Investigation of charge ratio variation in mRNA - DEAE-dextran polyplex delivery systems.

mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required. In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) - Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application.

Functional characterization of transgene integration patterns by halo fluorescence in situ hybridization: electroporation versus retroviral infection.

Gene transfer techniques are essential for bioengineering and gene therapy. Retroviral vectors constitute an important tool in this context as integrase-catalyzed genomic anchoring is one of the best defined results of nonhomologous recombination, occurring at loci with favorable expression properties. On the basis of a retroviral expression cassette, this study focuses on differences regarding the genomic targets after its transfer by either retroviral infection or electroporation. Fluorescence in situ hybridization (FISH) of clones generated by infection revealed for the majority an association of the transgene with the nuclear matrix while this was true only for a minority of single copy clones if the same construct was transferred by electroporation. Our results demonstrate that the process of integration is distinct for both gene transfer routes and confirm that retroviral survival strategies favor integration next to scaffold/matrix attachment regions. These results may be one key to explain recent consequences of gene therapy trials that have led to the deregulation of endogenous genes.