166 A Novel Ligand for Enhanced and Targeted Non Viral Gene Delivery to Pulmonary Epithelial Cells Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy[.]
CHEMICAL AND MOLECULAR CONJUGATES I 164 Chemical Modification of Polyethylenimine for Improved Gene Delivery Yajun Duan, Zhenfang Zhang, Cuihong Yang, Yaoting Yu, Deling Kong The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin, China Non-viral gene delivery has become a promising alternative to viral vectors since the vectors could be synthesized with high purity and quality, are less immunogenic, and safer As one of few commercially available polycations, polyethylenimine (PEI, molecular weight 25 kDa) was widely studied and used due to its superior transfection efficiency However, its cytotoxicity, non-degradability and nonspecific gene delivery limited its uses in clinical trials In our study, we focused on chemical modification of PEI into develop a suitable nonviral gene vector for in vivo application G250mAb, one of the most extensively studied monoclonal antibodies associated with renal cell carcinoma, was prepared and used to modify PEI G250 modification significantly improved the biocompatibility of PEI, and the conjugate G250-PEI was capable of condensing DNA into nano particles The FACS data showed that for G250 positive cells, the transfection efficiency of G250-PEI was 2-fold higher than that of PEI In addition, the expression of luciferase protein in G250 positive cancer cells is 15-fold and 61-fold higher than that in G250 negative cancer cells and normal smooth muscle cells, respectively However, the G250-PEI conjugate showed limited transfection efficiency in the presence of serum suggesting that further modification is needed In this regards, poly (ethylene glycol) (PEG) modification has been a common approach in improving the performance of gene or drug carriers in terms of less aggregation, longer life time in blood circulation and lower cytotoxicity In our study, PEI was modified with PEG (molecular weight 550 or 2000) prior to G250 conjugation The resultant conjugate G250-PEI-PEG was almost nontoxic at a wide range of concentrations In vitro experiment showed that the transfection efficiency in Hela cells was as high as 70% Interestingly, PEG modification significantly enhanced the transfection in the presence of serum and in vivo The expression of luciferase in tumors mediated by G250-PEI-PEG conjugate was folds of that by PEI Since the regular PEI (25 kDa) is non-degradable, we further developed a new class of polymeric gene vectors based on low molecular weight of PEI (800Da) Degradable PEG and D,L-Dithiothreitol (DTT) co-polymers were first synthesized, PEI 800 was then grafted into the main chain of PEG-DTTs and gave rise to a new class of polycations, (PEG-DTT)-g-PEIs These new gene vectors have shown excellent biocompatibility and higher transfection efficiency than PEI in Cos-7, HepG2 and MCF-7 cells Unlike the above mentioned PEI (25kDa) derivatives, these PEG containing co-polymers showed excellent transfection efficiency in the presence of serum (up to 30%) Further evaluation experiment has been performed with rat tumor models In summary, these results indicate that chemical modification of PEI improved its performance as nonviral gene vectors Development of new gene vectors with low cytotoxicity, high efficiency and targeted in vivo gene delivery is still of great importance for human gene therapy Keywords: polyethylenimine; G250 antibody; nonviral gene delivery; poly (ethylene glycol) Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 165 Characterization of Low Molecular Weight Folate-Conjugated Chitosan as Polymeric Nanocarriers for siRNA Delivery Julio C Fernandes,1 Christian Jreyssaty,1 Mohamed Benderdour,1 Patrick Lavigne,1 Xingpin Qiu,2 Francoise M Winnik,2 Xiaoling Zhang,3,4 Kerong Dai,3,4 Qin Shi.1 Orthopaedic Research Laboratory, Sacre-Coeur Hospital, University of Montreal, Montreal, QC, Canada; 2Faculty of Pharmacy and Department of Physical Chemistry and Polymer Sicence, University of Montreal, Montreal, QC, Canada; 3Insititue of Health Science and School of Medicine, Shanghai JiaoTong University, Chinese Academy of Sicences, Shanghai, China; Department of Orthopaedics, Ninth People’s Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China Low transfection efficiency of chitosan is one of drawbacks for its use as a gene delivery carrier Folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing cells In this study, folate-chitosan particles were investigated as an effective delivery system for small interfering RNA (siRNA) in different cell lines Chitosan or folate-chitosan-siRNA complexes were formed at varying weight ratios and characterized using gel electrophoresis Complex size and surface charge were determined by Zetasier and dynamic light scattering, respectively Cell viability and the transfection efficiency of chitosan or folatechitosan were examined in Hela, KB and OV-3 line cells, respectively