799. Expressed Long Hairpin RNA Sequences Inhibit HBV Replication In Vivo without Inducing an Interferon Response

OLIGONUCLEOTIDE THERAPIES: VIRAL TARGETS cotransfected with a luciferase expression plasmid into mouse liver by hydrodynamic transfection, this construct induced ∼90% silencing relative to a random-hairpin control. The construct was next adapted to target hepatitis B virus (HBV) s-antigen (sAg), and packaged as a double-strand in pseudotyped adeno-associated virus serotype 8 (dsAAV8). The hairpin sequence used was for the expression of a 25mer shRNA that had proven to be toxic when expressed from a U6 promoter. Previous work demonstrated that the toxic effect observed for this U6 construct was such that the ultimate result was either loss of the RNAiinduced silencing effect or the death of recipient mice due to liver failure less than a month following injection of expression cassetteharboring AAV. We tested the ApoE/hAAT expression construct in transgenic mice harboring an integrated HBV genome. Over the course of three months, the ApoE/hAAT construct was well tolerated and induced a stable level of sAg silencing of ∼80% of starting levels. Healthy FVB mice were used in a separate toxicity study for direct comparison of U6- and ApoE/hAAT-driven 25mers. Serum ALT levels were followed over three months as an indicator of liver toxicity. Mice that received the ApoE/hAAT-driven construct had stable, normal ALT levels, while those of mice that received the U6driven construct spiked to peak around three weeks before gradually returning to normal. While we continue to optimize its efficacy, we conclude that this pol II system is an effective and safer HBV therapeutic in a mouse model of human disease.

797. Long-Term Inhibition of Hepatitis B Virus in Transgenic Mice by Adeno-Associated VirusDelivered Short Hairpin RNA Chun-Chi Chen,1,2 Tai-Ming Ko,1,2 Hsin-I Ma,3 Hui-Lin Wu,4 Xiao Xiao,5 Mi-Hua Tao.1 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan; 3Department of Neurological Surgery, Tri-Service General Hospital and the National Defense Medical Center, Taipei, Taiwan; 4Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan; 5Molecular Therapy Laboratory, Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA. RNA interference (RNAi) was reported to block viral infections in animal models when treatment began before or shortly after viral challenge. However, it remains unknown whether RNAi can eliminate more established infections, such as chronic hepatitis B virus (HBV) infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver small hairpin RNA (shRNA). HBV transgenic mice, which produced large amounts of HBV in liver and maintained a high viral load in circulation, were used an animal model to evaluate the inhibition effect of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log10 decrease in HBV load in the circulation. Significant HBV suppression was still observed 120 days after administration of the vector. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. In conclusion, by combining the high liver transduction efficiency of the dsAAV2/8 vector and the power of RNAi silencing, a rapid, profound, and durable HBV inhibition could be achieved in animal models with a heavily ongoing HBV replication. These results support that dsAAV2/8 vector may be applicable to design an effective RANi-based therapy to treat chronic HBV infection as well as other persistent liver infections. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

798. Anti-Influenza-Viral Efficacy Attributed to siRNA Formulated as Simple Lipoplex or PEI Polyplexes Is a Result of Their Immunostimulatory Properties Marjorie Robbins, Ellen Ambegia, Matthew Fedoruk, Lorne Palmer, Kevin McClintock, Adam Judge, Ian MacLachlan. 1 Research and Development, Protiva Biotherapeutics, Burnaby, BC, Canada. A number of groups have published reports of antiviral efficacy associated with the delivery of siRNA directed against the influenza virus using either lipoplex or polyplex mediated siRNA delivery. In order to assess the extent to which the immunostimulatory properties of siRNA may have contributed to these results we developed nonimmunostimulatory analogues of the previously published NP1496 and PA2087 siRNA. siRNA were prepared containing minimal chemical modifications previously shown to abrogate immune stimulation while retaining the ability to mediate RNAi. The immunostimulatory properties of all siRNA were confirmed by intravenous injection of liposome encapsulated siRNA followed by determination of interferon alpha in the plasma 6h post-injection in mice. When tested in an in vitro cell-based system the modified, non-stimulatory siRNA potently inhibited influenza virus replication in MDCK cells. However, the modified siRNA lost all previously observed antiviral efficacy when delivered to mice infected with Influenza A/PR/8/34. Mice were treated either intranasally using lipoplex (Oligofectamine TM) at 1 mg/kg, intranasally as ‘naked’ siRNA at 12.5 mg/kg or by intravenous administration as PEI polyplex at 6 mg/kg. This was in contrast to previously reported studies yielding substantial antiviral efficacy when using the immunostimulatory parent molecules. When these studies were repeated using the immunostimulatory parent molecules we observed a significant reduction in viral titre in the mouse lung as measured by HA EIA. The results suggest caution when interpreting results obtained in vivo using immunostimulatory siRNA, in particular within the context of anti-viral applications.

