Project Title

Kinetic characterization of cellular entry, trafficking and cargo release by a novel TAT-derived cell-penetrating peptide

Presenters

Academic department under which the project should be listed

CSM - Chemistry and Biochemistry

Faculty Sponsor Name

Jonathan McMurry

Additional Faculty

Julia LeCher, Cell Biology, jwand@kennesaw.edu Daniel Morris, Biochemistry, dmorris@kennesaw.edu

Abstract (300 words maximum)

Kinetic characterization of cellular entry, trafficking and cargo release by a novel TAT-derived cell-penetrating peptide

Hannah Moorman, Robert L. Dickson, Julia C. LeCher, Daniel P. Morris and Jonathan L. McMurry

Commonly derived from viral proteins, cell-penetrating peptides (CPPs) confer onto other macromolecules to which they are attached the ability to cross membranes in a rapid, nontoxic manner. They have long held great promise as delivery vehicles for biomolecular cargos for research and therapeutic purposes. However, standard CPP technologies rely on covalent linkages between CPP and cargo that commonly result in endosomal entrapment and destruction of cargo. Our group developed a high affinity, reversible, non-covalent CPP-adaptor, TAT-CaM, that solves this problem, releasing cargos into the cytoplasm with high efficiency. TAT-CaM consists of the cell-penetrating sequence of HIV transactivator of transcription fused to human calmodulin. It binds calmodulin binding site (CBS)-containing cargos with nM affinity in the presence of Ca2+, but not at all in its absence. Prior studies utilizing TAT-CaM to deliver an array of protein cargos to living mammalian cells found that delivery was so rapid that the kinetics could not be described using standard assays. The present report describes development of an assay utilizing cell culture under flow to observe penetration and cargo delivery in real-time. Baby hamster kidney cells were cultured in flow chambers that were installed onto a confocal microscope stage under culture conditions. Subconfluent cells were exposed to fluorescently labelled TAT-CaM/cargo complexes as live images were collected from time 0. Parameters such as concentration and time of dosing were examined with respect to efficient cellular delivery as evidenced by cargo-induced increases in cytoplasmic fluorescence. Differential fates of CPP-adaptors and cargos were also observed using compatible fluorophores with TAT-CaM expected to traffic to lysosomes while cargo localized to the cytoplasm. The results of this work will increase our basic knowledge of CPP trafficking with respect to mode and kinetics of entry and as well as inform future efforts to develop effective CPP-based delivery methods for therapeutics.

Project Type

Poster

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Kinetic characterization of cellular entry, trafficking and cargo release by a novel TAT-derived cell-penetrating peptide

Kinetic characterization of cellular entry, trafficking and cargo release by a novel TAT-derived cell-penetrating peptide

Hannah Moorman, Robert L. Dickson, Julia C. LeCher, Daniel P. Morris and Jonathan L. McMurry

Commonly derived from viral proteins, cell-penetrating peptides (CPPs) confer onto other macromolecules to which they are attached the ability to cross membranes in a rapid, nontoxic manner. They have long held great promise as delivery vehicles for biomolecular cargos for research and therapeutic purposes. However, standard CPP technologies rely on covalent linkages between CPP and cargo that commonly result in endosomal entrapment and destruction of cargo. Our group developed a high affinity, reversible, non-covalent CPP-adaptor, TAT-CaM, that solves this problem, releasing cargos into the cytoplasm with high efficiency. TAT-CaM consists of the cell-penetrating sequence of HIV transactivator of transcription fused to human calmodulin. It binds calmodulin binding site (CBS)-containing cargos with nM affinity in the presence of Ca2+, but not at all in its absence. Prior studies utilizing TAT-CaM to deliver an array of protein cargos to living mammalian cells found that delivery was so rapid that the kinetics could not be described using standard assays. The present report describes development of an assay utilizing cell culture under flow to observe penetration and cargo delivery in real-time. Baby hamster kidney cells were cultured in flow chambers that were installed onto a confocal microscope stage under culture conditions. Subconfluent cells were exposed to fluorescently labelled TAT-CaM/cargo complexes as live images were collected from time 0. Parameters such as concentration and time of dosing were examined with respect to efficient cellular delivery as evidenced by cargo-induced increases in cytoplasmic fluorescence. Differential fates of CPP-adaptors and cargos were also observed using compatible fluorophores with TAT-CaM expected to traffic to lysosomes while cargo localized to the cytoplasm. The results of this work will increase our basic knowledge of CPP trafficking with respect to mode and kinetics of entry and as well as inform future efforts to develop effective CPP-based delivery methods for therapeutics.