Asialofetuin liposome-mediated human α1-antitrypsin gene transfer in vivo results in stationary long-term gene expression

Abstract

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Pig liver gene therapy by noninvasive interventionist catheterism

“…18 Briefly, goat anti-hAAT (0.2 mg/well) and goat anti-hAAT peroxidase conjugate (1.5 mg/well) were used as capture and detecting antibodies, respectively. In our experiments, the limit of human protein detection was 1 ng/ml.…”

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles

“…The assay was performed in 96-well microtitration plates (CoStar), as previously described. 19 In brief, goat antihAAT (0.2 mg/well) and goat anti-hAAT peroxidase conjugate (1.5 mg/well) were used as capture and detecting antibodies, respectively. Capture antibody (2 mg/ml) diluted in adsorption buffer (carbonate 0.05 M, pH 9.6) was distributed (100 ml/well) in the microplate and incubated at 41C overnight.…”

Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors

“…The thermal cycling conditions included an initial step at 951C for 10 minutes, followed by 40 cycles at 951C for 15 seconds and 601C for 1 min. 30,31 The method used for obtaining quantitative data of relative gene expression is the comparative Ct method (DDCt method) as described by the manufacturer. Ct is measured as the PCR cycle numbers at which the reporter fluorescent emission increases beyond a threshold level that is based on the background fluorescence of the system.…”