1101 Rational Design Leads to More Potent RNA Interference Targeting Hepatitis B Virus OLIGONUCLEOTIDE THERAPIES II 1098 Inhibition of HIV 1 Replication by Lentiviral Vectors Harboring Long Hairpin RN[.]
OLIGONUCLEOTIDE THERAPIES II 1098 Inhibition of HIV-1 Replication by Lentiviral Vectors Harboring Long Hairpin RNAs Masayuki Sano,!" Haitang Li,' Mahito Nakanishi,' John J cells, thereby creating a reservoir of cells which are permanently resistant to infection by the HlV virus 1100 Novel Cell Type-Specifical Aptamer-siRNA Delivery System for HIV-1 Therapy Rossl.'-' Jiehua Zhou, Haitang Li, John J Rossi I I Division ofMolecular Biology, Beckman Research Institute of the City ofHope Duarte CA; lBio/herapeutic Research Laboratory, National Institute ofAdvanced Industrial Science and Technology (A1ST) Tsukuba, Ibaraki, Japan; 'Dtvtston ofMolecular Biology; Graduate School ofBiological Sciences Beckman Research Institute ofthe City ofHope Duarte CA Small interfering RNA(siRNA) and short hairpin RNA(shRNA) targeted against human immunodeficiency virus type I (HIV-I) have been successfully employed to suppress viral replication However, the emergenceofsiRNA-resistant mutants remains problematic for a long-term inhibition of HIV-I To reduce the chance of emergence of siRNA-resistant mutants, we exploited 50 and 80 base-pair (bp) oflong hairpin RNAs, with multiple point mutations within the sense strand, targeted against essential viral regulatory genes, tat and rev , under the control of the human U6 promoter.To evaluate the potential anti-HIV-I activity of long hairpin RNAs, we made lentiviral vectors harboring long hairpin RNAexpression cassettes HIV-I susceptible cultured 'I' cells were transduced with these vectors, showing greater than 90% transduction efficiency Northern blotting analysis using extracted total RNA from these cells indicated that several kinds of siRNAs were expressed from the long hairpin transcripts, suggesting that generation of multiple siRNAs could contribute to simultaneously targetingseveral sites of I-IIV-J mRNAs and potentially reduce the chance ofthe emergence of escape mutants By semi-quantitative RT-PCR analysis, we revealed that the expression of long hairpin RNAs does not activate interferon-inducible genes, such as OASI and CDKL2 Moreover, we found that the long hairpin RNA targeted against tat/rev significantly suppressed HIV-I replication long-term This long hairpin RNAcould also effectively inhibited an HIV-I mutant in the tat/rev common exon Moreover, a single 21-bp shRNA targeting this exon failed to inhibit replication, supporting the validity of using the longer hairpins Our finding demonstrates that the long hairpin RNA-based strategy is a potent strategy for long-term control of HIV-I replication in a gene therapy setting 1099 Targeted Inactivation of the CCR5 Gene in Human Cells Using Peptide Nucleic Acids Ranjit S Bindra,' Erica B Schleifman,' Peter M Glazer,' 'Yale University School ofMedicine Department ofTherapeutic Radiology, New Haven Cr The ability to genetically manipulate human stem cells has the potential to enhance the utility of stem cell therapy for human disease One emerging approach to targeted genome modification is the use of triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) These molecules bind to duplex DNA in a sequence-specificmanner, and this binding can be used to stimulate recombination in mammalian cells when combined with donor DNA molecules Here, we report the development of PNAs that specifically bind to and stimulate recombination at the CCR510cus, resulting in site-specific inactivation of the CCR5 gene in several cell lines.The CCR5 gene encodes a major co-receptorfor R5-tropic strains ofthe human immunodeficiencyvirus-I (HIV-I), which are responsiblefor most cases ofinitial, acute HIV infection Individuals who possess a homozygous inactivating mutation in the CCR5 gene are resistant to infection by R5-tropic HIV-l strains Thus, PNAinduced mutation of the CCR5 gene ean be utilized to permanently inactivate the CCR5 co-receptor in human hematopoietic CD34+ S420 Division ofMolecular Biology Beckman Research Institute City ofHope Duarte CA The short double-strandedRNA,which is knownas short interfering RNA(siRNA),can be used to specificallydown-regulateexpression of the associated gene in a process known as RNA interference (RNAi) RNAi is rapidly becoming one of the main methods used to analyze gene functions and holds great promise for therapeutic applications However, whatever virus or non-virus vector for genc delivery, the successful use of small syntheticsiRNA for therapeutic purposes critically requires a safe and efficient delivery system capable of targeting specific cells Therefore, here we are designing novel anti-gp120 aptamer-siRNA chimeras to deliver siRNA for HlV-1 therapy, In our chimera system, the anti-gpl20 RNA aptamer binds with gp120, a glycoprotein exposed on the surface of the HIV envelope, which first binds to CD4 