Nanoparticles of Compacted DNA Transfect Postmitotic Cells

Abstract

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900 -360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a ϳ10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.Although nonviral gene transfer methods transfect dividing cells, these technologies fail to transfect most postmitotic cells (1-10), with the principal exceptions of naked DNA gene transfer into muscle (11) and large volume hydrodynamic gene transfer into liver (12, 13). In dividing cells, nuclear membrane disintegration during mitosis allows plasmid DNA to enter the nucleus prior to membrane reformation. Otherwise, the intact nuclear membrane restricts transfer of naked DNA into the nucleus. The nuclear membrane pore (NMP) 1 has an internal channel diameter of 25 nm (14, 15) and does not permit naked DNA to effectively cross into the nucleus, probably due to the extended size of hydrated DNA and its negative charge density (4,16,17). The NMP does permit passive transfer of gold particles less than 9 -10 nm in diameter and linear DNA fragments up to ϳ300 bp (18 -22) as well as facilitated transport of proteins and small DNA segments (up to ϳ1 kbp) having nuclear localization signals (7,(22)(23)(24)(25)(26)(27)(28). The relative inefficiency of naked DNA, liposome-DNA complexes, and protein-and polymer-based DNA conjugates to transfect nondividing cells productively remains a significant barrier for in vivo gene therapy. Electrostatic interactions between polycationic polymers and DNA can result in conjugates consisting of one or more molecules of DNA and a sufficient number of polycations to produce a nearly charge-neutral complex (29 -31). The ratio of positive to negative charges, buffer components, polycation counterion, DNA concentration, and pH, among other variables, influence the composition, size, and shape of these DNA conjugates (29,32). Based on specific fo...

Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles

“…In contrast, normalization of the luciferase activity per mg protein extract and pmol of DNA dosed demonstrated no difference of gene transfer activity among the different size plasmids for either type of formulation. Unlike the in vitro microinjection data, 1 there was no evidence for decreased gene transfer activity for ellipsoidal nanoparticles having a minor diameter 425 (up to a diameter of 32 nm). There was also no significant difference in gene transfer activity when comparing a given plasmid for either formulation.…”

“…We have developed nanoparticles consisting of single molecules of plasmid DNA condensed with polyethylene glycol (PEG)-substituted lysine peptides. 1 The volume, shape and size dimensions of these DNA nanoparticles depend on several parameters, including plasmid size and the lysine counterion present at the time of DNA mixing. 2 For example, lysine polymers containing trifluoroacetate and acetate counterions result in the formation of ellipsoidal and rod-like nanoparticles, with the volume of these complexes closely predicted by the partial specific volumes of the constituent components.…”

“…2 For example, lysine polymers containing trifluoroacetate and acetate counterions result in the formation of ellipsoidal and rod-like nanoparticles, with the volume of these complexes closely predicted by the partial specific volumes of the constituent components. 1,2 For ellipsoidal nanoparticles, microinjection studies indicated a size limitation for nuclear access and transgene expression, with nanoparticles having a minor diameter approaching 25 nm having decreased efficiency. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids.…”

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…Also, compact DNA particles were shown to transfect postmitotic cells, showing that at least some nanoparticle types are able to reach the nucleus of nondividing cells [110]. Furthermore, Escriou et al [15] although confirming that mitosis is an important upregulator of expression efficiency, also observed expression in cells which had not divided, albeit to a lower extent.…”

Toward Development of Artificial Viruses for Gene Therapy: A Comparative Evaluation of Viral and Non‐viral Transfection

“…100 Many polymeric or peptide-based vectors condense DNA, improving its stability and mobility in the cytoplasm, as well as its ability to penetrate the nucleus. 68,[101][102][103] Though the processes involved in nuclear translocation of complexes are not clearly understood, confocal microscopy studies provide considerable evidence that they reach the nucleus. 6,8,11,15,64,88 Some studies observe DNA in the nucleus with no evidence of carrier co-localization, suggesting that complexes release DNA prior to nuclear transport, 6,8 while others demonstrate entire complexes entering the nucleus.…”