1023 Chitosan Oligomers as siRNA Delivery Systems In Vitro 1020 Why Simple Cationic Liposome Formulations Fail To Deliver siRNA Efficiently In Vivo Kevin Buyens, Stefaan C De Smedt, Joseph Demeester,[.]
1020 Why Simple Cationic Liposome Formulations Fail To Deliver siRNA Efficiently In Vivo Kevin Buyens, Stefaan C De Smedt, Joseph Demeester, Niek N Sanders 'Lab General Biochemistry & Physical Pharmacy Ghent Univer- sity Ghent Belgium Purpose In order to find suitable non-viral carriers for systemic siRNA-delivery, we developed a microscopy based technique which allows fast determination ofthe dissociation ofsiRNA/carrier complexes in full human serum Methods The dissociation of siRNA from both non-pcgylatcd and pegylated DotaplDope Iiposomes was studied in buffer and human serum via non-denaturing PAGE and the newly developed fluorescence fluctuation spectroscopy (FrS) based technique Results Both non-pegylated and pegylated liposomes resulted in an efficient binding of siRNA in Hepes buffer (20 mM, pH 7.4) at a +/- ratio of and 10 In contrast, poor complexation occured at a +/- ratio of I Addition of the siRNAIliposome complexes to human serum resulted, independent of the +/- ratio and presence of PEG , in an immediate and massive release of siRNA By evaluating the dissociation of the siRNAIliposome complexes in PBS and a solution of albumin (40 mg/ml in PBS), we found that siRNA dissociation is mainly caused by albumin Discussion These data suggest that lipidic carriers that are succesful for in vitro transfections may not be suited for siRNA-delivery after systemic application We also show that in the screening of possible siRNA-carrier systems, it's important to evaluate their behaviour and biological activity in the presence of the biological matrices they will encounter on their way to the target cells In this view, the FFS technique is more straightforward and reliable than PAGE, which does not show good compatibility with complex media like human serum We showed that serum albumin is the major cause of siRNA-dissociation from liposomes This is probably due to competition ofalbumin with the siRNA for the possitive charges on the Iiposomes The massive amounts of albumin present in human serum also explains why the extent ofsiRNA release is more or less independent of the +/- ratio 1021 Highly Effective Bi-Functional shRNAs for Cancer Gene Therapy Donald Rao,' Padmasini Kumar.P Viviana Mangual; lIa Oxendine, I Chris Jay, 1.2 Alex W Tong,I,2 Phillip B Maples, 1.2 Neil Senzer,I.2,3,4 John J Nernunaitis.P:' 'Cancer Research, Murex Pharmaceuticals Inc Dallas TX; lCancer Research, MOlY Crowley Cancer Research Centers Dallas TX; JCancer Research Baylor Sammons Cancer Center; Dallas, TX; "Cancer Research Texas Oncology; P.A" Dallas TX Altered gene expression plays a key role in the pathogenesis and maintenance of most pathological conditions including cancer In addition to the well described changes in caretaker (e.g., p53) and gatekeeper genes (e.g., RB) , other cancer-related genes, which can either be differentially over- or under-expressed, have been correlated with key cancer functions and disease behavior (Hanahan and Weinberg, 2000) Specifically, in silico network analysis has shown that ofthose genes that are over-expressed, a limited number appear to be high-degree hubs (i.e have a high inter-gene connectivity) suggesting functional dependence RNA interference (RNAi) is a powerful new method for silencing the expression of targeted genes We are actively developing a clinically applicable systemic targeted RNAi therapeutic strategy Mechanistically, RNAi can be mediated either through synthetic small interfering RNA (siRNA) or vector based short hairpin RNA (shRNA) Intrinsically, siRNA is quite different from shRNA Our comparative studies have shown a number of advantages of shRNA over siRNA including durability S390 and potency We have developed a novel proprietary " bi-functional" shRNAstrategy designed to simultaneously enter into multiple RNAi pathways (cleavage dependent and cleavage independent) to further increase efficacy of the formulation This, in tum, provides a clear advantage in progress toward clinical application ofshRNA Wc will report and update our findings at this meeting These observations will add to our understanding of the molecular mechanism of RNA interference and allow refinement of future designs ofeffective and safe shRNAs for cancer gene therapy 1022 Delivery of a lacZ shRNA to Muscles of the ROSA26 Mouse Erin S Kirkegaard,' Leonard Meuse,' Eric Finn," Joel R Chamberlain.' 