67. Subcutaneous Delivery of Plasmid DNA to Hepatocytes by Ligand-Targeted Sub-50 nm Capsules

67. Subcutaneous Delivery of Plasmid DNA to Hepatocytes by Ligand-Targeted Sub-50 nm Capsules

CHEMICAL AND MOLECULAR CONJUGATES delivery in vivo. As a result, we compared the unmodified H3K4b polymer with two modified H3K4b polymers for their a...

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CHEMICAL AND MOLECULAR CONJUGATES delivery in vivo. As a result, we compared the unmodified H3K4b polymer with two modified H3K4b polymers for their ability to deliver siRNA to luciferase-expressing MDA-MB-435 cells, both in vitro and in a tumor-bearing mouse model; one modified HK polymer, (RGD-PEG)4-H3K4b, had four RGD-PEGs conjugated to each molecule, while the other polymer, (RGD-PEG)-H3K4b, had one RGD-PEG per molecule. For in vitro experiments, the H3K4b siRNA nanoparticle targeting luciferase decreased its activity by 90% compared with negligible down-regulation by the modified H3K4b carriers. In contrast, the two RGD-PEG modified H3K4b peptides administered intravenously were significantly more effective than H3K4b in silencing luciferase in a tumor-bearing animal model. Specifically, H3K4b, (RGD-PEG)-H3K4b, and (RGD-PEG)4-H3K4b carriers of Luc siRNA decreased luciferase activity in the lysates of tumor xenografts by 10%, 35%, and 80%, respectively. Furthermore, this result was validated by a real time bioluminescence imaging system. Thus, (RGD-PEG)4-H3K4b carrier with the greatest number of modifications was the most effective carrier of siRNA in vivo. These studies demonstrate that selected modified HK polymers are promising candidates to target oncogenes with siRNA.

67. Subcutaneous Delivery of Plasmid DNA to Hepatocytes by Ligand-Targeted Sub-50 nm Capsules

B. T. Kren,1 V. L. Korman,2 D. Tolbot,2 G. M. Unger.2 University of Minnesota, Minneapolis, MN; 2GeneSegues, Inc., Chaska, MN. 1

Gene augmentation for hepatocyte based metabolic disease has been a long-term goal of gene therapy. Subcutaneous (sq) administration of plasmid DNA is a desirable goal for patient-friendly genetic manipulation of target cells in chronic disease. However, two major delivery challenges that have impeded its development are avoiding sequstration at the injection site and in the target cells endosomes with subsequent degradation. A novel nonviral delivery technology developed by GeneSegues utilizes sub-50 nm crystalized capsules for intact delivery of nucleic acids (NA) from 19 bp to 15 kb. The capsule’s crystalized shell formulated from any appropriate targeting protein, peptide or carbohydrate ligand specifies the uptake of the capsule by the specific cell-type in vivo via the receptor targeted by the capsule ligand shell. The nanocapsule’s size, structure and charge mediates efficient in vivo penetration of tissues and cellular uptake via the non-endosomal lipid rafts pathway, delivering the NA cargo intact to the nucleus and cytoplasm of the target cell. We have previously demonstrated that asialoorasomucoid (ASOR) s50 capsules mediate hepatocyte specific delivery of transgenes in vivo following intravenous (iv) administration (J Clin Inv 119:2086, 2009). Here we show data comparing various sq regimens to iv administration for delivery of a secreted alkaline phosphatase (SEAP, pCpgSEAP) reporter packaged into ∼ 10-15 nm ASOR s50 capsules. We found that RNA production at 48 hrs after final dose was approximately 15 – 25% of that produced by a maximum volume iv regimen. Target RNA production levels were similar between equivalent dose sq regimens with different schedules. Importantly for clinically relevant delivery, fluorescence microscopy showed siimilar uniform gene expression only in hepatocytes for both iv and sq liver sections. In contrast, higher expression levels were observed with a second reporter pcDNA3.1/LacZ in sq liver sections potentially reflecting differences in gene regulation between the plasmids. To study sq administration of s50 capsules independently of cargo transgene expression we used ASOR capsules carrying Dysprosium (Dy)-chelated dextran. Dy is a large nuclei, fluorescent lanthanide enabling sensitive isotopic measurements of tissues by neutron activation analysis of Dy content. A cohort of mice were injected sq over a 5 day period held in metabolism cages for the final 24 hrs. Over 5 days, ∼ 4.3% of the injected dose accumulated in the liver (based Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

on summation of 24-hr collection points for the 5 days) peaking at 3 days postinjection. Of note, no uptake was observed in spleen, kidney, lung, heart or testes. These data support the view that sq delivery of ASOR targeted s50 capsules to hepatocytes provides a depot effect, with prolonged availability of cargo and possible avoidance of duration-adverse effects such as saturation of cellular uptake, intracellular enzymatic degradation of cargo, etc. In conclusion, these studies provide a solid framework for further development of the nonviral ASOR s50-mediated sq nucleic acid based gene therapies for hepatocyte-based liver disease.

68. Methods To Observe Intracellular Trafficking of Non-Viral Delivery Vehicles in 3D

Nilesh P. Ingle,1 Theresa M. Reineke.1 Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA. 1

In this study, we present methods that our lab has developed to quantitatively examine the intracellular trafficking of polyplexes in 3D. We have studied the trafficking of four carbohydrate-based polymeric delivery vehicles that have been previously developed in our lab that consists of trehalose and pentaethylenetetramine subunits along with two commercial controls Glycofect TM and JetPEI TM. Two lengths of the trehalose polymer vehicles were examined with degrees of polymerization of 55 (Tr4-55) and 77 (Tr4-77). Cell culture and Transfection: HeLa cells were cultured (15,000 cells/well) on acid washed glass coverslips coated with poly L-Lysine, 24-hours prior to transfection. The polyplexes were formulated in DNase/RNase-free H2O, 1 hour prior to transfection at the following formulation ratios: 5, 20, 7 and 7 N/P for JetPEI TM, Glycofect TM, Tr4-55, and Tr4-77 respectively. The polymer solution (50 µl/well) was added to FITCpDNA solution (50 µl/well at 0.02 µg/µl) to make a 100µl polyplex solution. The transfections were done in OptiMEM. Four hours after transfection, cells were washed with PBS at pH 7.4 and supplemented with 1 ml DMEM. Confocal Preparation, Image Acquisition, and Analysis: The cells were fixed at 4, 8, 12 and 24 hours post transfection. The nuclei were stained with DAPI. Zeiss LSM Meta 510 confocal microscope containing an Argon laser (488 nm) to excite FITC and UV laser (364 nm) to excite DAPI was used for imaging. The scaling was X: 0.070 µm, Y: 0.070 µm and Z: 0.140 µm. The raw Z-stack images were first deconvoluted based on theoretical point spread function and the Classical Maximum Likelihood Estimation algorithm using proprietary software and then volume rendered. Results and Discussion: The 3D images were analyzed for three parameters: (1) polyplex volume, (2) distance between polyplexes and (3) distance of polyplexes from the surface of nucleus. The volumetric images revealed that these intracellular polyplexes may exhibit nonspecific globular shapes as they approach the nucleus. The distance distribution plots suggested that after endocytosis the polyplexes appeared to traffic in groups (and not as single polyplexes), which move with the transport machinery of a cell along both actin and microtubules. After uptake, the polyplexes have similar 3D-positions in the cell, which lasts for about 4 hours. The onset of this dormant period varies with polymer type. In general all the polyplexes appear to traffic toward the nucleus with similar kinetics. However, after this dormant period, some of the polyplexes appear to traffic away from the surface of the nucleus, while others do not; this apparent exocytosis-like behavior is seen at an earlier time point of 4 to 8 hours for Tr4-55 and Tr4-77 than the commercial controls. We also observed that as the average volume of all the polyplexes within the cell increased, the surface area of these complexes increased within the cell suggesting that the complexes appear to undergo fission within the cell (increasing surface area) and the complexes that are divided appear to increase in volume (possibly swelling). This affect was noticed with all of the polymer-vehicles. We thus propose a method to visualize & quantify intracellular polyplexes in 3D. S27