- Email: [email protected]
718. PTD-Fusion Peptide as a Delivery Vehicle for siRNA To Target HIV Reservoirs
OLIGONUCLEOTIDE THERAPIES A cloning site of pSHAG-MAGIC2/pSM2 vector (Open Biosystems). IB3-1 (∆F508/W1282X) bronchial epithelial cells were transiently transfected with VCP and gp78 shRNA/siRNA constructs for selective inhibition of ERAD. CFTR levels and chloride efflux were measured by metabolic labeling and MQAE fluorescence assay, respectively. Proteasome inhibition in IB3-1 cells was carried out using PS-341 (Bortezomib/Velcade) for 6hrs. The IL8 cytokine levels were measured in IL1-β induced IB3-1 cells after ERAD or proteasome inhibition. Findings: We show here that p97/VCP and gp78 form complexes with CFTR during translocation from the ER for degradation by the cytosolic proteasome. Interference in the VCP/CFTR complex promotes accumulation of immature CFTR in the ER and partial rescue of functional chloride channels to the cell surface. Moreover, under these conditions, the cytokine whose expression is regulated by the proteasome, IL8, is reduced. Inhibition of the proteasome with bortezomib (PS-341/Velcade) also rescues CFTR and suppresses IL8 levels, but with less efficiency. Interpretation: We propose that selective inhibition of ERAD by VCP short hairpin RNA is superior to gp78 siRNA or proteasome inhibition in the correction of the ∆F508 CFTR trafficking defect.
718. PTD-Fusion Peptide as a Delivery Vehicle for siRNA To Target HIV Reservoirs Ashok Chauhan,1 Avindra Nath,1 Daivd Levy,2 Suzanne Gartner.1 1 Neurology, Johns Hopkins University, Baltimore, MD; 2 Medicine, University of Alabama at Birmingham, Birmingham, AL. Viral reservoirs are established early during an HIV infection; remain unaffected by highly active antiretroviral therapy (HAART) for their whole life and virus reemerge after treatment failure or interruption in treatment. It is often proposed that the brain serves as an important HIV reservoir. While macrophages/microglia (MM) in the brain undergoes a productive infection following virus entry, HIV infection in astrocytes becomes dormant after a brief period of viral replication. Given their lifespan and low rate of turnover, astrocytes may harbor the viral genome for long periods of time. In the presence of HAART, viral transcription and translation proceeds normally in the infected cells, except that virus is not assembled into fully infectious particles. Moreover, HIV infected macrophages sustain viral replication for their whole life and may be responsible for rebound of the virus in antiviral treatment interruption or failures. To reduce the impact of viral proteins, viral replication and emergence of drug resistance, inhibition of HIV transcription is of the utmost importance. To inhibit HIV transcription and replication we used Tat siRNA to target HIV in latently infected monocytic, HIV infected primary macrophages and astrocytic (SVGA) LTR-GFP reporter cells. We used a peptide based approach to deliver siRNA. Peptide based transduction of siRNA was very efficient in macrophages and astrocytes without any toxicity as compared to Lipofectamine 2000 and PEI transfection reagents. Tat siRNA strongly inhibited Tat mediated LTR transactivation activity up to 120 hrs in SVGALTR-GFP reporter cells in a dose dependent manner (0.4 µM-0.1 µM). Further, Tat siRNA strongly inhibited TNF-α reactivated HIV in latently HIV infected monocytic cells (THP89) for 72 hrs. To verify further whether Tat siRNA can inhibit HIV infection in primary macrophages, therefore, we established HIV infection in macrophages and then Tat siRNA was transduced on 2nd day post infection. In different experiments, we observed strong inhibition of HIV replication from day 12 to day 21 post infection in macrophages
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
using Tat-siRNA. Conclusion: HIV transcription and replication is strongly inhibited by Tat-siRNA in cells which acts as a long term reservoir of HIV in the human body.
