- Email: [email protected]
1023. Targeted Inactivation of the CCR5 Gene Via PNA Induced Homologous Recombination
Oligonucleotide Therapies (Including siRNA and shRNA) for Infectious Diseases and Inflammation require engraftment of ~25% of autologous hematopoietic stem cells expressing a β-like globin gene at ~20% of the level of endogenous α-globin. The current generation of globin vectors express the globin gene from a β-globin promoter. To express globin at therapeutic levels, these vectors include discrete Locus Control Region (LCR) enhancers, which inherently carry a risk of oncogene activation. Inclusion of the chicken β-globin insulator (ch5’HS4) in these vectors blocks the effects of the LCR, reducing the likelihood of oncogene activation, but also results in low virus titer. Our alternative approach to the development of safer, more efficient globin vectors, is to use erythroid-specific, non-globin promoters to express globin genes independent of additional enhancers. Our group has shown that a 1750bp Slc4a1 (Band 3/AE1) promoter fused to the human γ-globin gene (pSlc4a1/γ) and flanked by the ch5’HS4 insulator expresses γ-globin mRNA and protein at therapeutic-levels (~19.8% α-globin/ copy) in transgenic mice. First generation HIV-based lentiviral vectors containing pSlc4a1/γ flanked by distinct combinations of ch5’HS4 and/or barrier elements from the ankyrin (ANK) and α-spectrin (α-Sp) gene loci were evaluated To maximize the transduction efficiency of mouse progenitor cells our vectors were pseudotyped with an ecotropic envelope. Ecotropic first generation vectors were and produced at high titer (>1 x 106 IU/ml) and γ-globin mRNA and protein were detected at levels as high as 17% of endogenous α-globin/vector copy in spleen colonies and repopulated mice. We conducted an extensive (119 kilobase) survey for transcriptional regulatory elements associated with DNaseI hypersensitive sites (HS) within and surrounding the Slc4a1 locus. We have identified a single HS upstream of the transcription start site (-355 to-112 bp) that increases reporter gene expression in transient transfection assays independent of orientation and position; activities suggestive of a classical enhancer. Analysis of an adjacent HS (-112 to 0 bp) indicates that this site is a transcriptional silencer. Enhancer-blocking activity has been displayed by a 3’HS within intron 1, ~0.7 kb downstream, as well as a HS cluster located ~7 kb upstream of the enhancer. Finally, a HS that displays barrier activity is located ~43 kb downstream of the Slc4a1 promoter which marks the boundary between Slc4a1 and a neighboring locus. We are currently testing a second generation of lentiviruses in which the silencer has been deleted and pSlc4a1/γ is flanked by combinations of a barrier (Slc4a1, ANK or α-Sp) and the newly characterized Slc4a1 enhancer-blockers. We hypothesize that this vector strategy will safely produce therapeutic levels of γ-globin.
Scintillation measurement and gel shift assays showed that the selected RNA aptamers specifically bind to the target protein with a nanomolar affinity. Flow cytometry data indicated that the aptamers are able to specifically bind to the cell surface expressing gp160. In addition, these aptamers also have been shown to neutralize HIV-1 infectivity. Our results demonstrate that the aptamers are not only expected to provide a potential lead inhibitor to fight HIV-1, but also act as delivery molecules for siRNAs and perhaps other small RNA inhibitors. Further structural characterization, optimization and delivery applications are underway in our laboratory.
1022. Bifunctional Small Interfering RNAs and a Multifunctional Platform for HIV-1 Inhibition
Jane Zhang,1 Pal Saetrom,2 Lars Aagard,2 Haitang Li,2 Ali Ehsani,2 John Rossi.1,2 1 Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA; 2Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA.
