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372 Cell-Penetrating Peptides: Processes and Applications
Heart Muscle
Control TAT-β-Gal (120 kDa)
FIGURE 17.2 In vivo treated mice with Tat–β-galactosidase protein. Tissue sections of heart
muscle from mice treated with intraperitoneally administered Tat–β-galactosidase protein or
nontransducible control-β-galactosidase were assayed by X-gal overlay 4 h post-injection.
toneal (i.p.) injection of an Antp-antigenic peptide activated endogenous T cells in
mice. Pooga et al. 48 have shown the ability of Antp-antisense PNA oligomers to
penetrate the blood–brain barrier in rats. Additionally, Tat–GFP protein has been
shown to transduce into Drosophila by painting the eyes with a solution containing
Tat–GFP (C. Brachmann, carrie@pharmdec.wustl.edu; personal communication).
The therapeutic cache of recombinant Tat proteins, however, will be realized if
the effectiveness of this delivery modality can be reproduced in vivo to introduce
active proteins throughout all tissues. Recently, we have delivered a biologically
active enzyme to all cells and tissues of mouse models. 21 Intraperitoneal delivery of
200 to 500 µg of Tat–β-galactosidase into mice, equivalent to 10 to 25 mg/kg body
weight of protein, resulted in readily detectable β-galactosidase enzymatic activity
in the majority of tissues assayed 4 h after injection, although maximal β-galactosidase
loading of the tissues was found to occur within minutes. This difference is
likely to represent the time required for refolding and subsequent enzymatic activity
of the enzyme.
β-galactosidase activity was strongest in the liver, kidney, lung, heart, and spleen
and, significantly, could cross through the blood–brain barrier and be found in the
cell body layers of the brain (Figure 17.2). Importantly, Tat–β-galactosidase did not
disrupt the blood–brain barrier as assayed by coinjection with Evan’s blue dye. Thus,
protein transduction technology has the demonstrated potential to introduce a
120-kDa enzyme into many, if not all, cells and tissues of a mammal. Importantly,
this represents a molecule approximately 200 times larger than the current bioavailability
size restriction.
Another study differentiated between normal and HIV-1-infected cells by inclusion
of a viral-specific proteolytic cleavage site created in the Tat transducing protein.
This so-called “Trojan horse” strategy selectively induced apoptosis in HIV-infected
cells by exploiting the HIV protease. 26 A transducible Caspase-3 pro-apoptotic Tat
PTD fusion zymogen was engineered that substituted HIV proteolytic cleavage sites