title: Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery.
abstract: The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II topoisomerases. The technology for such self-entanglement can be an avenue for the improvement of gene delivery with high-molecular-weight naked DNA using therapeutically important methods associated with considerable shear stress. Priority applications include in vivo muscle electroporation and sonoporation for Duchenne muscular dystrophy patients, aerosol inhalation to reach the target lung cells of cystic fibrosis patients and bio-ballistic delivery to skin melanomas with the vector DNA adsorbed on gold or tungsten projectiles.
authors.affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, UK. o.tolmachov@imperial.ac.uk
authors.forename: Oleg E
authors.initials: OE
authors.lastname: Tolmachov
mesh_terms: D001665:Binding Sites / Q000235:genetics; D059371:DNA, B-Form / Q000737:chemistry / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D006801:Humans; D008957:Models, Genetic; D008970:Molecular Weight; D009690:Nucleic Acid Conformation
chemical_list: D059371:DNA, B-Form