Oligonucleotide Therapies (including siRNA and shRNA) for Infectious Diseases and Inflammation
RNA interference is a mechanism that utilizes double-stranded RNA and the RNA-induced silencing complex (RISC) for the regulation of gene expression. The guiding strand of the small interfering RNAs (siRNAs) serves as a template for mRNA target recognition and is incorporated into RISC, resulting in the cleavage of the mRNA target. MicroRNAs (miRNAs), do not direct cleavage of the mRNA and instead direct translational repression via binding to 3’ UTRs of target messages with near-perfect complementarity to the seed region (nucleotides 2-8 from the miRNA’s 5’ end). In order to evaluate the mechanistic downregulation of target mRNAs, we have designed multi-targeting siRNAs against the human immunodeficiency virus type 1 (HIV-1) which can function as both siRNAs and miRNAs on HIV transcripts. These siRNAs utilize both the cleavage and the “miRNA-like” mechanisms to downregulate HIV-1 gene expression. A goal of this research is to create a system that will minimize viral escape mutants to a single antiviral agent. We have also designed and optimized a tri-cistronic miRNA expression system. Here, the endogenously expressed miRNAs have been replaced with antiHIV RNAs and are processed as a part of the endogenous miRNA pathway. For an additive suppression of HIV-1 replication, we have also combined the expression of a small nucleolar RNA-TAR decoy within the construct. The ability of the bifunctional si/miRNAs to inhibit HIV replication and avert the emergence of HIV resistant virus will be tested. This strategy of multiplexing si/miRNA mimics within a single gene construct represents a novel approach for inhibiting HIV replication in a gene therapy setting.
1021. Selection of the RNA Aptamers Against the HIV-1 Gp120 Protein
1023. Targeted Inactivation of the CCR5 Gene Via PNA Induced Homologous Recombination
Jiehua Zhou,1 Haitang Li,1 John J. Rossi.1 1 Molecular Biology, City of Hope, Beckman Research Institute, Duarte, CA.
Envelope glycoproteins of human immunodeficiency virus (HIV), which consists of an exterior glycoprotein (gp120) and a transmembrane domain (gp41), play an important role in the viral entry into cells. The entry process begins with binding of gp120 to the cellular CD4 receptor and thereby triggers cell fusion. Therefore, HIV-1 entry has been validated as a clinically relevant anti-viral target for drug discovery. In our previous work, we described a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera delivery system for HIV-1 therapy. In order to increase applicability and efficacy of aptamers in clinical therapy, in the present of study, new 2’-F substituted RNA aptamers that bind to the HIV-1Ba-L gp120 protein were isolated from a RNA library by using a process called SELEX (Systematic evolution of ligands by exponential enrichment). Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Erica B. Schleifman,1 Ranjit S. Bindra,3 Peter M. Glazer.2 Genetics, Yale University, New Haven, CT; 2Therapeutic Radiology, Yale University School of Medicine, New Haven, CT; 3 Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY.
1
The chemokine receptor 5, CCR5, encodes a major co-receptor for R5-tropic human immunodeficiency virus-1 (HIV-1) and must be present at the cell surface for R5-viral entry. Individuals that possess a homozygous delta32 mutation in CCR5 express a truncated protein, reducing its expression at the cell surface and inhibiting HIV-1 from entering the cell. These individuals are almost completely resistant to R5-tropic HIV-1 infection and show no significant adverse phenotypes. One therapeutic strategy to mimic this naturally occurring inactivating mutation is targeted genome modification using peptide nucleic acid (PNA) induced homologous recombination. These DNA binding molecules bind sequence specifically to duplex DNA and S383
Oligonucleotide Therapies (Including siRNA and shRNA) for Infectious Diseases and Inflammation when combined with donor DNA molecules stimulate recombination in mammalian cells. We have designed a bis-PNA that specifically binds and enhances recombination in the CCR5 gene in the human monocytic acute leukemia cell line, THP-1. In combination with donor DNA molecules, targeted inactivation of CCR5 has been achieved. Single cell heterozygote clones have been isolated and the presence of the inactivating mutation has been confirmed at the DNA level by allele-specific PCR, at the RNA level by allele-specific reverse transcriptase PCR, and by sequencing. We have also shown persistence of the mutation in culture for over 3 months. Thus, this demonstrates that PNA-induced mutation of the CCR5 gene is a potentially powerful tool in targeted gene-inactivation. Furthermore, this approach can be utilized to permanently inactivate the CCR5 co-receptor in human hematopoietic CD34+ cells, thus creating a reservoir of cells which are permanently resistant to infection by the HIV virus.
