666 phase i study results of pbi shRNAâ„¢ STMN1 nanoplex via intratumoral injection in advanced cancer

666 Phase I Study Results of pbi shRNAâ„¢ STMN1 Nanoplex Via Intratumoral Injection in Advanced Cancer Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Ce[.]

CANCER-TARGETED GENE AND CELL THERAPY: NON-VIRAL APPROACHES, AND SCREENING/NEW TARGET IDENTIFICATION reduced in vitro angiogenesis and cell migration in HUVECs by combination knockdown of neuropilins Polymerase chain reaction RT-PCR, western blotting and immunohistochemistry showed that both the neuropilins are widely co-expressed on BxPc-3 cells Conclusion: In conclusion, our in vitro studies suggest that gene therapy, targeted against NRP-1 and NRP-2 to combine antiangiogenic and anti-lymphangiogenic effects, could potentially have a synergistic anti-cancer effect against human pancreatic adenocarcinoma Future studies will focus on anti-cancer ultrasound mediated gene therapy targeting NRP-1 and NRP-2 to suppress tumor angiogenesis and limit tumor growth/metastases in an orthotopic model of human pancreatic adenocarcinoma in RNU nude rats 665 Comparison of Ultrasound Targeted Minicircle DNA Versus Standard Plasmid DNA Delivery for Cancer Gene Therapy Pratiek N Matkar,1 Saleh A Alghadeer,1 Dmitriy Rudenko,1 Wei J Cao,1 Hao H Chen,1 Michael A Kuliszewski,1 Howard LeongPoi.1 Cardiology, Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St Michael’s Hospital, University of Toronto, Toronto, ON, Canada Background: Ultrasound-mediated gene delivery (UMGD) of plasmid DNA results in targeted gene transfection However, applicability of conventional plasmid is limited by low and transient expression Minicircle DNA has been shown to be a more efficient vector for gene transfection We hypothesized that UMGD of minicircle DNA will be more effective compared to conventional plasmid and can be successfully delivered to the pancreas for developing a safe and effective anti-cancer therapy Methods: In vitro: Human umbilical vein endothelial cells (HUVECs) were transfected with μg of plasmid carrying the GFP reporter gene or molar equivalent of μg of minicircle, using Lipofectamine LTX GFP expression was assessed by phase-contrast fluorescent microscopy until fluorescent signal became undetectable Prior to in vivo studies, minicircle was compared to conventional plasmid for its binding capacity to cationic microbubbles (100 million cationic microbubbles with 12.5, 25, 37.5 and 50 μg DNA vector) using spectrophotometer In vivo hindlimb: 500 μg of plasmid and molar equivalent of 214 μg of minicircle was charged-coupled with 1000 million cationic lipid microbubbles and intravenously infused into Sprague-Dawley rats (n= 30) during UMGD to the left proximal hindlimb adductor muscle Transfection using two ultrasound powers were evaluated: 1.3 MHz at 0.9W, 120V, mA and 0.2W, 67V, 4mA The transfection efficacy and durability of minicircle and conventional plasmid was determined using immunohistochemistry and RT-PCR In vivo tumor: million BxPc-3 (human pancreatic adenocarcinoma) cells were injected into the flanks of RNU nude rats to form a heterotopic tumor Fragments of heterotopic tumor were implanted onto the pancreas of RNU rats and were delivered by minicircle GFP using UMGD, and weeks post implantation Prior to sacrificing the animals for biochemical analyses, tumors were imaged by contrast ultrasound Results: In vitro results showed robust gene expression in HUVECs from minicircle with increased transfection efficiency at 7-10 fold compared to plasmid Minicircle binding to cationic microbubbles was significantly greater as compared to conventional plasmid For conventional plasmid, saturation occurred at 250-500 μg plasmid resulting in ~6,000 copies per microbubble For minicircle, saturation occurred at 125-250 μg minicircle resulting in ~20,000 copies per microbubble Using UMGD, minicircle vector showed significantly greater expression by RT-PCR and immunohistochemistry, compared to conventional plasmid using similar ultrasound settings Minicircle gene expression under lower power was comparable to the gene expression by conventional plasmid under higher power Minicircle Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy GFP was successfully delivered to orthotopic tumors implanted in the pancreas as demonstrated by RT-PCR, western blotting and immunohistochemistry Conclusions: UMGD using minicircle results in greater and more durable transfection compared to conventional plasmids, and may further the development of UMGD techniques for anti-cancer gene therapy 666 Phase I Study Results of pbi-shRNA™ STMN1 Nanoplex Via Intratumoral Injection in Advanced Cancer John Nemunaitis,1,2,3,4,7 Minal Barve,1,3 Zhaohui Wang,4,7 F C Brunicardi,6 Nancy S Templeton,4,7 Neil Senzer,1,4,7 Padmasini Kumar,4 Gladice Wallraven,4 Chris M Jay,4,7 Joseph Kuhn,5 Peter Beitsch,2 Robert G Mennel,3 Haskell M Kirkpatrick,3 Douglas Orr,3 Scott Stone,3 Tammy Roque,3 Phillip B Maples,4 Donald D Rao.4,7 Mary Crowley Cancer Research Centers, Dallas, TX; 2Medical City Dallas Hospital, Dallas, TX; 3Texas Oncology, P.A., Dallas, TX; 4Gradalis, Inc., Dallas, TX; 5WLS Surgical Associates, P.A., Dallas, TX; 6David Geffen School of Medicine at UCLA, Los Angeles, CA; 7Strike Bio, Inc., Dallas, TX STMN1 is a microtubule destabilizing protein critical in the control of mitosis and is differentially overexpressed in malignant versus nonmalignant tissue Using our novel RNA interference construct, pbishRNA™-STMN1 nanoplex, we previously demonstrated STMN1 expression knockdown in surgically obtained human tumors as well as safety and efficacy in animal xenograft and tumor graft models thereby justifying Phase I testing (BB-IND 14938) Eleven patients (angiosarcoma, anal, colorectal, sarcoma, head and neck, ovarian, melanoma x2 and breast cancer x3) have entered into our dose-escalation trial Design is shown in Figure PK analyses of circulating plasmid of evaluable patients in cohorts and is shown in Figure No toxic effects were observed All patients demonstrated stable disease during the first month post-treatment Intratumoral cleavage product was demonstrated in cohorts and by next generation sequencing and by RLM RACE assay in cohort These results support continuation of Phase I testing and establish the basis for transition to systemic treatment starting at a dose level equivalent to demonstrated safe plasmid levels in circulation S257 CANCER-TARGETED GENE AND CELL THERAPY: NON-VIRAL APPROACHES, AND SCREENING/NEW TARGET IDENTIFICATION 667 Multiple Cycles of Liposomal Rat Insulin Promoter-Thymidine Kinase and Ganciclovir (L-RIP-TK/GCV) Increases the Therapeutic Effect on Human Pancreatic Cancer (PDAC) Tumor in SCID Mice Shi-He Liu,1 James X Wu,1 John Nemunaitis,2 F Charles Brunicardi.1 Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Mary Crowley Cancer Research Centers, Dallas, TX Introduction: We previously demonstrated that RIP can be used for PDAC targeted gene therapy, since PDX1 is overexpressed in PDAC and activates RIP We showed that a single cycle of adenoviral RIP-TK/GCV therapy suppressed human pancreatic cancer tumor volume in SCID mice, however repeated cycles were limited by significant toxicity, hyperglycemia and immunogenicity In this study, we hypothesized that multiple cycles of L-RIP-TK/GCV efficiently ablate human PDAC in SCID mice without toxicity Methods: SCID mice two weeks after intraperitoneal injection of 0.5M PANC-1 cells were given either a single cycle of iv L-RIPTK/GCV (35 μg), versus four biweekly cycles of 35, 30, 20, 10, or μg iv L-RIP-TK (n=10-20 mice per group) Each treatment cycle consisted of L-RIP-TK on day followed by intraperitoneal GCV 40mg/kg twice daily for 14 days Tumor volume, survival, insulin and glucose levels were measured TUNEL assay was performed on tumors and islets Results: Multiple cycles of 10-35μg L-RIP-TK/GCV significantly reduced or completely ablated PANC-1 tumor volume as compared to single cycle with 35μg; multiple cycles of 35μg L-RIP-TK/GCV resulted in smallest tumor volume among all groups (p