Covalent attachment of multifunctional chimeric terminal proteins to 5' DNA ends: A potential new strategy for assembly of synthetic therapeutic gene vectors.

The generation of synthetic therapeutic gene vectors requires the coupling of DNA to transfer-promoting peptides including cellular receptor ligands, protein transduction domains, hydrophobic peptides for attachment to lipid membranes, nuclear localisation signals, cytoskeleton attachment motifs, nuclear matrix association elements and immune evasion moieties. Existing methods of peptide-DNA joining often interfere with transgene expression and, therefore, are inadequate for production of effective therapeutic vector complexes, particularly destined for gene delivery in the challenging environment in vivo. However, there is a natural mechanism for rigid coupling of polypeptides with DNA. Some bacterial and eukaryotic linear plasmids, adenoviruses and a number of bacteriophages including phi29 of Bacillus subtilis and PRD1 of Escherichia coli use terminal proteins covalently bound to 5' DNA ends to prime replication. Inverted terminal DNA repeats, normally short DNA sequences, contain all the sequences required in cis for the covalent coupling reaction. The complex of the terminal protein, DNA polymerase and some known auxiliary proteins supplies sufficient trans-functions, thus enabling simple linking of the terminal proteins to DNA in vitro. We hypothesise that chimeric fusion proteins, constructed on the basis of terminal proteins of adenoviruses, linear plasmids or bacteriophages with protein-primed replication, can on the one hand retain the ability to bind covalently 5' DNA termini in conditions established previously for protein-primed replication in vitro, and on the other hand confer gene transfer facilitating properties and enhanced longevity of efficient transgene expression. Terminal localisation of the chimeric proteins can ensure that they do not interfere with transgene transcription. At the same time a covalent bond between polypeptide and DNA can provide rigid coupling ensuring their stable association en route to nuclei. Bound to 5'-ends of the delivered DNA, terminal protein-based chimeras could also protect the vector DNA from 5'-3' and possibly 3'-5' exonuclease attack, thus limiting its intracellular degradation and increasing longevity of transgene expression. Our hypothesis can be tested by measuring the gene transfer efficiency of the novel complexes containing linear DNA fragments with covalently linked multifunctional chimeric terminal proteins, using previously described synthetic gene vectors as standards.