Non-invasive gene targeting to the fetal brain after intravenous administration and transplacental transfer of plasmid DNA using PEGylated immunoliposomes.

Research was undertaken to establish transplacental delivery of active genes to fetal brain by a non-viral vector, antibody-specific targeted therapeutic procedure. PEGylated immunoliposomes (PILs) containing firefly luciferase DNA under the influence of the SV40 promoter injected intravenously into near-term pregnant mice produced luminometric evidence of CNS tissue luciferase activity at 48-h post-injection in all newborn pups. In utero delivery of this pGL3 DNA was shown after a single i.v. injection in maternal and neonatal brains, spleen and lesser amounts in lungs, with only negligible background levels in negative controls exposed to unencapsulated pDNA. In addition to studies of normal wild-type mice, we similarly injected pregnant Lafora Knockout (EPM2a null-mutant) and demonstrated luciferase activity days later in the maternal and newborn pup brains of both types. Delivery of PILs containing a second reporter gene (the pSV40 beta-galactosidase transgene) transplacentally by the same procedure was also successful. Histochemical and biochemical demonstration of beta-galactosidase was documented for all mutant and non-mutant neonates. Brain areas of highest Lafora body development (such as the hippocampus and pontine nuclei) showed intraneuronal beta-glucosidase activity. We conclude that receptor-mediated transport of PIL-borne gene therapeutics across both the placental barrier as well as the fetal BBB in utero is feasible.

Location of molecules in layer-by-layer assembled microcapsules influences activity, cell delivery and susceptibility to enzyme degradation.

Layer-by-layer assembled microcapsules have the potential to be versatile cell delivery systems incorporating multiple activities and functions. However, it is necessary to determine the influence that different capsule locations have on activity of bioactive molecules in order to optimise delivery and for generation of multifunctional capsules. In this study we examine the influence that locating the bioluminescent enzyme luciferase in different microcapsule locations has on activity in intact synthetic and biodegradable microcapsules before and after cell delivery as well as its susceptibility to protease degradation. We also examine the effect of microcapsule position on cell transfection with plasmid DNA. Based on the findings of experiments in this study we also demonstrate co-delivery of luciferase protein and plasmid DNA encoding a fluorescent protein from two different locations within the same microcapsule. Our studies confirm that, the core, subouter layer, and outer layer are optimal for cell delivery but these positions offer least protection from protease activity. By contrast middle layer molecules remain entangled with capsule layers preventing their release which is inefficient for cell delivery but this provides better protection from protease degradation. The findings of this study will enable more rationale layer-by-layer assembly of microcapsules containing biologically active molecules for cell delivery and aid in the generation of multifunctional microcapsules.

Chitosan-interferon-ß gene complex powder for inhalation treatment of lung metastasis in mice.

We prepared gene powders with chitosan as a non-viral vector and mannitol as a dry powder carrier to compare their gene expression and therapeutic efficacy to intravenous or intratracheal gene solutions using mice burdened with pulmonary metastasis prepared by injecting CT26 cells. The expression of a luciferase expression plasmid driven by the cytomegalovirus promoter (pCMV-Luc) and plasmid DNA encoding farnesylated enhanced green fluorescent protein (pEGFP-F) suggested that the genes expressed in both normal and tumorous tissues and the intratracheal powder resulted in higher expression than the intravenous or intratracheal solution. The intravenous and intratracheal solutions and the intratracheal powder of pCMV-Muß encoding murine interferon-ß were administered the day after the inoculation of mice with CT26 cells. Lung weight and the number of pulmonary nodules at day 21 were significantly suppressed by the three formulations at a dose of 10 µg (N/P = 5). Reducing the dose to 1 µg resulted in a loss of effect by the intravenous solution; however, the intratracheal formulations, especially the powder, were still effective. The intratracheal powder of pCMV-Muß at a dose of 1 µg administered on day 1 significantly extended mean survival time compared to the untreated control. These findings showed that therapeutic gene powders are promising for gene therapy to treat lung cancer or metastasis.

Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins.

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. Tumour-associated endothelium offers an additional mechanism for enhanced viral uptake into tumours which is accessible for systemic gene delivery. Building on expertise in using polymer 'stealthed' viruses for targeting in vivo, adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to ablate normal infection pathways. Direct linkage of a monoclonal antibody against E-selectin (MHES) demonstrated E-selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells. A two-component targeting system using protein G was developed, to provide optimal antibody orientation. We report an enhancement in transduction of TNF-α-activated endothelium in vitro and ex vivo in a human umbilical vein cord model using the MHES antibody. Similarly a virus retargeted using a chimeric P-selectin Glycoprotein Ligand-1-Fc fusion (PSGL-1) protein showed better circulation kinetics and significant uptake into HepG2 xenografts following systemic administration in mice, with 36-fold higher genome copies, compared with non-modified virus. Immunohistochemistry staining of tumour sections from mice treated with PSGL-1-retargeted virus showed a co-localisation of firefly luciferase with CD31 suggesting selective endothelial targeting. Employment of optimal viral modification using protein G will enable exploration and comparison of alternative targeting ligands targeting tumour-associated endothelium.

Vascular endothelial growth factor broadens lentivector distribution in the heart after neonatal injection.

For some applications, the success of gene therapy depends on the efficiency of gene transfer into target organs, however, delivery to many tissues is limited. Efforts have been made to improve the efficiency of gene transfer into target organs such as the brain by using mannitol or vascular endothelial growth factor (VEGF) prior to gene delivery, since these treatments have been reported to increase vascular permeability in experimental animals. Here, we investigated the effect of VEGF pretreatment of neonatal mice on the ability of injected lentivirus (LV)--engineering expression of firefly luciferase (luc)--to enhance the transduction of various organs, including the brain and heart. LV/luc was delivered to VEGF-treated neonatal mice via the temporal vein. Whole-body bioluminescence imaging (WBLI) of luciferase expression showed that VEGF pretreatment does not diminish transgene expression over time since it remained steady for up to 12 weeks. Ex vivo imaging of the organs and assessments of organ luciferase activity showed that VEGF pretreatment resulted in significantly increased luciferase expression not only in the heart, but also in the brain, lung, and kidney. This study shows that VEGF may have therapeutic importance to enhance the efficiency of viral gene delivery to the heart, as well as to other target organs.

Hydrodynamics-based gene delivery to the liver by bile duct injection of plasmid DNA--the impact of lasting biliary obstruction and injection volume.

BACKGROUND/AIMS: Increased biliary pressure may mediate an opening of intrahepatic tight junctions. The biliary pressure is related to the duration of biliary obstruction and the injected volume. Their effects on the efficiency of gene delivery to the liver were studied. METHODOLOGY: The luciferase gene with a volume of 0.5, 1.0, 1.5 or 2.0mL was injected into the bile duct of Sprague-Dawley rats. After the injection, the bile ducts were occluded in some of the rats, while left to drain in the others. RESULTS: The activity of luciferase was the highest in rats that received 2.0mL of DNA injection and biliary occlusion, while the lowest in the rats that received 0.5mL of DNA only. CONCLUSIONS: The bile duct can be an efficient route for gene delivery to the liver depending on the volume of injected DNA. Biliary occlusion can enhance the expression of genes but it may induce a certain degree of liver injury, which should be taken into concern.