719. Specific Cancer Regression by TransSplicing Ribozyme-Mediated Replacement of KRASV12 Transcript Ju Hyun Kim,1 Sung Jin Kim,1 Sun Young Jang,1 Seong-Wook Lee.1 1 Dept of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul, Republic of Korea. RAS genes encode guanine nucleotide binding proteins and are ubiquitously expressed although different level of expression in each tissue. RAS proteins play essential roles in transducing several crucial signaling pathways that regulate normal cellular proliferation. However, RAS genes are frequently mutated in human cancers, particularly in pancreatic and colon carcinoma. Mutant RAS oncogenes often harbor point mutations, leading the oncogenic proteins in a persistently activated GTP-bound state. Especially, point mutations in codon 12, 13, and 61 of the K-RAS gene are necessary for an early event in lung cancer. Such K-RAS point mutations are found in 30% to 56% of pulmonary adenocarcinomas. Therefore, K-RAS is a good target for cancer therapy. Several approaches specifically targeting mutant RAS genes have been performed for cancer therapy. However, they have some limitations in target specificity and/or therapeutic efficacy. Here, we describe a novel approach to human cancer gene therapy that is based on targeted replacement of cancer-specific mutant K-RAS (K-RASV12) transcripts using Tetrahymena group I intron-based trans-splicing ribozyme. RNA replacement approach with trans-splicing ribozyme will have the advantage of cumulative effects such as mutant KRAS level reduction and simultaneous anti-cancer gene induction specifically in the mutant RNA-expressing cancer cells. These ribozymes were designed to specifically recognize mutated codon 12 site of K-RAS RNA, cleave the downstream sequence of the targeted site, and ligate with and hence induce the specific expression of reporter gene which was tagged at 3’ exon of the ribozymes in human cells as well as in vitro. Importantly, expression of the specific ribozyme with HSV-tk gene as 3’ exon caused efficient and specific cell regression with GCV treatment only in cancer cells expressing K-RASV12 transcript. Taken together, K-RASV12-targeting transsplicing ribozyme could be a specific and potent agent for the treatment of tumors which express the mutant K-RAS transcript.
720. Inhibition of Coxsackievirus B3 (CVB3) Replication by Self-Complementary AAV VectorBased Expression of shRNAs Directed Against the RNA-Dependent RNA Polymerase of CVB3 Isaac Sipo,1 Xiaomin Wang,1 Jens Kurreck,2 Stefan Weger,3 Heinz Zeichhardt,3 Wolfgang Poller,1 Henry Fechner.1 1 Cardiology and Pulmology, Charite - Universitaetsmedizin Berlin, Berlin, Germany; 2Institute of Chemistry (Biochemistry), Freie Universitaet Berlin, Berlin, Germany; 3Institute of Infectious Diseases, Department of Virology, Charite - Universitaetsmedizin Berlin, Berlin, Germany. Coxsackieviruses are associated with liver, pancreatic and heart diseases. In the heart coxsackievirus B3 (CVB3) induces acute myocarditis but is also involved in the development of inflammatory dilatative cardiomyopathy (DCMi). Here we investigate whether AAV vector mediated expression of short hairpin (sh) RNAs can
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718. PTD-Fusion Peptide as a Delivery Vehicle for siRNA To Target HIV Reservoirs Ashok Chauhan,1 Avindra Nath,1 Daivd Levy,2 Suzanne Gartner.1 1 Neurology, Johns Hopkins University, Baltimore, MD; 2 Medicine, University of Alabama at Birmingham, Birmingham, AL. Viral reservoirs are established early during an HIV infection; remain unaffected by highly active antiretroviral therapy (HAART) for their whole life and virus reemerge after treatment failure or interruption in treatment. It is often proposed that the brain serves as an important HIV reservoir. While macrophages/microglia (MM) in the brain undergoes a productive infection following virus entry, HIV infection in astrocytes becomes dormant after a brief period of viral replication. Given their lifespan and low rate of turnover, astrocytes may harbor the viral genome for long periods of time. In the presence of HAART, viral transcription and translation proceeds normally in the infected cells, except that virus is not assembled into fully infectious particles. Moreover, HIV infected macrophages sustain viral replication for their whole life and may be responsible for rebound of the virus in antiviral treatment interruption or failures. To reduce the impact of viral proteins, viral replication and emergence of drug resistance, inhibition of HIV transcription is of the utmost importance. To inhibit HIV transcription and replication we used Tat siRNA to target HIV in latently infected monocytic, HIV infected primary macrophages and astrocytic (SVGA) LTR-GFP reporter cells. We used a peptide based approach to deliver siRNA. Peptide based transduction of siRNA was very efficient in macrophages and astrocytes without any toxicity as compared to Lipofectamine 2000 and PEI transfection reagents. Tat siRNA strongly inhibited Tat mediated LTR transactivation activity up to 120 hrs in SVGALTR-GFP reporter cells in a dose dependent manner (0.4 µM-0.1 µM). Further, Tat siRNA strongly inhibited TNF-α reactivated HIV in latently HIV infected monocytic cells (THP89) for 72 hrs. To verify further whether Tat siRNA can inhibit HIV infection in primary macrophages, therefore, we established HIV infection in macrophages and then Tat siRNA was transduced on 2nd day post infection. In different experiments, we observed strong inhibition of HIV replication from day 12 to day 21 post infection in macrophages
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
using Tat-siRNA. Conclusion: HIV transcription and replication is strongly inhibited by Tat-siRNA in cells which acts as a long term reservoir of HIV in the human body.
