EP2099497A2 - Administration rétinienne d'un acide nucléique améliorée par ionophorèse - Google Patents

Administration rétinienne d'un acide nucléique améliorée par ionophorèse

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Publication number
EP2099497A2
EP2099497A2 EP07873352A EP07873352A EP2099497A2 EP 2099497 A2 EP2099497 A2 EP 2099497A2 EP 07873352 A EP07873352 A EP 07873352A EP 07873352 A EP07873352 A EP 07873352A EP 2099497 A2 EP2099497 A2 EP 2099497A2
Authority
EP
European Patent Office
Prior art keywords
iontophoresis
nucleic acid
rdl
injection
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07873352A
Other languages
German (de)
English (en)
Inventor
Therese De Bizemont
Florian Sennlaub
Francine Behar-Cohen
Yves Courtois
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EyeGate Pharma SAS
Original Assignee
EyeGate Pharma SAS
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Filing date
Publication date
Application filed by EyeGate Pharma SAS filed Critical EyeGate Pharma SAS
Publication of EP2099497A2 publication Critical patent/EP2099497A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • This invention relates to devices and methods for the enhanced delivery of a nucleic acid to tissues of the eye. More specifically, the methods of the present invention utilize iontophoresis to evoke a transient elongation of the Muller Cells of a mammalian eye to enhanced retinal delivery of a nucleic acid therapeutic or diagnostic agent.
  • Methods are known for introducing drug, such as nucleic acid, into target cells or tissues such as by topically applying to or injection into tissue, the use of techniques such as electroporation, iontophoresis, the encapsulation of nucleic acid in colloidal systems, such as liposomes or polymeric spheres or other chemical carriers or the use of a viral or non-viral vector.
  • nucleic acid While the use of conventional delivery systems has been widely investigated, there still exist many problems that are often associated with the in vivo introduction of nucleic acid into eukaryotic cells. Typically only a small percentage of cells targeted for transfection with a heterologous nucleic acid actually express the gene of interest at satisfying levels, notably the protein of interest. In addition, some therapeutic compositions, such as those that include synthetic oligonucleotides, are very expensive, toxic and degradable, consequently, requiring localized application and efficient internalization into the target cells.
  • Electroporation is a means of increasing the permeability of a cell membrane and/or at a portion of cells of a targeted tissue, to a chemical agent such as nucleic acids, wherein the increased permeability is caused by application of high pulse voltage across the cell or at least a portion of the tissue.
  • the increased permeability allows transport, or migration, of chemical agents through the tissue or across cell membranes into cells if the tissue or the cells are in the presence of a'-'sui table chemical agent.
  • Electroporation is typically carried out by applying high voltage pulses between a pair of electrodes that are applied to the tissue surface.
  • the voltage must be applied in proportional to the distance between the electrodes. When the space between the electrodes is too great, the generated electric field penetrates deep into the tissue where it causes unpleasant nerve and muscle reaction.
  • Iontophoresis is a technique that was proposed in 1747 by Verrati and consists in the administration, in particular of medicaments, into the body through the tissues using an electric field involving a small voltage.
  • An electrode is arranged at the site to be treated while a second electrode, intended to close the electric circuit, is placed at another site on the body.
  • the electric field facilitates the migration of the active products, and/or increases cellular permeability to the products that are preferably ionized.
  • This is commonly used transdermal technique for treating skin or rheumatologic diseases. It has also been shown that gene induction can be achieved through the ex vivo iontophoretic delivery of oligonucleotides in an ocular rabbit model.
  • the methods of the present invention utilize iontophoresis to evoke a transient elongation of the Muller Cells of a mammalian eye to enhanced retinal delivery of a nucleic acid therapeutic or diagnostic agent.
  • the method comprises transiently elongating Muller cells of a mammal retina by a step of iontophoresis; and administering a composition comprising the nucleic acid to the mammalian eye, wherein the step of iontophoresis transiently elongates the Muller cells of the mammal retina and enhances the in vivo delivery of the nucleic acid into the retinal cells of the mammalian eye.
  • the step of iontophoresis can be carried out prior to, during, or after the step of administering the nucleic acid composition.
  • the nucleic acid can be either a therapeutic agent or a diagnostic agent.
  • the nucleic acid can be a deoxyribonucleic acid ("DNA”), a ribonucleic acid (“RNA”), and a chimeric nucleic acid comprising both DNA and RNA bases, including but not limited to, an oligonucleotide DNA, an anti-sense DNA, a plasmid DNA, a component of a plasmid DNA, a vector, an expression cassette, a chimeric DNA sequence, a chromosomal DNA, a stabilized DNA, an aptamer, a stabilized aptamer, an oligonucleotide RNA, a transfer RNA (tRNA), a short interfering RNA (siRNA), a small nuclear RNA (snRNA), a ribosomal RNA (rRNA), an mRNA (messenger RNA), a micro RNA (miRNA), a short hair-pin RNA (shRNA
  • the nucleic acid is an oligonucleotide DNA or an oligonucleotide RNA, optionally with phosphorothioates linkages.
  • the nucleic acid is a single-stranded nucleic acid, a double-stranded nucleic acid, a triple-stranded nucleic acid, or a quadruple-stranded nucleic acid.
  • the nucleic acid is in a linear or circular form.
  • the nucleic acid is a single stranded oligonucleotide DNA (ssODN) or a single stranded oligonucleotide RNA (ssORN).
  • the nucleic acid composition can be administered by a topical instillation on the eye, by a topical instillation on the eyelid, or by an injection into the mammalian eye (should we include by direct ocular iontophoresis?).
  • the topical instillation can be administered in the form of a liquid solution, a paste, of a hydrogel.
  • the topical instillation can be embedded in a foam matrix or supported in a reservoir.
  • the injection into the mammalian eye can be an intracameral injection, an intracorneal injection, a subconjonctival injection, a subtenon injection, a subretinal injection, an intravitreal injection, and an injection into the anterior chamber.
  • the step of iontophoresis can be an ocular or a transpalpebral iontophoresis.
  • the transpalpebral iontophoresis is an anionic or a cationic iontophoresis performed with a current of about 1 - 5 mA for about 1 - 7 minutes.
  • the transpalpebral iontophoresis is an anionic or a cationic iontophoresis performed with a current of about 1 - 3 mA for about 3 - 6 minutes.
  • the transpalpebral iontophoresis is a cationic iontophoresis performed with a current of about 2 mA for up to 5 minutes.
  • FIGS. 1 A-C provide photomicrographs of histological sections of rat retina stained with hemalun.
  • FIG. IA Control retina.
  • FIG. IB Retina after injection into the vitreous of the biotynilated chimeroplast. No staining is observed in the retina or in the RPE showing that no chimeroplast has penetrated into the retina.
  • FIG. 1C Retina after injection into the vitreous of the chimeroplast, followed by the iontophoresis of saline. There is a clear brown DAB staining in the retinal layers, in the RPE and in the choroid, showing that the penetration of the chimeroplast has been enhanced by the application of the current.
  • FIG. 2 provides photomicrographs of a restriction fragment length analysis of ⁇ -cGMP phosphodiesterase cDNA.
  • RT-PCR were performed with rd ⁇ -PDE mRNA specific primers on extracted retinae at postnatal day 27 (except for lanes 4-7 analyzed at postnatal day 10).
  • the rd nonsense point mutation in codon 347 creates a Ddel restriction site and removes a BsaAI site from the wild-type sequence.
  • Digesting the 359 bp ⁇ -PDE cDNA with BsaAI or Ddel yields two diagnostic fragments of 120 bp and 239 bp.
  • the gel in FIG. 2 represents the restriction fragment length analysis by electrophoresis separation: lanes 1-3: for the wild-type cCDA sequence (+/+) without treatment; and lanes 4-18: for the mutated sequence (rd/rd) without treatment (lanes 4-6), with water injection treatment (lanes 7-9), with chimeroplast injection without iontophoresis transfer (lanes 10-12), with chimeroplast injection with iontophoresis transfer (lanes 13-15), with control chimeroplast injection with iontophoresis transfer (lanes 16-18).
  • FIGS. 3A and 3B provide photomicrographs illustrating rod survival by immunostaining.
  • FIG. 3 A The amount of rod-photoreceptors was counted on flat-mounted retina of chimeraplast treated animals ("active chimera”) and control ("scrambled chimera") at postnatal day 27 (P27). Results are expressed as mean ⁇ standard error of the mean (SEM).
  • FIG. 3B Opsin-immunohistochemistry has been performed on whole-mounted retina. Scanned photograph by fluorescence microscopy of flat-mounted retina of chimeraplast treated animals ("active chimera", right picture) and control ("scrambled chimera", left picture).
  • FIG 4 provides photomicrographs illustrating penetration of ODNs to retinal cells at one hour after injection of labeled ODNs with or without prior saline iontophoresis.
  • PN7 rdl/rdl eye section after intravitreal injection of labeled ODNs without prior saline iontophoresis (Panel A).
  • PN7 rdl/rdl eye section after intravitreal injection of Hex without prior saline iontophoresis (Panel B).
  • Iontophoresis device is composed by an eye-glass-shaped electrode made with aluminum foil and single-use disposable medical grade hydrophilic polyurethane sponge and a return electrode connected to the neck of the mouse (arrow) (Panel C).
  • PN7 rdl/rdl eye section after intravitreal injection of labeled ODNs with prior cathodal saline iontophoresis (Panel D).
  • PN7 rdl/rdl eye section after intravitreal injection of Hex with prior cathodal saline iontophoresis (Panel E).
  • PN7 rdl/rdl eye section after intravitreal injection of PBS with prior cathodal saline iontophoresis (Panel F).
  • Higher magnifications showing retina structures from eyes (A), (D) and (F) are shown in (a), (d) and (f), respectively. Insets show high magnification of the correspondent picture.
  • ONL outer nuclear layer
  • INL inner nuclear layer
  • GCL ganglion cell layer.
  • Scale bars A, D, and F ( ⁇ 2.5), 1 mm; a, B, d, E and f (> ⁇ 25), 100 ⁇ m; insets, 10 ⁇ m.
  • FIG. 5 depicts the analysis of varied iontophoresis conditions on the ODNs delivery to the ONL.
  • Relative fluorescent intensities in ONL were represented by histograms expressed as means ⁇ SD (vertical bars). Fluorescence in the ONL showed a significant increase of intensities when using any current application condition as compared to injection without iontophoresis (P ⁇ 0.05) or no treatment (PO.05) (*, **). ONL fluorescence showed a significant increase of intensities when using cathodal saline iontophoresis as compared to anodal saline iontophoresis (PO.05) (**).
  • FIG. 6 depicts the analysis of the iontophoresis intensity on the penetration of ODNs in the ONL. Relative fluorescent intensities in ONL were represented by histograms expressed as means ⁇ SD (vertical bars). Fluorescence in the ONL shows a significant increase of intensities when using cathodal saline iontophoresis at 1.5 mA as compared to cathodal saline iontophoresis at 0.5 mA (PO.05) (*). [0020] FIG.
