Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.
Telomerase, which is highly activated in neoplastic cells, can be a target for antisense therapy, and for that purpose, antisense oligonucleotides (AS ODNs) have to be effectively delivered into cellular nucleus where the target telomerase is present. The present work shows a new strategy to deliver AS ODNs to nucleus by use of a novel complex made from a natural polysaccharide schizophyllan (SPG) and AS ODNs. Nuclear transport is strictly regulated by the nuclear pore size and the related proteins. If the molecular weight of SPG is decreased, the SPG/AS ODN complex should be easily transported, although the stability of the complex decreases with a decrease in the molecular weight. We optimized the molecular weight of SPG to be 25 K. Furthermore, we attached importin-(a nuclear transport protein) to the side chain of SPG by use of a streptavidin-biotin interaction. When this complex was added to Jurkat cells, the telomerase activity was more suppressed than the naked dose, indicating that the importin-in the complex induced the nuclear transport of the complexed AS ODN and the AS ODN inhibited the telomerase. The present work shows a new methodology for nuclear anti-sense therapy that should be important in future anti-cancer therapies.Telomere shortening determines cellular lifetime and eventually induces apoptosis; therefore, the activation of telomerase and the resultant extension of telomere are essential for immortalization of neoplastic cells. This fact implies that telomerase can be a target for anti-cancer therapy. Telomerase is a ribonucleoprotein complex containing an RNA subunit that serves as a template for reverse transcription of telomere DNA. Corey et al. 1,2 showed that the oligonucleotides (ODNs) complementary to the template RNA can block the transcription process and therefore suppress neoplastic growth. This event happens only when ODNs are effectively delivered into cellular nucleus, because the target telomerase is present in nucleus. This strategy can be regarded as an antisense therapy, although the target RNA is present in nucleus, while the target RNAs for conventional antisense therapy are present in cytosol. To make this strategy practicable, three issues should be considered: (1) improving instability of AS ODNs in biological fluids; (2) increasing the cellular uptake of AS ODNs; (3) delivering AS ODNs to target organelle.
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