799. Expressed Long Hairpin RNA Sequences Inhibit HBV Replication In Vivo without Inducing an Interferon Response Abdullah Ely,1 Sergio Carmona,1 Carol Crowther,1 Steven Mufamadi,1 Samantha Barichievy,1 Marc Weinberg,1 Patrick Arbuthnot.1 1 Hepatitis B Virus Research Unit, University of the Witwatersrand, Private Bag 3, Wits, Johannesburg, Gauteng, South Africa. Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the global population and HBV carriers are at an increased risk for cirrhosis and hepatocellular carcinoma. Licensed therapies against HBV are only partially effective and development of improved treatment modalities remains an important medical objective. Exploitation of the RNA interference (RNAi) pathway shows promise as a novel antiviral treatment strategy for the management of HBV. Traditionally short hairpin RNAs (shRNAs) of 21-23 bp in the stem region have been used to induce RNAimediated gene silencing and avoid inducing the sequence non-specific interferon response. The use of longer (> 30 bp) hairpin RNAs affords the advantage of generating multiple siRNA from a single Dicer template, which target different sites. This potentially limits the problem of viral escape mutants that evade the silencing effects of a single siRNA. We generated U6 promoter-driven cassettes that encode long hairpin RNAs (lhRNAs) comprising a 60 bp stem. The duplex RNA was designed to target the HBx sequence of the A1 subgenotype of HBV. G:U pairings, which occurred at approximately every 6 bp, were incorporated in the stem sequence to circumvent induction of the interferon response by RNA-dependent protein S309

OLIGONUCLEOTIDE THERAPIES: VIRAL TARGETS kinase and RNase L pathways. One of the two lhRNA targeted to HBx successfully inhibited markers of HBV replication in cultured cells and in the murine hyperdynamic model of HBV replication. HBV surface antigen, HBV mRNA and viral particles were diminished by approximately 80% by the lhRNA. Knockdown was comparable to that achieved by a shRNA targeted against a region covered by the lhRNA and was observed over a period of 1 week. Control lhRNA targeted to the HIV-1 Tar region did not knock down HBV gene expression significantly. As an index of activation of the interferon response genes, OAS1, MxA and IFN-β mRNA concentrations were measured in transfected cells and in hepatocytes of mice treated using the hydrodynamic injection procedure. None of the lhRNA cassettes induced expression of these genes. These data indicate that U6 promoter-driven lhRNA are capable of effectively inhibiting HBV replication without inducing the unwanted interferon response. These lhRNA sequences have potential therapeutic application and may be useful to counter viral escape.

800. Complete Suppression of CCR5 Expression and Inhibition of HIV-1 Infection by Transfected and Lentiviral Vector Expressed shRNAs Joseph S. Anderson,1 Annaleen Vermeulen,2 Jon Karpilow,2 Ramesh Akkina.1 1 Microbiology, Immunology, Pathology, Colorado State University, Fort Collins, CO; 2Dharmacon, Inc, Lafayette, CO. SiRNAs have been shown to be highly potent and sequence specific in targeted gene silencing with great potential for use as therapeutics in combating HIV infection. By targeting the critical coreceptor CCR5, infection can be inhibited at the level of viral entry. The chemokine receptor, CCR5, has been shown to be fully dispensable for normal homeostasis and is, therefore, an excellent target for gene therapy applications. Several reports have so far described the use of CCR5 siRNAs showing varied levels of gene knockdown. In the present study, a new generation of anti-CCR5 siRNAs was designed with the goal of achieving complete silencing. Based on rational target sequence identification criteria, new and more effective CCR5 siRNAs were identified. Complete knockdown of CCR5 expression as determined by FACS and quantitative real time PCR was observed. To achieve stable and constitutive knockdown of CCR5, siRNA coding sequences were cloned individually and in tandem into a lentiviral vector. Vector transduced cells showed similar impressive levels of CCR5 knockdown. When challenged with R5-tropic HIV–1, the siRNA transduced cells exhibited resistance to infection. These results are the first to demonstrate complete constitutive knockdown of CCR5 expression by the use of siRNAs after years of efforts in several laboratories.

801. RNA Gene Therapy Targeting Herpes Simplex Virus Jia Liu,1 Sonal S. Tuli,2 David C. Bloom,1 Gregory S. Schultz,3 Steve C. Ghivizzani,4 Alfred S. Lewin.1 1 Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL; 2Ophthalmology, University of Florida College of Medicine, Gainesville, FL; 3Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL; 4Orthopaedics and Rehabilitation, University of Florida College of Medicine, Gainesville, FL. Purpose: Herpes Simplex Keratitis (HSK) is the leading cause of corneal blindness in developed nations. Despite active antiviral drugs (nucleoside analogues), there is no effective method preventing recurrent HSK. We tested the ability of ribozymes targeting the mRNAs of essential HSV genes to inhibit viral infection in vitro and in vivo. Methods: Hammerhead ribozymes were designed against S310

mRNAs of four essential HSV genes and those with the best cleavage kinetics were packaged in an adenoviral vector containing a CMV promoter and an IRES-GFP element. HSV-1 viral yields (infection at low MOI) following Ad-ribozyme treatment were compared to control cells transduced with adenovirus vector without ribozyme. After ribozyme treatment and HSV-1 infection (high MOI) total RNAs were extracted and a reverse transcription reaction followed by Real-time PCR to quantify target as well as GAPDH level was conducted. The UL20 ribozyme was tested in vivo using a protected ribozyme containing 2’modifications of the ribose sugars of selected nucleotides. This modified ribozyme was delivered to the right eyes of rabbits by iontophoresis; and an inactive ribozyme served as a control in left eyes, followed by inoculation of HSV-1 replicating virus expressing β-galactosidase. Three days post- infection, rabbits were sacrificed and corneas were harvested for X-gal staining to visualize the viral infection. Results: The UL20 ribozyme delivered by adenoviral vector reduced HSV-1 viral yield by 83% in the cell culture when cells were infected with HSV-1 for 24 hours in the presence of the ribozyme. In a time course study, the UL20 ribozyme reduced viral replication by 98% after 6 days of HSV-1 infection, indicating a cumulative effect of ribozyme cleavage. The real-time PCR results indicated that UL20 ribozyme reduced target mRNA level by 64%. The chemically stabilized UL20 ribozyme reduced HSV-1 reduced dendrite lesion area by 57% compared with the rabbit eyes treated with an inactive ribozyme. Another ribozyme targeting UL30 mRNA, whose gene product is viral DNA polymerase, was able to reduce viral replication by 98% at one day post HSV-1 infection and maintain the inhibition effect by 99% at third day post infection. Conclusions: A hammerhead ribozyme targeting the mRNA of UL20 gene (a late essential HSV-1 gene) significantly reduced the severity of an HSV-1 ocular infection in rabbits as well as significantly knocking down viral yield in cell culture. And a ribozyme targeting UL30 mRNA achieved the similar effect in the cell culture. These results suggest a RNA gene therapy strategy targeting HSV-1 early/late genes that should lack the toxicity and reduce the risk of generating drug resistant virus. Further studies evaluating the long term efficacy of different viral vectors to deliver therapeutic ribozymes during acute or recurrent HSV-1 infection are currently underway.

802. Silencing of HIV-1 with RNA Interference: A Multiple shRNA Approach Olivier ter Brake,1 Ben Berkhout.1 1 Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, NH, Netherlands. Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). The double-stranded RNA can be expressed in the cell as a short hairpin RNA (shRNA) with a stem of 21-23 base pairs. Previously, we have shown that stable expression of a shRNA targeting the Nef gene of HIV-1 results in strong inhibition of HIV-1 replication. However, the application of this single shRNA was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished the inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs (ter Brake and Berkhout 2005). Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication (Figure 1). We show, for the first time, that the expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA as compared to single shRNA vectors. Thus, their combined expression results in an additive effect on inhibition of virus production. Moreover, when we expressed multiple shRNAs in a cell line that is susceptible for HIV-1 replication, Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy