Clinical and Experimental Ophthalmology 2005; 33: 628–635
Original Article D-TAT transporter as an ocular peptide delivery system Daniel F Schorderet MD,1,2 Violaine d’Allèves Manzi PhD,1 Kriss Canola BSc,2 Christophe Bonny PhD,3 Yvan Arsenijevic PhD,2 Francis L Munier MD2 and Fabienne Maurer PhD,3 1
IRO – Institut de Recherche en Ophtalmologie, Sion, 2Unit of Oculogenetics, Jules Gonin Eye Hospital, Lausanne, and 3Division of Medical Genetics, CHUV, Lausanne, Switzerland
ABSTRACT Background: Future treatment for genetic diseases may involve the replacement of malfunctioning genes through virus-mediated gene therapy. However, this approach is plagued with many problems, both ethical and scientific. Therefore, alternative treatments based on new molecules may represent a safer option. Molecular treatment of many eye diseases will need to bring active molecules into the photoreceptors. Recently, the trans-activator protein (TAT) human immunodeficiency virus type 1 (HIV-1) transcriptional factor has proven to be effective in transporting molecules across cellular membranes. The half-life of these molecules does not exceed 48 hours. The potential use of the retro-inverso form of the TAT (D-TAT) peptide, the protein transducing domain of the HIV-1 transcriptional factor, as a molecular transporter was investigated. Methods: FITC-labelled D-TAT (D-TAT FITC) was applied to the 661W murine photoreceptor cell line in culture. The labelled peptide was also injected into the vitreous body or the subretinal space of adult mice. Cells and cryosections of eyes were analysed under fluorescence microscopy at various time points after peptide treatment. Coimmunostaining with various antibodies was performed in order to characterize the transduces cells. Results: D-TAT was effective in transducing photoreceptor cells in culture. Transduction of D-TAT FITC was also effective when injected into the vitreous or subretinal space and was observed for a longer period of time than L-TAT FITC. Conclusions: The retro-inverso form of the TAT sequence is effective in transducing cells from various compartments of the eye. After 14 days, the D-TAT FITC was clearly visible in the retina whereas L-TAT FITC had almost disappeared. The D-TAT peptide represents an interesting molecular
transporter that, when coupled to a specific effector, may have potential therapeutic future, especially when a longlasting action is needed. Key words: gene therapy, peptide, TAT.
INTRODUCTION Gene therapy is a promising approach for the treatment of many genetic diseases and, over the last decades, various methods have been developed to transport the genes of interest into the specific tissues and the correct cells: transfection of naked or plasmid DNA, bacterial or yeast artificial chromosomes, bullet-based DNA transfer, transduction with adenovirus, adeno-associated virus, lentivirus, etc. The genes of interest can integrate or stay as episomal structures. Although all these approaches have been tried successfully, each one has potentials and limitations. For systems that rely on genomic integration, the sites of integration, especially if those sites are non-random, may represent potential problems.1 In addition, activity, duration of expression, inability to reach the tissue of interest, concerns about the safety of such procedures and safety regulations have prevented the general use of these delivery systems. Polymer-based or encapsulated delivery systems have also been successfully tried in various situations.2 Depending on the approach, such systems may have similar restrictions and it is unlikely that a general-purpose delivery system will be developed for all applications. Therefore, additional delivery systems should be explored. Recently, novel carrier systems based on membrane shuttling proteins have been identified and developed as drug delivery tools. The Drosophila melanogaster homeobox protein Antennapedia was the first cell-penetrating protein (CPP) reported.3 Dissection of the protein revealed that a small 16-amino-acid peptide corresponding to the third helix of the homeodomain was able to cross biological membranes and to accumulate in the nucleus. Since this
Correspondence: Dr Daniel F Schorderet, IRO – Institut de Recherche en Ophtalmologie, Avenue Grand-Champsec 64, 1950 Sion, Switzerland. Email: [email protected]
; URL: http://www.irovision.ch
D-TAT peptide as a molecular transporter first discovery, many CPPs have been identified.4–6 Protein transducing domains are not restricted to Antennapedia. VP22 from Herpes Simplex virus7 human immunodeficiency virus type 1 (HIV-1) transcriptional factor transactivator protein (TAT) and even synthetic peptides may have protein transduction properties. Conjugation of a stretch of seven arginine moieties to cyclosporin A was able to greatly facilitate the transport of this drug into mouse and human skin fibroblasts.8 One characteristic shared by many protein transducing domains is their strong positive net charge.9 The potential of HIV-1 transcriptional factor TAT to be taken up by cells and to represent a CPP has been well studied.10–12 The sequential analysis of the TAT protein allowed Vivès et al. to define its protein transducing domain to a small region that included residues 48–60.13 Since then, this peptide has been linked to many large and small proteins and has been used for cellular delivery,14–19 and no toxic effect has been associated to this sequence. The TAT peptide itself and proteins fused to TAT are also able to cross the cellular membrane of Y79 and RPE-J, two cell lines derived from a human retinoblastoma tumour and rat retinal pigment epithelium cells, respectively.20 As in other organs, transduction was not limited to the TAT peptide and p15INK4B, a cargo protein of about 25 kDa, has been transported across human corneal epithelial cells.21 TAT is very effective in all cell culture systems. In vivo experiments have shown that TAT still retained its transducting capacity when injected intraperitoneally22 or systemically.23 Intra-vitreous injection of TAT in 3-month-old C57BL/6J mice revealed transduction in ganglion cells of the inner nuclear layer of the retina and cells of the cornea.20 Despite a wide range of use, some organs or cell layers are less permeable to transduction by TAT. This may reflect the specific protective function that has been acquired by such epithelia, that is, skin and cornea. Recently Guo et al. showed that transduction of TAT-β-galactosidase was obtained in cultured corneal epithelial cells, but was much less efficient in intact corneas. TAT-β-galactosidase was easily transducted when the superficial layer was removed.24 Impaired corneal transduction may be due to the absence of coated pits in the cells of the superficial epithelial layers. Such coated pits have been shown to be required for TAT internalization.25 The TAT peptide, in its natural composition, is readily degraded by proteases present in all tissues. We recently showed that a synthetic all retro-inverso form of TAT (DTAT), while still retaining similar transducing capacities in β-TC3 cells and in neurones, exhibited a much longer time of action in these cells and could carry efficiently a therapeutic peptide.26,27 Here, we investigated whether the D-TAT molecule would also behave similarly in the eye and show that D-TAT is able to cross the membranes of various cellular compartments of the eye. Therefore, D-TAT may be considered as a good candidate for transporting therapeutic molecules into the eye.
METHODS Peptide synthesis The FITC-labelled retro-inverso D-enantiomer TAT peptide was synthesized by Auspep PLT, Victoria, Australia and described previously.26 It was a gift from S.A. Xigen, Lausanne, Switzerland.
Cell culture 661W cells were maintained in DMEM containing 4.5 g/L D-glucose, 3.7 g/L NaHCO3, 25 mM Hepes, GlutaMAX I (Invitrogen, Gibco No. 32430) supplemented with 1 mM sodium pyruvate, 10% calf serum and 20 µL β-mercaptoethanol. The cell line was kept at 37°C in an atmosphere of 5% CO2. For each experiment, 10 000 cells were plated in a six-well tissue culture flask and cultured for 24 h. In total, 1 µM D-TAT FITC was incubated for 60 min, the cells were then washed with PBS and fixed in a 4% paraformaldehyde solution for 15 min. Analysis was performed by fluorescence microscopy.
Animals NMRI wild-type mice were purchased from Iffacredo (France); the VPP mice were obtained from M.I. Naash, University of Oklahoma Health Sciences Center, OK, USA. All animals were maintained on a basal diet with free access to food and water. For subretinal injections, animals were anaesthetized with inhalant isoflurane (Baxter, Deerfile, IL, USA) and kept under isoflurane during the whole procedure. In addition, local anaesthesia was performed by topical application of Tetracaine (Tetracaine 1% SDU Faure, Novartis, Switzerland). The sclera was removed on a small surface in
Figure 1. Fluorescence microscopy of 661W in culture, transduced with 1 µM D-TAT FITC. Original magnification ×400.
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the superior half part of the eye and a 23-gauge needle (Beckton Dickinson, Franklin Lakes, NJ, USA) was used to gently open the choroids. A 5 µL Hamilton syringe with a 34-gauge needle was inserted and positioned in the subretinal space where 1 µL of D-TAT or L-TAT (1 mM or 0.2 mM) was slowly injected. Intravitreal injections (1 µL of 1 mM) were performed with similar anaesthesia and doses, but the needle was positioned in the vitreous cavity, taking care not to perforate the lens. Animals were allowed to recuperate and were killed after 1 h or 1 week. All procedures were authorized by the Office of the Veterinary Service of the Etat de Vaud, Lausanne, Switzerland.
Tissue preparation and immunohistochemical analysis All eyes were obtained from mice postmortem. They were washed in PBS, fixed with 4% paraformaldehyde and sucrose
30% overnight. In total, 14 µm cryosections were obtained and embedded in optimal cutting temperature compound. Sections were blocked in PBS containing 10% normal goat serum and 0.3% Triton for 1 h at room temperature and incubated with primary antibodies overnight at room temperature. Primary antibodies used for immunohistochemical analysis included mouse monoclonal antineural nuclei for ganglion cells (NeuN, 1/100, Chemicon, Temecula, CA, USA); rabbit polyclonal antiglial fibrillary acidic protein for Müller cells (GFAP, 1/400, DAKO, Carpinteria, CA, USA); rabbit polyclonal anticalbindin for horizontal cells and specific amacrine and ganglion cells (1/1000, Chemicon, Temecula, CA, USA). After incubation with primary antibody, sections were washed in PBS and successively incubated with second antibody for 1 h at room temperature. Second antibodies comprised goat anti-mouse (1/100) and goat anti-rabbit (1/1000, Jackson ImmunoResearch, West Grove, PA, USA) conjugated to Cy3. After the reaction with
Figure 2. Cryosections of a mouse eye after intravitreal injection, incubation time: 1 h. (a) DAPI staining and (b) fluorescence of the retina. (c) DAPI staining and (d) fluorescence of the cornea. (a,b): Original magnification ×200; (c,d): original magnification ×100. E, endothelium; EPI, epithelium; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; S, stroma; P, pupilla; L, lens.
D-TAT peptide as a molecular transporter
secondary antibodies, the specimens were washed with PBS and cell nuclei were stained with DAPI (4′,6-diamidino-2phenylindole, dilactate, Molecular Probes, Eugene, OR, USA). Immunofluorescence was observed with an optical microscope (Olympus, BX60).
RESULTS Cells in culture 661W cells were efficiently transduced with D-TAT FITC and more than 95% of the cells were fluorescent after 60 min (Fig. 1).
In vivo experiments We injected 1 µL of 1 mM D-TAT FITC solution into the vitreous cavity of two 5-month-old NMRI anaesthetized
mice. Animals were euthanized after 1 h and the eyes were collected and processed for histology. Strong fluorescence was observed in nuclei from the ganglion cell layer (GCL) and the inner nuclear layer (INL). Weak fluorescence was also seen in fibres of the bipolar or amacrine cells, in few nuclei of the outer nuclear layer (ONL) and in the external processes of the photoreceptors (Fig 2a,b). The fluorescence showed a gradient of intensity, with higher expression in layers closed to the vitreous. Some fluorescence was also observed in the stroma and endothelium of the cornea and in the lens. However, this fluorescence did not concentrate in the nuclei and was not much higher than autofluorescence except for some nuclei in the lens capsule (Fig. 2c,d). In order to better identify some of the cells that were transduced with D-TAT FITC, colocalization experiments were performed with cell-specific antibodies. Several doublelabelled glial and Müller cells were identified with GFAP (Fig. 3a–c), horizontal, amacrine and ganglion cells with cal-
Figure 3. Cryosections of a mouse eye after intravitreal injection of 1 mM D-TAT FITC. (a–c) Same cryosection: the FITC (green) coupled to the D-TAT colocalized with the GFAP (red), a specific marker for glial and Müller cells. (a) GFAP; (b) computer superposition; (c) FITC fluorescence. Arrows point to a Müller cell. (d–f) Labelling with calbindin (red), a marker for horizontal, amacrine and ganglion cells. In d and e, colocalization occurred in the outer border of the inner nuclear layer where horizontal cell are located (arrows show an example of an horizontal or amacrine cell). In f, colocalization occurred in a subset of the ganglion cell layer (arrow points towards a positive cell for both calbindin and FITC). (g–i) In these cryosections, FITC colocalized with NeuN (red), a marker specific for ganglion cells. Arrows indicate positive cells. Original magnification ×200.
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c Figure 5. Cryosections of a mouse eye after subretinal injection of D-TAT FITC of (a) 1 mM, incubation time: 7 days and (b) 0.1 mM, incubation time: 7 days. Original magnification ×100.
Figure 4. Cryosections of a mouse eye after subretinal injection of D-TAT FITC. (a,c) 1 mM, (b) 0.1 mM. Incubation time: 1 day. (a): Original magnification ×100; (b,c): original magnification ×200.
bindin (Fig. 3d–f) and other ganglion cells with NeuN (Fig. 3g–i). This indicates that a great variety of retinal cells were transduced with D-TAT FITC. Many potential applications will deal with local eye delivery. Therefore, we wanted to establish whether a site-limited delivery was possible and subretinal injections were evaluated. One microlitre of 1 or 0.1 mM D-TAT FITC solution was injected in four anaesthetized 2-month-old VPP mice. The eyes were observed after 1 (two animals) or 7 days (two animals). Fluorescence was very strong in the ganglion cell layer and the nuclei of the ONL (Fig. 4a). Labelling was also observed in some nuclei of the INL (Fig. 4b) and, at higher magnification, in the processes of cells that project from the GCL to the photoreceptors, presumably Müller cells (Fig. 4c).
D-TAT peptide as a molecular transporter
Figure 6. Cryosections of a mouse eye after subretinal injection of 1 mM D-TAT FITC (a,b) or L-TAT FITC (c,d). Incubation time: 2 weeks. (a,c) DAPI staining, (b,d) FITC fluorescence. Original magnification: ×200.
In 2-month-old VPP mice evaluated 7 days post injection, the retina was completely reattached and fluorescence was still visible in the nuclei of the INL (Fig. 5a). Even when a lower concentration of D-TAT FITC was used (0.1 mM) several cells still showed fluorescence after 7 days (Fig. 5b). We also tested the lifespan of both L- and D-TAT forms when injected in the subretinal space. Fourteen days after injection, D-TAT FITC was still clearly visible in numerous cells (Fig. 6b) whereas L-TAT FITC was barely visible in some cells (Fig. 6d). At 21 days post injection, no FITCpositive cells were observed in the L-TAT-injected animals (data not shown).
DISCUSSION Although various peptide delivery systems have recently been discovered, the truncated version of TAT HIV-1 has become the leading tool in cellular transduction. When this peptide is fused to a protein, it allows the complex to cross
the membrane using the coated-pit system.25 This system is effective even for high-molecular-weight proteins. Fawell et al. described the uptake of a fused TAT-β-galactosidase protein after intravenous injection in mice. They observed galactose activity in liver, spleen, heart, skeletal muscles and lung.23 Schwarze et al. showed that L-TAT FITC injected intraperitoneally in C57Bl/6 mice could also transduce cells from the bone marrow, the spleen, skeletal muscle and brain in less than 20 min.22 L-TAT alone or fused to a protein can also transduce cultures of corneal epithelial or retinal cells.24 All these experiments clearly indicate that the TAT system is an effective transporter and, up to now, has not interfered with intracellular pathway or it has shown any sign of toxicity. However, one drawback of this peptide is its short duration of action and when long-lasting action is needed, the TAT vehicle may have some limitation. Proteins can have two chemical forms, an L- and a Dform. Only the L-enantiomeres occur in nature and proteins made of L-amino acids are readily degraded by many pro-
634 teases that have been selected by evolution. In contrast, Dproteins are poor substrate for these nucleases and, therefore, chemically synthesized D-proteins have a longer half-life compared with their L-counterpart. However, and because of the change in steric conformation, the biochemical activity of the D-protein is not always identical to its L-form and each compound has to be experimentally checked. Recently, we showed that the L-TAT delivery system was very effective in transducing cultured cells.26 Almost no L-TAT FITC was visible in a β-TC3 cell culture after 2 days. As expected, the use of a D-TAT protein was accompanied by an increase in its half-life and the peptide was still observed after 14 days in culture. At the same time, the transduction efficacy was completely retained. Peptides with long-lasting action could be very beneficial for the treatment of many diseases, in particular ophthalmic diseases. For example, age-related macular degeneration, Doyne’s honeycomb retinal dystrophy of malattia leventinese28 may need the local delivery of an active drug centred on the macula. We therefore evaluated whether the D-TAT peptide was as effective in transducing cells from various compartments of the eye as did L-TAT when applied locally.21 Our results clearly indicate that DTAT is as effective in transducing these compartments as LTAT and that its increased biodisponibility might be of great interest in delivering therapeutic molecules to the various compartments of the eye. In summary, our data showed that the D-TAT transporter is effective and stable in the eye and that it could represent an efficient transporter system for drug or protein delivery in ophthalmology. Various routes of application could be proposed depending on the cells affected by the disease and the duration of action of the peptide could be modulated by using various ratios of the L- or D-form of the transporter and effector peptide. Additional toxic studies will need to be performed before this all retro-inverso peptide could be used in clinics.
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10. 11. 12.
ACKNOWLEDGEMENTS We wish to thank Valérie Buchillier for technical help. This work was supported by a grant from Telethon Action Suisse.
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