1024. Anti-HIV RNAi Targeting a Highly Conserved Viral Sequence Results in Novel Mechanisms of Escape
Priya S. Shah,1 Joshua N. Leonard,2 David V. Schaffer.1 Chemical Engineering, UC Berkeley, Berkeley, CA; 2Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD. 1
HIV has proven to be a virus capable of evading both immune surveillance and antiviral therapies with relative ease. RNA interference (RNAi) offers a promising mechanism to exploit for antiviral therapy; however, previous studies using anti-HIV RNAi have observed viral escape by direct mutation of the target sequence and in one case structural rearrangement of the target. Consequently, sequence conservation among clinical isolates is now the primary consideration used when designing antiviral RNAi targets and strategies. We have constructed an experimental and computational system to design and test anti-HIV-1 RNAi targets while considering some inherent limitations involved with RNAi therapies, such as incomplete RNAi protection of the susceptible cellular population. We identified a novel shRNA directed against the highly conserved TAR region that greatly inhibited viral replication; however, viral replication eventually recovered. Upon sequencing the resulting virus, we surprisingly found that these variants contained mutations not in the target sequence itself, but instead in regions involved in the regulation of viral gene expression. Using a novel single LTR platform for mutant, replication competent virus generation, we have shown that many of these mutations confer HIV with the ability to evade RNAi indirectly by a novel mechanism not to our knowledge previously observed in RNAi directed against any virus. Specifically, further characterization using a luciferase assay demonstrates that several of these escape mutants have enhanced promoter activity and likely function by overwhelming the RNAi machinery with an excess of viral RNAs. Since HIV gene expression is controlled by a balance of positive (e.g. Tat/TAR, NF-kB p50/p65, Sp1) and negative (e.g. YY-1, NF-kB p50/p50, HDACs) regulators, we hypothesize that targeting different regions of a viral gene regulatory network can readily be balanced by compensatory mutations in either the target site (as previously observed) or in other regions of the genome such as the viral LTR, such that the virus can potentially manipulate multiple loci in its genome to enhance resistance to antiviral therapy. Such novel viral escape mechanisms further highlight the need to design combination RNAi therapies that simultaneously block multiple compensatory functions within the HIV gene regulatory network. To this end, we subjected escape variants to a combination of RNAi and an existing nucleoside Reverse Transcriptase inhibitor (NRTI) and could show that combinations of these different classes of therapies can lower the required effective concentration of the chemotherapeutic. This combination with RNAi can both increase S384
patient compliance to existing therapies and increase the therapeutic potential of anti-HIV RNAi given its limitations. While HIV evolution and resistance is an ever-growing problem, as evident in this novel evolution of viral gene regulation to escape antiviral RNAi, the expanding range of therapies available may be utilized in combination to inhibit viral replication.
1025. Rational Design Leads to More Potent RNA Interference Targeting Hepatitis B Virus Kathy Keck,1 Anton P. McCaffrey.1 Department of Internal Medicine, University of Iowa, Iowa City, IA.
1
Hepatitis B virus (HBV) is a small DNA virus that replicates through an RNA intermediate. Despite an effective vaccine, 400 million people chronically infected with HBV have a 100-fold higher risk of developing hepatocellular carcinoma. Current treatments are effective in ~50% of cases. HBV is the 9th leading cause of death worldwide. Previously, we conducted proof-of-principle experiments using RNA interference (RNAi) to degrade HBV RNAs in mice, and reduce levels of viral proteins and replicated DNA genomes (McCaffrey et al. 2003 Inhibition of Hepatitis B Virus in Mice by RNA Interference. Nature Biotechnology 21, 639). Recently Grimm et al. expressed the short hairpin RNA (shRNA), HBVU6#2, described in our previous study using self-complementary adeno-associated virus in HBV transgenic mice (Grimm et al. 2006 Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537). While this RNAi trigger resulted in substantial HBV knockdown in mice, it also resulted in acute toxicity. The authors concluded that high levels of shRNA expression required to observe HBV knockdown oversaturated the RNAi machinery and prevented proper expression of endogenous microRNAs (miRNAs). Clearly, identification of more potent HBV RNAi would be desirable, since this could allow knockdown without saturating the miRNA machinery. We have utilized recent mechanistic insights to