CPPs. The chemistry and actions of CPPs have been extensively reviewed (65–69) and here we touch on only a few key aspects. The prototypical CPPs derived from the Tat and antennepedia transcriptional regulators have been joined by a large number of new moieties. Most of these are relatively short (9–30 amino acids) polycationic peptides rich in argine and lysine, although some include membrane-interactive hydrophobic sequences (Figure 2). CPPs have been linked to proteins by recombinant DNA techniques or chemically coupled to peptides, oligonucleo- tides or nanocarriers, which then comprise the ‘cargo’ for the CPP. Initially, CPPs were thought to convey their cargo directly across the plasma membrane. However, it is now clear that polycationic CPPs initially bind to cell surface glycosaminoglycans; this is followed by endocyto- tic uptake (possibly macropinocytosis) (70), and eventual release of cargo from the endosome to the cytosol. Although initial reports emphasized the great promise of CPPs for delivery of macromolecules, recently there has been some controversy as to just how eﬀective they are (71). Certainly, the nature of the cargo (in terms of size, charge and other molecular characteristics) has an important impact on the eﬀectiveness and the toxicity of CPP-mediated delivery (72–75). In a section below, we will discuss how CPPs have been applied to oligonucleotide delivery. CTLs. A promising strategy is to deliver antisense and siRNA oligonucleotides by use of a CTL that binds with high aﬃnity to a cell surface receptor that is capable of undergoing eﬃcient internalization. A wide variety of potential ligands are available including antibodies (76), polypeptides derived from phage display libraries (77) and small organic molecules. Since various receptors are often preferentially expressed on particular cell types, this approach oﬀers the possibility of improved selectivity for the oligonucleotide reagents. While a rich variety of cell surface receptors are expressed in the human body, work thus far involving delivery of oligonucleotides has primarily focused on lipoprotein receptors (particularly those in the liver) (78), integrins (79,80) and receptor tyrosine kinases (81). Another potentially rich source of Cell Penetrating Peptides (CPPs) Small peptides (usually less than 30 aa) Net positive charge (in most cases) Translocate across plasma or endo-membranes Transport cargos into cytoplasm and nucleus Name Sequence Length Charge Penetratin RQIKIWFQNRRMKWKK 16 7 (+) TAT GRKKRRQRRRPPQ 13 8 (+) Transportan GWTLNSAGYLLGKINLKALAALAKKIL 27 5 (+) R9 peptide RRRRRRRRR 9 9 (+) MPG peptide GALFLGWLGAAGSTMGAPKKKRKV 24 5 (+) KALA peptide WEAKLAKALAKALAKHLAKALAKALKACEA 30 7 (+) MW 2247 1719 2842 1428 2445 3132 Figure 2. Cell penetrating peptides. This describes some of the more commonly used cell penetrating peptides in terms of molecular weight and net charge. Nucleic Acids Research, 2008, Vol. 36, No. 12 4161 targets is the G-protein coupled receptor (GPCR) super- family, by far the largest family of receptors in the human genome with approximately 850 members (82). GPCRs have long been a major interest of the pharmaceutical industry and thus a vast number of high aﬃnity small organic molecule ligands for GPCRs are available or are emerging via high-throughput screening procedures (83). CELLULAR DELIVERY OF OLIGONUCLEOTIDE CONJUGATES AND COMPLEXES In this section, we discuss the use of CPPs and CTLs in the delivery of antisense and siRNA oligonucleotides. Emphasis here is on cellular studies, while in vivo work is discussed in a following section. As a prelude, it is important to recall that oligonucleotides do not permeate intact cell membranes to any signiﬁcant degree via simple diﬀusion, primarily because of the hydrophobic nature of the membrane lipid bilayer. This is true for both negatively charged siRNA or antisense moieties as well as for molecules with uncharged backbones such as methyl- phosphonates, PNAs and morpholinos (84,85). CPP—oligonucleotide conjugates or complexes A considerable eﬀort has gone into the preparation and evaluation of conjugates of CPPs and oligonucleotides; however, on the whole this has been only moderately successful (86,87). Our laboratory has reported biological eﬀects of conjugates of CPPs with anionic antisense oligonucleotides (88,89), and others have reported on CPP–siRNA conjugates (90,91). However, the bulk of the literature suggests that CPPs are primarily able to deliver oligonucleotides with uncharged backbones, such as peptide nucleic acids and morpholino compounds (92–96). CPPs have been studied in connection with both antisense and siRNA, and as chemical conjugates or noncovalent complexes with the oligonucleotide. In addition to the usual assays based on ‘knockdown’ of mRNA by antisense or siRNA, another popular approach has been the use of an assay based on the splice-correcting properties of certain types of antisense oligonucleotides (25). Brieﬂy, an aberrant intron is placed into a reporter gene (luciferase, GFP) cassette and stably expressed in cells. The aberrant intron results in incorrect splicing and failure to produce functional mRNA and protein. However, appropriate splice switching oligonucleotides (SSOs) can correct splicing leading to expression of the reporter gene. Since splicing only takes place in the nucleus, this splice correction assay provides a convenient positive read-out for delivery of the SSO to the nuclear compartment. Early work from our laboratory explored conjugates of the CPPs Tat and Antennepedia (also know as penetratin) with either standard phosphorothioate oligonucleotides targeting the MDR1 drug resistance gene (89,97) or with SSOs comprised of 20-O-Me phosphorothioates (88); in both cases, the peptide and oligonucleotide were joined by bio-reversible S–S bridges. In both types of assay, the presence of the CPP enhanced biological eﬀects over those