Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold.

作者：Otmane Boussif, Maria Antonietta Zanta, Jean‐Paul Behr · 发表于：PubMed · 年份：1996 · 被引用次数：345 · 研究领域：RNA Interference and Gene Delivery、Virus-based gene therapy research、CRISPR and Genetic Engineering

Reproducible and optimized complex formation between polyanionic DNA and a polycationic vector is a key aspect of nonviral gene transfer systems. To this end, several factors relevant to in vivo delivery have been tested repeatedly on several cell types. Gene transfer with a lipopolyamine (transfectam) in the presence of serum was increased over 10-fold by sequential addition of the lipid to DNA. Paradoxically, high complex concentrations (> 200 micrograms DNA/ml) led to large enhancements too, which points to the fact that formation of productive complexes is a slow process. Each parameter, more than compensates for the decreased efficiency generally observed with nonviral vectors in serum. Transfectam and PEI (polyethylenimine)-mediated transfection also improved after mild centrifugation of the complexes on to the cells. These individual factors were shown to be essentially multiplicative, leading altogether to approximately a 1000-fold transfection increase with a luciferase reporter gene. Finally, 25 cell lines and primary cells (including fibroblasts, hepatocytes and endothelial cells) were successfully transfected over a five orders-of-magnitude efficiency range, From this large set of data, a general relation between the overall transfection level (as measured by luciferase reporter gene expression) and the fraction of transfected cells (histochemically stained for beta-galactosidase) could be inferred. Finally, transfectam and PEI displayed similar trends over this l...