Session: Current Topics in Biological Chemistry:

Hijacking dysfunctional apoptotic pathways to circumvent unregulated cell proliferation is an attractive and powerful therapeutic strategy for cancer treatment. Nevertheless, conventional chemotherapeutic methods demonstrate substantial off-target effects. To circumvent this, targeted delivery of enzymes that play a critical role in propagating apoptosis, cysteine aspartate proteases (caspases), is a strategic alternative strategy. Compared to death-inducing small molecules, proteases offer numerous advantages beyond targeting including their biodegradability, catalytic turnover and substrate specificity. However, biologics require systematic formulation in vehicles capable of intracellularly translocating an active, unmodified protein for therapeutic effect. Utilizing a redox-responsive polymeric delivery system, we have intracellularly delivered native caspase-3, the most enzymatically efficient member of the caspase family responsible for cleaving substrates critical to DNA repair and cell structure. In addition to our group, others have investigated caspase-3 but to date, no other caspase cargo has been evaluated. Beyond caspase-3, our group has now delivered the other apoptotic caspases, caspase-6, -7, -8 and -9 and observed unique apoptotic outcomes for each cargo reinforcing the importance for cargo and vehicle compatibility investigations.
The majority of protein delivery systems hinge on their endosomal escape efficiency. Thus, estimating intracellular protein therapeutic concentrations, for example for caspase-3, remains a challenge. Traditional methods to qualitatively observe endosomal escape utilize conjugated fluorophores that often introduce confounding factors leading to over- or under-enhanced interpretations of cytosolic localization. In our approach, we have utilized a split-complementation endosomal escape (SEE) assay via an engineered casp-3 variant that resembles wild-type casp-3 in both activity and structure, but can reassemble with a fragment of GFP (i.e. split GFP). Only upon endosomal escape of the protein cargo into the cytosol will fluorescence be generated via the split GFP mechanism. SEE enables quantitative comparison of casp-3 delivery vehicles.

Dr. Francesca Edith Anson

UMass Amherst

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