1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo
Authors: Betsy T. Kren, Gretchen M. Unger, Mark T. Reding, Clifford J. Steer
Journal: Molecular Therapy (2006)

In contrast to the efficient delivery afforded by viral vectors, the use of nonviral vectors for gene therapy has been hindered by the lack of adequate in vivo delivery systems. Although hydrodynamic push has been used extensively for hepatic plasmid delivery, this method delivers DNA nonselectively to liver cell types. One approach for increasing delivery to a specific cell type is by targeting receptor(s) that are either unique or highly expressed by that cell. Hepatocytes (heps) express aisaloglycoprotein receptors (ASGPr), and liver sinusoidal endothelial cells (LSECs) express hyaluronan receptors (HAr) in high abundance providing ideal targets for ligand- mediated receptor uptake. The aim of this study was to determine if liver cell type specific delivery of DNA could be achieved in vivo using asialoorosomucoid (ASOR) targeting to the hep ASGPr and HA for the LSEC HAr. Using a novel dispersion atomization method (Mol Cell Biochem 274:77, 2005) that forms sub 50 nm capsules with the receptor ligand noncovalently bound to the capsule coating, a red fluorescent protein (DsRed2) reporter plasmid was encapsulated using either ASOR for hep or HA for LSEC uptake. Eight week (wk) old C57/BL6 mice received 100 μg of the encapsulated plasmid targeted using ASOR or HA via tail vein injection and were sacrificed 1 wk post-injection. Liver, spleen, kidneys, lung, heart and brain were excised and processed for histology and protein extracts. Immunohistochemical identification of the LSECs in cryosections was done using anti-CD14 antibody (Ab), a marker specific for the discontinuous endothelial cells in the liver and a Cy5 labeled secondary Ab for confocal microscopy. The merged confocal micrographs of the DsRed2 and Cy5 fluorescence demonstrated colocalization of DsRed2 expression and the LSEC specific CD14 marker when HA was used as the targeting ligand. In contrast, there was no detectable colocalization when ASOR was used for targeting DsRed2 to heps. The presence of the DsRed2 protein was confirmed by western blot (WB) analysis of total liver protein extracts using a polyclonal anti-DsRed2 Ab and detection by ECL. There was no detectable DsRed2 expression in the other major organs. Transgenic hemophilia A mice were administered HA nanocapsules containing a Sleeping Beauty (SB) transposon (Tn) expressing B-domain deleted coagulation factor (F) VIII using a plasmid that codelivers an expression cassette for the SB transposase required for genomic Tn insertion. The mice were bled 2 and 5 wks post injection and plasma activated partial thromboplastin time (aPTT) determined. The treated mice had aPTTs of 25.5 ± 3.1 (2 wks) and 26 ± 1.9 sec (5 wks) which were not significantly different from the wild type (wt) aPTT of 23.5 ± 1.3 sec. In contrast, untreated hemophilia A mice had aPTTs of 46.7 ± 3.5 sec (P < 0.001 from treated and wt mice). In conclusion, ASOR and HA targeted nanocapsules can deliver plasmids in vivo to heps or LSECs, respectively. SB-Tns in HA nanocapsules targeted to LSECs provided expression of a clinically relevant gene product, FVIII that improved the phenotype of hemophilia A transgenic mice. In contrast to the efficient delivery afforded by viral vectors, the use of nonviral vectors for gene therapy has been hindered by the lack of adequate in vivo delivery systems. Although hydrodynamic push has been used extensively for hepatic plasmid delivery, this method delivers DNA nonselectively to liver cell types. One approach for increasing delivery to a specific cell type is by targeting receptor(s) that are either unique or highly expressed by that cell. Hepatocytes (heps) express aisaloglycoprotein receptors (ASGPr), and liver sinusoidal endothelial cells (LSECs) express hyaluronan receptors (HAr) in high abundance providing ideal targets for ligand- mediated receptor uptake. The aim of this study was to determine if liver cell type specific delivery of DNA could be achieved in vivo using asialoorosomucoid (ASOR) targeting to the hep ASGPr and HA for the LSEC HAr. Using a novel dispersion atomization method (Mol Cell Biochem 274:77, 2005) that forms sub 50 nm capsules with the receptor ligand noncovalently bound to the capsule coating, a red fluorescent protein (DsRed2) reporter plasmid was encapsulated using either ASOR for hep or HA for LSEC uptake. Eight week (wk) old C57/BL6 mice received 100 μg of the encapsulated plasmid targeted using ASOR or HA via tail vein injection and were sacrificed 1 wk post-injection. Liver, spleen, kidneys, lung, heart and brain were excised and processed for histology and protein extracts. Immunohistochemical identification of the LSECs in cryosections was done using anti-CD14 antibody (Ab), a marker specific for the discontinuous endothelial cells in the liver and a Cy5 labeled secondary Ab for confocal microscopy. The merged confocal micrographs of the DsRed2 and Cy5 fluorescence demonstrated colocalization of DsRed2 expression and the LSEC specific CD14 marker when HA was used as the targeting ligand. In contrast, there was no detectable colocalization when ASOR was used for targeting DsRed2 to heps. The presence of the DsRed2 protein was confirmed by western blot (WB) analysis of total liver protein extracts using a polyclonal anti-DsRed2 Ab and detection by ECL. There was no detectable DsRed2 expression in the other major organs. Transgenic hemophilia A mice were administered HA nanocapsules containing a Sleeping Beauty (SB) transposon (Tn) expressing B-domain deleted coagulation factor (F) VIII using a plasmid that codelivers an expression cassette for the SB transposase required for genomic Tn insertion. The mice were bled 2 and 5 wks post injection and plasma activated partial thromboplastin time (aPTT) determined. The treated mice had aPTTs of 25.5 ± 3.1 (2 wks) and 26 ± 1.9 sec (5 wks) which were not significantly different from the wild type (wt) aPTT of 23.5 ± 1.3 sec. In contrast, untreated hemophilia A mice had aPTTs of 46.7 ± 3.5 sec (P < 0.001 from treated and wt mice). In conclusion, ASOR and HA targeted nanocapsules can deliver plasmids in vivo to heps or LSECs, respectively. SB-Tns in HA nanocapsules targeted to LSECs provided expression of a clinically relevant gene product, FVIII that improved the phenotype of hemophilia A transgenic mice.