719. Specific Cancer Regression by TransSplicing Ribozyme-Mediated Replacement of KRASV12 Transcript Ju Hyun Kim,1 Sung Jin Kim,1 Sun Young Jang,1 Seong-Wook Lee.1 1 Dept of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul, Republic of Korea. RAS genes encode guanine nucleotide binding proteins and are ubiquitously expressed although different level of expression in each tissue. RAS proteins play essential roles in transducing several crucial signaling pathways that regulate normal cellular proliferation. However, RAS genes are frequently mutated in human cancers, particularly in pancreatic and colon carcinoma. Mutant RAS oncogenes often harbor point mutations, leading the oncogenic proteins in a persistently activated GTP-bound state. Especially, point mutations in codon 12, 13, and 61 of the K-RAS gene are necessary for an early event in lung cancer. Such K-RAS point mutations are found in 30% to 56% of pulmonary adenocarcinomas. Therefore, K-RAS is a good target for cancer therapy. Several approaches specifically targeting mutant RAS genes have been performed for cancer therapy. However, they have some limitations in target specificity and/or therapeutic efficacy. Here, we describe a novel approach to human cancer gene therapy that is based on targeted replacement of cancer-specific mutant K-RAS (K-RASV12) transcripts using Tetrahymena group I intron-based trans-splicing ribozyme. RNA replacement approach with trans-splicing ribozyme will have the advantage of cumulative effects such as mutant KRAS level reduction and simultaneous anti-cancer gene induction specifically in the mutant RNA-expressing cancer cells. These ribozymes were designed to specifically recognize mutated codon 12 site of K-RAS RNA, cleave the downstream sequence of the targeted site, and ligate with and hence induce the specific expression of reporter gene which was tagged at 3’ exon of the ribozymes in human cells as well as in vitro. Importantly, expression of the specific ribozyme with HSV-tk gene as 3’ exon caused efficient and specific cell regression with GCV treatment only in cancer cells expressing K-RASV12 transcript. Taken together, K-RASV12-targeting transsplicing ribozyme could be a specific and potent agent for the treatment of tumors which express the mutant K-RAS transcript.
720. Inhibition of Coxsackievirus B3 (CVB3) Replication by Self-Complementary AAV VectorBased Expression of shRNAs Directed Against the RNA-Dependent RNA Polymerase of CVB3 Isaac Sipo,1 Xiaomin Wang,1 Jens Kurreck,2 Stefan Weger,3 Heinz Zeichhardt,3 Wolfgang Poller,1 Henry Fechner.1 1 Cardiology and Pulmology, Charite - Universitaetsmedizin Berlin, Berlin, Germany; 2Institute of Chemistry (Biochemistry), Freie Universitaet Berlin, Berlin, Germany; 3Institute of Infectious Diseases, Department of Virology, Charite - Universitaetsmedizin Berlin, Berlin, Germany. Coxsackieviruses are associated with liver, pancreatic and heart diseases. In the heart coxsackievirus B3 (CVB3) induces acute myocarditis but is also involved in the development of inflammatory dilatative cardiomyopathy (DCMi). Here we investigate whether AAV vector mediated expression of short hairpin (sh) RNAs can
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