A strategy to Improve Tropism of HIV-Tat for Smart Drug Delivery System

Modified HIV-Tat showed enhanced biotin uptake by cardiomyocytes while producing minimal detectable cargo uptake in HEK-293T cells.

Rationale

Protein transduction domain (PTD) is a class of small peptides or protein domains that mediates cellular uptake of molecular cargo through energy-, receptor-, and conventional endocytosis-independent manners. The term PTD is closely related to cell-penetrating peptide (CPP), these two terms are often used interchangeably. However, PTD is typically derived from naturally occurring protein, whereas CPP can be either naturally occurring or synthetically designed. Notably, PTD enriched in positively charged amino acids can also function as CPP, reflecting the substantial overlap between these two classes of cell-penetrating molecules.

PTD and CPP can be used to deliver a wide range of molecular cargoes—including peptides, proteins, nucleic acids, nanoparticles, and other therapeutic agents—into cells, including slow- or non-dividing cells that are often difficult to transfect or transduce. Their short peptide sequences also make them relatively easy to synthesize, modify, and conjugate to various cargoes. Despite these attractive properties for drug delivery, several challenges continue to hinder their clinical translation. One major limitation of PTDs and CPPs is their lack of tissue specificity or tropism, as they can facilitate cargo delivery across a broad range of cell types. In the following section, I propose a strategy to address this limitation and improve the tissue specificity of PTD- and CPP-based delivery systems.

Modified HIV-Tat Selectively Targets Cardiomyocytes

The arginine-rich domain of the human immunodeficiency virus (HIV) trans-activator of transcription (Tat) is a good example of the functional overlap between PTD and CPP. Here, I sought to enhance the cellular specificity of HIV-Tat for delivering biotin as a molecular cargo to cardiomyocytes. Notably, cardiomyocytes are terminally differentiated cells and are generally more difficult to transfect or transduce than actively dividing cells. For this preliminary study, primary neonatal mouse cardiomyocytes and human embryonic kidney (HEK) 293T cells were used.

I first designed two peptides: (1) Tat-Bio, consisting of the HIV-Tat domain conjugated to biotin; and (2) YS-Tat-Bio, consisting of a ligand-guiding sequence linked to HIV-Tat and biotin (Fig A). The ligand guide is a short peptide designed to bind specific cell-surface receptors or tissue markers, thereby promoting targeted delivery and supporting cellular uptake of the cargo. Furthermore, incorporating additional residues derived from endogenous proteins or molecules may also help reduce immunogenicity and minimize rapid clearance by the kidneys or liver. In this preliminary experiment, I selected ErbB receptors as the targeting receptor because several studies have reported relatively high ErbB expression in cardiomyocytes compared with HEK-293T cells. I therefore fused Tat-Bio with a six-amino-acid sequence derived from the biological ligand of the ErbB receptor to generate YS-Tat-Bio.

Briefly, cardiomyocytes and HEK-293T cells were incubated with 5 µM Tat-Bio, YS-Tat-Bio, or biotin alone as a control. For simplicity, the peptide concentration was calculated based on the molecular weight of biotin rather than that of HIV-Tat. At this concentration, Tat-Bio showed low cellular uptake in HEK-293T cells (data not shown). Cellular uptake of the biotin cargo was detected using streptavidin-conjugated GFP (green signal) by immunofluorescence microscopy. Neither Tat-Bio nor YS-Tat-Bio produced strong streptavidin signals in HEK-293T cells (Fig. B, upper panel). Similarly, cardiomyocytes treated with Tat-Bio showed barely detectable streptavidin signals, suggesting limited cellular uptake of the biotin cargo (Fig. B, lower panel). In contrast, strong streptavidin signals were observed in cardiomyocytes treated with YS-Tat-Bio, indicating substantially enhanced uptake of the biotin cargo (Fig. B, lower panel). Notably, brief incubation with YS-Tat-Bio at 4°C resulted in detectable streptavidin signals on the surface of cardiomyocytes, suggesting that the ligand-guided peptide can efficiently bind to the cardiomyocyte cell surface (Fig. C). Together, these preliminary findings suggest that incorporation of a ligand-guiding sequence may enhance the cardiomyocyte-specific binding and uptake of the HIV-Tat-based delivery peptide.

Take-Home Hypothesis & Clinical Implication

Although these findings are preliminary and require further validation, they raise the possibility of transforming broadly acting PTDs or CPPs into tissue-selective delivery platforms by incorporating a short ligand-guiding sequence. The ligand-guided strategy is potentially modular: by replacing the ligand-guiding sequence with a peptide that recognizes a receptor or tissue marker enriched in a specific cell type, the same PTD- or CPP-based platform could be adapted for tissue-selective delivery across different organs. This approach may therefore provide a relatively simple and versatile strategy for improving the therapeutic index of PTD- and CPP-based delivery systems while minimizing nonspecific cargo uptake.

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Dr. Yogy Simanjuntak-HyVir

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