Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells

Abstract

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Cationic corticosteroid for nonviral gene delivery

“…Spermine alone or dexamethasone alone had no detectable gene transfer activity (data not shown). Using a flow cytometry cutoff of 100 FI to define percent transfection, 12 we observed a 4.3-fold increase in percent transfection over Lipofectamine from 5.9 to 25.5% ( Figure 3). Lipofection of subconfluent (proliferating) BAEC with DS/DOPE yielded a 4.6-fold increase in percent transfection over Lipofectamine from 16.0% with Lipofectamine to 73.8% lipofection with DS/DOPE (data not shown).…”

“…Bovine aortic endothelial cells (BAECs, passages [4][5][6][7][8][9][10][11][12][13] were passed at a 1:3 split to 24-well culture plates, and then grown to dense confluence before lipofection. The growth medium was Dulbeccos's modified Eagle's medium containing 10% heat-inactivated charcoal-filtered (to remove steroid hormones) fetal calf serum (Hyclone), 0.30 mg/ml glutamine, 150 U/ml penicillin, and 0.15 mg/ml streptomycin (Gibco).…”

Transfection of large plasmids in primary human myoblasts

“…20 The virion is then sequentially disassembled 21 and the adenovirus DNA is directed to the nuclear pores by adenovirus proteins. 22 Nuclear localization signal peptides [23][24][25] and integrinbinding peptides 26 have been proved to enhance transfection. Two peptides, one comprising a SV40 large T antigen nuclear localizing signal (KKPNKKKRKE), the other comprising an integrin binding motif (KKKKKK GRGDTP) were therefore investigated to replace the Gene Therapy adenovirus.…”

Gene therapy as an alternative to liver transplantation

“…124,125 Once released from the endosome, a number of factors including particle size and delivery agent appear to influence the nuclear uptake of molecules greater than ϳ125 nucleotides. 100,126 As a rule, small particle size is associated with increased nuclear localization and transgene expression. In addition, unlike the cationic lipids, polyplex delivery agents do not appear to inhibit nuclear expression of the transgene.…”

“…Cationic lipid transfecting agents such as lipofectamine; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); and the cationic polymers, including poly-L-lysine and PEI, have significantly improved nucleic acid delivery into the cytoplasm. 99,100 The substantially enhanced transfection efficacy observed with these delivery vehicles in vitro is associated with small, stable uniform particle sizes; however, these nucleic acid/cationic complexes often exhibit some cellular toxicity as well as reduced transfection efficiency in serum.…”