Design of an acid labile traceless-cleavable click linker for use in a novel protein transduction shuttle

Intracellular protein delivery is offering numerous possibilities
in research and in therapy. Aside gene therapy, protein delivery
into living cells is one of the most promising tools for the
treatment of various so far immedicable diseases including cancer.
To develop a practicable protein delivery platform, a test system
which allows easy control of successful intracellular delivery is
needed. Therefore a test system based on two model proteins was
established. A nuclear localization signal tagged EGFP molecule is
enabling fast control of cellular uptake and endosomal release. The
second model protein ß-galactosidase is evidencing that protein
conformation is not irreversible disturbed by modification with the
carrier molecules. Protein transduction technology is opening the
door for a promising alternative to gene therapy, as it is lacking
of the potential malignant side effects of gene therapy. The most
limiting step in the development of a therapeutic drug remains the
delivery process. In the last decade, many techniques to deliver
proteins into living cells were developed. Although great efforts
were made, so far no all-purpose technique is available that
addresses all critical steps, like efficient uptake, endo-lysosomal
escape, low toxicity, while maintaining enzymatic activity. Each
method has got its limitation, for example cell type dependence.
Among the so far used carriers, the most effective ones are
cationic polymers like polyethylenimine. These carriers are lacking
of precise structure and often show high toxicity, dependent on the
molecular weight of the used polymer. In this thesis the properties
of the three arm cationic oligomer 386, which was previously
designed for siRNA delivery was investigated in regard of being
applicable as a transduction carrier for protein delivery. This
carrier molecule, in contrast to other cationic polymers used for
protein delivery, is of precise structure, of low molecular weight
and potentially degradable by proteases. The transduction oligomer
was covalently bound to the protein by a bioreversible bond. Our
results reveal that covalent coupling of the structure defined
cationic oligomer 386 to a protein leads to a high efficient, serum
insensitive and low toxic alternative to established protein
transduction technologies. For a general all-purpose delivery
system covalent coupling of the carrier to the cargo protein is
indispensable. Protein delivery requires special properties to the
linker molecule. Therefore in this work a new pH sensitive linker
was developed which combines the advantages of click reactions with
the implementation of a traceless cleavable bond between two
conjugated molecules. Three different click chemistries were
performed which all are compatible with the acid labile properties.
A traceless cleavage may be a particularly important feature in
protein transduction strategies, to maintain full bioactivity of
enzymes and other proteins. The current example of 386
carrier-mediated cytosolic delivery and subsequent nuclear import
of released nls-EGFP demonstrates the advantage of the traceless
linker. To demonstrate that the modification does not irreversibly
affect structure and biological activity of proteins,
386-AzMMMan-ßgalactosidase was delivered as a model enzyme. It
exhibited cytosolic activity in the transduced cells far higher
than without shuttle. Aside from these encouraging options for
protein delivery and modification, the linker might have broader
use in the design of novel programmed, acid labile and
biodegradable drug delivery systems. Targeted therapeutics could,
after delivery into acidic tumor areas or upon cellular uptake into
endosomes, be dismantled from their outer shell including targeting
ligands. Besides drug delivery, the linker may also be of interest
for other applications, such as reversible labeling of various
biological and also chemical molecules. The developed linking
strategy and the presented concepts for transduction shuttles may
help to get a step closer in the design of an all-purpose protein
delivery platform, applicable on bench as on bedside.