Tat-tetanus toxin fragment C: a novel protein delivery vector and its use with photochemical internalization

Abstract

Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.

A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment

“…Unless a linker region was specifically designed into the expression vector, only the AgeI restriction site separated the 5′ domain and GBA1 . The TTC and Tat-TTC sequences were amplified from the pGEX4T3 TatTTCGFP vector (Gramlich et al, 2013) using PCR. Each of the Tat , Tet1 , and Tat-Tet1 sequences were made as duplex oligonucleotides by Integrated DNA Technologies (IDT) and cloned into either pTag GBA or pTag GBA GFP.…”

“…While a large number of different CPPs exist, Tat, an 11 amino acid peptide originally derived from the transactivator protein of HIV (Tat), was the first to be described (Frankel and Pabo, 1988) and remains one of the more widely used CPPs. Tat is taken up by receptor-independent macropinocytosis and following uptake, most of the Tat-linked cargo protein is sequestered in endosomes (Chauhan et al, 2007; Gillmeister et al, 2011; Gramlich et al, 2013). …”

“…Novel enzymes included binding domains Tat, RDP, Tet1, and combinations of Tat and the receptor-dependent peptides. The dual-peptide binding domains were investigated because the combination of Tat and TTC has been shown to work better than either Tat or TTC alone (Gramlich et al, 2013). Design of the candidate genes and enzymes reflected goals of high level expression, enzyme activity, and neuronal binding.…”

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research

“…It has however been estimated that more than 90% of Tat-linked cargoes remain in the endolysosomal compartments [90]. Gramlich and coworkers have reported that tetanus toxin fragment C linked to the Tat-peptide along with GFP as a fluorescent marker was much more efficiently translocated to the cell cytosol following the application of PCI [91]. Accordingly, it was found that PCI of peptide nucleic acids (PNA) towards hTERT (telomerase) expression was substantially more efficient than TAT-linked hTERT PNA [92] (in the absence of PCI).…”

Strategies to Stabilize Cell Penetrating Peptides forIn VivoApplications

“…Since endosomal entrapment is a common bottleneck in intracellular delivery of bioactive agents, a number of different strategies have been developed offering a good arsenal of tools to apply to CPPs [61]. Using biologymimicking strategies, CPPs have been modified with fusogenic viral peptides such as hemagglutinin HA2 fusion peptide [60,62] and bacterial toxin translocation domains improving cytoplasm entry [63,64].…”