Gel electrophoresis results indicated that the binding of chitosan or folate-chitosan and siRNA depended on molecular weight and weight ratios of chitosan (or folate-chitosan) to siRNA The particule sizes of chitosan or folate-chitosan/siRNA complexes were nano-size at the weight ratio of 10:1 and 50:1 The introduction of folate ligand into chitosan increased lightly the size of particles Compared to chitosan-siRNA, folate-chitosan-siRNA nanoparticles were less cytotoxic and improved gene silencing transfection efficiencies By quantitative analysis of target gene inhibition with reverse transcription-polymerase chain reaction (RT-PCR), folate-chitosanmediated siRNA silencing of the SSB (Sjogren syndrome antigen B) gene occurred maximally with 85-89% efficiency with molecular weight of 25 and 50 kDa Therefore, folate-chitosan nanoparticles show much potential as viable vector candidates for safer and costeffective siRNA delivery 166 A Novel Ligand for Enhanced and Targeted Non-Viral Gene Delivery to Pulmonary Epithelial Cells Johannes P Geiger,1,2 Manish K Aneja,1 Grit Kummerloewe,3 Burkhard Luy,3 Tina Romer,4 Ulrich Rothbauer,4 Carsten Rudolph.1,2 Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany; 2Department of Pharmacy, Free University of Berlin, Berlin, Germany; 3Department of Chemistry, Technical University Munich, Munich, Germany; 4Department of Biology II, Ludwig-Maximilians University, Munich, Germany One of the major prerequisite for successful non-viral gene therapy is the delivery of gene vectors to the target cells within a tissue Majority of polymers or lipids used for non-viral gene delivery lack the desirable feature of cell targeting In the present study a novel ligand was investigated for mediating specific delivery to bronchial epithelial cells Polyethylenimine (PEI) is one of the most effective, commercially available polymeric gene transfer agents which has been successfully used for gene delivery both in vitro and in vivo For this reason PEI was used as a polymer backbone for covalently coupling the ligand Initially the ligand was conjugated to Fluorescein-BSA and the resulting molecular conjugates were applied to different bronchial and alveolar epithelial cells Uptake of S65 METABOLIC DISEASES I Fluorescein-BSA-Ligand in the nanomolar range was observed only in bronchial epithelial cells and could be inhibited both by excess of free ligand and specific antagonist The uptake of FluoresceinBSA-Ligand conjugates was confirmed by confocal laser scanning microscopy We next investigated if this route of uptake could be used to successfully transfect the bronchial epithelial cells For this, the ligand was covalently coupled to PEI and the resulting conjugate was compared to unmodified PEI with respect to the transfection efficiencies Higher (15-fold) transfection efficiencies could be observed with ligand modified PEI at lower N/P-ratios without any significant toxicity Ligand-mediated cell specific gene delivery is currently being investigated in vivo Metabolic Diseases I 167 Inducing Immune Tolerance to a Therapeutic Protein Using Intrathymic Gene Delivery Qiuming Chu, Rod Moreland, Lan Gao, Elizabeth Meyers, Kristin Taylor, Jennifer Nietupski, Scott Bercury, Robin Ziegler, John Marshall, Seng H Cheng, Ronald K Scheule Applied Discovery Research, Genzyme Corporation, Framingham, MA Pompe disease is an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) Enzyme replacement therapy is being used to treat patients with Pompe disease However, the generation of neutralizing antibodies against recombinant human GAA (rhGAA) in patients may result in a decrease in treatment efficacy We have shown previously that immune tolerance can be induced to rhGAA in a mouse model of Pompe disease by systemic gene delivery of an AAV2/8 vector bearing hGAA, where expression is almost exclusively from hepatocytes Here, we have evaluated intrathymic gene delivery of AAV2/8-hGAA as an alternative method of inducing immune tolerance Expression of promiscuous genes, tissue restricted or peripheral antigens in the thymus plays a critical role in self-tolerance The generation of thymic tolerance has been evaluated previously by the intrathymic delivery of cells, proteins and genes T cells recognize antigens in the thymus as self, and go through negative (such as depletion) as well as positive selection (becoming T regulatory cells, Tregs) We hypothesized that expressing hGAA in the thymus by intrathymic delivery of a vector, namely, AAV2/8-DC190-hGAA, containing a liver specific promoter would induce immune tolerance to the protein through both selection mechanisms We have shown previously that the kinetics of tolerance induction following systemic gene delivery to the liver depend on the expression level attained, viz higher levels lead to more rapid tolerance induction In contrast, inducing tolerance by intrathymic gene expression may require relatively low expression, which could suggest an advantage for this approach in the presence of preexisting antibodies against the vector In a direct comparison of immune tolerance induction in mice by intrathymic and systemic gene delivery approaches, we have quantified antibodies to GAA after sequential challenges with rhGAA in Freund’s adjuvant Direct intrathymic injection of a low vector dose (2E10 drp/mouse) resulted in induction of a level of immune tolerance equivalent to that seen after systemic delivery of a high vector dose (5E11 drp/mouse) to the liver, ie no significant increase in anti-GAA antibody titers upon rhGAA challenge Compared to systemic delivery, intrathymic injection led to higher thymus GAA mRNA levels and much lower liver mRNA and serum GAA levels, suggesting a different mechanism for tolerance generation Like systemic delivery, intrathymic delivery also led to an increase in CD4+Foxp3+ T cells, suggesting a role for Tregs in tolerance induction Importantly, intrathymic delivery did not induce tolerance to the AAV vector itself Although these initial results are encouraging, additional preclinical studies will be required S66 to determine whether an intrathymic gene delivery approach is truly feasible in patients 168 Administration of rAAV2/8 and rAAV2/1 Vectors Mediates Sustained Correction of a Canine Model of Glycogen Storage Disease Type Ia Catherine Correia,1 David Weinstein,1 Laurie Fiske,1 John Verstegen,2 Andrew Specht,3 Maggie Struck,4 Harvey Ramirez,4 Karine Onclin-Verstegen,2 Stacy Porvasnik,1,6 Janice Chou,5 Barry Byrne,1,6 Cathryn Mah.1,6 Pediatrics, University of Florida, Gainesville; 2LACS, University of Florida, Gainesville; 3SACS, University of Florida, Gainesville; ACS, University of Florida, Gainesville; 5Section on Cellular Differentiation, NIH, NICHD, Bethesda; 6Powell Gene Therapy Center, University of Florida, Gainesville Glycogen storage disease type Ia (GSDIa) is an autosomal recessive disorder resulting from a lack of functional glucose-6-phosphatase-α (G6Pase-α) that catalyzes the hydrolysis of glucose-6-phosphate to glucose in the terminal step of gluconeogenesis and glycogenolysis GSDIa patients are unable to maintain glucose homeostasis and as a result, suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis The canine model of GSDIa recapitulates almost all aspects of the human form of disease Historically, untreated GSDIa dogs not survive past weeks of age and only with intense dietary supplementation and management have a few survived longer than that No cure exists for GSDIa and care is palliative Gene therapy may provide an alternate therapy for this disease We investigated the potential of recombinant adeno-associated virus vector-based therapy to treat the canine model of GSDIa Initially, 5x1013 vg/kg therapeutic rAAV2/8 vector was delivered intravenously into a one-day-old GSDIa dog At two weeks-post vector administration, blood glucose and lactate levels remained within normal range after hours of fasting, whereas by hour of fasting, the untreated GSDIa dog had blood glucose levels significantly below normal and elevated lactate levels At one month post-treatment, the rAAV2/8-treated dog still maintained normal blood glucose levels after a hour fast, however, blood lactate levels were elevated at the end of the fasting period By two months posttreatment, the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after one hour of fasting and intense dietary glucose supplementation had to be resumed These results were similar to other murine studies in which neonatal delivery of livertargeted rAAV2/8 vector could mediate only short-term correction The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein Two months post rAAV2/1 dosing, the animal was able to sustain near-normal blood glucose levels after hours of fasting Both blood glucose and lactate levels were normal at hrs post-fasting, however, lactate levels were elevated by hours, indicating that partial correction was achieved Absolute hepatic G6Pase-α activity was 6.3% wild-type levels (as compared to 0.4% in untreated control) at months post rAAV1 treatment All dietary glucose supplementation was discontinued starting at one month post rAAV1 delivery and the dog continues to thrive with minimal labwork abnormalities currently at 16 months of age This initial study suggests that gene therapy may provide a viable alternative for the treatment of GSDIa Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... exists for GSDIa and care is palliative Gene therapy may provide an alternate therapy for this disease We investigated the potential of recombinant adeno-associated virus vector-based therapy to. .. gene delivery of an AAV2/8 vector bearing hGAA, where expression is almost exclusively from hepatocytes Here, we have evaluated intrathymic gene delivery of AAV2/8-hGAA as an alternative method of... homeostasis and as a result, suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis The canine model of GSDIa recapitulates almost all aspects of the human