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translocation of full-length Tat fusion constructs, , and that (2) the amphipathi-city of the Tat peptide (suggested by modeling studies104) is not required for import. This latter point is supported by observations that partial d-amino acid analogs of the Tat PTD also possess highly efficient translocation activity.20

13.2.4. Homeoproteins: Penetratin

The nonclassical transport activity of the homeoprotein family of proteins was first demonstrated by Perez and colleagues during their studies of the Drosophila anten-napedia homeoprotein (Antp).105 A class of transactivating factors, the homeoproteins interact with DNA through a small (^60aa), highly conserved helical region called the "homeodomain."106 It was the homeodomain of Antp that was originally thought to enable the nonclassical internalization and nuclear localization observed for this protein.105,107 Additional studies, however, localized the activity to a 16-amino acid polycationic peptide (residues 43-58; Table 13.2), now designated Penetratin.49,107,108 (Interestingly, although the Penetratin peptide now serves as the model for nonclassical homeodomain-derived transport, it has been suggested that an extended sequence actually internalizes more efficiently than the minimal peptide vector.108) Since the discovery of the Penetratin peptide, similar activity has been observed in other members of the homeoprotein family, including Hoxa-5,109 Engrailed,110 and Islet-1.39 The Engrailed protein also exhibits nonclas-sical secretory activity,111 which may be true for the many members of the homeoprotein family that lack secretion signal peptides.105 This suggests that the potential for nonclassical import may exist throughout the homeoprotein family, especially in light of the high sequence conservation of the transport-competent homeodomain region.

Similar to the Tat PTD, Penetratin is a highly basic peptide possessing helical character, although once again, it has been shown that the intrinsic secondary structure of the peptide is not required for internalization.106,107 As shown in Table 13.1, Penetratin has been successfully employed to deliver protein and DNA-based macromolecules both in vivo and in vitro.112 Unlike other nonclassical transport-based vectors, however, the delivery capabilities of Penetratin appear to be limited by cargo size (100 residues for peptides and 55 base pairs for oligonucleotides).112 Two exceptions to this rule are the translocation of the parent Antp protein as well as a study by Kilk and colleagues that demonstrates transport of a ^63 kDa strep-tavidin-biotin complex.39 It is therefore not entirely clear that cargo size limitation is indeed a property of Penetratin delivery, nor is it known if this limitation will apply to other homeoprotein-derived vectors. Unlike other vectors, although the precise transport pathway has not been conclusively established for Penetratin, a specific internalization mechanism has been postulated, involving the formation of an inverted hexagonal phase in lipid bilayers107 (see Section 13.4.2.1).

13.2.5. Cell-Penetrating Peptides

The number of peptides demonstrating nonclassical import activity has risen dramatically in recent years (see the reviews by Lindgren et al.,19 Wadia and Dowdy,113 and others114-117). This now extensive group of potential delivery vectors can be divided into two general groups: (1) cationic peptides and (2) amphi-pathic helices, although some miscellaneous peptides are also included in the latter group. In order to focus on the central subject of this chapter, polycation-based delivery, we choose to include in this discussion only the former group of cationic peptides. For information on the latter group, the reader is referred to selected amphipathic peptides in Table 13.2 and references therein.

The cationic peptide family includes both synthetic and naturally derived pep-tides, all of which have been successfully employed for both in vitro and in vivo delivery of a variety of macromolecules (Table 13.1). The prototypic example of a naturally derived cationic peptide is the Tat PTD, described above. Other examples include the pVEC peptide, which has been successfully used for both protein and oligonucleotide delivery,40 as well as a series of small, RNA-binding peptides discovered by Futaki and colleagues.20 The Penetratin vector is also included in this group. The inclusion of Tat and Penetratin in the cationic peptide group arises from the intrinsic definition of these peptides. In general, the cationic delivery peptides

TABLE 13.2 Select Cell-Penetrating Peptides

Vector

Source

Sequence

Type

NLa

Ref.

Tat

HIV-1 Tat

0 0

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