- Email: [email protected]
571. Downregulation of CCR5 and Inhibition of HIV-1 with RNAi, Ribozyme and Single Chain Fv Antibody Delivered by SV40-Derived Vectors To Protect CCR5-Expressing Primary Cells: Monocyte-Derived Macrophages and Microglial Cells from R5-Tropic HIV-1
570. Multi-Targeting, Bifunctional Small Interfering RNAs
Jane Zhang,' Pal Saetrom. ?Ali Ehsani,' Lars Aagard.? John J.
Rossi.!-'
'Graduate School ofBiological Sciences. Division ofMolecular Biology, Beckman Research Institute ofthe City ofHope. Duarte. CA; 2Division ofMolecular Biology, Beckman Research Institute ofthe City ofHope. Duarte, CA. RNA interference (RNAi) is a mechanism that utilizes doublestranded RNA and the RNA-induced silencing complex (RISe) for the regula tion of gene expression. The guiding strand ofthc small interfering RNAs (siRNAs) serves as a template for mRNA target recognition and is incorporated into RISC, resulting in the cleavage ofthe mRNA target. MicroRNAs (miRNAs), do not direct cleavage ofthe 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 oftarget mRNAs, we have designed multi-targeting siRNAs against the human immunodeficiency virus type I (HIY- I) which can function as both siRNAs and miRNAs on HIY transcripts. These siRNAs utilize both the cleavage and the "miRNA-like" mechanisms to downregulate I-IlV-1 gene expression. Our approach is to generate siRNAs with perfect complemental)' target sites within the mRNA as well as target sites within the HIY 3'. We have identified several such bi-functional siRNAs, and show that they function both as siRNAs and as miRNAs with HIY target sequences cloned in a reporter construct. A goal of this research is to create a system that will minimize viral escape mutants to a single antiviral agent. These bi-functional sequences have been inserted in combination in a tri-cistronic microRNA expression system. In this backbone , the anti-HIV RNAs are processed as a part of the endogenous microRNA pathway. The tri-cistronic miRNA mimics are inserted within a lentiviral vector which will be used to transduce HIY infcctible T-cclls and monocytes/macrophages. The ability of the bi-funetional si/miRNAs to inhibit HIY replication and avert the emergence of HIY 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 .
571, Downregulation of CCR5 and Inhibition of HIV-1 with RNAi, Ribozyme and Single Chain Fv Antibody Delivered by SV40-Derived Vectors To Protect CCR5-Expressing Primary Cells: Monocyte-Derived Macrophages and Microglial Cells from R5-Tropic HIV-1 Alena A. Chekmasova, I David S. Strayer. I 'Pathology, Thomas Jefferson University, Philadelphia . PA. Chemokines and chemokine receptors play a crucial role in trafficking of leukocyte populations throughout the body, and are involved in the development of a large variety of human diseases. CCR5 is the main coreceptor used by macrophage (Mj-tropic strains of human immunodeficiency virus type I (I-IIV-I) and HIV-2. Individuals who have reduced or absent CCR5 due to naturally occurring polymorphisms in the CCR5 gene arc apparently otherwise healthy, but arc resistant to HIY infection. If these people become infected , their HIY-I infections tend to progress more slowly to clinical AIDS than those with wild type CCR5. Thus , since it appears to be dispensable to the host but importan t in mediating initial I-IlY infection and controlling progression of an established HIY-I infection , CCR5 is an excellent therapeutic target. With the goal ofreducing CCR5 and protecting CCR5+ cells from R5-tropic HIY, we have used recombinant Tag-deleted SY40-derived vectors to deliver several anti-CCR5 transgenes: siRNA against CCR5 S220
SV(siRNAR5); 2C7, a single chain Fv antibody - SV(2C7); YCKAI , a hammerhead ribozyme - SV(VCKA I) and combined siRNA plus SFv - SY(siRNAR5/2C7). Expression ofthe siRNA and ribozyme is driven by the adenovirus YA pol III promoter. Expression of the SFv is driven by the cytomegalovirus immediate early promoter (CMV-IEP). These transgenes were delivered using rSY40s to human CCR5-exprcssing cell lines , primary monocyte-derived macrophages (MOMs) and primary microglia. The effects ofthese transgenes on CCR5 were measured by cytofluorimetry (FACS). To determine whether the anti-CCR5 transgenes delivered by rSY40 vectors protected susceptible cells from infection with HIY-I entering via CCR5, SupTl/CCR5cclls, MOMs and microglia were transduccd with all these vectors individually. Cells were then challenged with several CCR5-dependent strains of HIY-I R5-tropic HIY-I. HIY replication was followed by measuring p24 antigen levels in culture supernatants by ELISA. All transgenes decreased CCR5, as assayed by FACS. In addition, all transgenes protected SupTIICCR5 cells, MOMs and microglia from R5-tropic strains HIY-I: Ba-L, JR-CSF and AOA-M (for MOM). Transduction of SupTi/CCR5 cells and MOMs with thc bifunctional SY(siRNAR5/2C7), or with SY(YCKAI), gave the greatest reductions in CCR5 and provided the best protection from R5-tropic strains of HIY-I, compared with SV(siRNAR5) and SY(2C7) individually. Therefore, using SY40 derived vectors, we show high efficiency transduction, simultaneous down-regulation ofCCR5 coreceptor and high levels ofresistance to R5-tropic HIY-1. Stable downregulation ofCCR5 by gene transfer may be an effective strategy for treating HIY-I-infccted people to slow disease progression, and for protecting high risk populations from HIY-I infection.
572. Complete Knockdown of CCR5 by Lentiviral Vector Expressed siRNAs for HIV Gene Therapy Joseph S. Anderson, I Ramesh Akkina. ' 'Microbiology. Immunology, Pathology, Colorado State University. Fort Collins. CO. RNAi has proven to be highly potent and effective in inhibiting HIV-I infection. Various reports have utilized siRNAs targeted to viral genes as well as cellular molecules essential for HIV-I infection and replication. Of'the cellular molecules, CCR5, a major coreceptor used by R5-tropic strains of I-II V-I , has been of particular interest due to a naturally occurring mutation in the CCR5 gene. A segement of the human population contain ing either a homozygous or heterozygous 32-bp deletion in the CCR5 gene arc less susceptible to HIV-I infection compared to individuals containing wild-type CCR5. Individuals containing the CCR5 mutation were also shown to be physiologically normal. Due to these characteristics of the CCR5 mutation, CCR5 has been a popular target for gene therapeutics. Silencing ofCCR5 expression by siRNAs has been tested by many groups with varied levels ofknockdown ranging from 55-91%. Complete knockdown of CCR5 expression is necessary to prevent even a low level of infection to occur. Near complete knockdown was achieved in our previous studies with six siRNAs driven by a polymerase-III H I promoter. To further improve knockdown levels, these specific siRNAs were tested under the control of a stronger polymerase-III U6 promoter. Using this strategy, complete silencing ofCCR5 expression was achieved. Complete knockdown ofCCR5 was successfully obtained in both cultured cell lines and C034 cell derived macrophages. The expression ofthese specific siRNAs also did not invoke an interferon response as measured by QRT-PCR. These highly effective CCR5 siRNA constructs can now be evaluated in a clinical setting.
Molecular Therapy Volume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr
Jane Zhang,' Pal Saetrom. ?Ali Ehsani,' Lars Aagard.? John J.
Rossi.!-'
'Graduate School ofBiological Sciences. Division ofMolecular Biology, Beckman Research Institute ofthe City ofHope. Duarte. CA; 2Division ofMolecular Biology, Beckman Research Institute ofthe City ofHope. Duarte, CA. RNA interference (RNAi) is a mechanism that utilizes doublestranded RNA and the RNA-induced silencing complex (RISe) for the regula tion of gene expression. The guiding strand ofthc small interfering RNAs (siRNAs) serves as a template for mRNA target recognition and is incorporated into RISC, resulting in the cleavage ofthe mRNA target. MicroRNAs (miRNAs), do not direct cleavage ofthe 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 oftarget mRNAs, we have designed multi-targeting siRNAs against the human immunodeficiency virus type I (HIY- I) which can function as both siRNAs and miRNAs on HIY transcripts. These siRNAs utilize both the cleavage and the "miRNA-like" mechanisms to downregulate I-IlV-1 gene expression. Our approach is to generate siRNAs with perfect complemental)' target sites within the mRNA as well as target sites within the HIY 3'. We have identified several such bi-functional siRNAs, and show that they function both as siRNAs and as miRNAs with HIY target sequences cloned in a reporter construct. A goal of this research is to create a system that will minimize viral escape mutants to a single antiviral agent. These bi-functional sequences have been inserted in combination in a tri-cistronic microRNA expression system. In this backbone , the anti-HIV RNAs are processed as a part of the endogenous microRNA pathway. The tri-cistronic miRNA mimics are inserted within a lentiviral vector which will be used to transduce HIY infcctible T-cclls and monocytes/macrophages. The ability of the bi-funetional si/miRNAs to inhibit HIY replication and avert the emergence of HIY 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 .
