Improved performance of protein‐based recombinant gene therapy vehicles by tuning downstream procedures

Abstract

Protein engineering offers a robust platform for the design and production in cell factories of a plethora of protein-based drugs, including nonviral gene therapy vehicles. We have determined here that a protein nanoparticle, formed by highly cationic protein monomers, fails to bind exogenous DNA and to promote detectable gene expression in target cells despite recruiting all the needed functions. Removal of DNA and RNA with nucleases previous to forming complexes with exogenous DNA dramatically enhances the ability of the protein to bind and transfer DNA to target cell nuclei. These data point out contaminant nucleic acids deriving from the cell factory as a major factor impairing the performance of protein-based artificial viruses and stress the need of a nuclease step in the downstream of proteins whose function is based on cationic domains.

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

“…Since the mere addition of cationic peptides to pre-existing protein nanoparticles did not alter cell penetrability per se 26 , the formation of nanoparticles rather that the single amino acid addition in Seq-1-8-GFP-H6 would be responsible for enhanced cell penetrability. A higher penetrability of protein nanoparticles compared with individual building blocks is in agreement with previous results obtained in our laboratory 27 and it is probably related with the multivalent presentation of the cell ligands (Seq-1, in the present case) on the surface of nanoparticles that favours endosomal entrapment 28 .…”