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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 13, 2005 as doi:10.1096/fj.05-4737fje. |
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soluble receptor in uveitis
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* INSERM, U640, CNRS, UMR8151, René Descartes Paris 5 University, Faculté des Sciences Pharmaceutiques et Biologiques, Chemical and Genetic Pharmacology Laboratory, Ecole Nationale Supérieure de Chimie de Paris, Paris, France;
INSERM, U598, René Descartes Paris 5 University, Physiopathology of Ocular Diseases: Therapeutic Implications, Paris, France;
Hadassah Hebrew University Hospital, Jerusalem, Israel; and
Laboratoire d’Innovations Thérapeutiques, Rothschild Foundation, Paris, France
1 Correspondence: Chemical and Genetic Pharmacology Laboratory, INSERM U640, CNRS UMR8151, René Descartes University, 4 avenue de l’Observatoire, Paris 75270, France. E-mail: daniel.scherman{at}univ-paris5.fr
SPECIFIC AIMS
Development of specific ocular drug delivery systems is currently a major research avenue in ophthalmology. Due to the existing ocular barriers, systemic drug administration requires high drug concentrations, enhancing the potential for secondary complications. On the other hand, because of its relatively small size and its particular isolating characteristics, the eye is an ideal target for development of local delivery therapeutic means. In several chronic diseases, the constant delivery of a therapeutic protein is required for an extended period of time. In that context, the use of ocular gene transfer leading to local therapeutic protein production seems very appealing, since repeated invasive injections of recombinant protein could be avoided.
The ciliary muscle is composed of smooth muscle fibers oriented in two directions: transversal fibers are directed from the ciliary body to the scleral spur while circular fibers form a ring around the ciliary body. The ciliary muscle is located just below the sclera, 1 mm posterior to the limbus of the rat eye, and its prolongations reach the anterior retina. It is located at the crossroad between the anterior and posterior segment, allowing for possible protein expression in ocular media.
Our purpose was thus to develop a novel nondamaging, electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle for the local production of therapeutic proteins in the ocular medias. The therapeutic potential of this technology has been evaluated in rats with endotoxin-induced uveitis (EIU) by using a gene encoding for a chimeric variant of human TNF-
soluble receptor I (hTNFR-Is/mIgG1).
PRINCIPAL FINDINGS
To use the ciliary muscle for therapeutic electrotransfer strategies, we have designed novel electrodes and developed suitable intraciliary muscle injection techniques. To carry out controlled plasmid injection into rat eye ciliary muscle, a tunnel was created using a 30G needle. The tunnel path was initiated in the cornea extending toward the limbal area, and was further continued backward under the sclera to the ciliary muscle. The cathode needle electrode, covered with an insulating material for its entire length except for the part inserted into the muscle, was then introduced into the tunnel. An anodal annular electrode was placed on the eye surface. After DNA injection (in 10 µL of saline solution), electric pulses were delivered (8 pulses, 20 ms, 10 V, 5 Hz). The localization of these controlled electric fields was ideally positioned to minimize the risk of electrical burning at the level of the retina or the cornea.
1. Tolerance of ciliary muscle electrotransfer
Clinical examination of treated eyes at the slit lamp either immediately, or on days 1 and 8 after GFP plasmid or saline injection, followed by electric pulses delivery, showed the absence of clinical signs of intraocular inflammation or of gross structural damage.
After rat sacrifice, histology of the treated eyes on day 8 did not show any observable damages. Moreover, in an inflammatory model of endotoxin-induced uveitis (EIU), rat eyes undergoing electrotransfer pulses after injection of saline solution did not show any increased TNF- level in their aqueous humor, when compared with control EIU rats (P=0.10, n=12). Thus, electrotransfer per se does not enhance TNF- production in rats’ eyes with an inflammatory status, such as uveitis.
2. Restricted gene transfer to the ciliary muscle
Eight days after GFP-encoding plasmid electrotransfer (n=8), histological analysis of longitudinal sections demonstrated a specific fluorescent signal strictly localized in the ciliary muscle. Elongated fluorescent cells correspond to longitudinal myofibers of the ciliary muscle (Fig. 1
A, inset a, B) as demonstrated by of smooth muscle actin (-sm-1) colocalization (Fig. 1C)
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On anterior frontal sections, circumferential myofibers were identified surrounding the ciliary body just below the sclera (Fig. 1D
). The GFP expression was high on both anterior sections, in circumferential fibers (Fig. 1E
), and on more posterior sections, in round tubes corresponding to radial and longitudinal fibers (Fig. 1F
). Circumferential fibers of the ciliary muscle were well identified by -sm-1 immunostaining (Fig. 1G
) and colocalization with GFP confirmed that transgene expression was limited to ciliary smooth muscle fibers (Fig. 1H
).
3. Long lasting electrotransfered gene expression
Using a luciferase reporter gene, no significant activity was detected in the ciliary muscle of rats injected with 3 µg of luciferase encoding plasmid without electrotransfer. On the other hand, high and sustained luciferase activity was observed using electrotransfer. This luciferase expression lasted for at least 30 days.
4. Ciliary muscle secretion of TNF- chimeric receptor in the eye aqueous humor
We then studied the electrotransfer of the pVAX2 hTNFR-Is/mIgG1 plasmid encoding a chimeric protein of human TNF- soluble receptor type I linked to the Fc fragment of immunoglobulin G1 (hTNFR-Is/mIgG1).
We injected a large amount of plasmid to detect hTNFR-Is/mIgG1 produced in the eye. In the aqueous humor of normal rats, 6 days after ciliary muscle injection of 30 µg pVAX2 hTNFR-Is/mIgG1 plasmid in the absence of electrotransfer, the hTNFR-Is/mIgG1 soluble receptor was detected at a concentration of 274 ± 39 pg/mL (n=4). In electrotransfered eyes, hTNFR-Is/mIgG1 level was 691 ± 121 pg/mL (P<0.01, n=4). No hTNFR-Is/mIgG1 was detected in contralateral eyes of plasmid injected eyes, without or with electrotransfer. The hTNFR-Is/mIgG1 level was below detection in the serum of all groups of rats, demonstrating that systemic diffusion of intraocular hTNFR-Is/mIgG1 was negligible.
In uveitis EIU rat eyes receiving a 30 µg injection of pVAX2 hTNFR-Is/mIgG1, the mean hTNFR-Is/mIgG1 aqueous humor concentration was 181 ± 108 pg/mL (n=8), a value which was markedly increased by electrotransfer, up to 1070 ± 218 pg/ml (P<0.005, n=8). As in normal rats, no hTNFR-Is/mIgG1 was detected in the aqueous humor of saline-injected EIU rats, whether in the absence or presence of electric pulses delivery. No hTNFR-Is/mIgG1 was detected in the serum of EIU rats whose eyes had been injected with plasmid alone or followed by electrotransfer.
5. Effect of TNF- chimeric receptor secretion by ciliary muscle on endotoxin-induced uveitis
No TNF- was detected in the eyes of naive healthy rats. The mean level of TNF- in the aqueous humor of EIU rat eyes was 510 ± 44 pg/mL (n=8). This level was not significantly different in saline-injected rat eyes submitted to electric pulses delivery (374±65 pg/mL, P=0.10, n=8). Electrotransfer of an "empty" noncoding plasmid had no effect on TNF- level in the aqueous humor, when compared with the control saline treated group (478±33 pg/mL, P=0.14, n=8). Simple ciliary muscle injection of a low dose of 3 µg pVAX2 hTNFR-Is/mIgG1 led to no statistically significant TNF- concentration decrease (250±45 pg/mL; P=0.07, n=8). On the other hand, the TNF- aqueous humor concentration was significantly reduced in the group of rat eyes treated with a low 3 µg dose of pVAX2 hTNFR-Is/mIgG1, followed by electric pulses delivery (126±16 pg/mL, n=8) compared with that observed in rat eyes receiving electrotransfer of saline (P<0.002) or to EIU control rat eyes (P<0.0005).
Similarly, when simply injecting this low 3 µg hTNFR-Is/mIgG1 plasmid dose into the ciliary muscle, the mean clinical score of EIU rats was 3.7 ± 0.2 (n=12), which was statistically identical to the clinical score of untreated EIU rats (3.8±0.2; P=0.81, n=12), or of saline electrotransfered EIU rats (3.9±0.1; P=0.62, n=12), or of rats electrotransfered with an "empty" noncoding plasmid (3.8±0.2, n=8). On the other hand, the EIU score was significantly reduced in the group of rats who were treated with 3 µg of pVAX2 hTNFR-Is/mIgG1, followed by electrotransfer (1.2±0.2, P<0.0001, n=12). Thus, plasmid injection followed by electrotransfer significantly reduced clinical uveitis when compared with simple plasmid injection (P<0.0001, n=12) or to absence of treatment (P<0.0001).
These results are in agreement with the number of eye infiltrating cells. In EIU rats, values of infiltrating cells per section of 316 ± 14 and 272 ± 66 (n=4) were observed in the anterior segment and in the posterior segment, respectively. No significant differences were observed in the anterior (369±65, P=0.77) or in the posterior (261±32, P=0.99) eye segment of rats treated with simple injection of 3 µg of pVAX2 hTNFR-Is/mIgG1. Similarly, intraciliary injection of the empty plasmid combined with electrotransfer had no effect on inflammatory cell infiltration neither in anterior segment (322±26, P=0.99), nor in the posterior segment (255±13, P=0.98). However, in the rat eyes electrotransfered with 3 µg of pVAX2 hTNFR-Is/mIgG1, a drastic significant reduction in the number of infiltrating cells was observed both in the anterior segment (49±1, P<0.002) and in the posterior segment (88±3, P<0.05).
CONCLUSIONS AND SIGNIFICANCE
Proof of the concept of ocular gene therapy was previously obtained using viral vectors. However, viral-associated risks may limit their applications for eye diseases, which are non-life threatening. To circumvent the use of viral vectors, nonviral systems for the delivery of proteins, genes or cells have been developed, such as the transscleral delivery of proteins using mini pumps or implants, the subretinal implantation of genetically modified cells, or the intravitreous implantation of encapsulated genetically modified retinal pigment epithelial cells. Until now, electrotransfer has been described in retinal photoreceptor cells in neonatal rodents, in ganglion and RPE cells from adult rodents, and in rat corneal keratocytes and endothelial cells.
To our knowledge, our work is the first study to target the ciliary muscle, which, due to its location at the crossroad between the anterior and posterior segments, we think is an ideal candidate for the electrotransfer of genes encoding secreted therapeutic proteins to be available in aqueous humor and vitreous. The electrotransfer method developed was safe. Protein production was restricted to the ciliary muscle and lasted for at least a month after electrotransfer (Fig. 2
A).
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The TNF- soluble receptor experiments show ocular secretion of the plasmid encoded protein, with no detection in the serum and contralateral eye, along with drastic inhibition of clinical and histological inflammation scores in rats with EIU (Fig. 2B
).
These findings demonstrate that local production of proteins possessing potential therapeutic applications can be achieved and that the produced proteins remain confined within the treated eye.
Studies are under way to evaluate long-term electrotransfer effects and human applications. Our work points to plasmid delivery to ciliary muscle as an ideal platform for the treatment of various ocular diseases that could benefit from proteic therapy, such as inflammatory or degenerative pathologies.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4737fje;
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