894. Ornithine and Tryptophan as an Efficient Polycation for siRNA Delivery to Tumor Cells

OLIGONUCLEOTIDE & RNAI THERAPEUTICS III 892. Development of a Novel, RNAi-Based Therapeutic Targeting Pancreatic Duodenal Homebox-1 (PDX-1) for Insulinoma and Pancreatic Cancer

Zhaohui Wang,1 Shi-he Liu,5 Donald D. Rao,1 Phillip B. Maples,1 Neil Senzer,1,2,3,4 John Nemunaitis,1,2,3,4 F. C. Brunicardi.5 1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3Texas Oncology, P.A., Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX; 5Department of Surgery, Baylor College of Medicine, Houston, TX.

Insulinoma is the most common type of islet cell tumor. Malignant insulinomas are devastating from hyperinsulinemia and result in uncontrollable hypoglycemia. Pancreatic duodenal homebox-1 (PDX-1) belongs to a homeodomain-containing transcription factor family and plays a primary role in pancreatic organogenesis. PDX-1 maintains beta-cell function by regulating transcription of insulin, glucokinase and glucose transporter type 2. PDX-1 is overexpressed in insulinomas resulting in hyperinsulinemia. PDX-1 is also found to be commonly overexpressed in pancreatic tumors. Metastatic pancreatic cancer has a 4-6 month survival from diagnosis. Silencing of PDX-1 expression represents an attractive approach to inhibit tumor growth. A proprietary tandem “bi-functional” short hairpin RNA (shRNA) was designed to silence gene expression of PDX-1. The bi-functional shRNA cassette has been demonstrated to be highly effective. To investigate the efcacy and specicity of PDX-1 bi-shRNA in insulinoma and pancreatic cancer, miR30-based bi-functional shRNA cassettes against either human PDX-1 or mouse PDX-1 were cloned into pUMVC3 vector (currently used in clinical studies of the TAG and FANG cancer vaccines). Bi-functional shRNAs were electroporated into either a mouse insulinoma cell line with signicant endogenous expression of mouse PDX-1 or a human colon cancer cell line with overexpressed PDX-1. Stem-loop PCR was used to detect processed mature shRNAs, while RACE-PCR was employed to examine potential cleavage products of human and mouse PDX1 mRNA. RT-QPCR and immunoblotting were used to examine the knockdown of expression of either human PDX-1 or mouse PDX-1. The processed mature shRNAs were detected 24 hours after transfection and sequence conrmed. RACE-PCR analysis showed that both human PDX1 mRNA and mouse PDX1 mRNA were precisely cleaved in the center of target region as predicted by corresponding bi-functional shRNAs. In addition, the bi-functional shRNA targeting human PDX-1 did not cause the cleavage of the mouse PDX-1 mRNA and vice versa. The silencing effect of bifunctional shRNA on human PDX-1 was observed 24 hours after transfection and lasted for at least 96 hours. The maximum silencing effect, 80% knockdown of human PDX-1, was achieved 72 hours after transfection. Moreover, bi-functional shRNA targeting mouse PDX-1 did not affect the expression of human PDX-1. Similarly, expression of mouse PDX-1 was silenced 24 hours after transfection and the silencing effects lasted for at least for 96 hours. The maximum silencing effect, 95% of mouse PDX-1 expression, was observed 48 hours after transfection. Moreover, bi-functional shRNAs targeting human PDX-1 did not alter the expression of mouse PDX-1 either. This study demonstrates the efcacy and species specicity of bifunctional shRNAs targeting either mouse or human PDX-1.

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893. Systemic Delivery of Highly Active Functionalized Lipopolyamine-Formulated siRNA to Murine Lung with Reduction of Inammation by Dexamethasone Pre-Treatment

Jeff Sparks, Kevin J. Polach, Richard T. Congo, Gregory Slobodkin, Angela Rea-Ramsey, Casey Pence, Majed Matar, Diane McClure, Jennifer Rice, David Ulkoski, Elaine Brunhoeber, Leslie Wilkinson, Khursheed Anwer, Jason G. Fewell. EGEN, Inc., Huntsville, AL. The efcient delivery of siRNA to target cells is critical for the application of RNAi to the treatment of human disease. To date, some liposomal systems have been shown to give a practical balance of activity and toxicity in the context of systemic application. However, the primary hurdle in the advancement of this technology is the tendency toward generation of an acute inammatory response and in many cases liver toxicity when therapeutic levels of formulated material are used. Several approaches have been utilized to abrogate these responses, including the use of siRNA duplexes with modied bases, reduction of overall particle size and positive charge of the formulation, and pretreatment of animals with anti-inammatory drugs. To this end, we have developed a novel chemically exible cationic lipopolyamine core structure that when modied with serumstabilizing agents can be formulated with siRNA into nanoparticles for efcient in vitro and in vivo delivery. In mice at 48 hours following a single intravenous administration of formulated CAV-1 siRNA (∼2mg/kg siRNA), signicant target transcript knockdown was noted in the lungs with no transcript knockdown observed in the livers. Serum chemistry and cage side observations indicated little or no systemic toxicity associated with administration. A transcript array was performed on lung tissue harvested from the animals to look for indications of local inammation at the site of transcript knockdown. The results from this analysis showed increases in some transcripts associated with inammation relative to dextrose treated animals. Pretreatment with dexamethasone served to reduce the local inammatory response without adversely affecting target mRNA knockdown and in fact transcript knockdown was slightly increased over levels seen in animals not treated with dexamethasone. The retention of silencing activity of the administered siRNA in combination with anti-inammatory treatment suggests a specic RNAi-mediated response. The lung-specic response observed with this formulation is also not related to the “rst pass” phenomenon common to aggregation of traditional highly cationic delivery systems upon exposure to serum. In addition, the delivery system is envisioned as a platform for the ongoing development of highly specialized targeted structures utilizing organ- and tumor-specic ligands.

894. Ornithine and Tryptophan as an Efcient Polycation for siRNA Delivery to Tumor Cells Yusuke Sato,1 Hiroto Hatakeyama,1 Hideyoshi Harashima.1 Faculty of Pharmaceutical sciences, Hokkaido University, Sapporo, Hokkaido, Japan.

1

Cancer-targeted nucleic acid delivery is expected as one of the effective strategies for a cure of it. We developed a multifunctional envelope-type nano device (MEND) for efficient delivery of plasmid DNA (pDNA). In addition, we succeeded in delivering short interference RNA (siRNA) into cytoplasm by MEND which contains siRNA nano particles formed by using stearyl octaarginine (STR-R8). In the present study, to achieve further gene silencing activity compared with STR-R8, we performed the screening of the various polycations. We chose protamine and 10 kinds of random sequence polypeptides containing basic amino acids. First, the ability of these polycations to form nano particles (<100 nm) with siRNA were evaluated by measuring size and zeta-potential of these nano particles, and as a consequence, 6 kinds of polycations were selected. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

OLIGONUCLEOTIDE & RNAI THERAPEUTICS III Next, we prepared MENDs containing siRNA nano particles which were formed with the 6 kinds polycations. The lipid composition of MENDs consisted of DOPE/PA (7/2). For effective cellular uptake and endosomal escape, MENDs were modied with PPD (PEG-peptideDOPE), STR-R8 and GALA and the resulted MENDs indicated 120-200 nm in diameter and 15-30 mV in zeta-potential. These MENDs were transfected to HeLa cells stably expressing luciferase and we compared the knockdown ability of polycations. Most of the polycations failed to knockdown luciferase activity, on the other hand, only the polypeptide containing ornithine and tryptophan (Orn/Trp) could induce higher knockdown ability than STR-R8. Furthermore, Orn/Trp induced knockdown effect at lower dose than STR-R8 based on the dose-response experiment. In conclusion, our results suggested that Orn/Trp is superior polycation to STR-R8 for siRNA delivery.

895. Inuence of 5’ Terminal Triphosphate in Short Hairpin RNA Targeting HCV Replicon on Antiviral Activity and Innate Immune Response

Kyungbo Kim,1 Bokhui Lee,1 Soo-Ryoon Ryoo,2 Dal-Hee Min,2 Dong-Eun Kim.1 1 Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea; 2Chemistry, KAIST, Daejeon, Republic of Korea. Hepatitis C virus (HCV) infection is a major cause of worldwide liver-relating diseases like brosis cirrhosis, chronic hepatitis and human hepatocellular carcinoma. The viral genome is a positive stranded 9.6kb RNA genome and is translated as a single polypeptide which is processed into several structural and nonstructural proteins. Because of multi-functionalities of HCV nonstructural protein 3 (NS3) in viral replication and evasion of intracellular host defense, it is promising target in HCV therapy. Previously, our laboratory utilized novel in vitro screening method to screen accessible sites on RNA by using combinatorial deoxyribozyme (DNAzyme) pool. In that study we discovered specic DNAzymes that potently cleave HCV NS3 RNA and inhibit HCV replication in cells. At present, we proved that target sites of the DNAzymes in the HCV genome RNA are also available to another kind of antisense oligonucleotide (AON) such as a short hairpin RNA (shRNA) that is processed into RNAinduced silencing complex (RISC). shRNA is usually synthesized by in vitro transcription reaction using bacteriophage RNA polymerase and the transcribed RNA has 5’ terminal triphosphate. Unfortunately, the 5’ triphosphate RNA can be recognized as a foreign RNA by retinoic acid inducible gene I (RIG-I) in eukaryotic cells. RIG-I is one of the major detector and protector in antiviral immunity of eukaryotic cells against RNA virus infection. To avoid the innate immune response, calf intestinal alkaline phosphatase (CIAP)treated shRNA that is transcribed by using T7 RNA polymerase was used. The dephosphorylated shRNA at 5’ terminus exhibited a higher efcacy of HCV gene silencing in Huh7 cells carrying HCV subgenomic replicon RNA than the shRNA bearing the 5’ terminal triphosphate. We hypothesized that the phenomenon is caused by competitive binding for shRNA bearing the 5’ terminal triphosphate between RISC and RIG-1. To investigate this hypothesis, we tested for binding afnity of each shRNA with RIG-I by using electrophoretic mobility shift assay (EMSA). We observed that 5’ triphosphate shRNA exhibited signicantly higher afnity for RIG-I than the CIAP-treated shRNA. In addition, stimulation of innate immune response was tested by monitoring the amount of interferon-β (IFN-β) expression that is triggered by the RIG-I activation. 5-fold higher up-regulation of IFN-β was observed in the 5’ triphosphate shRNA-transfected Huh7 cells without HCV replicon RNA (naïve Huh7) than in the CIAP-treated shRNA transfected one. In contrast, expression of IFN-β was not detected when the shRNA bearing 5’ triphosphate was transfected into the Huh7 cells carrying HCV replicon RNA, suggesting that viral factors may abrogate host immune surveillance of Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

external RNA. Thus, shRNA with or without innate immune response triggering via RIG-1 helicase activation should be evaluated for a better understanding of antiviral oligonucleotide therapy.

896. Enhanced Delivery of GFP siRNA Using New Amino Lipid-Based Cationic Liposomes

Hyun-Ki Kim,1 Enkhzaya Davaa,1 Chang-seon Myung,2 JeongSook Park.1 1 Department of Physical Pharmacy, College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea; 2 Department of Pharmacology, College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea. Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufcient cellular uptake and poor stability have limited its usefulness. Here, we report efcient delivery of siRNA via the use of cationic liposomes that contain a new amino lipid. The new amino lipid, poly-l-arginine-conjugated polyethylene glycol (PLR-PEG), was synthesized. To conrm the synthesis of the amino acid, 1H-NMR and gel permeation chromatography (GPC) were performed. Cationic liposomes as non-viral vectors were formulated using the cationic lipids 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolaminepropane (DOPE), cholesterol (Chol) and the amino lipid PLR-PEG. Physicochemical properties of cationic liposomes were investigated. A GFP siRNA was used as a model siRNA to test the efciency of cationic liposomemediated siRNA delivery. The liposomes could enhance delivery efciency and decrease cytotoxicity at an optimized lipid composition. The new cationic liposome formulation using a new amino lipid showed not only enhanced intracellular delivery of siRNA but also decreased cytotoxicity in H4II-E and HepG2 cell lines. The GFP siRNA delivered by new cationic liposomes using PLR-PEG was effective in reducing the GFP protein expression levels of the gene. These results suggest that the new cationic liposomes could be used for efcient delivery of siRNA therapeutics.

897. Gene Editing Activity in C. elegans Directed by Single-Stranded Oligonucleotides Kerry A. Falgowski, Eric B. Kmiec. Marshall Institute for Interdisciplinary Research, Marshall University, Huntington, WV.

Gene editing using the process of targeted nucleotide exchange (TNE) has been successful in reversing genotype and phenotype in mammalian cells, plant cells and animal models of human disease. TNE utilizes single-stranded DNA oligonucleotides (ODNs) to direct the exchange of a single base at a designated site in the genome, a reaction executed by the cell’s DNA repair and recombination pathways. While the reversal of a mutation is a signicant characteristic, gene editing can also be applied to the eld of functional genomics through the creation of mutations or polymorphisms in a particular gene. We chose the genetically welldened model organism, C. elegans, as a host for exploring the use of gene editing for functional genomics. To gain insight into whether TNE activity exists in C. elegans, we developed a protocol to evaluate the exchange reaction using a genetic readout system in E. coli. In this in vitro genetic readout assay system, a kanamycin antibiotic resistance gene containing a nonsense mutation serves as the target site for nucleotide exchange. Our results have shown that site specic ODNs can direct the exchange of a nucleotide in a plasmid to restore the antibiotic resistance phenotype in a dose dependent manner when incubated in C. elegans cell-free extract. The reaction also depends on the specic sequence in the ODN, the length of the ODN, and exhibits a strand bias for early detection of the change. TNE activity was conrmed by RFLP analyses and direct DNA sequencing of S345