receptor The entry of HlV-1 into target cells and elements of pathogenesis of AIDS are largely determined by gp 120 Moreover, the siRNA part targets the expression of Tar/Revgenes of HIV-I Our studies demonstrate that the aptamer-siRNA chimeras can bind cells expressing HIV-I gp120and selectivelyenter intothese cells.The chimera is processed by Diccr and the resulting siRNA results in inhibition of HIV gene expression Moreover, these chimeras not bind to or function in control cells not expressing gp 120 Interestingly, thc 27-mcr duplex RNA showed greater efficiency in gene silencing than the transitional Zl-rner siRNA, consistent with the results from in vitro Dicer assays In addition to the siRNA, the anti-gp120 aptamer can strongly neutralize HIV-I infection These results demonstrate the potential for systemic, cell-type specific, double-functional siRNA delivery system for HIV-I therapy 1101 Rational Design Leads to More Potent RNA Interference Targeting Hepatitis Virus Kathy Keck, I Anton P McCaffrey.' Department ofInternal Medicine University ofIowa Iowa City IA I 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 9°' leading cause of death worldwide Previously, we conducted proof-of-principle experimentsusing RNA interference(RNAi) to degrade I-mv RNAs in mice, and reduce levels of viral proteins and replicated DNA gcnomes (McCaffrey et al 2003 Inhibition of Hepatitis B Virus in Mice by RNA Interference Nature Biotechnology 21,639) Recently Grimm et al expressedthe short hairpin RNA(shRNA), HBVU6#2, describedin our previousstudy usingself-complementaryadeno-associatedvirusserotype8 in HBV transgenicmice (Grimm et al 2006 Fatality in mice due to oversaturation of cellular microRNAIshort hairpin RNA pathways Nature 441, 537) While this RNAi trigger resulted in substantiall-IBV 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 Molecular Therapy Yofume 15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr triggers than our I" first generation trigger, HBVU6#2 Khovorova et al and Schwarz et al demonstrated that an siRNA's internalthermodynamic stability profile (ISP) determines whether the desired antisense strand is efficiently incorporated into the RNA Induced SilencingComplex (RISC) Weused the webtool, SFOLD to identify HBV RNAi triggers most closely conforming to the ISPdescribed by Khovorova et al Wethen incorporated GU base pairs into the sense strand that altered the thermodynamic profile to more closely match the consensus These triggers were designed to conform to reported sequence preferences for efficient RNAi described by Reynolds et at Wealso 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 ofthe endogenous miRNA, miR30.This will also allow expression using liver-specific and inducible promoters 14 out of 15 of the rationally designed HBV RNAi triggers were more potent in cultured cells than our I" generation construct, HBVU6#2 Northern blots indicated that our rationally designed RNAi triggers favored incorporation of the desired antisense guide strand into the RISC complex However,our data also suggest that other downstream parameters influencesilencing efficiency These results demonstrate that rational approaches can be used to reliably design more potent RNAi triggers Studies with these optimized triggers in mice will be discussed Because ofthe general nature of these approaches, they could be adapted to the treatment of diverse infections and diseases 1102 Novel "Bifunctional" shRNA Knockdown Therapeutics to Individualized Cancer Targets John Nemunaitis.P" Phillip B Maples.P Donald Rao,' Joseph Kuhn,' Yuqiao Shen.! Alex W Tong.t-' Chris Jay" ,2 Padmasini 2,3,~ Kumar,' Neil Senzer.I [Cancer Research, Mary Crowley Cancer Research Centers , Dallas, TX; 2Cancer Research, Murex Pharmaceuticals, lnc , Dallas, TX; JCancer Research, Baylor Sammons Cancer Center; Dallas, TX; 'Cancer Research, Texas Oncology; I~A , Dallas, TX We have created a novel process of identifying cancer specific and, potentially critical, molecular targets in individual patients and have constructed corresponding RNA interference therapeutics to test clinical impact Malignant and non malignant tissue from 81 cancer patients were harvested Differentially overexpressed protein and correlated mRNA signals were determined (mRNAArray, UTSWMC, Dallas, TX and 2D DIGE/MS; Applied Biomics, San Francisco, CA) in 32 of these patients Gene expression pathway modeling(GNS, Boston, MA) of prioritizedproteinswith pro-cancer function overexpressed in cancer allowed for determination of intra pathway protein connectivity in IS patients Novel "bifunctional" shRNA constructs, which demonstrate both cleavage dependent and cleavage independent RISC (RNA induced silencing complex) mediated inhibition of the targeted homologous mRNA, were constructed and tested The "bifunctional" shRNA has produced up to a 260 percent enhanced knockdown of the target protein (compared to traditional siRNA knockdown to the same sequence at optimal dose), at 48 hours after clonal cancer cell exposure Furthermore, consequent functional modulation (e.g., apoptosis) was observed Duration of target knockdown, measured by sequential Western blot and alkaline phosphatase reporter assay, demonstrated a further temporal advantage of bifunctional shRNA over siRNA (at optimal doses) beyond 48 hours Subsequent treatment of malignant clonal cells with known high expression ofthe target protein confirmed increased cell death over time following exposure to the "bifunctional" shRNA A clinical grade plasmid containing a unique promoter for tumor specificcontrolled shRNAexpression was engineered.Animal efficacy and toxicology testing are underway using the target vector Molecular Therapy Volume 15, Supplement I May 2007 © Th e American Socie ty o f G ene Th erap y Copyright packaged in a tumor antigen targeting immunoliposome nanoparticle for delivery The clinicallND justifying patient treatment is in process Update of results will be presented 1103 Using Let-7 microRNA To Inhibit Pancreatic Cancer Cells Proliferation Laurie Parmentier,' Jerome Torrisani,' Barbara Bournet,' Anny Souque,' Michele Bouisson,' Jean Escourrou,' P Senesse,' Marc Barthet,"N Lesavre,' Philippe Ruszniewski,' Dermot O'Toole,' A Aubert,' Pascal Hammel,' Philippe Levy,' Janick Selves," Lucien Pradayrol,I Louis Buseail, I Pierre Cordclicr.' IINSERM U858, 12MR, Toulouse, France; 2Service de Gastroenterologie, CflU Rangueil, Toulouse, France; 'Service de Gastroenterologie, Val d'Aurelle, Montpelliet; France; 'Service de Gastroenterologie, CHU Nord et CIG, Marseille, France; ' Service de Gastroenterologie, Hopital Seal/jon, Clichy; France; 61NSERM U563, CHU Purpan, Toulouse, France Background; pancreatic ductal carcinoma is the fifth leading cause of cancer related deaths in Westerncountries, with an increasing incidence over the last 40 years So far, no effective therapies have been established to alleviate this devasting and often fatal endstage condition Thus, there is an urgent need for the development of new modalities to improve diagnosis and treatment of pancreatic cancer MicroRNAs (miR) are small non-coding transcripts that regulate gene expression by inhibiting translation Emerging studies suggest that many miRNAs may participate in human disease, including oncogenesis The aim of present work was to establish the pattern of expression of miR known to potentiate or to inhibit oncogenesis in PC-derived cell lines, tumors and fine needle aspiration (FNA) Methods: Total RNA was purified from PC-derived cell lines, tumors and FNA RNA collected by aspiration was amplified using Full Spectrum amplification kit As controls, we used RNA purified from normal, adjacent pancreas to pancreatic cancer miR levels were ascertained by Taqman RT-PCR Results: using Taqman-based RT-PCR, we identified a global down regulation of anti-oncogenicmiR (miR-21, miR-22I, miR-222,miR-16, miR-15a, miR 17-5-p, Let-7a) in pancreatic cancer samples, when oncogenic miR were elevated (miR-211, mir-30I) Let-7 miR down regulation was further confirmed by Northern blotting and in situ RT-PCR on pancreatic adenocarcinoma tissue micro array Eventually, we identified a global down expression of let-7 miR family members in 30 FNA of pancreatic cancer tumors Because ki-RAS oncogene is a validated target of Let-7 miR, we transfected pancreatic cancerderived cells with Let-? miR We observed a dramatic decrease in PC-derived cells proliferation, with a eoncomittant inhibition ofkiRAS protein expression Conclusion: a better understanding of the molecular mechanisms involved in pancreatic cancer oncogenesis could lead to the development of new molecular markers for either early diagnosis or targeted therapy Here we show that anti-oncogenic miR expression, such as let-7, is strongly down regulated in pancreatic cancer samples Taken together, this study describes the potential clinical and therapeutic interest of delivering Let-7 for treating pancreatic cancer 1104 Argonaute-2 Is a Key Limiting Factor for Therapeutic RNAi in the Liver Dirk Grimm, Lora Wang, Joyce S Lee, Theresa A Storm, Mark A Kay I Pediatrics and Genetics, Stanford University, Stanford, CA We recently reported efficient and persistent suppression of Hepatitis B virus (HBV) in mice using double-stranded AAV-8 vectors expressing anti-HBV shRNAs A striking finding was that shRNA over-expression from a robust U6 promoter frequently caused hepatocellular toxicity, rapid organ failure and lethality S421 ... described by Reynolds et at Wealso 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... incorporated into the RNA Induced SilencingComplex (RISC) Weused the webtool, SFOLD to identify HBV RNAi triggers most closely conforming to the ISPdescribed by Khovorova et al Wethen incorporated GU base... inducible promoters 14 out of 15 of the rationally designed HBV RNAi triggers were more potent in cultured cells than our I" generation construct, HBVU6#2 Northern blots indicated that our rationally