  • FIG. 8 depicts the analysis of the penetration of ODNs to retinal cells at different time point after injection of labeled ODNs with prior saline iontophoresis.
  • DAPI staining in blue of top panels is shown in corresponding middle panel (F-J).
  • Double staining with DAPI in blue and Hex-labeled ODNs is shown in corresponding lower panel (K-O).
  • FIG. 9 provides photomicrographs of eye sections from PN7 rdl/rdl mice at different time point after saline iontophoresis.
  • FIG. 10 provides photomicrographs of eye semi-thin eye sections at different time points after injection of labeled ODNs with or without prior saline iontophoresis.
  • Semi-thin section of control untreated PN7 rdl/rdl eye section (Panel A).
  • Higher magnification showing retina structure from eyes (B) and (C) are shown in (b) and (c) respectively.
  • PN7 rdl/rdl eye section at 24 hours after intravitreous injection of labeled ODNs without prior saline iontophoresis (D) or with prior cathodal saline iontophoresis (E). Arrow represents vacuoles.
  • RPE retinal pigment epithelium cells
  • ONL outer nuclear layer
  • INL inner nuclear layer.
  • Scale bars A, B, C, D, and E (x25), 50 ⁇ m; b, and c, 25 ⁇ m.
  • FIG. 11 provides photomicrographs of ultra-thin eye sections observed by TEM at different time point after injection of labeled ODNs with or without prior saline iontophoresis.
  • Ultra-thin section of control untreated PN7 rdl/rdl eye section (Panel A).
  • PN7 rdl/rdl eye section at 24 hours after intravitreous injection of labeled ODNs without prior saline iontophoresis (Panel D) or with prior cathodal saline iontophoresis (Panel E). Arrow represents vacuoles.
  • RPE retinal pigment epithelium cells
  • IS inner segments
  • ONL outer nuclear layer.
  • Scale bars A, B, C, and D (> ⁇ 25), 5 ⁇ m.
  • FIG. 12 depicts the iontophoresis device and eye sections from PN7 rdl/rdl mice one hour after treatment.
  • Iontophoresis device (Panel A) an eye-glass-shaped electrode made with aluminum foil and single-use disposable medical grade hydrophilic polyurethane sponge, (Panel B) iontophoresis generator and the return electrode. Eye section one hour after transpalpebral iontophoresis: (Panel C) hematoxylin and eosin stained eye section showing integrity of the eye structures after iontophoresis (inset: high magnification).
  • FIG. 13 provides photomicrographs of eye sections of treated and control PN28 rdl/rdl mice and ONL cell counting. Hematoxylin-eosin stained sections of rdl/rdl eyes showing an increased number of nuclei rows in the ONL of ODN-treated eye (arrows): (Panel A) untreated mouse, (Panel B) PBS-treated mouse, (Panel C) WTAS ODN-treated mouse, (Panel D) WTS ODN-treated mouse.
  • FIG. 14 depicts rhodopsin irnmunohistochemistry on wild-type eye section and rdl/rdl whole flat-mount retinas, reflecting the time course of the retinal degeneration and the treatment efficacy.
  • Panel A Wild-type eye section from a mouse at PN28.
  • FIG. 15 depicts the responsiveness of rhodopsin immunoreactivity to the number of ODN treatment.
  • PBS Three treatments with PBS
  • Panel B One treatment with ODN at PN 4.
  • Panel C Two treatments with ODN (PN 4 and 6).
  • Panel D Three treatments with ODN (PN 4, 6, and 8).
  • Scale bars A, B, C and D, 1 mm.
  • FIG. 16 depicts rhodopsin irnmunohistochemistry on eye sections from PBS- or ODN- treated rdl/rdl mice at PN28.
  • Panel A DAPI staining in blue and rho-4D2 immunostaining in green (arrows) on section from PN28 PBS-treated rdl/rdl retina.
  • Panel B DAPI staining in blue and rho-4D2 immunostaining in green (arrows) on section from PN28 ODN-treated rdl/rdl retina.
  • Scale bars A and B, 150 ⁇ m.
  • FIG. 17 depicts ⁇ -PDE immunohistochemistry and western blot.
  • ONL Outer Nuclear Layer
  • INL Inner Nuclear Layer
  • lane 1 is the molecular weight marker (sizes given on left)
  • lane 2 is the polypeptide antigen against which the antibody was grown
  • lane 3 is protein from a C3H (rdl/rdl) mouse retina
  • lane 4 is protein from a rdl/rdl mouse retina
  • lane 5 is protein from an FVB (rdl/rdl) mouse retina
  • lane 6 is protein from a CCRC (wild-type +/+) mouse retina
  • lane 7 is protein from a Baln/C (wild-type +/+) mouse retina.
  • Scale bars A, B, C and D, 100 ⁇ m. [0031)
  • FIG. 19 depicts representative plots of allele-specific real time PCR.
  • the graph shows the real-time detection of fluorescence resulting from intercalation of SybrGreen fluorescent dye into double-stranded PCR products.
  • Template DNA was isolated from BALB/c mouse (WT), retinas of rd 1 /rd 1 mice treated with WTS ODN (ODN-treated), or retinas of rd 1 /rd 1 mice treated with PBS (PBS-treated). Primers were specific for wild-type allele. Each experimental sample was assayed in 5-10 replicates.
  • FIG. 20 depicts photoreceptors targeting was significantly increased when the saline transpalpebral iontophoresis is applied before ODN injection as compared with its application after ODN injection.
  • FIG. 21 shows the results of iontophoresis in target retinal cells
  • FIG. 22 shows specific coding phosphorothioate oligonucleotide showing a dose- dependant rescue of photoreceptors.
  • FIG. 23 eye sections from mice after treatment (shows / ⁇ -phosphodiesterase protein was detected in the eye section).
  • FIG. 24 is a qualitative evaluation of iontophoresis of labeled oligonucleotide on rat.
  • FIG. 25 shows reactions with wild-type /3-PDE DNA reached threshold in many fewer cycles than those with untreated rd template.
  • a SRT-PCR analysis shows statistically significant leftward shift in the reaction profiles of ODN-treated samples, indicating that treatment induces repair of genomic DNA.
  • the present invention provides devices and methods for the enhanced delivery of a nucleic acid to tissues of the eye.
  • the methods of the present invention can be used to deliver various different types of nucleic acids to various tissues of the eye. These nucleic acids can be used as diagnostics or therapeutics.
  • nucleic acid is a term of art that refers to a polymer containing at least two nucleotides.
  • Nucleotides contain a sugar deoxyribose (in DNA) or ribose (in RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and synthetic derivatives of purines and pyrimidines, or natural analogs.
  • nucleic acid also encompasses nucleic acids containing known nucleotide analogs, modified nucleotide (or modified nucleoside or modified base) or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid.
  • nucleic acid is also understood to mean an isolated natural, or a synthetic, a DNA and/or RNA fragment comprising natural and/or non- natural nucleotides, designating a precise succession of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 nucleotides, optionally modified.
  • modified nucleotide or “modified nucleoside” or “modified base” refer to variations of the standard bases, sugars and/or phosphate backbone chemical structures occurring in ribonucleic (i.e., A, C, G and U) and deoxyribonucleic (i.e., A, C, G and T) acids.
  • Gm represents 2'-methoxyguanylic acid
  • Am represents 2'-methoxyadenylic acid
  • Cf represents 2'-fluorocytidylic acid
  • Uf represents 2'-fluorouridylic acid
  • Ar represents riboadenylic acid.
  • the oligonucleotide can include cytosine or any cytosine-related base including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, 5-hydroxymethyl cytosine, 2-thiocytosine, 5-halocytosine (e.g., 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, and 5-iodocytosine), 5-propynyl cytosine, 6-azocytosine, 5-trifluoromethylcytosine, N4, N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine, carbazole cytidine or pyridoindole cytidine.
  • cytosine or any cytosine-related base including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, 5-hydroxymethyl cytosine, 2-thiocytosine, 5-halocytosine (e
  • Modifications can further include guanine or any guanine-related base including 6-methylguanine, 1 -methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 2-propylguanine, 6-propylguanine, 8-haloguanine (e.g., 8-fluoroguanine,
  • the oligonucleotide can further include adenine or any adenine-related base including 6-methyladenine, N6-isopentenyladenine, N6-methyladenine, 1 -methyladenine, 2-methyladenine, 2-methylthio-N6-isopentenyladenine, 8-haloadenine (e.g., 8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and 8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine, 8-thioalkyladenine, 8-hydroxyladenine, 7-methyladenine, 2-haloadenine (e.g., 2-fluoroadenine, 2-bromoadenine, 2-chloroadenine, and 2-iodoadenine), 2-aminoadenine, 8-azaadenine, 7-deazaadenine or 3-deazaadenine.
  • uracil or any uracil-related base including 5-halouracil (e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil), 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, 1 -methylpseudouracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methylamin
  • 6-azouracil or 4-thiouracil.
  • modified base variants known in the art include, without limitation, those listed at 37 C.F.R. ⁇ 1.822(p)(l), e.g., 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2'-methoxycytidine, 5-carboxymethylaminomethyl- 2-thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, ⁇ -D-galactosylqueosine, inosine, N6-isopentenyladenosine, 1 -methyladenosine,
  • Nucleotides also include any of the modified nucleobases described in U.S. Patent Nos. 3,687,808, 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711 , 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096 and 5,681,941.
  • modified nucleoside and nucleotide sugar backbone variants include, without limitation, those having, e.g., 2' ribosyl substituents such as F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, ONO2, NO2, N3, NH2, OCH2CH2OCH3, O(CH2)2ON(CH3)2, OCH2OCH2N(CH3)2, 0(Cl -IO alkyl), O(C2-10 alkenyl), O(C2-10 alkynyl), S(Cl-IO alkyl), S(C2-10 alkenyl), S(C2-10 alkynyl), NH(Cl-IO alkyl), NH(C2-10 alkenyl), NH(C2-10 alkynyl), and O-alkyl-O-alkyl.
  • Desirable 2' ribosyl substituents include 2'-methoxy
  • the 2'-substituent may be in the arabino (up) position or ribo (down) position.
  • nucleic acid also encompasses nucleic acids containing 5 '-5' and 3 '-3' inverted nucleotide caps.
  • nucleic acid cap means a first nucleotide covalently linked to the 5' end of an oligonucleotide via a phosphodiester linkage between the 5' position of the first nucleotide and the 5' terminus of the oligonucleotide as shown below.
  • 3 '-3' inverted nucleotide cap is used herein to mean a last nucleotide covalently linked to the 3' end of an oligonucleotide via a phosphodiester linkage between the 3' position of the last nucleotide and the 3' terminus of the oligonucleotide as shown below.
  • Further modifications include conjugation at either the 5' or 3' end with; polyethylene glycol, polyesters, polyanyhdrides, polycarbonates, polyurethanes, methacrylates, lipids, biopolymers (hyaluronic acid, cellulose derivatives, chitosan, alginate, etc.), thermoreponsive polymers (pluronix), dendrimers, poly-amines, proteins, poly-peptides, antibodies, metals (gold, silver, etc.), chelating agents, molecules to aid in detection (fluorophores and chromophores).
  • polyethylene glycol polyesters, polyanyhdrides, polycarbonates, polyurethanes, methacrylates, lipids, biopolymers (hyaluronic acid, cellulose derivatives, chitosan, alginate, etc.), thermoreponsive polymers (pluronix), dendrimers, poly-amines, proteins, poly-peptides, antibodies, metals (gold, silver, etc.), chelating
  • nucleic acid is also understood to include nucleic acid molecules that modulate the expression or function of one or more target genes including, but not limited to, antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, allozymes, aptamers, decoys and siRNA (RNAi).
  • antisense and enzymatic nucleic acid molecules such as hammerhead ribozymes, DNAzymes, allozymes, aptamers, decoys and siRNA (RNAi).
  • Oligonucleotide agents have been shown to have functional activity in vitro and thus the promise of therapeutic potential. High sensitivity to nuclease digestion, however, makes oligonucleotide agents unstable and thus impracticable for in vivo administration.
  • methods for stabilizing nucleic acid or oligonucleotides have been developed in the art that can be used to produce high binding, nuclease-resistant oligonucleotide that retain their specificity.
  • US Patent No. 6,423,493, issued July 23, 2002 discloses a random combinatorial selection method is disclosed for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides.
  • the modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides.
  • mRNAs are stabilized by modifying the sequence and optimizing for translation. See US Patent Application Nos. 2005/0032730 Al and 2005/0250723 Al .
  • the term "nucleic acid” is also understood to include stabilized nucleic acid molecules.
  • the oligonucleotide(s) of the present invention can be incorporated into pharmaceutical compositions suitable for administration.
  • the pharmaceutical compositions generally comprise at least one oligonucleotide and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject.
  • Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the oligonucleotide compositions (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th ed., 1990).
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • pharmaceutically-acceptable excipient means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the oligonucleotide are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine and the like.
  • Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid).
  • Pharmaceutically-acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the oligonucleotide, e.g., C 1-6 alkyl esters.
  • a pharmaceutically-acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salif ⁇ ed or esterif ⁇ ed.
  • the oligonucleotide of the invention can be present in unsalified or unesterified form, or in salified and/or esterif ⁇ ed form, and the naming of such oligonucleotide is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically-acceptable salts and esters.
  • Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the oligonucleotide, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the oligonucleotide of the invention are administered as a sustained release composition or device, such as a MedipadTM device.
  • the oligonucleotide of the invention can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various oligonucleotide-related diseases.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0055] In all cases, the composition must be sterile. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.
  • Sterile solutions can be prepared by incorporating the oligonucleotide in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze- drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • the oligonucleotide is formulated into ointments, salves, gels, or creams as generally known in the art.
  • the oligonucleotide is prepared with carriers that will protect the oligonucleotide against rapid elimination, such as a controlled-release formulation, including implants and microencapsulated delivery systems.
  • a controlled-release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, e.g., intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (Chen et al., Proc. Natl. Acad. Sci. USA, 91 :3054-3057, 1994).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • "Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • a liquid aqueous medium or other material is ophthalmically acceptable when it is compatible with ocular tissue, that is, it does not cause significant or undue detrimental effects when brought into contact with ocular tissue.
  • An ophthalmic composition or pharmaceutical composition of the present invention is a composition that is compatible with ocular tissue, e.g., a composition that is suitable for administration to the eye.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • a “therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
  • the terms “subject” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the term “subject” or “patient” as used herein means any mammalian patient or subject to which the compositions of the invention can be administered. In some embodiments of the present invention, the patient will be suffering from a condition that causes lowered resistance to disease, e.g., HFV.
  • HFV a condition that causes lowered resistance to disease
  • accepted screening methods are employed to determine the status of an existing disease or condition in a subject or risk factors associated with a targeted or suspected disease or condition. These screening methods include, for example, ocular examinations to determine whether a subject is suffering from an ocular disease. These and other routine methods allow the clinician to select subjects in need of therapy.
  • ophthalmic compositions for storing, cleaning, re-wetting and/or disinfecting a contact lens, as well as artificial tear compositions and/or contact lenses will contain one or more collectins and/or surfactant proteins thereby inhibiting the development of ocular disease in contact-lens wearers.
  • each linkage possesses a negative charge.
  • the average molecular weight of a nucleotide is 335 (330 for RNA and 340 for DNA), each oligonucleotide possesses 29 or more negative charges. Any modifications to the sugar moiety (2' modifications) does not diminish the charge density and any increase in lipophilicity brought about by the modification is more than compensated for by this inherent negative charge.
  • the sclera has been shown to passively allow transscleral delivery of proteins and polysaccharides with molecular weights up to 150 kD. Also, Asahara demonstrated the transcorneal delivery of a 4.7 kB plasmid (-174 kD). The eye has clearly been demonstrated to be exceedingly more permeable to therapeutics than the skin. Any observed retardation of flux derived from sequence specific secondary structures within an oligonucleotide series in the skin would not be a factor in ocular tissue. One could reasonably expect that the main factor governing iontophoretic mobility would be charge density, regardless of the specific sequence.
  • Example 1 The enhanced retinal delivery of oligonucleotides is demonstrated in Example 1 utilizing a chimeric oligonucleotide.
  • a chimeric oligonucleotide being composed of both RNA and DNA nucleotides, represents an all encompassing example of the types of oligonucleotides one would deliver.
  • the successful delivery of the chimeric oligonucleotide would lead one skilled in the art to predict the successful delivery of a wide range of oligonucleotides belonging to the subcategories of: aptamers, antisense, siRNA, etc. (Jayasena, S., Clinical Chemistry, 45: 1628-1650, 1999; Henry, S. et al., Exp. Opin. Pharmacother., 2: 1-15, 2001 ; Marro, D. et al., Drug Delivery Pharm. Res., 18: 1701 -1708, 2001).
  • the present invention is also directed to methods wherein a nucleic acid comprised in a composition of the invention is capable of specifically hybridizing with part of target nucleic acid, preferably a target gene (genomic DNA), or target protein belonging to said target cells.
  • a target gene genomic DNA
  • nucleic acids that can delivered by the method of the present invention are oligonucleotide sense or antisense or a triple helix capable of modulating the expression products of a target gene of cells can be cited, in addition to the oligo- or polynucleotide (DNA or RNA) or the chimeric oligonucleotides relating to the correction of a functionally deficient gene, or to the creation of a deficient gene disclosed in the above cited documents or in the present specification, as below.
  • the invention relates to a method wherein a nucleic acid, particularly an oligo- or polynucleotide (DNA or RNA) or a chimeric oligonucleotide as defined above, comprised in a composition is a polynucleotide containing at least a sequence complementary to a target gene of cells with the exception of at least one nucleotide that is desired to be inserted, or deleted or substituted in said target gene.
  • a nucleic acid particularly an oligo- or polynucleotide (DNA or RNA) or a chimeric oligonucleotide as defined above
  • a sequence complementary to a target gene means a sequence forming Watson- Crick base pairing with part of the target gene sequence, part of the target gene sequence that particularly comprises, in the context of this invention, the fragment of the target sequence wherein said at least one nucleotide is desired to be inserted (or deleted) or changed.
  • Guanine/cytosine or adenine/thymine (or /uracil) are examples of complementary bases that are known to form hydrogen bonds between them.
  • chimeric oligonucleotide is defined as a polynucleotide having both ribonucleotides, modified or not and deoxyribonucleotides in a first strand and solely deoxyribonucleotides in a second strand wherein the strands have a Watson-Crick complementarity and are linked by oligonucleotides so that the polynucleotide has at most a single 3' and a single 5' end, and wherein these ends can be ligated so that the polynucleotide is a single continuous circular polymer.
  • Nucleotides are the monomeric units of nucleic acid polymers.
  • a "polynucleotide” is distinguished here from an “oligonucleotide” by containing more than 80 monomeric units; oligonucleotides contain from 2 to 80 nucleotides.
  • the term nucleic acid includes deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA").
  • DNA can be in the form of antisense, plasmid DNA, parts of a plasmid DNA, expression vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • RNA can be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), a shRNA (short RNA), miRNA (microRNA), a siRNA (small interfering RNA), antisense RNA, ribozymes, chimeric sequences, or derivatives of these groups.
  • DNA and RNA can be single, double, triple, or quadruple stranded, in a linear or circular form and eventually closed.
  • Antisense is a nucleic acid that interferes with the function of DNA and/or RNA. This may result in suppression of expression.
  • Natural nucleic acids have a phosphate backbone
  • artificial nucleic acids may contain other types of backbones, nucleotides, or bases. These include PNAs (peptide nucleic acids), phosphothioates, and other variants of the phosphate backbone of native nucleic acids such as phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire protein coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can be antisense to a non-coding region of the coding strand of a nucleotide sequence encoding the target protein.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 or 75 nucleotides in length.
  • the administered nucleic acid of the method of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an oligonucleotide or polynucleotide nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the nucleic acid molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives.
  • the nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned.
  • Expression cassette refers to a natural or recombinantly produced nucleic acid that is capable of expressing protein(s).
  • a DNA expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins.
  • the expression cassette can include trancriptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals.
  • An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins.
  • the expression cassette may include translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non-coding sequences.
  • siRNA, shRNA, miRNA, like antisense nucleic acid refer to a nucleic acid that has the ability to reduce or inhibit expression (transcription and/or translation) of a target nucleic acid sequences when said nucleic acid is introduced or expressed in the same cell as the target nucleic acid.
  • Said nucleic acid has substantial or complete identity to the complementary sequence of the target gene and can so hybridizes with it.
  • said nucleic acid is at least about 15-80 nucleotides in length, preferably about 15-50 nucleotides in length, 20-30 nucleotides length is more preferred.
  • nucleic acid is also intended to designate ribozymes, which are capable of selectively destroying target RNAs (see the document EP 321 201).
  • the invention relates to a method according to the present invention, wherein said nucleic acid comprised in the composition is an aptamer.
  • aptamer means any polynucleotide, or salt thereof, having selective binding affinity for a non-polynucleotide molecule (such as a protein) via non-covalent physical interactions.
  • An aptamer is a polynucleotide that binds to a ligand in a manner analogous to the binding of an antibody to its epitope.
  • Aptamers are chemically synthesized short strands of nucleic acid that adopt specific three-dimensional conformations and are selected for their affinity to a particular target through a process of in vitro selection referred to as systematic evolution of ligands by exponential enrichment (SELEX).
  • SELEX is a combinatorial chemistry methodology in which vast numbers of oligonucleotides are screened rapidly for specific sequences that have appropriate binding affinities and specificities toward any target. Using this process, novel aptamer nucleic acid ligands that are specific for a particular target may be created.
  • the SELEX process in general, and VEGF aptamers and formulations in particular, are described in, e.g., U.S. Patents Nos.
  • Anti-VEGF aptamers are small stable RNA-like molecules that bind with high affinity to the 165 kDa isoform of human VEGF.
  • aptamer sequences have been developed that target various other biological targets.
  • aptamer sequences have been developed that target PDGF (U.S. Patents Nos. 5,668,264, 5,674,685, 5,723,594, 6,229,002, 6,582,918 and 6,699,843), basic FGF (U.S. Patents Nos. 5,459,015 and 5,639,868), CD40 (U.S. Patent No. 6,171,795), TGF ⁇ (U.S. Patents Nos. 6,124,449; 6,346,611 ; and 6,713,616), CD4 (U.S. Patent No. 5,869,641), chorionic gonadotropin hormone (U.S.
  • Patents Nos. 5,837,456 and 5,849,890 HKGF
  • U.S. Patents Nos. 5,731 ,424, 5,731,144, 5,837,834 and 5,846,713 ICP4 (U.S. Patent No. 5,795,721), HIV- reverse transcriptase (U.S. Patent No. 5,786,462), HIV-integrase (U.S. Patents Nos. 5,587,468 and 5,756,287), HIV-gag (U.S. Patent No. 5,726,017), HIV-tat (U.S. Patent No. 5,637,461), HIV-RT and HTV-rev (U.S. Patents Nos.
  • HIV nucleocapsid U.S. Patents Nos. 5,635,615 and 5,654,151
  • neutophil elastase U.S. Patents Nos. 5,472,841 and 5,734,034
  • IgE U.S. Patents Nos. 5,629,155 and 5,686,592
  • tachykinin substance P U.S. Patents Nos. 5,637,682 and 5,648,214
  • secretory phospholipase A2 U.S. Patent No.
  • the aptamer is directed to an adhesion molecule, such as ICAM-I , or its binding LFA-I .
  • the aptamer is directed to any known ligand or its receptor.
  • ligands and/or their receptors for targeting with the sterically-enhanced aptamer conjugates of the invention include TGF, PDGF, IGF, and FGF.
  • ligands and/or their receptors for targeting include: cytokines, lymphokines, growth factors, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, ILl 8, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, GM-CSF; thrombopoietin, stem cell factor, and erythropoietin, hepatocyte growth factor/NKl or factors that modulate angiogenesis, such as angiopoietins Ang-1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular
  • compositions of the invention include angiogenin, BMPs such as bone morphogenic protein- 1 , etc., bone morphogenic protein receptors such as bone morphogenic protein receptors IA and IB, neurotrophic factors, chemotactic factor, CD proteins such as CD3, CD4, CD8, CDl 9 and CD20; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-alpha, - beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-I to IL-IO; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; regulatory proteins;
  • the invention further includes compositions comprising any of the known aptamer nucleic acid sequences that target, for example, a ligand or its receptor, such as those compiled in the aptamer database, which is available at the website, aptamer.icmb.utexas.edu).
  • the nucleic acid molecules used in the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic ("PNAs").
  • the invention relates to a method according to the present invention wherein said nucleic acid comprised in the composition is a chimeric oligonucleotide DNA/2' OMeRNA type wherein at least part of that DNA/RNA sequence is complementary to a genomic DNA fragment sequence of a target mutated gene of said cells with the exception of the mutation that is desired to be reverted in said target mutated gene.
  • the nucleic acid is an oligonucleotide DNA or an oligonucleotide RNA, optionally with phosphorothioates linkages.
  • the nucleic acid is a single stranded oligonucleotide DNA (ssODN) or a single stranded oligonucleotide RNA (ssORN).
  • the nucleic acid is capable of specifically hybridizing with a sequence of a genomic DNA contained in said retinal cells.
  • the nucleic acid is capable of specifically hybridizing with a sequence located into the nucleus of said retinal cells.
  • the nucleic acid is capable of binding to an extracellular protein, thereby inhibiting the protein binding with its designated receptor.
  • the target cells are photoreceptor cells or retinal pigment epithelium cells (RPE cells), photoreceptor cells are preferred.
  • the nucleic acid target cells/proteins are located in the outer nuclear layer of the retina.
  • the step of injection of said composition comprising said nucleic is selected from the group consisting of periocular (subconjunctival, peribulbar, laterobulbar, subtenon), subretinal, supra choroidal, intracameral, intracorneal, and intravitreous injection of the composition comprising said nucleic acid.
  • the administration of the composition containing the nucleic acid is carried out by a step of topical instillation of said composition.
  • a preferred application would be a topical application at the time of iontophoresis utilizing the device disclosed in the U.S. Patent No. 6,154,671, issued November 28, 2000.
  • the iontophoresis is an ocular or a transpalpebral iontophoresis.
  • Devices for delivery of therapeutic or diagnostic agents into target cells of an eye tissue through ocular or transpalpebral iontophoresis are commonly used and thus have been already disclosed. The skilled artisan could easily choose and determined the iontophoresis device and its use conditions, particularly the current density, the period of time of applying the current and the electrodes form and location etc., adapted to the tissue containing the target cells where the nucleic acid transfer is desired to be done.
  • the ocular iontophoresis system used in the methods of the present invention is a device selected in the group consisting of the devices disclosed in the following patents: U.S. Patent No. 4,141,359, issued February 27, 1979; U.S. Patent No. 4,250,878, issued January 17, 1981; U.S. Patent No. 4,301 ,794, issued November 24, 1981; U.S. Patent No.
  • the ocular iontophoresis system used in the methods of the present invention is a device selected in the group consisting of the devices disclosed in the U.S. Patent No.
  • said device being characterized in that it comprises a reservoir configured to receive an aqueous solution, optionally buffered and including various electrolytes, and having an internal wall, an external wall, and an end wall bridging the internal wall and the external wall, the internal wall and the external wall being annular and having a free end configured to be applied to an eyeball, said device further comprising at least one active electrode arranged in the reservoir, a passive electrode and a current generator, wherein the at least one active electrode is a surface electrode arranged on an interior surface of the end wall and wherein the internal wall has an outer diameter that is configured to be at least equal to a predetermined diameter, whereby the predetermined diameter represents a diameter of a human cornea.
  • a transpalpebral iontophoresis system is used in the methods of the present invention is a transpalpebral iontophoresis device disclosed in the U.S. Patent Application No. US 2004/267188 on December 30, 2004.
  • the device comprises a main electrode having an insulating layer and an adhesive layer able to bond the insulating layer to a conductive layer characterized in that the main electrode has an area able to come into contact with an eyelid.
  • the transpalpebral iontophoresis can be an anionic or cationic iontophoresis, although cationic iontophoresis is also preferred for transpalpebral iontophoresis.
  • Transpalpebral iontophoresis is preferably performed with a current of about 1 - 5mA for about 1 - 7 min; more preferably with a current of about 1 -3 mA for about 3 - 6 min; and more preferably with a current of about 2 mA for up to 5 min being more preferred.
  • the present invention is directed to a method for the treatment of an eye disease resulting from a deficient protein with a therapeutic nucleic acid that is capable of specifically hybridizing with a target DNA or RNA sequence encoding said deficient protein.
  • the invention relates to a method of the invention for the treatment of an eye inherited pathology resulting from a mutated gene expressed in the target cells with a nucleic acid composition containing at least a sequence complementary to a genomic DNA sequence of the gene, preferably wherein at least part of the nucleic acid composition is complementary to a genomic DNA sequence of the mutated gene expressed in the target cells, with the exception of the mutation that is desired to be reverted in said target gene.
  • the present invention also encompasses a method to treat an eye disease with a nucleic acid capable of reverting or inducing a mutation in a gene of target eye cells, gene expression of which is associated to that disease, in a non-human animal or in a human subject in need of such treatment.
  • mutated gene is understood to mean a gene whose sequence comprises at least one mutation or polymorphism relative to a wild-type reference.
  • the mutation can be, for example, a deletion, addition or substitution of at least one nucleotide compared to the wild-type gene.
  • a mutation can be at least partially responsible of a pathology or affection, notably associated with the loss of the normal function of the protein encoded by the wild-type functional gene.
  • the murine gene encoding the cGMP-phosphodiesterase ⁇ -subunit wherein the non-sense C to A mutation in the codon 347 of the cDNA of part of said gene leads to retinitis pigmentosa disease can be cited.
  • the RPl gene can be particularly cited. Indeed, in that RPl gene, the missense mutation of the active-site Lys-296 in that rhodopsin gene, such as K296E, has been found to produces an opsin with no chromophore binding site and therefore not activated by light, causing autosomal dominant retinitis pigmentosa (ADRP), or a nonsense mutation R677- STOP has also been found to be associated with retinitis pigmentosa in family linked to the RPl locus (Payne et al., Invest. Ophthalmol. Vis.
  • ADRP autosomal dominant retinitis pigmentosa
  • Hypoxia inducible factor- 1 is a transcription factor composed of HEF-I alpha and HIF-I beta subunits. HIF-I transactivates multiple genes whose products play key roles in oxygen homeostasis (Ozaki et al., Invest. Ophthalmol. Vis. Sci., 40: 182-189, 1999).
  • the gene encoding the transcription factor HIFalpha for example, which governs the expression of several genes involved in inflammation and neovascularization, can be targeted to cure patients with ocular neovascularization, mainly retinal neovascularization (Wenger, J. Exp. Biol., 203:1253-1263, 2000).
  • PCDHG Its normal sequence, PCDHG, is conserved in humans (439-464) and in mice (669-693).
  • a chimeroplast (term used in the present specification to also designate a chimeric oligonucleotide) bringing a codon stop can be designed in order to have the expressed protein not be able to promote hypoxia induced neovascularization in human or in mice.
  • the present invention is directed to a method to treat a disease comprising the administration of an acid nucleic, preferably an oligo- or polynucleotide (DNA or RNA) or chimeric oligonucleotide as defined above, capable of reverting or inducing a mutation in a target gene of target cells, gene expression of which is associated to that disease, in a human or animal host in need of such treatment, wherein the method used for delivering in vivo said nucleic acid into said target cells is the method for delivering in vivo nucleic acid according to the present invention.
  • the disease treated by the method of the invention is an inherited pathology, including an inherited retinopathy.
  • Eye diseases that can be treated by the methods of the present invention include, but are not limited to, Bardet-Biedl syndrome, autosomal recessive; chorioretinal atrophy or degeneration, autosomal dominant; cone or cone-rod dystrophy, autosomal dominant; cone or cone-rod dystrophy, autosomal recessive; cone or cone-rod dystrophy, X-linked; congenital stationary night blindness, autosomal dominant; congenital stationary night blindness, autosomal recessive; congenital stationary night blindness, X-linked; Leber congenital amaurosis, autosomal recessive; macular degeneration, autosomal dominant; macular degeneration, autosomal recessive; ocular-retinal developmental disease, autosomal dominant; optic atrophy, autosomal recessive; optic atrophy, X-linked; retinitis pigmentosa, autosomal dominant; retinitis pigmentosa, autosomal dominant; autosomal dominant
  • the said retinal disease is selected from the group of autosomal dominant, autosomal recessive or X-linked Retinitis pigmentosa.
  • retinal neovascular diseases can be also particularly cited.
  • the retinal neovascularization to be treated or inhibited can be caused by diabetic retinopathy, vein occlusion, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ischemia or trauma.
  • the intravitreal neovascularization to be treated or inhibited can be caused by diabetic retinopathy, vein occlusion, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ischemia or trauma.
  • the choroidal neovascularization to be treated or inhibited can be caused by retinal or subretinal disorders of age-related macular degeneration, presumed ocular histoplasmosis syndrome, myopic degeneration, angioid streaks or ocular trauma.
  • the nucleic acid that is administered is capable of reverting or inducing a mutation of the RPl gene.
  • the present invention also comprises a method according to the present invention, wherein the nucleic acid that is administered is capable of reverting or inducing a mutation of the gene encoding the transcription factor HIF l ⁇ .
  • the present invention is directed to a method to obtain an animal model comprising the administration of an acid nucleic, preferably an oligonucleotide or polynucleotide (DNA or RNA) or chimeric oligonucleotide as defined above, capable of reverting or inducing a mutation in a target gene of target cells of that animal, wherein the method used for delivering in vivo said nucleic acid into said target cells is the method for delivering in vivo nucleic acid according to the present invention.
  • an acid nucleic preferably an oligonucleotide or polynucleotide (DNA or RNA) or chimeric oligonucleotide as defined above, capable of reverting or inducing a mutation in a target gene of target cells of that animal, wherein the method used for delivering in vivo said nucleic acid into said target cells is the method for delivering in vivo nucleic acid according to the present invention.
  • the present invention is directed to a method for the screening of pharmaceutical or cosmetic compounds comprising a step wherein said pharmaceutical or cosmetic compound to be tested is administered to an animal model obtain by the method of the present invention, animal model that has been modified by the administration of an acid nucleic, preferably an oligo- or polynucleotide (DNA or RNA) or a chimeric oligonucleotide as defined above and capable of reverting or inducing a mutation in that target gene, wherein the method used for delivering in vivo said nucleic acid into said target cells is the method for delivering in vivo nucleic acid according to the present invention.
  • an acid nucleic preferably an oligo- or polynucleotide (DNA or RNA) or a chimeric oligonucleotide as defined above and capable of reverting or inducing a mutation in that target gene
  • the method used for delivering in vivo said nucleic acid into said target cells is the method for delivering in viv
  • Muller cells The integral nature and electrophysiology of Muller cells are described in the review by Bringmann and Reichenbach (Frontiers in Bioscience, 6:77-92, 2001). They describe the structural as well as the functional roles these cells play in order to maintain normal retinal function. In particular, they describe the active maintenance of a negative membrane potential brought about by the efflux of potassium and influx of sodium (by the appropriate ion channels). Due to the inherent membrane potential, it is expected that an applied electric current would have an affect on theses cells, potentially causing activation of these ion channels Cho et al.
  • the M ⁇ ller Cells span the entire depth of the retina and are in direct contact with the vitreous body. It follows that while the channels are artificially opened and/or compromised, molecules residing in the vitreous, such as oligonucleotides, would passively enter the Muller cells, which by their integral nature, would allow diffusion into the remaining retinal layers.
  • iontophoresis is a simple and efficient method for the temporary elongation of Muller Cells and that this transient elongation facilitates the intraretinal penetration of nucleic acids without altering the photoreceptors.
  • eyes analyzed 1 hour after an iontophoretic current application demonstrated enlargement of Muller Cells prolongations with normal integrity of the photoreceptor nuclei. Enlargement of the Muller Cells prolongations were no not detected when the treated eyes were examined 24 hours after iontophoresis.
  • the observed Muller Cells changes induced by the electric current were temporary and that no permanent ultra-structure change was induced by iontophoresis. Particularly, no alteration of the photoreceptors could be detected.
  • EXAMPLE 1 Treatment of the retinal degeneration of the rd mouse by iontopherically transferring in vivo a chimeric oligonucleotide into retina cells
  • mice homozygous for the rd mutation display hereditary retinal degeneration and serve as a model for human retinitis pigmentosa.
  • the retinal rod photoreceptor cells begin degenerating at about postnatal day 8 and by four weeks no cones are left.
  • Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod cGMP-phosphodiesterase. This enzymatic defect is due to the presence of a nonsense C ⁇ A mutation in the rd ⁇ -PDE gene.
  • the nonsense mutation creates an ochre stop codon (position 347) within exon 7 and leads to the truncation of the resulting cGMP-phosphodiesterase ⁇ -subunit.
  • the absence of a functional cGMP-phosphodiesterase protein in rd/rd mice is responsible for retinal degeneration.
  • the chimeric oligonucleotides were delivered into the targeted tissue using the combination of both, local injection and iontophoresis.
  • the DNA/2'OMeRNA chimeric oligonucleotides were synthetisized and purified by high pressure liquid chromatography by GensetOligos (France). The oligonucleotides were resuspended in distilled water and quantitated by ultra-violet absorbance at 260 nm. The sequences of the chimeric oligonucleotides are follows:
  • Control chimeric oligonucleotide (named Ctr) (the 2'OMe RNA nucleotides are underlined):
  • C3H/HeN mice with a nonsense mutation (position 347) were purchased (Iffa Credo). Genotyping to verify the absence or presence of the rd/rd mutation was accomplished by PCR of DNA from tail biopsies and subsequent restriction fragment analysis. The animals were given food and water ad libitum and maintained under pathogen-free conditions of 12h- light/12h darkness.
  • Iontophoresis was performed using the drug delivery device designed by OPTIS France (as disclosed in U.S. Patent No. 6,154,671, issued November 18, 2000).
  • a container was designed to allow transcorneoscleral iontophoresis.
  • a platinium electrode was placed at the bottom of the container and two silicone tubes were settled laterally. One tube was used to infuse saline buffer and the other to aspirate bubbles.
  • the CCI electronic unit can delivered up to 2,500 ⁇ A for 600 sec.
  • An audio-visual alarm indicated each disruption in the electric circuit ensuring a calibrated and controlled delivery of the product.
  • the CCI ocular cup was placed on the eye and the other electrode was maintained in contact with the animal.
  • Biotinylated chimeric oligonucleotide were injected and followed by iontophoresis as described above.
  • the eyes were enucleated Ih after the treatment, immediately frozen in OCT (Tissue Tek, USA) and sectioned (10 ⁇ m). They were fixed in methanol at -20 0 C for 10 min.
  • the sections were then washed in 1 % Triton X-100 PBS and incubated in a 1/100 streptavidin horseradish-peroxidase PBS solution for 2 h at room-temperature.
  • the sections were washed and the complex was revealed using 3.3' diaminobenzidine tetrahydrochloride in the presence of H2O2. Finally, the sections were counterstained with Hemalun. 3) rd-mutation test by restriction fragment analysis of RT-PCR products
  • MMV Moloney leukemia virus
  • Oligonucleotide primers included the sequences 5'-GGC CGG GAA ATT GTC TTC TAC-3' (SEQ ID NO:3) and 5'-CCC CAG GAA CTG TGT CAG AGA-3' (SEQ ID NO:4), located at nucleotide positions 921 to 943 and 1258 to 1279 of the ⁇ -cGMP- phosphodiesterase cDNA respectively.
  • RT product was amplified by PCR in a volume of 100 ⁇ l using 3U of Taq polymerase and primers described above.
  • PCR cycles were performed in thermal cycler with an initial denaturation of 5 min at 94°C, denaturation temperature of 94°C for 1 min, annealing temperature of 55°C for 1 min, extension temperature of 72°C for 1 min and a final extension of 10 min at 72°C.
  • the PCR buffer contained ⁇ -32P dCTP.
  • products were digested with 2.5 units BsaAI and/or 5 units Ddel in the provided buffer at 37°C overnight, then ethanol precipitated, washed and resuspended in 10 ⁇ l gel loading buffer.
  • the products were run on an 8 % nondenaturing polyacrylamyde gel at 500 volts for 3 hours. The gel was exposed to a film for 3 days.
  • RNA from +/+ retinae and from untreated rd/rd retinae served as controls.
  • opsin-immunohistochemistry was performed on whole-mounted retina.
  • the antibody Rho4D2 recognizes specifically opsin, which is the photo-pigment of rod-photoreceteptors.
  • Eyes were enucleated and fixed for 30 min in PBS/Paraformaldehyde 4 %.
  • the retinae were dissected and fixed in methanol at -20 0 C for 10 min, washed three times in 1 % Triton X-100 PBS, incubated over-night in a 1/100 Rho4D2, 1 % Triton X-100 PBS solution at room temperature.
  • the retina were then washed, incubated for 2h at room temperature with an 1/250 anti-mouse Alexa 40 antibody, washed and flat-mounted in glycerol/PBS. They were viewed and photographed by fluorescence microscopy (see FIG. 3B). The photographs were all taken with the same film (Illford 400ASA), exposure time (1 h 30 min), and developed in exactly the same manner. The photographs of flat-mounted retinae were scanned. The number of rod- photoreceptors was measured using a computerized image-analysis system (NIH).
  • NIR computerized image-analysis system
  • Results were expressed as mean ⁇ standard error of the mean (SEM) (see FIG. 3A). Statistical analyses were performed using the non parametric Man Whitney U test.
  • a DNA/RNA2'OMe oligonucleotide (named Chi) has been designed, which has the potentialities to revert the C ⁇ A point mutation located within codon 347 in the mouse rd ⁇ -PDE gene.
  • a control oligonucleotide (named Ctr) contains the same base composition as the active chimeric oligonucleotide but a different sequence. Photoreceptor transfection by chimeric oligonucleotides
  • RT-PCR were performed with rd ⁇ -PDE mRNA specific primers on extracted retinae.
  • the rd nonsense point mutation in codon 347 creates a Ddel restriction site and removes a BsaAI site from the wild-type sequence.
  • Digesting the 359 bp ⁇ -PDE cDNA with BsaAI or Ddel yields two diagnostic fragments of 120 bp and 239 bp. This method allows the differentiation of the mutated sequence (Ddel sensitive) from the wild-type one (BsaAI sensitive) at the mRNA level.
  • Iontophoresis is known to be a non-invasive process to deliver drugs using a low- intensity current. It uses an electrode of the same polarity as the charge on the drug to drive ionic drugs into the tissues.
  • the present inventors have demonstrated that iontophoresis can be used to enhance the nucleic acid penetration into cells tissue, such as chimeric oligonucleotide DNA/2'OMeRNA type, particularly into ocular cells after intra- or peri-ocular injection and to enhance retinal transfer or penetration after or before or simultaneously to intraocular injection.
  • EXAMPLE 2 Combination of oligonucleotides (ODNs), intravitreous injection, and saline transpalpebral iontophoresis on the delivery of ODNs to photoreceptors in the new born rdl/rdl mice
  • Direct iontophoresis enhances the intraocular levels of locally applied drugs both in experimental models and in patients.
  • Different types of devices have been designed to apply the current on the cornea, the sclera or both, with drug application on the eye surface in containers of various forms and materials.
  • This type of iontophoresis procedure can be qualified as "direct ocular iontophoresis".
  • Direct ocular iontophoresis has also been used to enhance the intra-tissue and intra-cellular penetration of oligonucleotides (ODNs).
  • ODNs oligonucleotides
  • the mechanisms of drug penetration facilitation by iontophoresis include electrorepulsion, electroosmosis and current-induced tissue permeation.
  • C3H/HeN mice homozygous for the nonsense mutation (amino acid position 347) in the ⁇ -PDE gene (Janvier, Le Genest, France) were used (36 mice, 72 eyes). Mice were maintained in clear plastic cages and subjected to a standard light: dark cycle of 12 hours.
  • ODN Oligonucleotide
  • Transpalpebral iontophoresis system (patent # FR2830766) was used. Eye-glass- shaped aluminum foil and disposable medical grade hydrophilic polyurethane sponge (3.2 mm thick, 1.5 x 0.7 cm length by width, Optis, Levallois, France), were soaked in PBS (phosphate buffered saline: 0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, 2.16 g/L Na2HPO4 7H2O, pH7.4) and used as the active electrode (FIG. 1C). The electrode covered both closed eyelids of the treated newborn mouse. The return electrode was connected to the neck of the mouse. An audio-visual alarm indicated any disruption of the electric circuit ensuring a controlled delivery of the current.
  • PBS phosphate buffered saline: 0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, 2.16 g/L Na2HPO
  • Intraocular injections were carried out with ES TransferTips microcapillaries (Leica, Rueil Malmaison, France) and cut at 2 mm from their extremity, leading to a 60 ⁇ m injecting hole.
  • Microcapillaries were linked to a Micro4TM microsyringe pump controller (World Precision instruments, Sarasota, USA).
  • ODN 500 ⁇ M
  • the position of the needle was monitored by observation with a dissecting microscope through a glass cover slip placed on the corneal surface.
  • the micropipette needle was left in place for 10 sec after the injection before withdrawal.
  • the ODN distribution was evaluated at one hour after the end of the procedure.
  • Anodal or cathodal transpalpebral saline iontophoresis (respectively positive or negative electrode connected to the eyelids) was performed with a current of 1.5 mA for 5 min (1.43 mA/cm2) before the intravitreous injection of ODN (4 eyes for each experiment).
  • Cathodal iontophoresis was then tested when applied immediately after the intravitreous injection of ODNs (4 eyes). The results derived from this initial set of experiments showed that cathodal iontophoresis performed prior to the intravitreal injection led to the highest ODN penetration in the ONL.
  • This condition was therefore used to evaluate the effect of lower current intensity (0.5 mA for 5 min) or higher current intensity (2.5 mA for 5 min), as well as the duration of current-induced permeation by injecting ODN at various time point after iontophoresis (acute, 1 , 3 and 6 hours) (4 eyes for each experiment).
  • the optimal conditions defined the kinetics of fluorescent ODN distribution was evaluated at 1 , 6 and 24 hours after treatment (4 eyes for each condition).
  • Control animals received intravitreous injection of 1 ⁇ L Hex (500 mM) (Invitrogen, Cergy Pontoise, France) or PBS with or without previous cathodal iontophoresis (4 eyes for each condition). Four additional eyes were treated and sacrificed at one hour to evaluate the integrity of the injected ODN by acrylamide gel electrophoresis.
  • mice were sacrificed by an intraperitoneal lethal dose of pentobarbital (6 g/100 ml; Ceva Sante Animale, Libourne, France). The eyes were enucleated and processed for the various tests as described below.
  • digestion buffer 50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS
  • mice received iontophoresis followed by ODN injection, 4 eyes received ODN injection without iontophoresis and 4 other eyes received iontophoresis alone.
  • mice were sacrificed, the eyes enucleated and fixed in 2.5% glutaraldehyde of cacodylate buffer (Na 0.1 M, pH7.4). After 1 hour, the globes were dissected at the limbus, the posterior eye ball post fixed for 3 hours and cut in 4 parts. Tissues were post-fixed in 1% osmium tetraoxyde in cacodylate buffer (Na 0.1M, pH7.4) and dehydrated in graduated ethanol solution (50, 70, 95, 100 %).
  • Results were expressed as means ⁇ SD and compared using the analysis of variance (ANOVA) test with post Fisher test. P ⁇ 0.05 was considered as significant.
  • ODN integrity was confirmed by acrylamide gel electrophoresis of the DNA extracted from retinas of treated eyes one hour after iontophoresis followed by injection (data not shown).
  • Iontophoresis was performed prior to ODN injection in order to limit the risk of infection and of potential reflux of ODNs from the globe by mechanical pressure of the probe on the eyelids.
  • Cathodal or anodal iontophoresis (1.5 mA for 5 min) prior the intravitreous injection of ODNs showed that the application of current enhanced ODN penetration when compared to injection without iontophoresis (P ⁇ 0.05) or no treatment (P ⁇ 0.05) (FIG. 5: * and **).
  • prior cathodal saline iontophoresis significantly enhanced ODN penetration in the ONL cells when compared to anodal saline iontophoresis (P ⁇ 0.05: **).
  • cathodal saline iontophoresis immediately prior to the intravitreous ODN injection significantly enhanced the ODN penetration in ONL when compared to application of cathodal iontophoresis immediately following ODN injection (PO.05:**). From all tested conditions, cathodal iontophoresis performed immediately prior to ODN injection yielded the highest ODN penetration in the ONL of treated mice eyes. The later condition was therefore used to evaluate further parameters.
  • nuclei in the INL and the ONL have normal structure and do not show any sign of apoptosis or necrosis (FIG. 1OC and 10c). No such changes were observed in untreated control retinas or in eyes without current application (FIG. 1OA, 1OB and 10b). Twenty four hours after iontophoresis application, internuclear spacing was no more observed and the ONL has regained a normal architecture (FIG. 10E).
  • TEM analysis showed that in eyes receiving injection without electric current applied or in untreated control eyes, the retinas retained a normal structure without any detectable changes (FIGs. 1 IA and 1 IB). Eyes analyzed 1 hour after iontophoretic current application demonstrated enlargement of RMG prolongations (FIG. 11C, arrow) with normal integrity of the photoreceptor nuclei. Enlargement of the RMG prolongations were no more detected when the treated eyes were examined 24 hours after iontophoresis (FIG. 1 IE). Thus, the observed RMG changes induced by the electric current were temporary and that no permanent ultra- structure change was induced by the optimal iontophoresis parameters used in this study. Particularly, no alteration of photoreceptors could be detected.
  • Electroosmosis is another mechanism of drug penetration acting through a flow process (vol/distance/time). Electroosmosis-induced drug penetration is particularly important for larger molecules. Increased "passive" permeability for a limited period of time after the application of current was also observed in the skin. In these experiments, it was demonstrated that the slow recovery of skin impedance following iontophoresis was due to the movement of ions in response to electric field and that the resulting post-iontophoretic enhanced-diffusion was not associated with damage to the skin barrier.
  • ODNs used in this study were negatively charged and with a molecular weight of 7591 g/mol. These characteristics can allow them to penetrate through the internal limiting membrane. However, the exact mechanisms responsible for the transport from the inner retina to the photoreceptor cells remain ill understood. Such transport is probably highly regulated and does not follow passive diffusion. Simple direct vitreous injection of ODNs does not lead to their penetration into photoreceptors. On the other hand, when saline iontophoresis is performed with the intravitreous injection of ODNs, penetration in the photoreceptors cell nuclei is observed.
  • EXAMPLE 3 Single-stranded oligonucleotide mediated in vivo gene repair in the rdl/rdl retina [0185]
  • the purpose of this study was to test iontophoresis enhanced the penetration of oligonucleotide in the photoreceptors nuclei and induce targeted gene repair of a point mutation in vivo following iontophoretic delivery of the oligonucleotide.
  • the rd/rdl mouse is a model of rapid retinal degeneration. It results in part from a point mutation in the gene encoding the ⁇ -subunit of rod receptor cGMP-phosphodiesterase ( ⁇ -PDE), leading to a stop codon (Tyr347Ter) and subsequent truncated protein. In rd/rdl mouse, rod photoreceptor loss is complete by postnatal day (PN)21. Mutations in the same gene are responsible for retinal degeneration in patients with retinitis pigmentosa.
  • Targeted gene repair is a non-viral gene therapy strategy, which aims at correcting mutations in genomic DNA by using RNA/DNA oligonucleotides (RDOs) or single-stranded DNA oligonucleotides (ssODNs).
  • RDOs RNA/DNA oligonucleotides
  • ssODNs single-stranded DNA oligonucleotides
  • This gene therapy strategy should allow for a permanent correction of the genomic DNA and for normal physiologic regulation of the corrected gene by its endogenous promoter.
  • Targeted gene repair has been effective in inducing genotypic and phenotypic corrections both in vitro and in several animal models of various disorders such as hemophilia, Crigier-Najjar syndrome type 1 , albinism, Duchenne muscular dystrophy, hyperlipidemia type 2, and sickle cell disease.
  • mice homozygous for the nonsense mutation (amino acid position 347) in the ⁇ -PDE gene (Janvier, Le Genest, France) were used. Wild-type mice C57B16/Sevl29 served as positive controls. Mice were maintained in clear plastic cages and subjected to a standard light: dark cycle of 12 hours. Experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmologic and Vision Research and the institutional guidelines regarding animal experimentation in Ophthalmic and Vision Research.
  • ODNs used were synthesized and purified by high pressure liquid chromatography by Proligo (Paris, France). ODNs in distilled water were quantified by absorbance at 260 nm.
  • Sense (S) and antisense (AS) wild-type allele of the ⁇ -PDE gene sequence were assayed [WTS (5 '-C + C + T + T + C + C + AACCTACGTAGCA + G + A + A + G + TO ' SEQ ID N0:6), WTAS (5'- A*C*T*T*T*C*TGCTACGTAGGTT*G*G*A*A*G*G-3' SEQ ID N0:7)] as well as scrambled ODNs [WTSscr7 (5 '-C + C + T + T + C + C + AACAACGTCTGCA + G + A + A + A + G + TO ' SEQ ID NO:8), WTSscr25 (5 '-A + A + T + C + A + C + AGTTGCCTATAGG + A + C + C + C + C*A-3'
  • Transpalpebral (across closed eyelids) iontophoresis system (patent # FR2830766) vas used. It was found that applying transpalpebral iontophoresis immediately after or before intravitreal injection of ODNs leads to the same penetration efficiencies. Therefore, iontophoresis was performed immediately prior to the intravitreal injection of ODNs to avoid manipulation of the injected pups' eyes and reduce the potential danger of secondary infection. Prior to iontophoresis, tetracaine 1 % drops (Novartis Ophthalmics SA, Rueil Malmaison, France) were instilled.
  • Eye-glass-shaped aluminum foil and disposable medical grade hydrophilic polyurethane sponge (3.2 mm thick, 1.5 x 0.7 cm length by width, Optis, Levallois, France), were soaked in PBS (phosphate buffered saline: 0.2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, 2.16 g/L Na2HPO4 7H2O, pH 7.4) and used as the active electrode (FIG. 12A).
  • the electrode covered both closed eyelids of the treated newborn mouse.
  • the return electrode was connected b the tail and hind foot pads of the mouse.
  • Anionic iontophoresis (negative electrode connected to the eyelids) was performed with a 1.5 mA current for 5 min (1.43 mA/cm2 (FIG. 12B). An audio-visual alarm indicated any disruption of the electric circuit ensuring a controlled delivery of the current.
  • the pup's eyelids were then opened with a scalpel (Swann Morton, Peynier, France) and intraocular injections were carried out with borosilicate micropipette needles (Phymep, Paris, France) pulled with a pipette puller (model 720, Kops lnstrument,Tujunga, CA, USA) and cut at 2 mm from their extremity, leading to a 60 ⁇ m injecting hole.
  • Micropipette needles were linked to an Eppendorf microinjector 5242 (Roucaire, Velizy, France).
  • One ⁇ L of PBS or ODN was injected into the vitreous.
  • the position of the needle was monitored by observation under a dissecting microscope through a glass cover slip placed on the corneal surface.
  • the micropipette needle was left in place for 10 sec before withdrawal.
  • mice were sacrificed by a lethal dose of pentobarbital (6 g/100 mL; Ceva Sante Animale, Libourne, France) injected intraperitoneally.
  • rdl/rdl mice at PN7 underwent a single transpalpebral iontophoresis (anionic, 5 min, 1.5 mA) followed by an intravitreal injection of 1 ⁇ L of CY3-labeled WTS ODN (1 ⁇ L of 272 ⁇ M).
  • rdl/rdl mice al PN7 received either iontophoresis followed by PBS injection, ODN injection without iontophoresis, iontophoresis without injection, or had no treatment (8 eyes for each condition). Animals were sacrificed one hour after treatment.
  • the eyes were enucleated, rinsed in PBS, and embedded with Tissue-Tek OCT compound (Bayer Diagnostics, Puteaux, France) for cryo-sectioning. Sections (10 ⁇ m) were fixed in 4 % paraformaldehyde (Merck Eurolab, France) for 5 min at room temperature, washed in PBS, counter-stained for 2 min with DAPI (4',6-diamino-2- phenylindole) (1/3000 dilution) (Sigma-Aldrich, Saint-Quentin Fallavier, France), washed in PBS, mounted in Gel Mount (Microm Microtech, Francheville, France) and examined under a fluorescence microscope (Aristoplan, Leica, Rueil Malmaison, France) with FIBOl 03 w lamp and a digital SPOT camera (Optilas, Evry, France). For each eye, sections at the optic nerve level were counter-stained with hematoxylin and eosin for structural analysis.
  • ODN (1 ⁇ L of 500 ⁇ M) intravitreal injections.
  • Rhodopsin immunohistochemistry on whole flat-mount retinas Rhodopsin immunohistochemistry performed at PN19 and 28 on flat-mount rdl/rdl retinas of untreated (control) mice allows to follow the progressive loss of rhodopsin signal )see Results section (FIGs. 14C and 14E)). Therefore, rho-4D2 immunohistochemistry of PN28 whole flat-mounts was used as a global and rapid method to evaluate the potential rescue of photoreceptors and was therefore used to evaluate dosing effects and assayed of scrambled ODNs.
  • Rhodopsin immunohistochemistry was assessed on whole flat-mount retinas as previously described. Briefly, at PNl 9 and 28, ocular globes were fixed in 4 % paraformaldehyde (Merck Eurolab) for 1 hour. Retinas were isolated, placed in PBS in 1.5 mL microcentrifuge tubes, permeabilized in PBS, 0.1 % Triton X-IOO (Sigma-Aldrich) for 5 min, and incubated in blocking buffer (PBS containing 0.1 % bovine serum albumin (Sigma- Aldrich), 0.1% Tween 20 (Sigma-Aldrich) and 0.1% sodium azide (Sigma-Aldrich)) for 15 min.
  • blocking buffer PBS containing 0.1 % bovine serum albumin (Sigma- Aldrich), 0.1% Tween 20 (Sigma-Aldrich) and 0.1% sodium azide (Sigma-Aldrich)
  • Retinas were incubated in rod photoreceptor specific monoclonal mouse antibody rho-4D2 (1/100 dilution in blocking buffer; kindly provided by Dr. Robert Molday, University of British Columbia, Vancouver BC, Canada.
  • rho-4D2 monoclonal mouse antibody
  • As negative controls normal mouse serum (Nordic Immunological Laboratories, Tebu-bio, Le Perray en Yvelines, France) or mouse monoclonal antibody Leu-M5 directed against macrophages and monocytes (BD Biosciences, Pont-de- Claix, France) were used instead of rho-4D2 antibody (1/100 dilution in blocking buffer, 1 hour).
  • the retinas were washed 3 times for 5 min in blocking buffer and incubated with a secondary goal anti-IgG mouse antibody conjugated to Alexa Fluor 488 (1/250 dilution in blocking buffer; Molecular Probes, Leiden, Neitherlands).
  • the incubation volumes were 0.2 mL for antibody incubations and 1.5 ml for blocking and washing steps.
  • retinas were mounted in PBS-glycerol (1/1), with the photoreceptor layer facing up, and examined by fluorescence microscopy with a 2.5 objective and photographed using a digital SPOT camera (Optilas). All pictures were taken with an exposure time of 5 seconds. For each retina three pictures were taken to cover the whole retinal surface. Photographs of flat-mounts were merged in Photoshop 7.0 to reconstruct the whole retina.
  • Intensity of rhodopsin immunoreactivity was quantified by using the luminosity feature of Photoshop for raw pictures. Tissue and background regions were manually selected. Any residual pigmented epithelium was excluded. Mean pixel brightness was determined for each region by using the "Histogram" imaging feature. To normalize background levels among images, the mean brightness level per pixel of the tissue region was divided by the background region from each flat-mount image.
  • mice serum Normal mouse serum (Nordic Immunological Laboratories) or mouse monoclonal antibody Leu-M5 (BD Biosciences) replaced the primary antibodies (1/100 dilution in PBS).
  • Slides were washed 3 times in PBS and incubated with mouse anti-IgG conjugated b Alexa Fluor 488 (1/250 dilution in PBS; Molecular Probes). Then, the slides were washed 3 times in PBS, mounted in PBS/glycerol (1/1) and examined under a fluorescence microscope (Leica). ⁇ -PDE immunohistochemisty on eye sections
  • ⁇ -PDE was assessed in eye sections from PN28 untreated and PBS- or ODN-treated rdl /rdl mice previously labeled with rho-4D2. Immunohistochemistry was performed with rabbit IgG PDE6 ⁇ antibody (1/100 dilution; Affinity Bioreagents, Golden, CO, USA) or non-immune rabbit serum (1/100 dilution) as primary antibodies and goat anti-IgG rabbit antibody conjugated to Texas red Fluor (1/100 dilution; Molecular Probes) as secondary antibody. Sections were mounted in PBS/glycerol (1/1) and examined under a fluorescence microscope (Leica).
  • Genomic DNA from rdl /rdl retinas was extracted from individual whole flat-mount retinas (PN28) using a DNeasy Tissue kit and eluted in 200 ⁇ L of AE buffer as per manufacturer instructions (Qiagen, Courtaboeuf, France and Valencia, CA, USA). Genomic DNA from wild-type retinas, untreated, and PBS-treated rdl/rdl retinas served as controls. [0205] Allele-specif ⁇ c real-time PCR was used to detect small amounts of wild-type sequence resulting from ODN treatment. DNA samples isolated from treated and untreated retinas were used as template DNA in PCR reactions with primers designed to preferentially amplify wild- type rather than mutant ⁇ -PDE sequence.
  • the 3' base of one primer was complementary to wild-type sequence, but not rdl/rdl sequence, at position 1048 of GenBank accession no. X60133.
  • Primers used for preferential amplification of wild-type ⁇ -PDE sequence were 5 '-TGCAAGCATTCATTCCTTCGAC-S ' (SEQ ID NO: 10) and 5'-
  • AAGCC ACTTTCTGCTACG-3' (SEQ ID NO: 1 1).
  • parallel reactions using aliquots of the same source of template DNA were run using primers designed to nonspecifically amplify ⁇ -PDE sequence:
  • Reactions were run in a Bio-Rad iCycler iQ real time PCR detection system with melt curve analysis (Bio-Rad, Hercules, CA). Reactions of 20 ⁇ l final volume included template DNA (10 ng), primers (50 nM), and QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA), which is composed of SYBR Green I (a dye that fluoresces strongly when bound to dsDNA), HotStarTaq DNA polymerase, dNTPs, and buffer components optimized by the manufacturer. Poly (dI:dC), 20 ng per assay, was added to reduce nonspecific PCR products.
  • the limit of detection for quantification was considered 10 times the root mean square noise of fluorescence intensity across a window usually spanning cycles 2 through 10.
  • the cycle number at which product accumulated past this detection threshold (Ct) was related to beginning copy number of a specific template allele in a reaction by a calibration curve created with standard amounts of the wild-type ⁇ -PDE gene.
  • Ct cycle number at which product accumulated past this detection threshold
  • a lower Ct compared to untreated rdl/rdl controls indicates the presence of a specific allele, in this case, a presumed rdl/rdl allele repaired to wild-type sequence (mutant adenine converted to wild-type cytosine).
  • Ct data are means ⁇ SEM of 5-6 experimental samples assayed in 5-10 replicates. To determine the efficiency of the assay, calibration curves of gene copy versus threshold cycle were made using increasing amounts of wild-type genomic DNA (1 pg to 10 ng) mixed with 10 ng of rdl/rdl genomic DNA.
  • Results were expressed as means ⁇ SD and compared using the non-parametric Mann Whitney test and the analysis of variance (ANOVA) test with post-hoc Student-Newman-Keuls. P ⁇ 0.05 was considered as significant.
  • Transpalpebral iontophoresis was performed with an eye-glass-shaped electrode made with aluminum foil and sponge (FIG. 12A) and connected to a power supply (FIG. 12B). It did not cause any detectable clinical or histologic lesions to the mice eyes (FIG. 12C).
  • FTC ganglion cell layer
  • INL inner nuclear layer
  • No fluorescence was detected in the outer nuclear layer (ONL) of PN7 mice (FIG. 12D).
  • the antibody directed against ⁇ -PDE specifically labeled the rod outer segments in the wild-type mice at PN28 (FIG. 17, top panels).
  • the specificity of the anti- ⁇ -PDE antibody was confirmed by Western blot with a specific signal on wild-type retinas and an absence of signal on rdl/rdl retinas (FIG. 17E).
  • the estimated surface area of a retina is two-thirds the surface of a sphere (4 ⁇ r2), and fora radius of 1.25 mm at PN28, this is 13.1 mm2.
  • Double labeling with rho-4D2 showed that cells positive for ⁇ -PDE were also positive for rhodopsin (FIG. 18B). However, rhodopsin expressing cells were more numerous than those expressing ⁇ -PDE (FIGs. 16B and 18A). No significant fluorescence was observed when ODN was omitted in otherwise complete treatments (data not shown). Analysis of conversion of genomic DNA from rdl/rdl to wild-type
  • DNA extracted from ODN- or PBS treated rdl/rdl retinas and from wild-type retinas was used as template in allele-specific real-time PCR with primers designed to amplify only wild-type DNA.
  • the Threshold Cycle values (Ct) were 35 ⁇ 0.6 (6 retinas) for the ODN-treated group and 37 ⁇ 0.5 (5 retinas) for the negative control PBS group.
  • the Ct for the same amount of pure wild-type genomic DNA was 26 ⁇ 0.1 (6 retinas) (FIG. 19).
  • the adequate delivery of a sufficient amount of ODNs to the target cells is critical.
  • the direct intravitreal injection does not allow enough intact ODNs to reach photoreceptor nuclei.
  • the application of iontophoresis prior to the injection of ODNs results in an increased penetration of ODNs in photoreceptor nuclei.
  • the repetition of injections two or three times induces a significant rescue of photoreceptors. Therefore, photoreceptor cell rescue requires repeated delivery and/or a critical mass of intravitreal ODNs combined with an enhanced penetration efficiency to the target cells.
  • FIG. 13A While a single row or less of sparse cells was observed at PN28 in the ONL of untreated rdl/rdl retina (FIG. 13A), discontinuous areas containing two or three rows of cells were observed over the entire ONL of mice treated with WTAS ODN and WTS ODN, FIG.13C and 13D represent two of those areas where maximal rescue was observed. In PBS-treated eyes, a limited increase of cells was observed in the ONL (FIG. 13B).
  • the number of cells in the ONL was not significantly different in WTAS ODN- and WTS ODN- treated retinas with previously described conditions, (respectively 101 ⁇ 15 and 103 ⁇ 14 on a 400 ⁇ m length; 8 eyes for each condition, P>0.05). But there were significantly increased when compared to the PBS-treated retinas (74 ⁇ 5; 6 eyes) or to the untreated retinas from rdl/rdl mice (55 ⁇ 8; 6 eyes) (PO.05 and PO.01 respectively).
  • Rhodopsin immunostaining at PN28 was increased by specific oligonucleotides [0217] Extensive positive immunoreactive signal for rhodopsin was observed in wild-type eye sections at PN28 when rho-4D2 is used as primary antibody (FIG. 14A). Sections reacted with normal mouse serum in place of rho-4D2 yielded negative results (FIG. 14D). In rdl/rdl flat- mount retinas, rhodopsin-positive signal was observed throughout at PNl 9 (FIG. 14C).
  • the intense fluorescence observed at low magnification at PN 19 corresponds to dispersed immunoreactive photoreceptors as shown at a higher magnification (inset in FIG. 14C).
  • the rhodopsin signal was extremely low, reflecting the advanced and nearly complete degeneration of rods in these retinas al this time point (FIG. 14E), paralleling the time course of the rdl/rdl retinal degeneration.
  • Rhodopsin is the most abundantly-expressed photorecep tor-specific protein and is frequently used as a marker to detect the existence of rod photoreceptor cells.
  • rhodopsin immunohistochemistry was performed on treated flat-mount rdl/rdl retinas at PN28.
  • Gene repair treatment consisting of iontophoresis followed by injection with 1 ⁇ L of 500 ⁇ M specific ODNs, was performed on rdl/rdl mice at PN4, 6, and 8. As shown in Table 1 , the calculated ratios of tissue-to-background fluorescence (Tissue
  • Fl/Bkgd Fl were less than 1.75 for no treatment, treatment with PBS alone, or treatment with any of the scrambled ODNs (WTSscr25, and WTSscr7; ODN nomenclature is given in the legend of Table 1).
  • WTS ODN yielded the most intense rhodopsin immunostaining, resulting in a reproducible Tissue Fl/Bkgd Fl ratio of 2.57 (FIG. 14F). This increase was statistically significant compared to all other treatments as determined by the non parametric Mann-Whitney test (P ⁇ 0.004 for comparisons between WTS and any other treatment groups).
  • ⁇ -PDE ⁇ -subunit of rod photoreceptor cGMP-phosphodiesterase
  • PN postnatal day
  • ODN oligonucleotide
  • GCL ganglion cell layer
  • INL inner nuclear layer
  • ONL outer nuclear layer
  • CY3 cyanin 3
  • PBS phosphate buffer saline
  • chim chimeric
  • WTS/ AS wild-type sense/antisense
  • o 2'-O-methyl RNA bases
  • 25 or 45 25 or 45 mers
  • PO phosphodiester
  • polyU polyU-protected
  • 6x6PS six phosphorothioate linkages at each 5' and 3' positions
  • scr7/25 scrambled sequence of the 7 central bases/the entire 25 mers ODN.
  • Targeting gene repair is a new gene therapeutic that can corrects a punctual mutation by targeting oligonucleotide (ODNs) to the genomic DNA sequence where alteration is required.
  • ODNs oligonucleotide
  • One of the main limiting step in this technique is the need for an efficient delivery of high ODN amounts in the targeted cells. After intravitrous injection, ODNs concentrate in inner nuclear layers and finally accumulate in RPE cells. However, their penetration in the photoreceptor nuclei is very poor, compromising any potential gene repair.
  • Cationic or anionic transpalpebral iontophoresis (respectively positive or negative electrode connected to the eyelids) was performed immediately before or after the intravitreous injection of ODN labeled using a current of 1.5 mA for 5 min (4 eyes for each combination). Conditions with lower current intensities (0.5 mA for 5 min) were also tested (4 eyes for each condition). The effect of timing (1 , 3 and 6 hours) between iontophoresis and ODN intravitreous injection was carried out (4 eyes for each condition). For the control groups, rdl/rdl mice at PN7 received either iontophoresis followed by PBS injection, ODN injection without iontophoresis or had no treatment (4 eyes for each condition).
  • Fluorescence intensity of the outer nuclear layer fluorescence was quantified by image analysis of cryosections. Three microphotographs taken from 5 sections from each eye with a fixed exposure time and expressed in mean pixel brightness ⁇ SD served as the basis for this analysis. Relative OLN intensity values obtained were compared and analyzed by statistic program.
  • transpalpebral saline iontophoresis all conditions of transpalpebral saline iontophoresis increase the penetration of ODNs in the retinal layers when compared to injection without iontophoresis. Intact ODNs were extracted from the retina at one hour after treatment. As illustrated in FIG. 20, photoreceptors targeting was significantly increased when the saline transpalpebral iontophoresis is applied before ODN injection as compared with its application after ODN injection. Anionic iontophoresis is more efficient than cationic iontophoresis. Of all tested conditions, optimal photoreceptor targeting is achieved with application of transpalpebral saline anionic iontophoresis using 1.5 mA for 5 min before ODN injection. The facilitation of intraocular penetration by iontophoresis remains apparent at least for 3 hours with a marked decrease observed later. No intraocular tissue damage was observed after the iontophoresis application.
  • mice homozygous for the rdl mutation display hereditary retinal degeneration and serve as a model for human retinitis pigmentosa
  • rdl mice present a C ⁇ A mutation in the rod c-GMP-phosphodiesterase (PDE) /3-subunit gene, which creates a stop codon leading to the truncation of the protein.
  • PDE rod c-GMP-phosphodiesterase
  • the retinal rod photoreceptor cell death begins at about postnatal day 8 and complete degeneration is achieved by 4 weeks.
  • the purpose of this work is to evaluate the genoplasty strategy on the rdl mice and study the intra-retinal penetration and action of different types of ODNs designed to correct the rdl mutation.
  • Enhanced delivery of injected labeled-oligonucleotides to retinal layers by iontophoresis a process delivering drugs using low current density, in a safe way
  • FIG. 21 iontophoresis was very efficient to target retinal cells.
  • FIG. 22 the best results were obtained with specific coding phosphorothioate oligonucleotide showing a dose-dependant rescue of photoreceptors.
  • Treated rdl mice were injected by coding phosphorothioate oligonucleotides following iontophoresis at PN 4, 6 and 8. Eye sections from mice at PNl 2 were used for immunohistochemistry anti-/3 PDE (Affinity Bioreagents) using a secondary antibody conjugated to Alexa Fluor 488. They were examined under a Confocal microscope. As illustrated in FIG. 23, / 3-phosphodiesterase protein was detected in eye sections from mice after treatment. The enhanced retinal expression of ⁇ -phosphodiesterase protein in treated rdl mice further supports iontophoresis mediated retinal delivery.
  • a qualitative evaluation of iontophoresis of labeled oligonucleotide on rat is also provided in FIG. 24.
  • DNA was extracted at PN28 from ODN- or PBS-treated mice retinas. DNA was used as template in real-time PCR with allele-specific primers designed to amplify only wild-type 0-PDE DNA. Control PCRs were run with non allele-specific 0-PDE primers to compensate for any errors in template concentration measurement.

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Abstract

La présente invention concerne un dispositif et un procédé d'administration rétinienne d'agents thérapeutiques de type acide nucléique améliorée utilisant l'ionophorèse afin de provoquer une élongation transitoire des cellules de Muller d'un oeil de mammifère. L'augmentation du dépôt rétinien peut être obtenue soit par une application topique, soit par une injection sous-conjonctivale ou intravitréenne de la composition d'acide nucléique suivie par, précédée par, ou administrée simultanément avec l'application ionophorétique. La présente invention concerne ainsi un procédé particulièrement avantageux pour le traitement de maladies oculaires qui comprend l'administration in vivo d'un acide nucléique qui est capable d'atténuer les symptômes d'une maladie, l'administration de l'acide nucléique étant améliorée par l'utilisation de l'ionophorèse. Ce procédé peut être appliqué en particulier aux maladies de la rétine résultant d'une altération de l'expression d'un gène et/ou de la surexpression de facteurs de croissance particuliers. Les maladies comprennent, mais ne sont pas limitées à, les rétinopathies oculaires humaines comprenant les maladies néovasculaires (l'œdème maculaire lié à l'âge, les rétinopathies diabétiques, l'œdème maculaire diabétique, etc.) et les rétinopathies héréditaires telles que la rétinite pigmentaire.
EP07873352A 2006-12-05 2007-12-05 Administration rétinienne d'un acide nucléique améliorée par ionophorèse Withdrawn EP2099497A2 (fr)

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