4162 Nucleic Acids Research, 2008, Vol. 36, No. 12 attained with unconjugated ‘free’ antisense oligonucleo- tide; additionally, a limited amount of microscopy was done to evaluate intracellular distribution and evidence was found for nuclear accumulation of the oligonucleo- tide. In contrast, a later study from another group examined a number of disulﬁde bridged conjugates between various CPPs and 20-O-Me or LNA oligonucleo- tides complementary to the HIV-1 TAR element (98). In this case, there was little biological eﬀect unless endosome disrupting agents were used; further, the oligonucleotides were observed by ﬂuorescence microscopy to be restricted to cytoplasmic vesicles, with no sign of nuclear localiza- tion. The reason for the discrepancies between these two sets of early studies is unclear. One possibility is that, in our early studies, the peptide–oligonucleotide conjugates became aggregated during use, and this actually enhanced their eﬀectiveness. Interestingly, there have been several reports of noncovalent complexes or aggregates between anionic siRNA oligonucleotides and cationic CPPs that seem to have provided moderately eﬀective delivery to cells in culture. In one case, a modiﬁed version of the CPP penetratin having endosomolytic properties was superior to other CPPs that bound the siRNA equally well but lacked endosome lytic ability (99). In another case, a designed peptide comprised of both positively charged residues and a fusion peptide sequence was found to complex with siRNA and deliver it to the cytosol (100); this approach has also been followed by other groups (101). Whether conjugates or complexes are likely to provide more eﬀective delivery of anionic oligonucleotides in vivo is an important issue and will be further explored below. A number of investigators have evaluated conjugates of CPP with oligonucleotides having uncharged backbones. In one study, both stable and bioreversible disulﬁde linkages were used to produce conjugates between various CPPs and a PNA targeting the HIV-1 Tar motif (92). Certain conjugates, particularly an R6–penetratin version, demonstrated clear-cut biological eﬀects, although little nuclear localization was seen by ﬂuorescence microscopy. Several other CPP–oligonucleotide conjugates, with either stable or disulﬁde linkages, were able to attain biological eﬀects when chloroquine was used to enhance their release from endosomes. An additional study used an R6– penetratin conjugate of a PNA SSO to activate a luciferase reporter gene; good eﬀects were attained at micromolar concentrations (96). Another study also utilized PNA SSOs coupled via disulﬁde bridges to various CPPs to activate luciferase (93); here the transportan CPP was found to be particularly eﬀective. Confocal microscopy and use of endosomal markers indicated that the CPP– PNA conjugates were most likely taken up by macro- pinocytosis, but there was little evidence of nuclear localization despite the observed eﬀects on splicing. Studies from another group examined additional con- jugates between PNA SSOs and various CPPs; they also found that a transportan–PNA conjugate linked via a bioreversible disulﬁde bridge was most eﬀective (94). Recent studies have described a novel CPP termed ‘M918’ (63). When conjugated to PNA SSOs, M918 did not require binding to cell surface glycosaminoglycans for uptake (in contrast to Tat or penetratin). Nonetheless, it entered cells by endocytosis and attained good biological eﬀects. An interesting variation used CPP–PNA conju- gates to target chromosomal DNA and cause eﬀects at the transcriptional level (102). Similar studies have also been done with CPP con- jugates of morpholino oligonucleotides. In one very comprehensive investigation, a variety of linkages were formed between a morpholino SSO and several CPPs (103). A peptide containing nine arginines was particularly eﬀective and resulted in splice correction activity at micromolar concentrations; ﬂuorescence microscopy indi- cated some delivery of the oligonucleotide to the nucleus as well as to intracellular vesicles. More recently, this group has investigated the properties of conjugates comprised of morpholino SSOs linked to CPPs containing 6-aminohexanoic acid residues (104,105), ﬁnding that these have superior properties in terms of stability and eﬀects on splicing. The strategy of using CPP conjugates of SSOs having uncharged backbones has recently been reviewed (106). The overall picture seems to be that conjugates of various CPPs with uncharged backbone oligonucleotides can enter cells and eﬀectively alter RNA splicing processes. Various CPPs diﬀer somewhat in their potency in this regard; however, in most cases biological eﬀects are only attained when the conjugates are used at micromolar concentra- tions. This may indicate that most of the material taken up by cells remains in endosomal compartments, with only a tiny fraction reaching the nucleus where RNA splicing occurs. One technical issue with many of these studies is their reliance on a single model system involving correc- tion of splicing in a modiﬁed HeLa cell line. As discussed earlier, this system allows facile evaluation of whether a splice switching oligonucleotide can reach the nucleus and correct splicing of the aberrant reporter gene present there. However, it seems unwise to rely so heavily on a single cell type. CTL–oligonucleotide conjugates or complexes A number of studies have appeared recently using CTLs for the delivery of antisense or siRNA. Some of these studies had in vivo components that will be more fully discussed in a section below. Here, we will focus on the cell targeting and uptake aspects. An aptamer-siRNA chimera targeting prostate-speciﬁc membrane antigen (PSMA) was able to eﬀectively deliver the associated siRNA to LNCaP prostate cancer cells; use of plk-1 siRNA triggered apoptosis and resulted in cell death both in culture and in a prostate tumor model (107). In this case, concentrations in the 2–400 nM range were eﬀective in attaining gene silencing in cultured LNCaP cells, but not in PC-3 cells that lack PSMA. Other interesting approaches to aptamer-mediated siRNA deliv- ery have also been described (108). The conjunction of nucleic acid aptamer technology and siRNA could potentially be a very powerful avenue for developing reagents for cell type selective regulation of gene expression.