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Protein Transport 373
for endogenous Caspase cleavage sites. Tat–Caspase-3 was processed into an active
form by the HIV protease in infected cells, resulting in initiation of the apoptotic
cascade in infected cells, while uninfected cells were spared. These preclinical
approaches to harness the therapeutic potential of protein transduction likely represent
the tip of the iceberg as to what lies in store for the future, with significant
improvements in transduction potential and creative approaches to target specific
disease cells and tissues.
17.5 FUTURE DIRECTIONS AND CONSIDERATIONS
Although unrestricted access of proteins into cells is now possible, we have a poor
grasp of how this technology works. Based on whole animal studies, all cells appear
to be susceptible to protein transduction. At the molecular level, it is unclear how
proteins behave when interacting with the cell membrane or if any particular molecule
(such as a specific phospholipid) is necessary to mediate entry. Currently
available limited data suggest that transduction across the cellular membrane results
in a partial or complete unfolding of the protein that will likely differ from one
protein to another. Therefore, once inside the cell, the transduced protein requires
refolding to obtain biologically active proteins.
Another consideration is the intracellular half-life of the protein. As has already
been observed with several genetically altered proteins, protein half-life can be
dramatically extended. In our animal studies the Tat–β-gal protein served as an
excellent “tester” enzyme to begin initially characterizing the ability to transduce
large proteins in model mammalian organisms. However, administration of additional
Tat fusion molecules to model organisms that generate phenotypic changes is
necessary to determine how much promise in vivo protein transduction really holds.
Basic pharmacological questions of tissue distribution, protein half-life, immunogenicity,
and modes of delivery also need to be addressed in a quantitative fashion.
Therefore, a complete understanding of protein transduction would not only facilitate
rational drug design, but also improve the efficacy of in vitro experiments.
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