'Division ofMedical Genetics Department ofMedicine University of Washington Seattle 11':4; lDepartment ofNeurology, University ofWashington Seattle 1JI:4 RNA interference (RNAi) is a conserved cellular process whereby double-stranded RNAs direct post-transcriptional gene sileneing through sequence-specific degradation ofthe target RNA Inhibitory RNAs can be expressed in cells as naturally occurring microRNAs or small interfering RNAs (siRNAs) An efficient alternative to transfection of siRNAs synthesized in vitro is the introduction of recombinant viral vectors expressing short hairpin RNAs (shRNAs) that take advantage of the cellular RNAi pathways for subsequent processing to active siRNAs in vivo AAV vectors have proven useful for systemic delivery of genes to a variety of tissues We are investigating the potential of systemic delivery ofMV vectors for gene knockdown to study gene funct ion and for gene therapy of dominant genetic disorders In the present study we have developed a recombinant AAV6 vector expressing an shRNA directed against E coli lacZ that allows efficient knockdown oflacZ mRNA We asked whether total l3-galactosidase (l3-gal) activity could be decreased following systemic delivery of the shRNA targeting laeZ mRNA in a variety of tissues in the ROSA26 mouse Our initial studies focus on the reduction of constituitively expressed l3-gal in cardiac and skeletal muscles Intravenous injection of the vector led to a time-dependent decrease in l3-gal activity compared to uninjected controls with a greater reduction in cardiac versus skeletal muscle This difference most likely reflects the variability in transduction of the heart and quadriceps muscles The ability to decrease l3-gal activity with RNAi in vivo suggests that AAV6 vectors delivering siRNAs could be a useful approach for domin antly inherited diseases of muscle and other tissues 1023 Chitosan Oligomers as siRNA Delivery Systems In Vitro Mohamed M Issa,' Sabina P Strand,' Kjell M Varum,' Per Artursson.' 'Pharmacy; Uppsala University, Uppsala, Sweden; 2BiotechnologJ', Norwegian Biopolymer Laboratory (NOB/POL) Norwegian University ofScience and Technology; Trondheim, Norway Objective To investigate the potential of chitosan oligomers as novel delivery systems for small interfering RNA (siRNA) in vitro Background Cationic lipid-b ased formulations were shown to be effective for ill vitro and ill vivo delivery ofsiRNA In contrast, cationic polymers were initially considered unsuitable for oligonucleotides delivery However, recent studies have shown that cationic polymers such as polycthylcnciminc (PEl) and polyamidoamine (PAMAM) dendrimers can be used for siRNA delivery Possibly, the contradictory reports on the efficiency ofcationic polymers as deliver)' systems for oligonucleotides could be attributed to formulation issues since in most studies these oligonucleotides were formulated and delivered under conditions optimized for pDNA We, therefore, Molecular Therapy Volume 15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene Tllcr.lpr investigated the critical requirements for siRNA formulation by ehitosan oligomers More specifically, the role ofthe structural variables of chitosan oligomers, the formulation parameters and the effect of serum were studied and correlated to the efficiency of the gene silencing activity in vitro Methods Using polyethyleneimine (PEl) and Iipofeetamine 2000 (LF2000) as controls, linear and branched chitosan oligomers of various chain lengths were complexed with siRNA and their physical stability, protection against enzymatic degradation and particle size were examined The cellular toxicity and luciferase gene silencing activity ofthe siRNA complexes were investigated in stably luciferase-expressing 293 cells (293-Luc) in vitro Results Chitosan oligomers were able to complex siRNA into nanoparticles (34-86 nm) that provided protection against RNase degradation A higher number of positive charges provided by longer chitosan chains and/or higher charge ratios between chitosan oligomers and siRNA was shown to be essential not only for obtaining physically stable complexes but also for mediating the highest luciferase silencing activity in vitro Unlike PEl and LF2000, siRNA complexes formulated with chitosan oligomers retained their luciferase silencing activity when the transfection medium contained 10% serum Chitosan oligomers also displayed minimal cellular toxicity compared to PEl and LF2000 Linear chitosan oligomers having an average-number degree of polymerization (DP,) of 85 monomer units (DP.85) required a siRNA concentration as low as 44 nM to obtain 95% silencing of the luciferase gene expression that was sustained for days in 293-Luc cells Conclusion Our findings forward chitosan oligomers as safe, efficient alternative delivery systems for siRNA in vitro and possibly in vivo 1024 Enhanced RNA Interference Using Reducible Poly(Amido Ethylenimine) Ji Hoon Jeong,' Lane V Christensen, I James W Yockman,' Zhiyuan Zhong,' Johan F J Engbcrscn.' Won Jong Kim,' Jan Fcijcn.? Sung Wan Kim ICCCD/Department ofPharmaceutics and Pharmaceutical Chemistry, University ofUtah, Salt Lake City, UT; l Department ofPolymer Chemistry and Biomaterials and lnstitute for Biomedical Technology (BMTI), University ofTwente, Enschede, Netherlands RNA interference (RNAi), a post-transcriptional gene silencing mechanism , has been considered a promising therapeutic tool for the treatment ofmany genetic disorders, infectious diseases and cancer Short interfering RNA (siRNA)-mediated RNAi is initiated by the incorporation of one of the siRNA strands into an endonuclease complex, called RNA-induced silencing complex (RISC) , which specifically binds to target mRNA sequence to perform endonucleolytic digestion ofthe mRNA However, due to its instability and poor permeability across biological membranes, the development of safe and efficient siRNA delivery systems is a prerequisite for the successful application ofsiRNA in mammalian cells To address the needs, a reducible poly(amido ethylenimine) (SS-PAEI) was synthesized by addition copolymerization of triethylenetetramine and cystamine bis-acrylamide (TETA-CBA) TETA-CBA was used as a carrier for siRNA based on the hypothesis in which TETACBA/siRNA complexes would be destabilized once located within the reductive eytosolie environment, leading to the release of intact siRNA into the cytoplasm where the siRNA would be efficiently incorporated into RISC without any interference ofa cationic polymer TETA-CBA could form stable complexes upon interacting with siRNA The complexes were also readily destabilized to release siRNA under reductive force VEGF siRNAlTETA-CBA complexes showed significantly higher suppression of VEGF expression in human prostate cancer cells (PC-3), compared to PEl 25 kDa formulations Confocal microscopy revea led that siRNA formulated with TETA-CBA was dissociated much more efficiently from the Molecular Therapy Volume15.Supplement ~by Cop yright © The Americ m Society o r Gene Therapy 2007 comp lexes in the eytosolie space than one with PEl 25kDa This results suggests that the increased bioavailability of siRNA by the triggered release mechanism ofreducible TETA-CBA polymer could facilitate the access of siRNA to RISC, resulting in the enhanced RNAi activity 1025 Silencing of Microphage Migration Inhibitory Factor (MIF) Via siRNA Causes Attenuation of Growth and Induction of Apoptosis in Human Prostate Cancer Cells Nan Jiang,' Hongwei Lj,! Yongxin Gao,' Colin Sumners.' I Department ofPhysiology and Functional Genomics, Unlveristy ofFlorida, Gainesville, FL Macrophage migration inhibitory factor (MIF) has an established role in proinfiammatory and immune responses More recently it has begun to be recognized as a pro-tumorigenic factor and has been defined as a novel oncogene Increased expression ofMIF has been demonstrated in human melanomas, breast carcinomas, adenocarcinomas of the lung, bladder cancer, hepatocellular carcinomas, gastric cancer and neuroblastoma The expression of MIF is also increased in prostate cancer (PCa), one ofthe most common cancers in men Each year approximately 543,000 new cases are reported worldwide, and the disease kills 200,000 (mostly older men) in developed countries Exist ing therapies and surgical interventions have not been able to effectively manage this form of cancer and, therefore, efforts are ongoing to explore novel targets and strategies for the management ofPCa.The aim ofthis study was to test whether tumor cell growth could be inhibited by a reduction of endogenous MIF expression in prostate cancer cells To define the role of MIF in PCa, we used the technique of RNA silencing via small interfering RNA (siRNA) We transfected the androgen-independent prostate cancer cell line Dul45 with MIF siRNA , which resulted in a significant inhibition ofMIF expression from 48h to 72 h posttransfection MIF depletion resulted in a decrease in cell proliferation and an induction of apoptosis in Ou-145 cells Oownregulation of MIF expression significantly decreased the expression of cyclin D I, c-Met (proto-oncogene) and p21 (cyclin-dependent kinase inhibitor 1A) at the mRNA level at 48 h post-transfection However, expression of these genes returned to baseline (e-Met and p21) or increased (cyclin D I) at the mRNA level at 72 h post-transfection Interestingly, p53 mRNA level was significantly increased at 72 h post-transfection although it was not significantly changed at 48 h post-transfection These studies may help in understanding the molecular mechanism(s) associated with MIF function in prostate cancer cells In summary our data demonstrate that blocking MIF expression via siRNA is a potential gene therapeutic strategy for androgen-independent prostate cancer 1026 Gene Silencing for Epidermal Growth Factor Receptor Variant-III Induces Cell-Specific Cytotoxicity Shay E Park,' Vidya Bodempudi,' Weihong Pan,' Mary Jean Mauzy, I Farnaz Yamoutpour, I Tuba Esfandyari,' Robert Kratzke, I Arkadiusz Dudek,' Donald M O'Rourke,' Donald J Tindall.i Faris Farassati.' I Department ofMedicine , Division ofHaematology, Oncology and Transplantation, University ofMinnesota, Minneapolis, MN; lMayo Clinic Rochester; Rochester; MN; 'University ofCalifornia San Francisco, San Francisco, CA; "University ofPennsylvania, Philadelphia, PA Epidermal growth factor receptor (EGFR) undergoes a deletion resulting in a constitutively active version named EGFR variant-Ill (EGFRvlII) which is expressed in differen t malignancies including glioma In this work, by designing a siRNA molecule against the S391 ... siRNA concentration as low as 44 nM to obtain 95% silencing of the luciferase gene expression that was sustained for days in 293-Luc cells Conclusion Our findings forward chitosan oligomers as. .. recently it has begun to be recognized as a pro-tumorigenic factor and has been defined as a novel oncogene Increased expression ofMIF has been demonstrated in human melanomas, breast carcinomas, adenocarcinomas... silencing activity in vitro Methods Using polyethyleneimine (PEl) and Iipofeetamine 2000 (LF2000) as controls, linear and branched chitosan oligomers of various chain lengths were complexed with siRNA