL rather Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
attenuated anti-PEG IgM production by causing apoptosis in the splenic cells including B cells. In conclusion, our study indicating that PEG-coated cationic liposome (PCL) needs to be used with caution for applications involving siRNA-based therapeutics, increases the importance of evaluating the carrier-mediated effect in siRNA delivery system.
664. Generation of Epstein Barr Virus Specific Cytotoxic T Lymphocytes (EBV-CTLs) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)
Biagio De Angelis,1 Gianpietro Dotti,1 Concetta Quintarelli,1 Leslie E. Huye,1 Lan Zhang,1 Ming Zhang,1 Helen E. Heslop,1 Malcolm K. Brenner,1 Cliona M. Rooney,1 Barbara Savoldo.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant and solid organ transplant recipients is safe and effective for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD). However, CTLs expansion, persistence and efficacy can be limited by immunosuppressive drugs, which can often be tapered in patients developing PTLD, but not completely withdrawn due to the risk of graft rejection. One of the most used immunosuppressive agents is FK506 whose effects are highly dependent on binding of FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506. We therefore hypothesize that EBV-CTLs can be rendered resistant to FK506 by knocking down FKBP12, using a small interfering RNA (siRNA) stably expressed from a retroviral vector. After extensive screening of potential target sequences, we identified by Western blotting that siRNA4 knocked down>90% of FKBP12 in CTLs. We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively. These vectors were used to transduce established CTLs generated from 7 EBV-seropositive donors. Transduction efficiency was 46%±22% for siRNA4 and 55%±27% for irrelevant-siRNA. We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures. Using a thymidine uptake assay, we found that the inhibition of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41%±4% inhibition vs 74%±2%, respectively). To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+CTLs were stimulated weekly with autologous LCL, low dose IL-2 (20U/mL) and in the presence or absence of FK506 (5ng/ml). The proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46%±22% to 89%±5% after 5 stimulations) but also numerically (median fold expansion: 39, range 5-111). In contrast, control EBVCTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 55%±27% to 57%±23%) and CTLs ceased to proliferate (median fold expansion: 2, range 0-11). In addition, siRNA4+ CTLs kept their MHC-restricted cytotoxic activity, assessed by Cr release assay (66%±22% killing of autologous LCL S253
GENE SILENCING vs 16%±12% of allogenic LCL at an E:T ratio of 20:1). Finally, in 4 CTL lines, we ensured by that the broad EBV reactivity necessary for effective activity was maintained using tetramer against lytic and latent EBV antigen. We confirmed that EBV responses were functional for both lytic and latent antigens with IFN-g ELISPOT assays. We are currently evaluating the in vivo expansion of siRNA4+CTLs in the presence of FK506. In conclusion, we have developed a strategy that produces EBV-CTLs resistant to FK506. This strategy may be beneficial to improve EBV immune reconstitution in patients at high risk of developing post transplant lymphoma.
665. Multiplexing of snoRNAs for HIV-1 Inhibition
Jane Zhang,1 John J. Rossi.1 Graduate School of Biological Sciences, Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA.
1
Small nucleolar RNAS (snoRNAs) are a group of small RNAs that are shuttled between the nucleus and the nucleolus to regulate RNA gene expression. Early HIV gene expression is known to occur within the nucleolus with the nucleolar trafficking of Tat and other regulatory factors to be critical for HIV-1 viral replication. We have designed various HIV-1 inhibitory molecules that are inserted onto the stem of a C/D box snoRNA. By doing so, we can localize these inhibitory RNAs to the nucleolus to capture the nucleolar trafficking of HIV proteins or with the use of a ribozyme, interfere with the nucleolar trafficking of HIV unspliced RNAs. To minimize viral escape mutants to a single antiviral agent, we have incorporated into a tri-cistronic miRNA expression system 3 snoRNA-chimeric RNA molecules: a Rev decoy, a U5 HIV-1 targeting hammerhead ribozyme, and a TAR decoy. Alternatively, we have also designed a construct that replaces one of the snoRNA molecules with a miRNA mimic targeting the HIV tat/rev transcript. Our approach disrupts the HIV replication cycle by simultaneously targeting HIV proteins and RNAs within the nucleolus. This novel strategy of multiplexing snoRNAs within a single gene construct represents a new approach for inhibiting HIV replication in a gene therapy setting.
666. A Combination of an Effective shRNA and the Weak H1 Promoter Is Required for Efficient and Non-Toxic Antiviral Effects in Hepatitis B Virus Transgenic Mice with Heavy Viral Load
Cheng-Pu Sun,1 Chun-Chi Chen,1 Tze-Hui Wu,1 Pin-Yi Wu,1 MiHua Tao.1 1 Institute of Biomedical Sciences, Taipei, Taiwan, Province of China.
Background & Aims: Using double-stranded adeno-associated virus serotype 8 (dsAAV8) as a delivery vehicle, we previously showed that HBV-S1 is the only shRNA which can achieve profound and sustained HBV suppression in hepatitis B virus (HBV) transgenic mice without causing toxicity (1, 2). However, Grimm et al. reported that shRNAs delivered by a similar dsAAV8 vector caused acute hepatitis and death in the majority of animals (3). This report aims to identify factors which might cause differences in these two studies. Methods: We noted that different promoters (U6 in Grimm’s and H1 in ours) and shRNAs were used in these two studies. For a direct comparison, we constructed dsAAV8 vectors containing U6 or H1 promoters to drive expression of HBV-S1 or two anti-HBV shRNAs used in Grimm’s study, the non-toxic sAg19 and toxic sAg25. These dsAAV8 vectors were compared for their anti-HBV efficacy and toxicity using our HBV transgenic mice, which produce at least 10fold more serum HBV than mice used in the other study. Results: In vitro co-transfection and hydrodynamic assays showed that U6 promoter produced more shRNAs than H1 promoter, and under the S254
weak H1 promoter HBV-S1 is equal to sAg25 but better than sAg19 in HBV suppression. When testing in HBV transgenic mice, all shRNAs expressed by dsAAV8 vectors with U6 promoter were highly toxic, and induced only transient HBV suppression. In contrast, none of the dsAAV8 vectors containing H1 promoter caused apparent liver damage, but only HBVS1, but not sAg19 or sAg25, could achieve profound and persistent HBV suppression under the weaker H1 promoter. Activation of type I interferon and down-regulation of endogenous miRNAs were observed in mice treated with U6-based dsAAV8 vectors which developed severe liver toxicity. However, the fact that toxicity remained in STAT-1-deficient mice, which are not responsive to IFN signaling, argues against the importance of IFN pathway in the RNAi-mediated toxicity. Conclusions: We conclude that a combination of an effective shRNA with the weak H1 promoter in dsAAV8 vectors is required to achieve effective and not-toxic anti-HBV effect in animal models with heavy viral load. 1. Chen et al., Gene Ther. 2007, 14: 11-9. 2. Chen et al., Mol Ther. 2009, 17: 352-9. 3. Grimm et al., Nature. 2006, 441: 537-41.
667. Therapeutic Effect of Ribbon Type NFkB Decoy Oligonucleotides on Inflammatory Bowel Disease Model Rats
Kazunari Ozaki,1,2 Hirofumi Makino,1 Yasushi Takeya,1 Takashi Miyake,1 Hiromi Rakugi,2 Ryuichi Morishita.1 1 Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Background: The pathogenesis of inflammatory bowel disease (IBD) involves with local expression of inflammatory cytokines. Nuclear factor-kB (NFkB), a key transcriptional factor, plays an important role in the coordinated transactivation of the inflammatory cytokines. We have developed decoy oligonucleotide (ODN) targeting NF-kB which offers a novel gene therapy approach for prevention and treatment for inflammatory diseases. In addition, we developed a novel Ribbon-type NFkB decoy ODN (RODN) with loop ends that increase the stability. RODN is resistant to digestion by endonucleases, whereas double-stranded decoy ODN ( dsODN) is easily digested. Aim: To investigate the therapeutic potential of a locally administered RODN in dextran sulphate sodium (DSS)-induced colitis, experimental model of IBD. Methods: We administered NFkB decoys (RODN, dsODN) in rats with DSS-induced IBD. Scrambled decoy was used as a control. Mixture of decoy ODNs and echo contrast microbubble (Optison) was administrated intrarectally with the use of ultrasound on day 0. The therapeutic effects of these ODNs were evaluated on day 3,7,10 with the survival rate, length of large intestine, and disease activity index (DAI), representing clinical symptoms such as body weight loss, bleeding in stool, and stool consistency. Furthermore, expression of inflammatory cytokines was assessed using real-time RT PCR. Results: The survival rate, the length of large intestine and DAI significantly improved in the rat treated with dsODN or RODN than in the control group. Although expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the IBD group significantly increased, treatment with dsODN or RODN significantly reduced expression of these cytokines. In addition, RODN was more effective than dsODN. Conclusions: These data suggest that intracolonic administration of RODN may be effective in DSS-induced colitis, experimental model of IBD, and support the potential use of RODN that increase the stability as a future therapeutics in human IBD.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy