Molecular Vision 2005; 11:184-191 <http://www.molvis.org/molvis/v11/a21/> Received 28 May 2004 | Accepted 28 February 2005 | Published 4 March 2005

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VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo

Nadia Normand,¹ Fatemeh Valamanesh,^1,2 Michèle Savoldelli,^1,3 Frédéric Mascarelli,¹ David BenEzra,⁴ Yves Courtois,¹ Francine Behar-Cohen^1,2
 
 

¹INSERM U598, Paris, France; ²Laboratoire d'Innovation Thérapeutique en Ophtalmologie, Fondation Ophtalmologique A. de Rothschild, Paris, France; ³Department of Ophthalmology, Hôtel-Dieu Hospital, Paris, France; ⁴Hadassah Hebrew University Hospital, Jerusalem, Israel

Correspondence to: Francine Behar-Cohen, INSERM U598, Institut Biomédical des Cordeliers, 75006 Paris; Phone: (331) 40 46 78 46; FAX: (331) 40 46 78 55; email: behar@idf.inserm.fr

Abstract

Purpose: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo.

Methods: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination.

Results: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells.

Conclusions: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Introduction

Antisense oligonucleotides (ODNs) displaying base sequence complementary to a specific mRNA are able to selectively modulate the expression of a given gene [1]. Based on these observations, treatment of viral diseases and cancers was attempted [2-6]. The eye is a small and closed organ with limited diffusion of locally applied drug into the circulation. Therefore, antisense strategy for localized gene therapy of ocular diseases might be considered. To prolong the very short half life of ODNs in biological fluids, phosphorothioate oligomers resistant to nucleases have been tried as potential therapeutic agents [7,8]. The intravitreal injection of a phosphorothioate ODN, targeted to cytomegalovirus (CMV) immediate early mRNA, for the treatment of CMV retinitis (Vitravene, Isis, Carlsbad, CA), has gained FDA approval [9]. Several techniques have been developed to enhance both the intracellular delivery of ODNs and their sustained release. Cationic lipids, polymers, peptides and macromolecular carriers improved the cellular uptake of nucleic acids with variable success in vivo [10]. Encapsulation in block copolymers [11], liposomes [12,13] or nanoparticles [14] have been investigated as additional sustained release delivery systems.

A novel system utilizing VP22, a structural protein of herpes simplex virus, was described [15]. The C-terminal amino acids 159-301 of the purified VP22 protein could bind ODNs leading to the formation of spherical particles of 0.3 to 1 μm diameter that could be internalized by cells. These particles remained stable within the cell cytoplasm for weeks and could be destabilized with the release of their bound ODNs after illumination [15,16]. The light controlled delivery of ODNs to the intraocular structures is of particular interest due to the wide potential for the use of lasers in ophthalmology.

Recently, it was reported that the proliferation of the choroidal melanoma cell line OCM-1 is controlled by Raf-1/MEK/ERK signaling pathway [17]. Furthermore, long term regulation of c-Myc and p27Kip1 through over activation of Raf-1 and MEK/ERK modulate the proliferation of human choroidal melanoma cells [17].

In the present study, we confirm the feasibility of VP22 light induced delivery of ODNs in vitro in melanoma cells of ocular origin (OCM-1) and in cells of retinal pigment epithelial origin (ARPE-19). Furthermore, new electron microscopy observations regarding the vectosomes cellular internalization process and their fate following illumination in vitro and in vivo are studied.

Methods

Materials

All culture reagents were obtained from Invitrogen (Cergy Pontoise, France). All other drugs and chemicals, unless stated otherwise, were obtained from Sigma (Saint-Quentin Fallavier, France). HPLC purified fluorescein and hexachlorofluorescein 5'-labeled phosphorothioate ODNs were purchased from Proligo (Paris, France). The sequences of the ODNs used in this study were: 5'-TCC CGC CTG TGA CAT GCA TT-3', complementary to the 3'-untranslated region of human c-raf kinase gene and 5'-TCC CGC gca ctt gAT GCA TT-3', a control ODN for the anti-c-raf ODN containing a 7-base mismatch (lowercase letters) [15]. Human OCM-1 (Ocular Choroidal Melanoma-1) cells were grown in RPMI 1640 medium with Glutamax supplemented with 5% fetal calf serum, 2.5 μg/ml amphotericin B, 100 units/ml penicillin, 100 μg/ml streptomycin. Human ARPE-19 cells (ATCC CRL-2302), a spontaneous immortalized human RPE cell line was cultured in a mixture 1:1 of Dulbecco's modified Eagle's medium (DMEM) and Nutrient Mixture F12, with Hepes buffer (Gibco, Grand Island, NY), and supplemented with 10% fetal bovine serum (FBS). Cells were grown at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air.

VP22 protein production

The plasmid pVP24 provided by Dr P. O'Hare (Marie Curie Research Institute, Oxted, UK) coding for the C-terminal half of the VP22 protein (amino acids 159-301) with a 6xHis tag at the C-terminus was expressed in BL21 CodonPlus (DE3) RP strain cells (Stratagene, Amsterdam, Netherlands) as previously described. Cultures were grown in L-broth containing 34 μg/ml chloramphenicol and 10 μg/ml kanamycin. Expression was induced for 4 h by the addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside. The protein purification was performed as previously described [15] except that the protein was bound to a Ni-NTA resin in batches (Qiagen, Courtaboeuf, France) and washed. The protein was eluted in batches in 500 mM imidazole pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol and stored at -80 °C after dialysis in PBS. Protein concentration was measured by the Bradford assay using bovine serum albumin as standard (Pierce Protein Plus Assay kit; Perbio Sciences, Brebiéres, France).

Assembly of VP22-ODN complexes and monitoring of cellular delivery in vitro

For in vitro experiments, fluorescent ODNs were complexed to VP22 by adding 10 μM ODNs to 20 μM VP22 in PBS. Following 10 min incubation at room temperature the solution was diluted 10 times in cell culture medium containing 5% fetal calf serum and added to cells seeded the day before at 10⁴ cells/ml in 4 well Nunc Lab-Tek coverglass incubation chambers (Fisher Scientific Labosi, Elancourt, France). The final concentrations were 1 μM VP22 protein and 0.5 μM ODN. Cultures incubated with the free ODN at a final concentration of 0.5 μM served as controls. OCM-1 and ARPE-19 cell cultures were incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. At 24 h, the medium was removed and the cells were washed with PBS. Fresh medium was added before illumination of the cultures.

The intracellular localization of the fluorescein labeled ODN was documented using an inverted confocal fluorescence microscope (Zeiss LSM 510, Jena, Germany) equipped with a 488 nm argon laser. Images were annotated and presented using Adobe Photoshop software. Movies showing the redistribution of cell fluorescence after illumination were recorded by time lapse microscopy.

The effect of cold halogen light on the release of ODNs from the VP22 complexes was assessed. Cultured OCM-1 and ARPE-19 cells were washed in PBS and illuminated for 3 min using a fiber optic cold lamp (Schott KL2500LCD, Nikon, Champigny sur Marne, France) set to an intensity of 170 mW/cm². The light was focused with lenses to allow for a precise matching of the beam diameter to the size of a well in the 4 well Nunc lab-Tek chamber. The cultured cells were then examined by epifluorescence. A 543 nm helium-neon laser was also used to illuminate vectosomes in the same cell lines. In this experiment, the vectosomes were formed from hexachlorofluorescein labeled ODNs.

Electron microscopy

OCM-1 cells in 4 well Nunc Lab-Tek coverglass incubation chambers were incubated overnight with VP22/ODN complexes at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were washed with PBS 24 h later, and illuminated as described above. After illumination, cells were fixed with 2% glutaraldehyde for 30 min at room temperature, washed in 0.1 M cacodylate buffer pH 7.4 and post-fixed with 1% osmium tetroxide for 15 min at room temperature. Cells were dehydrated in a graded series of ethanol (70% -100%) and flat embedded in Epon. Ultra-thin sections (70-80 nm) were obtained using an ultramicrotome (Reichert OM UZ, Vienna, Austria), counterstained with uranyl acetate and lead citrate and examined using an electron microscope (Philips CM10, Philips Electronic Instruments Co., Mahwah, NJ). In a preliminary experiment, vectosomes were evaporated on formvar and directly examined by TEM without any staining. Vectsosmes were identified as round dense particles.

Cell proliferation assays

The efficacy of VP22 mediated delivery of ODNs to OCM-1 cells was assessed using vectosomes formed by incubation with an antisense 20-mer ODN targeting the 3'-untranslated region of human c-raf kinase. An ODN with a 7 base mismatch served as control.

OCM-1 cells were seeded (1.5x10⁴ cells per ml) in 24 well plates. After 24 h, antisense ODN vectosomes and mismatched ODN vectosomes or the free corresponding ODNs were added and the cultures incubated for an additional period of 12 h. After this incubation, cells were washed in PBS, illuminated for 3 min using the fiber optic cold lamp and further incubated for six additional days. Then, the cell cultures were washed with PBS and stained with crystal violet (0.2% in 2% ethanol), washed in water, solubilized by addition of 0.3% SDS and transferred to 96 well plates. The cell culture absorption was read at 570 nm with a microplate reader (Bio-Rad Model 450, Marnes-la-Coquette, France) and the number of cells per well was calculated according to the crystal violet calibration curve established with OCM-1 cells. Experiments were run in quadruplicate. Untreated and non-illuminated cultures were used as a reference for normal OCM-1 cell proliferation.

Intravitreal injection of vectosomes in rat eyes and in vivo illumination

Albino male Lewis rats, 6-7 weeks old and weighing 150-200 g (IFFA CREDO, Lyon, France), were used, to reduce autofluorescence in RPE cells and choroid. Experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rats were anesthetized by intraperitoneal pentobarbital injection (40 mg/kg) and the right eyes dilated using tropicamide 0.5% eye drops.

The oligodeoxynucleotide (ODN) 5'-TCC CGC GCA CTT GAT GCA TT-3' (with a HEX 5' label; HEX-ODN) was used for the preparation of vectosomes and for the free ODN control.

The right eye of six rats (Group I, n=6) was injected intravitreally with 5 μl of a vectosome preparation containing 0.8 μg HEX-ODN (25 μM) and 4.5 μg VP22 (50 μM) in PBS. For controls, six eyes of six rats (Group II, n=6) received 5 μl of free HEX-ODN (0.8 μg) in PBS. Additionally four eyes received 5 μl of PBS (Group III, n=4). The intravitreal injections were performed using a 30-gauge needle inserted at 11 o'clock, 2 mm posterior to the limbus.

After the intravitreal injection, the eyes were examined by indirect ophthalmoscopy. If lens trauma or vitreous hemorrhage occurred, the eyes were excluded from the study and replaced. The rats were anesthetized 24 h after intravitreal injection, and the eyes illuminated for 3 min using a fiber optic cold lamp (Schott KL2500LCD, Nikon, Champigny sur Marne, France) set to an intensity of 170 mW/cm². The rat eyes remained opened during the illumination period but no mydriatics were used for pupil dilation. For 532 nm laser illumination, the laser was applied through the cornea using a coverslip and projected on the posterior pole of the retina (10 impacts, 50 μm, 10 to 15 J/cm²). The light source was placed laterally on the temporal side of the eye at a distance of 3 cm to limit the effect of heat on the eye surface. In each group, half of the rats received transscleral illumination and the other half were used as non-illuminated controls. The effect of laser illumination with a 532 nm argon laser set at 10 to 15 J/cm² (Viridis, Quantel Medical, France), instead of cold light, was also evaluated. One h after illumination, all rats were sacrificed. The treated right eyes (and the control left untreated eyes) were enucleated, snap frozen in OCT compound, cryosectioned, and processed for fluorescence microscopy. The sections were fixed in 4% paraformaldehyde and treated with 1/6000 diamino-2-phenylindol (DAPI) to visualize the cell nuclei.

Results

VP22 mediated delivery of ODNs

Following 24 h incubation of OCM-1 or ARPE-19 cells with vectosomes, no toxic effect or vacuolization of the cultured cells was observed (Figure 1A,D). After washing of the original incubation medium, confocal fluorescence microscopy set at a low laser power demonstrated a punctate fluorescence pattern. The fluorescent dots (representing the intracellular vectosomes) were confined to the cell cytoplasm (Figure 1B). After illumination, the fluorescence in the cytoplasm was transformed from punctate to diffuse with strong nuclear staining (Figure 1C, arrows). Cells incubated with free ODNs showed no fluorescence due to the minimal uptake of free ODN by the cells. The lack of fluorescence was not influenced by illumination (Figure 1F).

The light triggered redistribution of fluorescence is best illustrated by time lapse microscopy (Figure 2). The time lapse photography clearly demonstrates the quick release and redistribution of ODNs following iIllumination. Cold light or argon laser had the same effect on vectosome dissociation and release of F-ODN in both OCM-1 and ARPE-19 cells.

Intracellular localization of vectosomes

Transmission electron microscopy of grids covered with VP22/F-ODN complexes (vectosomes) showed that they were electron dense particles (Figure 3A). OCM-1 cells incubated overnight with vectosomes harbored electron dense particles within their cytoplasm (Figure 3B). In some of the cells, images suggesting an endocytotic process were observed (Figure 3C). On illumination of cells that had internalized the vectosomes, the well formed electron dense particles lost their membrane integrity (Figure 3D, arrows) and electron lucent spots within the vectosomes became visible. Following these structural changes, fragments of the electron dense particles were observed in cell nuclei (Figure 3E,F, arrows). These fragments appear to correspond to ODN aggregates released from vectosomes and now localized within the nucleus.

Potential of VP22 to deliver functional ODNs

Without illumination, no significant effect on the number of viable cells was observed when the OCM-1 cell cultures were incubated with either mismatched ODN alone (p=0.1508), antisense ODN alone (p=0.4206), vectosomes formed with mismatched ODN (p=0.4206) or vectosomes formed with antisense ODN (p=0.0952, Figure 4). Illumination had no effect on cell viability of the control cultures (p=0.6905), cultures incubated with either the mismatched or free antisense ODN (p=0.8413 and p=0.6905, respectively). Illumination of cultures incubated with vectosomes formed with the mismatched ODN induced no significant reduction in cell number when compared to illuminated control cells (p=0.1508). However, Illumination of cultures incubated with vectosomes formed with the antisense ODN induced a significant decrease of cell viability of up to 60% when compared to the cell viability in non illuminated or non treated cell cultures (p=0.0079).

In vivo light controlled delivery of ODN

Vectosomes were observed in all retinal layers 24 h after their intravitreal injection (Figure 5A). Vectosomes were particularly well delineated at the ONL (outer nuclear layer) forming "migration lines" between the photoreceptors, accumulating at the external limiting membrane and within the cytoplasm of retinal pigment epithelial (RPE) cells (Figure 5A). This pattern of vectosome localization within the retinal layers is consistent with a transretinal migration along the retinal Müller glial (RMG) cells.

In the non-illuminated eyes, the fluorescence within the RPE cells was confined to the cell cytoplasm while the nuclei were not fluorescent (Figure 5A and inset a). After illumination, fluorescent ODNs redistributed and were most intense in the nuclei of the ganglion cell layer, the inner nuclear layer and the nuclei of RPE cells (Figure 5B and inset b). A few fluorescent vectosomes could also be detected in the choroid. Before illumination, the fluorescence was associated with punctate dots in cell cytoplasm (Figure 6A). After illumination, the fluorescence was transferred localizing more intensively in cell nuclei (Figure 6B).

When free fluorescent ODNs were injected in the vitreous, a diffuse fluorescence at the level of the rod outer segments (ROS) and RPE cells was observed 24 h later. The fluorescent pattern in these eyes is, most probably, due to the fluorescent ODN. Noteworthy is the fact that the fluorescent pattern in these free ODN injected eyes is similar between the non-illuminated (Figure 5C) and illuminated (Figure 5D) eyes.

No detectable fluorescence was observed in the retina of eyes injected with PBS. Illumination had no detectable effect on these eyes (data not shown).

Discussion

It was previously shown that the C-terminal half of the purified VP22 can bind to fluorescein labeled ODN and form vectosomes [15]. The exact mechanism of light induced ODN release is poorly understood but it requires that fluorochrome to be covalently linked either to the protein or to the ODN [15]. It is assumed that thermal effects resulting from the absorbance of light by the fluorochrome may cause the vectosome disruption and the ODN release throughout the cell.

In the present study, we evaluated the potential of OCM-1 and ARPE-19 cells to internalize vectosomes and the ability to induce a controlled light release of ODNs from these designed vectosomes. The feasibility and biological efficacy of this approach was demonstrated in vitro on OCM-1 cells using vectosomes of VP22 complexed with a raf antisense ODN. The raf serine-threonine kinase is upregulated in many human tumors and plays a pivotal role in cell proliferation and resistance to apoptosis [18]. Abrogation of raf expression by specific antisense ODNs inhibits tumor cell growth [19]. More recently, it was demonstrated that light activated VP22 delivery of the c-raf1 antisense ODN decreased the growth potential of subcutaneous A549 tumors implanted in nude mice [16].

The mechanism of vectosome internalization into cells had not yet been explored thus far. We analyzed the fate of vectosomes incubated with OCM-1 cells in vitro by electron microscopy and observed that the intracellular process probably is carried out by endocytosis. Once within the cell cytoplasm, the vectosomes form well delineated endosome-like vesicles without any apparent effect on the cell structure or function. When illuminated, disruption of the vectosome membranes occurred and was accompanied by the release of small dense particles migrating from the cytoplasm to the cell nuclei. These observations along with the high transfection rate of VP22 indicates that the use of specifically designed vectosomes may be a safe alternative to the use of cationic lipids for transfection of genes. Cationic lipid use is limited by their potential toxicity and their reduced efficacy in the presence of serum [20]. Transfection with VP22, on the other hand, is not affected by serum and can be used in vivo. Our results showed that light dependent OCM-1 and ARPE-19 cell growth inhibition could be achieved using vectosomes formed with a c-raf antisense ODN. These observations are in line with earlier data obtained with non-ocular derived cells [15].

In vivo, vectosomes follow a transretinal migration and are rapidly internalized in RPE cells after intravitreal injection. This intraocular pattern of migration within ocular tissues is similar to that previously observed with the injection of small polymeric nanoparticles [14,21]. Unlike the polymeric nanoparticles however, the vectosome retinal course appears to be associated with Müller glial cell distribution. This difference in behavior of VP22 particles in vivo may also be associated with their specific properties regarding intercellular trafficking [22].

From the data obtained in the present study, it is evident that the light controlled release of the complexed ODN within the designed vectosomes can be carried out as efficiently in cultured cells in vitro and in ocular tissues in vivo. Within the eye, the injected vectosomes were rapidly internalized by various cell types. In all, moreover, the vectosomes remained strictly confined to the cell cytoplasm. On transscleral illumination of the treated eyes, disruption of the light sensitive vectosomes was followed by the release of the complexed ODNs, which rapidly migrated to the cell nucleus. This was clearly demonstrated by the concurrent strong fluorescence of nuclei in the ganglion cell layer, inner nuclear layer, and RPE cell layer immediately following the controlled delivery of the light stimulus.

Although somewhat surprising, the presence of a small number of vectosomes within the choroid was consistently observed in all treated eyes. Similar to the pattern observed in the retina, the vectosomes were initially localized in the cell cytoplasm with migration of the fluorescent ODNs to the cell nuclei only after illumination of the treated eyes.

The present study demonstrates the feasibility of a concept for a precise light controlled delivery of genetic materials within the intraocular tissues. As lasers of 488 nm, 532 nm, and 543 nm induce vectosome disruption as efficiently as white light, it might be possible to design ODNs coupled to different fluorophores and complexed with VP22 for the controlled targeted delivery of genetic materials. These specifically designed ODNs might be differentially and sequentially released according to the desired planned therapeutic aim.

Despite the excellent capability for precise local delivery and targeting of the lasers and other lights to eye tissues, potential harmful collateral and secondary retinal damage may occur. Therefore, the wavelengths and energies least harmful to the retina have to be carefully evaluated in vivo before clinical application of this methodology can be practically considered.

Since the eye is the organ of choice for light induced delivery of active molecules, VP22 vectosome technology may open new perspectives in controlled ocular ODN delivery. To ascertain these assumed potential benefits, additional extensive in vivo studies are needed. These are now being planned and pilot studies on along these avenues are conducted by our group.

Acknowledgements

We thank Christophe Klein for confocal microscopy services. This work was supported by INSERM, Conseil Régional d'Ile-de-France (Programme Avenir) and Fondation de l'Avenir. This work was funded in part by EviGenoRet, EU project 512036.

References

1. Helene C, Toulme JJ. Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta 1990; 1049:99-125.

2. Cohen JS. Antisense oligodeoxynucleotides as antiviral agents. Antiviral Res 1991; 16:121-33.

3. Crooke ST. Therapeutic applications of oligonucleotides. Annu Rev Pharmacol Toxicol 1992; 32:329-76.

4. Milligan JF, Matteucci MD, Martin JC. Current concepts in antisense drug design. J Med Chem 1993; 36:1923-37.

5. Stein CA, Cheng YC. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science 1993; 261:1004-12.

6. Kasid U, Dritschilo A. RAF antisense oligonucleotide as a tumor radiosensitizer. Oncogene 2003; 22:5876-84.

7. Agrawal S, Zhao Q. Antisense therapeutics. Curr Opin Chem Biol 1998; 2:519-28.

8. Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, Dorr A, Sikic BI. Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 1999; 5:3357-63.

9. Orr RM. Technology evaluation: fomivirsen, Isis Pharmaceuticals Inc/CIBA vision. Curr Opin Mol Ther 2001; 3:288-94.

10. Lebedeva I, Benimetskaya L, Stein CA, Vilenchik M. Cellular delivery of antisense oligonucleotides. Eur J Pharm Biopharm 2000; 50:101-19.

11. Roy S, Zhang K, Roth T, Vinogradov S, Kao RS, Kabanov A. Reduction of fibronectin expression by intravitreal administration of antisense oligonucleotides. Nat Biotechnol 1999; 17:476-9.

12. Hangai M, Tanihara H, Honda Y, Kaneda Y. Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes. Invest Ophthalmol Vis Sci 1998; 39:509-16.

13. Bochot A, Fattal E, Boutet V, Deverre JR, Jeanny JC, Chacun H, Couvreur P. Intravitreal delivery of oligonucleotides by sterically stabilized liposomes. Invest Ophthalmol Vis Sci 2002; 43:253-9.

14. Bourges JL, Gautier SE, Delie F, Bejjani RA, Jeanny JC, Gurny R, BenEzra D, Behar-Cohen FF. Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Invest Ophthalmol Vis Sci 2003; 44:3562-9.

15. Normand N, van Leeuwen H, O'Hare P. Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery. J Biol Chem 2001; 276:15042-50.

16. Zavaglia D, Normand N, Brewis N, O'Hare P, Favrot MC, Coll JL. VP22-mediated and light-activated delivery of an anti-c-raf1 antisense oligonucleotide improves its activity after intratumoral injection in nude mice. Mol Ther 2003; 8:840-5.

17. Lefevre G, Calipel A, Mouriaux F, Hecquet C, Malecaze F, Mascarelli F. Opposite long-term regulation of c-Myc and p27Kip1 through overactivation of Raf-1 and the MEK/ERK module in proliferating human choroidal melanoma cells. Oncogene 2003; 22:8813-22.

18. Slater EP, Stubig T, Lau QC, Achenbach TV, Rapp UR, Muller R. C-Raf controlled pathways in the protection of tumor cells from apoptosis. Int J Cancer 2003; 104:425-32.

19. Dancey JE. Agents targeting ras signaling pathway. Curr Pharm Des 2002; 8:2259-67.

20. Dass CR. Cytotoxicity issues pertinent to lipoplex-mediated gene therapy in-vivo. J Pharm Pharmacol 2002; 54:593-601.

21. Sakurai E, Ozeki H, Kunou N, Ogura Y. Effect of particle size of polymeric nanospheres on intravitreal kinetics. Ophthalmic Res 2001; 33:31-6.

22. Cashman SM, Sadowski SL, Morris DJ, Frederick J, Kumar-Singh R. Intercellular trafficking of adenovirus-delivered HSV VP22 from the retinal pigment epithelium to the photoreceptors--implications for gene therapy. Mol Ther 2002; 6:813-23.