ReviewGene-to-screenTherapeutic applications of the cell-penetrating HIV-1 Tat peptide
Introduction
Many human diseases, including neurodegenerative disorders, are currently incurable. New therapeutic approaches able to correct identified causative genetic defects or early pathogenic mechanisms are strongly needed. The presence of cell and tissue barriers, such as the blood–brain barrier (BBB) in the specific case of neurodegenerative diseases, represents a real drawback for systemic drug delivery, precluding the ability of therapeutic molecules to reach their own targets. A promising strategy to increase tissue biodistribution of therapeutics is represented by their conjugation with cell-penetrating peptides (CPPs) derived from proteins that are able to cross biological membranes. CPPs would be able to bear different therapeutic molecules, conveying them to their specific target and increasing their concentration in difficult-to-access tissues. Consequently, their therapeutic efficiency might also be augmented. This approach has the potential to revolutionize the treatment of a wide spectrum of human disorders.
One of the most promising and most studied CPPs is the HIV-1 transactivator of transcription peptide (pTat). pTat can be efficiently linked to different potential therapeutic molecules, including small molecules and antibodies, peptides, liposomes, nanoparticles and nucleic acids; it represents an extremely powerful tool to increase tissue biodistribution and the efficiency with which targets are reached. pTat is a promising strategy for the treatment of various human diseases, and particularly for neurodegenerative diseases. In this review, we will first provide an analysis of CPP biology and we will discuss CPP chemical structure and classification as well as their mechanisms of internalization. Then, we will focus our attention on pTat, which represents one of the first peptides identified that is currently widely studied and used.
Section snippets
The biology of CPPs
The integrity of biological membranes is crucial for tissue homeostasis. Some membranes, such as the BBB, are physically selective barriers to pathogenic bacteria, viruses and large hydrophilic molecules, whereas they allow the penetration of small or hydrophobic molecules [1]. By contrast, tissue barriers represent an obstacle to the use of a systemic administration protocol; they impair the ability of therapeutics to reach their targets when administered in the bloodstream. Therefore, the
CPPs as biological delivery agents
During the past 20 years, studies about internalization mechanisms have identified more than 100 peptidic sequences in the range 5–40 amino acids in length that are able to conduct active molecules, cargo and drug delivery vectors [2]. In addition, high-throughput screening of DNA-encoded peptide libraries has resulted in the discovery of many CPPs, for a detailed review see [3]. pTat was the first CPP identified; pTat peptide is the basic domain of the Tat protein, the shortest amino acid
The physicochemical classification of CPPs
CPPs constitute a broad heterogeneous group of peptides derived from proteins, chimeric sequences resulting from the merger of two natural sequences or the synthetic result of structure–activity software prediction studies. Classification criteria are based on the physicochemical features of the sequences [8]; the main three classes are cationic (83%), amphipathic (44%) and hydrophobic (15%) sequences [3].
The cationic class is the largest, and the best-known member of this class derives from
Mechanisms of internalization
CPPs are translocated into cells by several mechanisms that are independent but can occur simultaneously (Fig. 1). The protein or peptide from which CPPs derive can often provide information about the mechanism of internalization. Short, positively charged, arginine-rich CPPs such as pTat increase cellular drug uptake by interacting with the negatively charged plasma membrane and activating permeabilization of the cell membrane through a receptor-independent pathway, which results in
HIV-1 Tat peptide
Since the discovery of CPPs, many studies have been conducted to transport a wide variety of therapeutic molecules within the target cells. After the characterization of cellular and molecular mechanisms needed to support HIV infection, Tat protein was identified for its great ability to move across cells. Tat protein is a 14 kDa, RNA-binding protein that recognizes the transactivator response element (TAR), a specific sequence from the viral genome (Fig. 2) [18]. Tat stimulates HIV-1 gene
Small molecules, antibodies and miscellaneous delivery agents
Small molecules (e.g. drugs and imaging agents) have been linked to pTat in an attempt to increase their bioavailability. The bioavailability of these molecules has been limited by their high degree of hydrophilicity, which impairs their ability to cross the lipid bilayer. In particular, the imaging agents oxotechnetium V and oxorhenium V have been conjugated with pTat with good results, as shown by high intracellular concentrations [24]. Paramagnetic labels complexed with pTat can be detected
Peptides and proteins
The conveyance of proteins and peptides in vivo is a difficult goal to reach, and many studies have been designed to investigate the possibility of using CPPs as a delivery system for these molecules. CPPs can be covalently linked or complexed with the cargo, although properties of the peptide and the cargo influence the efficiency with which the complex is delivered, and therefore the related toxicity. Many heterologous proteins, such as horseradish peroxidase, RNase A and a specific domain of
Liposomes
The conjugation of HIV-1 pTat with liposomes represents a very promising drug delivery system. The potential of this liposome-based approach lies in its ability to combine the membrane-crossing properties of CPPs with the specificity of these carrier systems loaded with drugs [10]. Liposomes are vesicles comprising phospholipids that can be filled with different cargos, reducing the intrinsic drug toxicity and improving molecule biodistribution. Because conventional liposomes are quickly
Nanoparticles
To overcome the BBB in a noninvasive way, different biodegradable supramolecular nanodevices have been developed. Liposomes, niosomes, nanogels and cyclodextrins are some colloidal drug delivery systems that can target and release bioactive specific molecules in the central nervous system (CNS) [51]. In the field of brain disorders, lipid nanoparticles have always been studied because they ensure improved drug loading, storage stability, ease of production and safety of formulations for
Nucleic acids
CPP-based gene therapy has been used to overcome difficulties with introducing genetic material into target cells, which arise because of the low safety profile of viral vectors and the inefficiency associated with nonviral methods, for a complete review see [63]. CPPs such as pTat have been exploited for cancer treatment with suicide gene therapy approaches, delivering an enzyme encoding a gene that is able to produce a cytotoxic effect [64]. CPPs have also been used for plasmid DNA
Antisense oligonucleotides
Antisense oligonucleotides (ASOs) are modified nucleotides that bind specific complementary mRNA sequences. This binding can mark a specific mRNA for degradation or linkage to specific cis-acting splicing regulatory motifs. Different ASOs utilizing various chemistries 71, 72, 73 have been studied in cell and animal models. A variety of sequences have been targeted by three different ASO chemistries: 2ʹ-O-methyl phosphorothioate (2OMePS) oligonucleotides; the more stable variant, 2ʹ-O
pTat and clinical trials
In the past decade, pTat has proceeded to various phases of human clinical trials, although no therapy employing pTat has been approved by the FDA. Revance Therapeutics has completed a Phase II clinical trial using pTat to deliver botulinum toxin type A in a topical ointment for the removal of wrinkles (RT-001). Subcutaneous infusion of protein kinase Cδ inhibitor conjugates with pTat has been studied in Phase I/II clinical trials to evaluate the efficacy of the drug in: pain caused by
Tat pitfalls
Although different strategies of pTat conjugation with several compounds have resulted in very interesting, selective delivery to target cells, many pitfalls and critical aspects have emerged from basic, preclinical and clinical studies. Generally speaking, the stability of the peptide is fundamental to ensure the delivery of the cargo molecule to the target site. In fact, CPP cleavage by extracellular proteases influences the peptide uptake the most, whereas intracellular metabolism in
Concluding remarks
As summarized in this review, many studies have been conducted with pTat. Despite several promising lines of preclinical evidence that have demonstrated its ability to overcome biological barriers to deliver several types of drugs to target tissues, many clinical trials involving pTat have failed and some critical issues have emerged. However, as a CPP, pTat represents an interesting and promising tool to improve the biodistribution of drugs and to allow their systemic administration owing to
Acknowledgments
We wish to thank the Associazione Amici del Centro Dino Ferrari for its support.
References (93)
- et al.
Cell-penetrating peptides: breaking through to the other side
Trends Mol. Med.
(2012) Cell-penetrating peptides: classes, origin, and current landscape
Drug Discov. Today
(2012)- et al.
Cellular uptake of the tat protein from human immunodeficiency virus
Cell
(1988) Cell-penetrating peptides. A re-evaluation of the mechanism of cellular uptake
J. Biol. Chem.
(2003)- et al.
Cell-penetrating peptides: 20 years later, where do we stand?
FEBS Lett.
(2013) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent
J. Biol. Chem.
(1996)Permeability of the blood–brain barrier to HIV-1 Tat
Exp. Neurol.
(2005)HIV-1 Tat protein increases the permeability of brain endothelial cells by both inhibiting occluding expression and cleaving occludin via matrix metalloproteinase-9
Brain Res.
(2012)Peptide derived from HIV-1 TAT protein, destabilizes a monolayer of endothelial cells in an in vitro model of the blood–brain barrier, and allows permeation of high molecular weight proteins
J. Biol. Chem.
(2012)Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein-derived peptide
Biochem. Biophys. Res. Commun.
(1993)
A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells
FEBS Lett.
Intracellular delivery of antibodies using TAT fusion protein A
Biochem. Biophys. Res. Commun.
Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency
Biochem. Biophys. Res. Commun.
Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides
Adv. Drug Deliv. Rev.
Tat peptide-mediated intracellular delivery of liposomes
Methods Enzymol.
Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain glioma in animals
Int. J. Pharm.
Supramolecular devices to improve the treatment of brain diseases
Drug Discov. Today
Lipid nanoparticles for drug targeting to the brain
Methods Enzymol.
Delivery of siRNA to the brain using a combination of nose-to-brain delivery and cell-penetrating peptide-modified nano-micelles
Biomaterials
Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells
J. Biol. Chem.
Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells
J. Biol. Chem.
The use of cell-penetrating peptides as a tool for gene regulation
Drug Discov. Today
Translocating peptides and proteins and their use for gene delivery
Curr. Opin. Biotechnol.
Prospects for the gene therapy of spinal muscular atrophy
Trends Mol. Med.
Correction of SMN2 pre-mRNA splicing by antisense U7 small nuclear RNAs
Mol. Ther.
Pip6-PMO, a new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment
Mol. Ther. Nucleic Acids
Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy
Biochim. Biophys. Acta
Safety and efficacy of NA-1 in patients with iatrogenic stroke after endovascular aneurysm repair (ENACT): a phase 2, randomised, double-blind, placebo-controlled trial
Lancet Neurol.
Cell-penetrating peptides: a comparative membrane toxicity study
Anal. Biochem.
Chances and pitfalls of cell penetrating peptides for cellular drug delivery
Eur. J. Pharm. Biopharm.
Human immunodeficiency virus infection of human astrocytes disrupts blood–brain barrier integrity by a gap junction-dependent mechanism
J. Neurosci.
Antennapedia homeobox peptide regulates neural morphogenesis
Proc. Natl. Acad. Sci. U. S. A.
The medicinal chemistry of peptides
Curr. Med. Chem.
Live-cell analysis of cell penetration ability and toxicity of oligo-arginines
J. Pept. Sci.
Cell-penetrating peptides for drug delivery across membrane barriers
Expert Opin. Drug. Deliv.
Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis
Biochemistry
Regulated portals of entry into the cell
Nature
Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells
FASEB J.
Mechanisms of cellular uptake of cell-penetrating peptides
J. Biophys.
A comprehensive model for the cellular uptake of cationic cell-penetrating peptides
Traffic
Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides
FASEB J.
HIV-1 tat trans-activation requires the loop sequence within tar
Nature
Tat acetylation: a regulatory switch between early and late phases in HIV transcription elongation
Novartis Found. Symp.
Antiretroviral therapy does not block the secretion of the human immunodeficiency virus tat protein
Infect. Disord. Drug Targets
Novel TAT-peptide chelates fordirect transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy
Bioconjug. Chem.
Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide
Bioconjug. Chem.
Cited by (171)
Cell-penetrating peptides for transmucosal delivery of proteins
2024, Journal of Controlled ReleaseInnovative drug delivery strategies to the CNS for the treatment of multiple sclerosis
2023, Journal of Controlled ReleaseRational design of an anti-cancer peptide inhibiting CD147 / Cyp A interaction
2023, Journal of Molecular StructureA peptide inhibitor that rescues polyglutamine-induced synaptic defects and cell death through suppressing RNA and protein toxicities
2022, Molecular Therapy Nucleic Acids