23 Facial Amphipathic Deoxycholic Acid Modified Polyethyleneimine Used as SHP 1 siRNA Carrier for the Treatment of Ischemia Reperfusion Cardiac Injury Molecular Therapy Volume 20, Supplement 1, May 20[.]
CHEMICAL AND MOLECULAR CONJUGATES relationships are concealed In contrast, solid-phase assisted synthesis allows precise and modular assembly of natural and artificial amino acids into monodisperse polyaminoamides (PAAs) The resulting polymers with small, but defined, structural variations were analyzed with respect to their ability of pDNA and siRNA delivery, in order to correlate structure and activity Methods and Results: A functioning T-shaped polymer was chosen and two essential motifs for oligonucleotide transfer were modified: i) the lytic domain, responsible for pH-responsiveness and hence, the release of the payload out of the endolysosomal compartment, and ii) the crosslinking domains, increasing polyplex stability All PAAs had a constant number of protonable 1,2-diaminoethane motifs within the artificial amino acids, providing electrostatic oligonucleotide binding and endosomal buffering capacity Furthermore, stability of the polyplexes was increased by cysteines, a dioleic acid motif or an oligotyrosine modification, or a combination of these, integrated into the polymer structure The endosomal pH-specific lytic potential was evaluated with an erythrocyte leakage assay Only PAAs modified with the dioleic acid motif were lytic, in contrast to T-shapes with the oligotyrosine motif To assess the influence of the proton sponge effect on endosomal release, the buffer capacity between pH 5.5 and 7.4 was determined with pH-titration In vitro pDNA transfection showed that all polyplexes led to ≥ 100-fold increase in luciferase expression, compared to HBG treated cells Additionally, all polymers, except the T-shaped polymer containing only the oligotyrosine motif, led to potent siRNA gene silencing of 70-90% This oligotyrosine containing polymer showed the lowest pDNA and siRNA binding capacity in an agarose gel shift assay, which explained the decreased efficacy in nucleic acid delivery Moreover, toxicity was negligible Selected DNA polyplexes were applied in vivo and showed tumor specific gene delivery Intravenous injection of EG5 siRNA polyplexes resulted in mitotic aster formation exclusively in the tumor of the mice Furthermore, intratumoral EG5 siRNA delivery led to reduced tumor growth of the treated animal compared to the reference groups Conclusion: The described defined T-shaped PAAs allowed studies of structure activity relationships and showed that different structural prerequisites were necessary for pDNA and siRNA delivery These new polyaminoamides provide the setup for the design and development of robust, well tolerated oligonucleotide delivery systems 22 Theoretical Model of Cationic Nanoparticles Dellivery and Margination under Convective Flow Virginia Pensabene,1 Todd D Giorgio.1 Biomedical Engineering, Vanderbilt University, Nashville, TN Exposure to dynamic flow can strongly influence the efficiency of nanoparticles in gene delivery Cardiovascular fluid forces are important in modulating the delivery of synthetic vehicles, but electrostatic and steric forces of the medium constituents also influences the firm adhesion on the cell membrane by non-specific interactions Harris et al.(2005) tested cationic lipoplex uptake by endothelial cells in a recirculating flow chamber reporting 9-fold increases at shear stress τ=2.3dyn/cm2 compared to the no-flow case Mennesson et al.(2006) observed that the binding rate of cationic polyplexes is enhanced by shear stress exposure (τ=0.3-1dyn/cm2) We modeled margination and adhesion of cationic DNA carriers under convective flow informed by these works to simulate the nanoparticle trajectories inside a parallel plate flow chamber This study evaluates the forces acting on the complexes approaching the cell layer to generate insight into new methods for improving the efficiency of gene delivery We confirmed that nanoparticle motion is influenced by particle radius and shear stress Flow improves the number of plasmids impacting on cells (0.32% for PEI-polyplex in 100% serum, τ=0.06-2dyn/cm2), more than carrier size ( 10-fold) or absent in many areas of the treated brain In long term therapeutic experiments, AAV-treated GM2 cats are currently 23.1, 21.3 and 20.0 months old All remain ambulatory but with diminished mobility due to hind limb weakness, and all are generally healthy and able to maintain body weight The other AAV-treated GM2 cats reached humane endpoint between 9.8 – 15.5 months old The humane endpoint, defined by inability to stand, is reached in untreated GM2 cats at 4.5 ± 0.5 months, n=11 AAV-treated animals did not reach the humane endpoint according to stereotypical disease progression in untreated animals, which consists of debilitating whole body tremors and severe balance disturbances In contrast, AAV-treated animals lost the ability to stand due to hind limb weakness, with or without joint abnormalities, but never developed debilitating tremors or balance problems In treated brains collected at humane endpoint, Hex activity was distributed throughout the entire anterior-posterior axis of the cerebrum (0.3-34.9 fold normal) and cerebellum (1.7-59.6 fold normal) The AAV vector was detected by qPCR at varying levels in each coronal block Evidence of an inflammatory cellular infiltrate was observed in some H&E-stained brain sections, and serum antibody titers were pronounced (up to 1:200,000) Furthermore, GM2 cats treated with a ten-fold lower dose lived to 13.0-16.9 months old Variable evidence of disease correction in the periphery has been documented, with up to 50% normal Hex activity in liver but extensive vacuolation and undetectable Hex activity in peripheral blood mononuclear cells These translational studies provide strong support for the initiation of AAV-based clinical trials for human GM2 gangliosidosis Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... from the injected heart region, there remained relatively large amounts of siRNA molecules after treatment with the DA-PEI /SHP- 1 siRNA polyplexes The cardiac administration of the DA-PEI /SHP- 1 siRNA. .. in SHP- 1 gene silencing, which can be explained by the improvement of in vivo cardiac gene delivery efficiency of DA-PEI based delivery system In addition, in vivo treatment of the DA-PEI /SHP- 1. .. vehicles for protein delivery and antigen presentation to effector T cells This work was supported by the Department of Defense grant W81XWH-09 -1- 0386 and National institutes of health grant R 01 CA 116 591