rationally design more potent HBV RNAi triggers than HBVU6#2. Khovorova et al. and Schwarz et al. demonstrated that an siRNA’s internal thermodynamic stability profile (ISP) determines whether the desired antisense strand is efficiently incorporated into the RNA Induced Silencing Complex (RISC). We used the webtool, SFOLD to identify HBV RNAi triggers most closely conforming to the ISP described by Khovorova et al. We then incorporated GU base pairs into the sense strand that altered the thermodynamic profile to more closely match the consensus. We also selected triggers that were predicted by SFOLD to target regions in HBV RNAs that are accessible to hybridization. RNAi triggers embedded in endogenous miRNA scaffolds are more efficiently processed into mature siRNAs than shRNAs. Therefore, we expressed our HBV RNAi triggers in the context of the endogenous miRNA, miR30. This will also allow expression using liver-specific and inducible promoters. All our rationally designed HBV RNAi triggers showed significant silencing and eight were significantly more potent thatnHBVU6#2. Three of these triggers still gave 50 % silencing at 200 fold lower doses. Northern blots indicated that our rationally designed RNAi triggers favored incorporation of the desired antisense guide strand into the RISC complex. A two step model was used to model the hybridization of the guide strand with the target RNA. A regression analysis identified several thermodynamic features that were highly correlated with RNAi activity. These results will be discussed. These results demonstrate that rational approaches can be used to reliably design more potent RNAi triggers. Studies with these optimized triggers in mice are currently being carried out. Because of the general nature of these approaches, they could be adapted to the treatment of diverse infections and diseases.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Scintillation measurement and gel shift assays showed that the selected RNA aptamers specifically bind to the target protein with a nanomolar affinity. Flow cytometry data indicated that the aptamers are able to specifically bind to the cell surface expressing gp160. In addition, these aptamers also have been shown to neutralize HIV-1 infectivity. Our results demonstrate that the aptamers are not only expected to provide a potential lead inhibitor to fight HIV-1, but also act as delivery molecules for siRNAs and perhaps other small RNA inhibitors. Further structural characterization, optimization and delivery applications are underway in our laboratory.
1022. Bifunctional Small Interfering RNAs and a Multifunctional Platform for HIV-1 Inhibition
Jane Zhang,1 Pal Saetrom,2 Lars Aagard,2 Haitang Li,2 Ali Ehsani,2 John Rossi.1,2 1 Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA; 2Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA.
Oligonucleotide Therapies (including siRNA and shRNA) for Infectious Diseases and Inflammation
RNA interference is a mechanism that utilizes double-stranded RNA and the RNA-induced silencing complex (RISC) for the regulation of gene expression. The guiding strand of the small interfering RNAs (siRNAs) serves as a template for mRNA target recognition and is incorporated into RISC, resulting in the cleavage of the mRNA target. MicroRNAs (miRNAs), do not direct cleavage of the mRNA and instead direct translational repression via binding to 3’ UTRs of target messages with near-perfect complementarity to the seed region (nucleotides 2-8 from the miRNA’s 5’ end). In order to evaluate the mechanistic downregulation of target mRNAs, we have designed multi-targeting siRNAs against the human immunodeficiency virus type 1 (HIV-1) which can function as both siRNAs and miRNAs on HIV transcripts. These siRNAs utilize both the cleavage and the “miRNA-like” mechanisms to downregulate HIV-1 gene expression. A goal of this research is to create a system that will minimize viral escape mutants to a single antiviral agent. We have also designed and optimized a tri-cistronic miRNA expression system. Here, the endogenously expressed miRNAs have been replaced with antiHIV RNAs and are processed as a part of the endogenous miRNA pathway. For an additive suppression of HIV-1 replication, we have also combined the expression of a small nucleolar RNA-TAR decoy within the construct. The ability of the bifunctional si/miRNAs to inhibit HIV replication and avert the emergence of HIV resistant virus will be tested. This strategy of multiplexing si/miRNA mimics within a single gene construct represents a novel approach for inhibiting HIV replication in a gene therapy setting.
1021. Selection of the RNA Aptamers Against the HIV-1 Gp120 Protein
1023. Targeted Inactivation of the CCR5 Gene Via PNA Induced Homologous Recombination
Jiehua Zhou,1 Haitang Li,1 John J. Rossi.1 1 Molecular Biology, City of Hope, Beckman Research Institute, Duarte, CA.
Envelope glycoproteins of human immunodeficiency virus (HIV), which consists of an exterior glycoprotein (gp120) and a transmembrane domain (gp41), play an important role in the viral entry into cells. The entry process begins with binding of gp120 to the cellular CD4 receptor and thereby triggers cell fusion. Therefore, HIV-1 entry has been validated as a clinically relevant anti-viral target for drug discovery. In our previous work, we described a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera delivery system for HIV-1 therapy. In order to increase applicability and efficacy of aptamers in clinical therapy, in the present of study, new 2’-F substituted RNA aptamers that bind to the HIV-1Ba-L gp120 protein were isolated from a RNA library by using a process called SELEX (Systematic evolution of ligands by exponential enrichment). Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Erica B. Schleifman,1 Ranjit S. Bindra,3 Peter M. Glazer.2 Genetics, Yale University, New Haven, CT; 2Therapeutic Radiology, Yale University School of Medicine, New Haven, CT; 3 Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY.
1
The chemokine receptor 5, CCR5, encodes a major co-receptor for R5-tropic human immunodeficiency virus-1 (HIV-1) and must be present at the cell surface for R5-viral entry. Individuals that possess a homozygous delta32 mutation in CCR5 express a truncated protein, reducing its expression at the cell surface and inhibiting HIV-1 from entering the cell. These individuals are almost completely resistant to R5-tropic HIV-1 infection and show no significant adverse phenotypes. One therapeutic strategy to mimic this naturally occurring inactivating mutation is targeted genome modification using peptide nucleic acid (PNA) induced homologous recombination. These DNA binding molecules bind sequence specifically to duplex DNA and S383
Oligonucleotide Therapies (Including siRNA and shRNA) for Infectious Diseases and Inflammation when combined with donor DNA molecules stimulate recombination in mammalian cells. We have designed a bis-PNA that specifically binds and enhances recombination in the CCR5 gene in the human monocytic acute leukemia cell line, THP-1. In combination with donor DNA molecules, targeted inactivation of CCR5 has been achieved. Single cell heterozygote clones have been isolated and the presence of the inactivating mutation has been confirmed at the DNA level by allele-specific PCR, at the RNA level by allele-specific reverse transcriptase PCR, and by sequencing. We have also shown persistence of the mutation in culture for over 3 months. Thus, this demonstrates that PNA-induced mutation of the CCR5 gene is a potentially powerful tool in targeted gene-inactivation. Furthermore, this approach can be utilized to permanently inactivate the CCR5 co-receptor in human hematopoietic CD34+ cells, thus creating a reservoir of cells which are permanently resistant to infection by the HIV virus.
1024. Anti-HIV RNAi Targeting a Highly Conserved Viral Sequence Results in Novel Mechanisms of Escape
Priya S. Shah,1 Joshua N. Leonard,2 David V. Schaffer.1 Chemical Engineering, UC Berkeley, Berkeley, CA; 2Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD. 1
HIV has proven to be a virus capable of evading both immune surveillance and antiviral therapies with relative ease. RNA interference (RNAi) offers a promising mechanism to exploit for antiviral therapy; however, previous studies using anti-HIV RNAi have observed viral escape by direct mutation of the target sequence and in one case structural rearrangement of the target. Consequently, sequence conservation among clinical isolates is now the primary consideration used when designing antiviral RNAi targets and strategies. We have constructed an experimental and computational system to design and test anti-HIV-1 RNAi targets while considering some inherent limitations involved with RNAi therapies, such as incomplete RNAi protection of the susceptible cellular population. We identified a novel shRNA directed against the highly conserved TAR region that greatly inhibited viral replication; however, viral replication eventually recovered. Upon sequencing the resulting virus, we surprisingly found that these variants contained mutations not in the target sequence itself, but instead in regions involved in the regulation of viral gene expression. Using a novel single LTR platform for mutant, replication competent virus generation, we have shown that many of these mutations confer HIV with the ability to evade RNAi indirectly by a novel mechanism not to our knowledge previously observed in RNAi directed against any virus. Specifically, further characterization using a luciferase assay demonstrates that several of these escape mutants have enhanced promoter activity and likely function by overwhelming the RNAi machinery with an excess of viral RNAs. Since HIV gene expression is controlled by a balance of positive (e.g. Tat/TAR, NF-kB p50/p65, Sp1) and negative (e.g. YY-1, NF-kB p50/p50, HDACs) regulators, we hypothesize that targeting different regions of a viral gene regulatory network can readily be balanced by compensatory mutations in either the target site (as previously observed) or in other regions of the genome such as the viral LTR, such that the virus can potentially manipulate multiple loci in its genome to enhance resistance to antiviral therapy. Such novel viral escape mechanisms further highlight the need to design combination RNAi therapies that simultaneously block multiple compensatory functions within the HIV gene regulatory network. To this end, we subjected escape variants to a combination of RNAi and an existing nucleoside Reverse Transcriptase inhibitor (NRTI) and could show that combinations of these different classes of therapies can lower the required effective concentration of the chemotherapeutic. This combination with RNAi can both increase S384
patient compliance to existing therapies and increase the therapeutic potential of anti-HIV RNAi given its limitations. While HIV evolution and resistance is an ever-growing problem, as evident in this novel evolution of viral gene regulation to escape antiviral RNAi, the expanding range of therapies available may be utilized in combination to inhibit viral replication.
1025. Rational Design Leads to More Potent RNA Interference Targeting Hepatitis B Virus Kathy Keck,1 Anton P. McCaffrey.1 Department of Internal Medicine, University of Iowa, Iowa City, IA.
1
Hepatitis B virus (HBV) is a small DNA virus that replicates through an RNA intermediate. Despite an effective vaccine, 400 million people chronically infected with HBV have a 100-fold higher risk of developing hepatocellular carcinoma. Current treatments are effective in ~50% of cases. HBV is the 9th leading cause of death worldwide. Previously, we conducted proof-of-principle experiments using RNA interference (RNAi) to degrade HBV RNAs in mice, and reduce levels of viral proteins and replicated DNA genomes (McCaffrey et al. 2003 Inhibition of Hepatitis B Virus in Mice by RNA Interference. Nature Biotechnology 21, 639). Recently Grimm et al. expressed the short hairpin RNA (shRNA), HBVU6#2, described in our previous study using self-complementary adeno-associated virus in HBV transgenic mice (Grimm et al. 2006 Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537). While this RNAi trigger resulted in substantial HBV knockdown in mice, it also resulted in acute toxicity. The authors concluded that high levels of shRNA expression required to observe HBV knockdown oversaturated the RNAi machinery and prevented proper expression of endogenous microRNAs (miRNAs). Clearly, identification of more potent HBV RNAi would be desirable, since this could allow knockdown without saturating the miRNA machinery. We have utilized recent mechanistic insights to rationally design more potent HBV RNAi triggers than HBVU6#2. Khovorova et al. and Schwarz et al. demonstrated that an siRNA’s internal thermodynamic stability profile (ISP) determines whether the desired antisense strand is efficiently incorporated into the RNA Induced Silencing Complex (RISC). We used the webtool, SFOLD to identify HBV RNAi triggers most closely conforming to the ISP described by Khovorova et al. We then incorporated GU base pairs into the sense strand that altered the thermodynamic profile to more closely match the consensus. We also selected triggers that were predicted by SFOLD to target regions in HBV RNAs that are accessible to hybridization. RNAi triggers embedded in endogenous miRNA scaffolds are more efficiently processed into mature siRNAs than shRNAs. Therefore, we expressed our HBV RNAi triggers in the context of the endogenous miRNA, miR30. This will also allow expression using liver-specific and inducible promoters. All our rationally designed HBV RNAi triggers showed significant silencing and eight were significantly more potent thatnHBVU6#2. Three of these triggers still gave 50 % silencing at 200 fold lower doses. Northern blots indicated that our rationally designed RNAi triggers favored incorporation of the desired antisense guide strand into the RISC complex. A two step model was used to model the hybridization of the guide strand with the target RNA. A regression analysis identified several thermodynamic features that were highly correlated with RNAi activity. These results will be discussed. These results demonstrate that rational approaches can be used to reliably design more potent RNAi triggers. Studies with these optimized triggers in mice are currently being carried out. Because of the general nature of these approaches, they could be adapted to the treatment of diverse infections and diseases.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy