Protein transduction - Nature

Gene Therapy (2001) 8, 1–4  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt

REVIEW

Protein transduction: an alternative to genetic intervention? KG Ford, BE Souberbielle, D Darling and F Farzaneh Department of Molecular Medicine, Guy’s, King’s and St Thomas’ School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK

Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alterna-

tive to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed. Gene Therapy (2001) 8, 1–4.

Keywords: protein transduction; antennapedia; TAT; VP22; gene therapy

Introduction The objective of gene therapy is the gene delivery and concomitant expression of gene products that either possess a therapeutic biological activity or induce an altered cellular phenotype. Gene therapy approaches to a number of genetic disorders, including enzyme deficiencies or diseases such as cystic fibrosis and Duchenne’s muscular dystrophy, require long-term and appropriately regulated expression of the transgene. In addition, efficient clinical application of a gene therapy strategy requires in vivo targeting, which encompasses both targeted delivery and tissue-specific expression of the transgene. However, for most current gene therapy approaches, especially gene therapy of cancer, sustained and regulated expression of the transgene is not a prerequisite for effective treatment, the objective being either the direct induction of tumour cell death or the induction/enhancement of immune-mediated elimination of the tumour. The short-term requirement for the presence of the therapeutic gene product raises the possibility of achieving the same objective by direct delivery of the gene product itself, rather than the gene. Recent developments in protein transduction (delivery of protein into cells) suggest this is now a realistic approach.

Protein transduction technology The observation that some proteins are capable of being taken up by the cell, when added exogenously, has prompted a more detailed study of the fundamental mechanisms in operation throughout this process. This has resulted in the identification of protein transduction domains, that when fused to other proteins, confer on

Correspondence: K Ford or F Farzaneh

them the ability to similarly enter the cell and even the nucleus. The three most widely studied of these sequences are the Drosophila antennapedia peptide, the herpes simplex virus VP22 protein and the HIV TAT protein transduction motif. However, other proteins may have similar properties as suggested for haemagglutinin from influenza1 or lactoferrin, fibroblast growth factor 1 and 2 and the homeodomain (HD) of engrailed, Hoxa-5, Hoxb-4 and Hoxc-8 proteins.2,3

Antennapedia peptide The antennapedia motif is derived from a family of Drosophila homeoproteins, a class of trans-activating factors involved in the developmental process.3 These proteins recognise and bind DNA through a 60 amino acid carboxy-terminal region arranged in three ␣-helical sequences, called the homeodomain. The homeodomain of antennapedia (AntpHD) is capable of translocating across neuronal membranes and is conveyed to the nucleus.4–6 The third helix of the homeodomain, comprising just 16 amino acids, also possesses this unusual property,2,7 enabling small molecules to be taken up with AntpHD or with the AntpHD peptide into live cells8 and has formed the basis for the family of peptides.3 Internalisation is independent of the ␣-helix configuration of the third domain of AntpHD, and occurs both at 37°C and 4°C and thus is not receptor-mediated.2 However, it has been demonstrated that the first tryptophan (W) residue in the 16 aa antennapedia-derived peptide (amino acids RQIKIWFQNRRMKWKK) is important for internalisation.3 The high arginine and lysine content of this peptide gives it a positive charge at physiological pH. Therefore, a model of internalisation based on the formation of an ‘inverted micelle’ across the plasma membrane has been proposed.2,3 In this model the positively charged antennapedia peptide recruits negatively charged cell

Protein transduction KG Ford et al

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membrane phospholipids, inducing the formation of a hydrophilic cavity (inverted micelle) which then travels across the plasma membrane, culminating in the translocation of the protein to the cytoplasm. This model may also apply to other protein transduction peptides such as HIV TAT, which shares similar charge characteristics (see below). A possible limitation of the antennapedia peptide, however, is that it has so far been reported to translocate only small peptides or proteins.9,10

Herpes simplex virus VP22 protein The herpes simplex virus type 1 (HSV-1) VP22 protein is a structural polypeptide forming the major component of the virus tegument situated between the envelope and capsid regions of the mature virion. It is a small basic protein, approximately 38 kDa in size, encoded by the UL49 gene. In vivo, VP22 exists in two distinct forms as assayed by gel electrophoresis, phosphorylated and nonphosphorylated.11 VP22 is exported from cells in which it is synthesised, despite lacking a signal sequence,12 by ‘nonclassical’ Golgi-independent secretion. Upon export, VP22 can enter cells with high efficiency. Moreover, such re-internalised VP22 is targeted to the nucleus (despite having no recognised nuclear localisation signal), binds to chromatin and segregates to daughter cells. It is now believed that nuclear localisation may be a consequence of cell division, rather than an active process, which may be a limiting factor in the application of VP22 as a protein delivery system. However, this is only a problem for proteins that exert their biological effects in the nucleus, and where such proteins do not have an intrinsic nuclear localisation motif or are not able to associate with cellular components that are themselves targeted to the nucleus (ie ‘piggybacking’). Recent experiments have demonstrated that VP22 is able to introduce large proteins, such as green fluorescent protein (GFP), p53, HSV-thymidine kinase and the 116 kDa ␤-galactosidase protein, into simian (COS-1, Vero), human (HeLa) and hamster (BHK-21) cells.12–14 Direct cellular uptake of VP22, when added as an exogenous protein, has also been clearly demonstrated.12 The mechanism of protein transduction is not clear although VP22 possesses relatively basic domains in its C-terminal region. Whether protein transduction is due to a similar mechanism to that proposed for Antennapedia is not known. HIV TAT Protein transduction domain The HIV-1 trans-activator gene product, TAT, has been shown to be a regulator of transcription in latent HIV and is essential for HIV replication. It is an 86 amino acid protein made from two exons of 72 and 14 amino acids, respectively. It was first demonstrated independently by Green and Loewenstein15 and Frankel and Pabo16 that TAT added exogenously in culture was taken up rapidly by cells. This protein transduction property was shown to reside in amino acids 37–72.15 In 1991, Mann and Frankel17 focused on the transduction property of TAT and demonstrated that the domain encompassing amino acids 38–58 (the basic region of TAT) retained the transducing ability, enabling both its nuclear and cytoplasmic accumulation. They showed that uptake was rapid, inhibited by heparin, dextran sulfate, or by incubation at 4°C in some cells (eg HeLa cells) but only partially inhibited in other cells (eg H9). In addition, uptake was not inhibited by treatment of the cells with trypsin, hepGene Therapy

arinase and neuramidase. They speculated that TAT binding to the cellular membrane was mediated through charge-interaction between the basic region of TAT and charged polysaccharides on the cellular membrane. Fawell and his colleagues,18 in 1994, demonstrated that large molecules (␤-galactosidase, horseradish peroxidase, RNAse A and domain III of Pseudomonas exotoxin A) when chemically cross-linked with TAT peptides (either amino acids 1–72 or 37–72) were taken up by cells in vitro. Using the ␤-galactosidase-TAT model they showed that uptake also took place in vivo after i.v. injection of the chimeric protein in mice and that the protein was present 20 min later in heart, liver, spleen, lung and skeletal muscles, but almost absent in kidney and brain.18 In 1997, Vives and his colleagues19 demonstrated that the still smaller HIV-1 TAT protein basic domain (37–47 aa) rapidly translocated through the plasma membrane and accumulated in the nucleus. In this study they did not observe inhibition of peptide uptake at 4°C in HeLa cells, and confirmed that protein transduction was rapid. The same group also reported efficient cellular uptake of a small peptide conjugated to the TAT peptide.20 Subsequent work by Stephen Dowdy and his colleagues improved TAT-mediated delivery by constructing fusion proteins between several polypeptides and proteins and a short 11 aa region of the TAT protein. This region corresponds to aa 47–57 of TAT (YGRKKRRQRRR) and has a high net positive charge at physiological pH with nine out of 11 of its amino acids being either arginine or lysine. In vivo experiments revealed the power of this strategy by demonstrating the presence of ␤-gal activity in several tissues 4 h after intraperitoneal injection of the fusion protein into mice. Beta-gal activity was highest in liver, kidney, lung tissues, heart muscle fibres and in the red pulp area of the spleen. Interestingly, strong ␤-gal activity was also detected in the brain mainly in cell bodies and to a lesser extent in the white matter. The blood– brain barrier remained intact as ascertained by Evans’ blue exclusion test. It was speculated that transport of the chimeric protein into the nuclei of nondividing cells may have been mediated by the integral nuclear localisation signal of the TAT peptide.21 Many proteins have since been successfully transported into a wide variety of human and murine cell types, using the TAT PTD methodology.21–25 One significant aspect of TAT-mediated protein transduction is the requirement for purified recombinant protein. Dowdy’s group have suggested that denatured proteins may be transduced more efficiently than correctly folded proteins. They have also proposed that once inside the cell, these proteins can be correctly refolded by chaperones. Protein purification protocols based on a denaturation step may therefore contribute to both an improved yield and more efficient protein transduction.22 Recently, it has been claimed that a poly arginine peptide may have even greater transduction properties than the 11 amino acid TAT PTD, strengthening the notion that regional charge may be a prominent factor in cellular uptake.26 However, the transactivation potential of endogenous cellular genes by the 11 aa TAT PTD is not known and will have to be monitored closely in the future. It is well established that the full-length TAT protein stimulates growth of Kaposi’s sarcoma-derived cells27 and that TAT transgenic mice develop Kaposi sarcoma.28 However, if the 11 aa TAT PTD is shown to be devoid of toxic effects in vivo, it


will be of tremendous use in a variety of potential applications.

Future prospects In addition to protein transduction, PTD may provide efficient means of intracellular delivery of not just proteins but macromolecules, such as DNA as well as cancer chemotherapeutic agents (eg methotrexate,29 or doxorubicin30). Ex-vivo applications include stem cell expansion, by stimulation of growth through intracellular introduction of proteins such as SV40 large T antigen. There is increasing evidence that such transiently growth-stimulated cells are likely to revert back to their nontransformed phenotype following the decay and/or dilution of the tranduced proteins, thus providing an expanded pool of stem cells for transplantation.31,32 Protein transduction may also prove useful for efficient antigen loading of dendritic cells for a range of vaccination purposes, including anti-tumour immune therapy. Long-term use of the PTD technology may also encompass clinical applications that necessitate sustained in vivo delivery of the gene product. Although gene therapy may be more suitable for such situations in that sustained expression of the gene product may be achieved following infrequent, or possibly even a single, delivery of the transgene. The fact that PTDs can cross the blood–brain barrier may also make them suitable for a range of neurological applications, including those refractory to gene therapymediated intervention. Potential therapeutic and research applications of protein transduction technology encompass both ex vivo and in vivo applications. Although the second option is more practical and potentially cheaper, possible side-effects may hamper its development to the clinic. Human administration of recombinant or purified proteins is used routinely as treatment for many diseases.33,34 One potential side-effect in the clinical administration of recombinant or purified proteins is induction of hypersensitivity. Recognised approaches to alleviating this problem include species switching of the therapeutic protein (or generation of functional mutants) as well as immune suppression. However, immune-mediated reactions are still likely to be significant, especially when the protein is administered long term. Protein transduction technology is still in its infancy, but is potentially the basis for an entirely new form of therapy. It may have particular applications where a therapeutic effect would not normally require sustained and regulated expression of the transgene, as is often the case in the gene therapy of cancer. In the long term, this approach may prove to be technically simpler, avoiding some of the possible side-effects of gene therapy. Protein transduction may also enable more efficient penetration and delivery at solid tumour sites, than can currently be achieved using viral vectors. However, the pharmacology of in vivo protein transduction is still poorly understood and requires extensive study before either the therapeutic potential or limitations of protein transduction technology can be adequately evaluated.

References 1 Plank C et al. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems J Biol Chem 1994; 269: 12918–12924.

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