571, Downregulation of CCR5 and Inhibition of HIV-1 with RNAi, Ribozyme and Single Chain Fv Antibody Delivered by SV40-Derived Vectors To Protect CCR5-Expressing Primary Cells: Monocyte-Derived Macrophages and Microglial Cells from R5-Tropic HIV-1 Alena A. Chekmasova, I David S. Strayer. I 'Pathology, Thomas Jefferson University, Philadelphia . PA. Chemokines and chemokine receptors play a crucial role in trafficking of leukocyte populations throughout the body, and are involved in the development of a large variety of human diseases. CCR5 is the main coreceptor used by macrophage (Mj-tropic strains of human immunodeficiency virus type I (I-IIV-I) and HIV-2. Individuals who have reduced or absent CCR5 due to naturally occurring polymorphisms in the CCR5 gene arc apparently otherwise healthy, but arc resistant to HIY infection. If these people become infected , their HIY-I infections tend to progress more slowly to clinical AIDS than those with wild type CCR5. Thus , since it appears to be dispensable to the host but importan t in mediating initial I-IlY infection and controlling progression of an established HIY-I infection , CCR5 is an excellent therapeutic target. With the goal ofreducing CCR5 and protecting CCR5+ cells from R5-tropic HIY, we have used recombinant Tag-deleted SY40-derived vectors to deliver several anti-CCR5 transgenes: siRNA against CCR5 S220
SV(siRNAR5); 2C7, a single chain Fv antibody - SV(2C7); YCKAI , a hammerhead ribozyme - SV(VCKA I) and combined siRNA plus SFv - SY(siRNAR5/2C7). Expression ofthe siRNA and ribozyme is driven by the adenovirus YA pol III promoter. Expression of the SFv is driven by the cytomegalovirus immediate early promoter (CMV-IEP). These transgenes were delivered using rSY40s to human CCR5-exprcssing cell lines , primary monocyte-derived macrophages (MOMs) and primary microglia. The effects ofthese transgenes on CCR5 were measured by cytofluorimetry (FACS). To determine whether the anti-CCR5 transgenes delivered by rSY40 vectors protected susceptible cells from infection with HIY-I entering via CCR5, SupTl/CCR5cclls, MOMs and microglia were transduccd with all these vectors individually. Cells were then challenged with several CCR5-dependent strains of HIY-I R5-tropic HIY-I. HIY replication was followed by measuring p24 antigen levels in culture supernatants by ELISA. All transgenes decreased CCR5, as assayed by FACS. In addition, all transgenes protected SupTIICCR5 cells, MOMs and microglia from R5-tropic strains HIY-I: Ba-L, JR-CSF and AOA-M (for MOM). Transduction of SupTi/CCR5 cells and MOMs with thc bifunctional SY(siRNAR5/2C7), or with SY(YCKAI), gave the greatest reductions in CCR5 and provided the best protection from R5-tropic strains of HIY-I, compared with SV(siRNAR5) and SY(2C7) individually. Therefore, using SY40 derived vectors, we show high efficiency transduction, simultaneous down-regulation ofCCR5 coreceptor and high levels ofresistance to R5-tropic HIY-1. Stable downregulation ofCCR5 by gene transfer may be an effective strategy for treating HIY-I-infccted people to slow disease progression, and for protecting high risk populations from HIY-I infection.
572. Complete Knockdown of CCR5 by Lentiviral Vector Expressed siRNAs for HIV Gene Therapy Joseph S. Anderson, I Ramesh Akkina. ' 'Microbiology. Immunology, Pathology, Colorado State University. Fort Collins. CO. RNAi has proven to be highly potent and effective in inhibiting HIV-I infection. Various reports have utilized siRNAs targeted to viral genes as well as cellular molecules essential for HIV-I infection and replication. Of'the cellular molecules, CCR5, a major coreceptor used by R5-tropic strains of I-II V-I , has been of particular interest due to a naturally occurring mutation in the CCR5 gene. A segement of the human population contain ing either a homozygous or heterozygous 32-bp deletion in the CCR5 gene arc less susceptible to HIV-I infection compared to individuals containing wild-type CCR5. Individuals containing the CCR5 mutation were also shown to be physiologically normal. Due to these characteristics of the CCR5 mutation, CCR5 has been a popular target for gene therapeutics. Silencing ofCCR5 expression by siRNAs has been tested by many groups with varied levels ofknockdown ranging from 55-91%. Complete knockdown of CCR5 expression is necessary to prevent even a low level of infection to occur. Near complete knockdown was achieved in our previous studies with six siRNAs driven by a polymerase-III H I promoter. To further improve knockdown levels, these specific siRNAs were tested under the control of a stronger polymerase-III U6 promoter. Using this strategy, complete silencing ofCCR5 expression was achieved. Complete knockdown ofCCR5 was successfully obtained in both cultured cell lines and C034 cell derived macrophages. The expression ofthese specific siRNAs also did not invoke an interferon response as measured by QRT-PCR. These highly effective CCR5 siRNA constructs can now be evaluated in a clinical setting.
Molecular Therapy Volume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr