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WO2011017313A1 - Method of administering non-viral nucleic acid vectors to the eye - Google Patents

Method of administering non-viral nucleic acid vectors to the eye Download PDF

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Publication number
WO2011017313A1
WO2011017313A1 PCT/US2010/044234 US2010044234W WO2011017313A1 WO 2011017313 A1 WO2011017313 A1 WO 2011017313A1 US 2010044234 W US2010044234 W US 2010044234W WO 2011017313 A1 WO2011017313 A1 WO 2011017313A1
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WIPO (PCT)
Prior art keywords
nucleic acid
viral nucleic
retinal
eye
compacted
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PCT/US2010/044234
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French (fr)
Inventor
Mark J. Cooper
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Copernicus Therapeutics Inc.
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Publication of WO2011017313A1 publication Critical patent/WO2011017313A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008Medicinal 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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025Medicinal 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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091Purification or manufacturing processes for gene therapy compositions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents

Definitions

  • the invention relates to the field of gene therapy. In particular, it relates to the field of ocular gene therapy.
  • subretinal injections have been used with both viral vectors and with non- viral vectors, such as nanoparticles.
  • Subretinal injection is a technique whereby a needle is directly placed in the deep retina in a potential space between the photoreceptor and retinal pigment epithelial (RPE) cell layers (Fig. 1).
  • RPE retinal pigment epithelial
  • subretinal injections are invasive, operative procedures in humans that cause retinal detachment, which usually reattaches within several days to a week.
  • the retinal detachment process can induce apoptosis in retinal cells from subjects having retinal diseases, such as retinitis pigmentosa.
  • retinal diseases such as retinitis pigmentosa.
  • intravitreal injection is a less invasive procedure that can be performed in a physician's office, and is far less likely to cause retinal detachment.
  • the ophthalmologist introduces a needle into the vitreous humor, which is contained in the posterior chamber between the lens and the retina (Fig. 1).
  • nucleic acid vectors evaluated to date including viral vectors such as adeno associated virus (AAV), do not transduce RPE cells following intravitreal injection, and photoreceptor transduction is limited (Hellstr ⁇ m, Petrs-Silva).
  • An aspect of the invention is a method of delivering nucleic acids to retinal pigment epithelium.
  • An effective dose of a non-viral nucleic acid is administered by intravitreal injection to an eye.
  • the non-viral nucleic acid encodes an RNA and/or protein product.
  • Retinal pigment epithelial cells are thereby transfected and express the RNA and/or protein product.
  • Another aspect of the invention is a single dose vial for delivery of the dose intravitreally to an eye.
  • the single dose is 0.05 to 0.5 ml of a non-viral nucleic acid which encodes an RNA and/or protein product.
  • the nucleic acid is compacted to achieve a minor dimension of less than or equal to 30 nm. Transcription of the product is controlled by a tissue- or cell type- specific promoter. The promoter is operably linked to the nucleic acid which encodes the product.
  • kits comprises a single dose vial for delivery of the dose intravitreally to an eye.
  • the single dose is 0.05 to 0.5 ml of a non-viral nucleic acid which encodes an RNA and/or protein product.
  • the nucleic acid is compacted to achieve a minor dimension of less than 30 nm. Transcription of the product is controlled by a retinal pigment epithelium-specific promoter. The promoter is operably linked to the nucleic acid which encodes the product.
  • the kit further comprises a filter needle and an injection needle.
  • the kit optionally comprises a product insert which details methods of administration, and contraindications.
  • Another aspect of the invention is a method of delivering nucleic acid to retinal pigment epithelium or retinal photoreceptor cells.
  • An effective dose of a non-viral nucleic acid is administered by intravitreal injection to an eye.
  • the non-viral nucleic acid is an siRNA.
  • Retinal pigment epithelium cells or retinal photoreceptor cells are transfected by the siRNA.
  • Yet another aspect of the invention is a method of delivering nucleic acid to retinal photoreceptor cells.
  • An effective dose of a non- viral nucleic acid is administered by intravitreal injection to an eye.
  • the non-viral nucleic acid encodes an RNA and/or protein product.
  • Retinal photoreceptor cells are trans fected and express a detectable amount of the RNA and/or protein product.
  • Still another aspect of the invention is a method of treating an ocular disease.
  • An effective dose of a non-viral nucleic acid is administered to an eye by intravitreal injection.
  • the non-viral nucleic acid encodes or is a therapeutic product. Vision by the eye is improved due to the administration.
  • Fig. 1 Diagram of retinal architecture, including deep (posterior) cell layers of retinal pigment epithelial (RPE) cells, and rod and cone photoreceptor cells.
  • Light passes through superficial (anterior) retinal layers, including ganglion, amacrine, bipolar, and horizontal cells, and is absorbed by various opsins in rods and cones, with subsequent electrical signals conveyed to ganglion cells, whose axonal processes constitute the optic nerve.
  • the posterior eye chamber between the lens and retina, contains vitreous humor fluid. The sites of subretinal and intravitreal injections are noted.
  • Fig. 2 Transmission electron micrograph of unimolecularly compacted pVMD_Y2 DNA nanoparticles formulated with CK3 OPEG 10k having an acetate counterion at the time of DNA mixing. Nanoparticles were concentrated to approximately 4 mg/ml of DNA with solvent exchange to normal saline. Bar indicates 200 nm.
  • FIG. 3A-3B Detection of EYFP protein in deep retinal RPE cells following intravitreal dosing. Shown are EYFP direct fluorescent images of retinal flat mounts from Balb/c mice dosed with approximately 1 ⁇ l volumes ( ⁇ 4 ⁇ g of pVMD_Y2 DNA nanoparticles) administered either subretinally (Fig. 3A) or intravitreally (Fig. 3B).
  • FIG. 4 Immunohistochemical localization of EYFP protein in RPE cells. Shown are retinal cross sections of Balb/c mouse eyes dosed intravitreally with ⁇ 4 ⁇ g pVMD_Y4 DNA nanoparticles or phosphate-buffered saline. Eyes were harvested 21 days post-injection. Primary antibody was a rabbit polyclonal anti-EYFP antibody (Santa Cruz 32897). Arrow indicates RPE cell layer in nanoparticle dosed eyes. Detection of EYFP protein was localized specifically in RPE cells. [16] Fig. 5.
  • FIG. 6A-6B Transmission electron micrographs of unimolecularly compacted pVMD_luc4 DNA nanoparticles formulated with CK3 OPEG 10k having either a trifluoroacetate (Fig. 6A) or an acetate (Fig. 6B) counterion at the time of DNA mixing. Nanoparticles were concentrated to approximately 4 mg/ml of DNA with solvent exchange to normal saline. The nanoparticles in (Fig. 6A) are ellipsoids and some short rod forms, whereas the nanoparticles in (Fig. 6B) are longer rods. Bar indicates 200 nm.
  • FIG. 7 Intravitreal dose response curves comparing ellipsoidal and rod-like DNA nanoparticles.
  • the pVMD_luc4 DNA nanoparticles shown in Figures 6A and 6B were administered intravitreally at a dose of approximately 1, 3, or 8 ⁇ g. Eyes were harvested 3 days post injection and eye lysates were assayed for luciferase activity (expressed as RLU/mg protein). The 7.1 ⁇ g dose of ellipsoids was significantly different than each of the other DNA nanoparticle groups (1 way ANOVA with Bonferroni's multiple comparison test, *).
  • FIG. 8 Ocular retinyl ester levels in lrat -I- mice 14 days after either subretinal or intravitreal dosing with compacted pVMD_lrat DNA nanoparticles.
  • FIG. 9 Electroretinogram (ERG) analysis of 6 week old lrat -I- mice dosed with pVMD_lrat3 DNA nanoparticles. Shown are representative ERGs in lrat -I- mice dosed 1 week after a subretinal injection or 1 month after an intravitreal injection of compacted DNA. For comparison is an ERG from an lrat -I- mouse 1 week after a subretinal dose of saline, which demonstrates minor ERG signal. In contrast, substantial electrical activity is noted in the lrat -I- mouse eyes dosed with pVMD Irat3 DNA nanoparticles. As a positive control, an age- matched wild type eye ERG (no injection) is also shown.
  • ERG Electroretinogram
  • RPE retinal pigment epithelium
  • photoreceptor cells by non- viral vectors.
  • One suitable form for delivery of non- viral vectors is as nanoparticles.
  • the nanoparticles may be formed by condensation between the nucleic acids and polycations.
  • One suitable form which can be used is a unimolecularly-compacted DNA nanoparticle.
  • the effective transfection and expression of non-viral vectors is unexpected because intravitreal injection of viral vectors has been shown to be ineffective for transfection of RPE.
  • Intravitreal injections of non- viral vectors can be used to treat retinitis pigmentosa and other diseases of RPE cells and photoreceptors. In experimental treatments, improved vision has resulted in as little as 1 week.
  • Such methods can be used to also transfect and obtain expression in retinal photoreceptor cells. Moreover, such methods are also useful for delivering nucleic acids which are non-coding but are themselves therapeutic, such as siRNA or antisense RNA. When therapeutic products are produced in the eye, improvement in the function of diseased eyes has been observed.
  • Doses per eye or per injection may be at least O.Olug, at least 0.1 ug, at least 1 ug, at least 10 ug, at least 10 2 ug, at least 10 3 ug, at least 2 x 10 3 ug, at least 4 x 10 3 ug, at least ⁇ x lO 3 ug, at least 8 x 10 3 ug, at least 10 4 ug, and up to about 10 5 ug of nucleic acid or nanoparticle. Dosings may be divided into multiple injections, or injections may be repeated, for example, if expression of the delivered gene declines over time. Volume that can be added to the vitreous humor is limited.
  • Typical injection volumes may be from 0.05, from 0.06, from 0.07, from 0.08, or from 0.09 up to 0.1, to 0.2, to 0.3, to 0.4, to 0.5 ml, to 1 ml, or to 5 ml.
  • a fine gauge needle can be used such as a 30 gauge, Vi inch for intravitreal injections. Other fine gauge needles can be used.
  • the volume of vitreous humor in a mouse eye is believed to be between about 3 and 5 ul.
  • the volume of vitreous humor in a human eye is believed to be between about 3 and 5 ml.
  • Other species may be treated as well, and the volumes of vitreous humor in their eyes may vary. If the additional volume necessary to treat is too great, then endogenous vitreous humor may be withdrawn and replaced with the volume of the dosing.
  • Posterior ocular conditions which can be treated according to the invention include acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; retinal
  • neovascularization diabetic uveitis; histoplasmosis; infections, such as bacterial, fungal or viral-caused infections; macular degeneration, such as acute macular degeneration, non- exudative age related macular degeneration and exudative age related macular degeneration; edema, such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma which affects a posterior ocular site or location; ocular tumors;
  • retinal disorders such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic opthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy, photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; other forms of optic neuropathy and optic neuritis; non-retinopathy diabetic retinal dysfunction; retinitis pigmentosa; and glaucoma.
  • Glaucoma can be considered a posterior ocular condition because the therapeutic goal is to prevent the loss of or reduce the occurrence of loss of vision due to damage to or
  • ocular disorders, conditions, and diseases that can be treated using the methods of the present invention are severe visual impairment (i.e., blindness), including diseases related to degeneration of cells of the retina and macula, including, but not limited to, Usher syndrome, Stargardt disease, Bardet-Biedl syndrome, Best disease, choroideremia, gyrate-atrophy, retinitis pigmentosa, macular degeneration, Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), Blue-cone monochromacy, retinoschisis, Malattia Leventinese, Oguchi Disease, or Refsum disease, or other diseases related to impairment of the function of the retina or macula.
  • severe visual impairment i.e., blindness
  • diseases related to degeneration of cells of the retina and macula including, but not limited to, Usher syndrome, Stargardt disease, Bardet-Biedl syndrome, Best disease, choroideremia, gyrate-atrophy, retin
  • Macular degeneration disorders may include but are not limited to any of a number of conditions in which the retinal macula degenerates or becomes dysfunctional, e.g., as a consequence of decreased growth of cells of the macula, increased death or rearrangement of the cells of the macula (e.g., RPE cells), loss of normal biological function, or a combination of these events such as North Carolina macular dystrophy, Sorsby's fundus dystrophy, pattern dystrophy, dominant drusen, and any condition which alters or damages the integrity or function of the macula (e.g., damage to the RPE or Bruch's membrane).
  • macular degeneration may involve retinal detachment, chorioretinal degenerations, retinal
  • the methods disclosed herein for delivering nucleic acids to the eye via non- viral nanoparticles may be used to treat or prevent ocular diseases or conditions, such as the following: maculopathies and retinal degeneration; macular degeneration, including age related macular degeneration (AMD), such as non-exudative age related macular degeneration and exudative age related macular degeneration; choroidal neovascularization; retinal
  • retinopathy including diabetic retinopathy, acute and chronic macular neuroretinopathy; central serous chorioretinopathy, and macular edema, including cystoid macular edema, and diabetic macular edema
  • uveitis retinitis; choroiditis; acute multifocal placoid pigment epitheliopathy; Behcet's disease; birdshot retinochoroidopathy; infectious (syphilis, lyme, tuberculosis, toxoplasmosis) uveitis; intermediate uveitis (pars planitis) and anterior uveitis; multifocal choroiditis; multiple evanescent white dot syndrome (MEWDS); ocular sarcoidosis; posterior scleritis; serpignous choroiditis; subretinal fibrosis; uveitis syndrome; Vogt-Koyanagi-Harada syndrome;
  • Stargardt's disease and fundus flavimaculatus Bests disease, pattern dystrophy of the retinal pigmented epithelium, X-lmked retinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti's crystalline dystrophy, and pseudoxanthoma elasticum; retinal tears/holes including retinal detachment, macular hole, and giant retinal tear; tumors including retinal disease associated with tumors, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined hamartoma of the retina and retinal pigmented epithelium, retinoblastoma, vasoproliferative tumors of the ocular fundus, retinal astrocytoma, intraocular lymphoid tumors; and miscellaneous conditions including punctate inner choroidopathy, acute posterior multifo
  • the invention may employ nucleic acid nanoparticles comprising one or more of the genes CA4, CRX, FSCN2, GUCAlB, IMPDHl, NR2E3, NRL, PRPF3, PRPF8, PRPF31, PRPH2, RHO, ROMl, RPl, RP9, SEMA4A, TOPORS, ABCA4, CERKL, CNGAl, CNGBl, CRBl, LRAT, MERTK, NRL, PDE6A, PDE6B, PRCD, PROMl, RGR, RLBPl, RPl, RPE65, SAG, TULPl, USH2A, RP2, and RPGR for use in treating autosomal dominant, autosomal recessive, or X-linked forms of retinitis pigmentosa.
  • These nucleic acids may be used to express other genes in other genotypes of retinitis pigmentosa not listed above.
  • the nucleic acids can also be used in other forms and formulations
  • nucleic acids which may be used in nanoparticles are one or more of genes
  • Genes which provide a supportive function, but do not replace a defective gene may be used.
  • Genes whose protein products inhibit neovascularization or anti-sense nucleic acids that inhibit expression of genes that stimulate neovascularization may be used.
  • Anti-sense nucleic acids that inhibit expression of genes that stimulate neovascularization may be used.
  • oligonucleotides, anti-sense plasmid constructs, siRNA, and shRNA plasmids can be used to provide a desired therapeutic effect.
  • the nucleic acids may be coding or non-coding.
  • the therapeutic product may be a nucleic acid or a protein.
  • Expression of the therapeutic product or persistence of the therapeutic product in the eye can be monitored once or multiple times.
  • the monitoring may be used to determine, e.g., if a sufficient dosing has been achieved or to determine if an additional dosing is required after passage of time.
  • a patient may be treated therapeutically or prophylactically by administering the compacted nucleic acid nanoparticles to the patient by at least one of intravitreal placement, subretinal placement, subconjuctival placement, conjuctival placement, anterior chamber placement, episcleral placement, sub-tenon placement, retrobulbar placement, suprachoroidal placement, and systemic injection via intravenous and/or intraarterial administration.
  • Placement methods may include injection and/or surgical insertion.
  • the compacted nucleic acid nanoparticle is administered via intravitreal injection.
  • the amount of nucleic acid per dosage is provided to the subject's eye at a concentration of 0.01 ug/ul to 15 ug/ul, depending on the desired level of expression in the ocular cells.
  • Individual dosage volumes may range (in non-limiting examples) for example from 1 ul to 5000 ul, or from 10 ul to 1000 ul.
  • the total amount of DNA administered per eye may be at least 1 ug, at least 3 ug, at least 8 ug, at least 10 ug, at least 12 ug, at least 15 ug, at least 20 ug, at least 50 ug, at least 100 ug, at least 500 ug, at least 1,000 ug, at least 5,000 ug, at least 10.000 ug, at least 50,000 ug.
  • the nucleic acid nanoparticles may be provided in a composition comprising any pharmaceutically acceptable carrier, such as a saline solution (e.g., PBS). Depending on the dose administered, a volume of vitreous humor may be removed to improve tolerance of the injected volume containing the nanoparticles.
  • a subject having an ocular disorder is treated by providing a compacted nucleic acid nanoparticle having a minor dimension equal to or below 30 nm or below 25 nm and a nucleic acid non-covalently linked to a cationic polymeric material.
  • the minor dimension may even be below 20, 15, or 11 nm as measured by transmission electron microscopy.
  • the compacted nanoparticle is administered to a tissue of the eye of the patient for treating the ocular disorder.
  • the ocular condition or disorder to be treated is related to retinal and macular degeneration, Usher syndrome, Stargardt disease, Bardet-Biedl syndrome, Best disease, choroideremia, gyrate-atrophy, retinitis pigmentosa, Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), various types of optic neuropathy and optic neuritis, Blue-cone monochromacy, retinoschisis, Malattia Leventinese, Oguchi Disease, and Refsum disease, retinal detachment, chorioretinal degenerations, retinal degenerations, photoreceptor degenerations, degeneration of the retinal pigment epithelium, mucopolysaccharidoses, rod-cone dystrophies, cone -rod dystrophies, cone degenerations, conditions involving decreased growth of cells of the macula, increased death or rearrangement of the retinal pigment epithelial cells of the macula, North Carolina macular dys
  • Any gene may be used that will be useful in the retina. These may encode at least one of opsin protein of rhodopsin (RHO), cyclic GMP phosophodiesterase ⁇ -subunit (PDE6A) or ⁇ - subunit (PDE6B), the ⁇ subunit of the rod cyclic nucleotide gated channel (CNGAl), RPE65, RLBPl, ABCR, ABCA4, CRBl, LRAT, CRX, IPl, EFEMPl, peripherin/RDS, ROMl, arrestin (SAG), ⁇ -transducin (GNATl), rhodopsin kinase (RHOK), guanylate cyclase activator IA (GUCAlA), retina specific guanylate cyclase (GUCY2D), the ⁇ subunit of the cone cyclic nucleotide gated cation channel (CNGA3), and cone opsins BCP, GCP, and
  • genes may also be used including those encoding ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), pigment epithelium-derived factor (PEDF), and ORE 15 variant of Retinitis Pigmentosa GTPase Regulator (RPGR).
  • CNTF ciliary neurotrophic factor
  • BDNF brain derived neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • PEDF pigment epithelium-derived factor
  • RPGR Retinitis Pigmentosa GTPase Regulator
  • genes which are related to macular degeneration include CFH (Complement Factor H), CFB (Complement Factor B), ABCR and ACBA4, C2 (Complement Component 2), C3 (Complement Component 3), HTRAl, T2-TrpRS, and RdCVF; any of these alone or in combination may be used in nanoparticles to
  • Nucleic acid nanoparticles can be made, for example, according to the methods disclosed in Hanson, Perales, and/or Cooper. See U.S. 6506890, 5877302, 5844107, the disclosures of which are expressly incorporated here. Nanoparticles may be 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 11 nm or less, as measured by transmission electron microscopy.
  • Nucleic acids may optionally contain a plasmid origin of replication. Nucleic acids may optionally contain coding sequences for RNA and/or protein products that are transcribed under the control of promoters that are cell-type or tissue-type specific.
  • One embodiment of the invention utilizes the promoter from the VMD gene, which is specifically expressed in the retinal pigment epithelium. Promoters that are specific for rods or cones may be used as well. Types of RNA molecules which may be used include siRNA.
  • Antisense oligonucleotides or antisense-producing constructs may be used as well, including DNA plasmids transcribing shRNA.
  • shRNA small hairpin RNA
  • shRNA small hairpin RNA
  • shRNA is a transcript that is processed by the cell to siRNA. The siRNA inhibits expression of a target mRNA.
  • Nucleic acids of the invention may be packaged in any suitable manner known in the art. They may be packaged in single dose vials, which are typically aseptic. The vials may be provided in a kit with additional components to facilitate the successful delivery to the eye. Additional components include an injection needle, a filter needle, a 1-cc tuberculin syringe, and a sterile, disposable eyelid speculum. Instructions and other information may be provided on a package insert, for example. Suitable types of needles for injection and for preparation include 30 gauge, Vi inch for injection and 19 gauge, 5 micron for preparation and withdrawal from a vial.
  • Improvement in the symptoms or phenotype of an ocular disease can be monitored in any way that the disease is usually assessed. These may involve neurological assessments, behavioral assessments, electronic assessments, reading or object recognition, imaging assessments, etc. Assessments may be combined. Assessments may be carried out once or many times to determine the trajectory of the disease process and/or the therapeutic process.
  • Example l ⁇ Intravitreal dosing of compacted DNA nanoparticles transfects the deep retina, including retinal pigment epithelial (RPE) cells.
  • RPE retinal pigment epithelial
  • Figure 1 presents a diagram of the retina, including deep retinal layers (retinal pigment epithelial cells, rod and cone photoreceptor cells), and location of injection sites for subretinal and intravitreal delivery.
  • Subretinal dosing of viral vectors, such as AAV transduces RPE and photoreceptor cells
  • intravitreal dosing of AAV fails to express well in photoreceptors and not at all in RPE cells (Hellstr ⁇ m, Petrs-Silva) in wild-type mice and most forms of retinitis pigmentosa evaluated to date.
  • AAV vectors to have improved transduction of photoreceptors in one type of retinitis pigmentosa, X- linked juvenile retinoschisis, where microcysts and internal retinal dissection disturbs the normal lamination of various neuronal and plexiform layers (Park).
  • proteolytic enzymes such as pronase
  • Delivery in the vitreous humor can facilitate deep retina transduction (Dalkara), although the use of such proteolytic enzymes in subjects with retinitis pigmentosa and other ocular diseases has unknown and potential deleterious side effects.
  • pVMD_Y2 enhanced yellow fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • Plasmid pVMD_Y2 was unimolecularly compacted with CK3 OPEG 10k polycation, a 30-mer lysine peptide with an N-terminal cysteine that is covalently modified via a S-C bond with 10 kDa polyethylene glycol (PEGlOk) having a maleimide reactive group.
  • Polycation preparation and the DNA compaction protocols were conducted essentially as previously published (Liu, Fink, Method of Nucleic Acid Compaction US 6,506,890). After compaction, the compacted DNA was processed with tangential flow filtration to remove excess uncompacted polycation, exchange water solvent with normal saline, and to concentrate the DNA nanoparticles to approximately 4 mg/ml of DNA.
  • the final compacted DNA preparation was then evaluated with a series of quality control assays, including transmission electron microscopy, turbidity parameter analysis, gel analysis, sedimentation analysis, serum stability assays, and other studies as previously described (Liu, Fink, Ziady, Konstan).
  • the compacted DNA met or exceeded all quality control assay end-release specifications, indicating compaction of the DNA, colloidal stability of the DNA nanoparticles in normal saline, and stability of the compacted DNA in DNAse-rich solutions.
  • FIG. 2 shows an image of an electron micrograph of the compacted pVMD_Y2 nanoparticles in saline, which are compacted into rod-like shapes by using CK30PEG10k with an acetate lysine counterion prior to mixing of the polycation with DNA (see Fink et al. for discussion of rod-like nanoparticle dimensions).
  • pVMD_Y2 nanoparticles were dosed subretinally and intravitreally into wild-type Balb/c mice. Approximately 4 ug was administered to the mice dosed intravitreally. The DNA concentration in the vitreous humor would be about 0.8 ug/ul, assuming a typical 5 ul volume of vitreous humer per mouse eye. Two days post injection, eyes were harvested, retinal flat mounts were prepared, and eyes were imaged for EYFP direct fluorescence.
  • Fig. 3 A shows robust EYFP expression in RPE cells at the site of subretinal injection, which is an expected result for these nanoparticles. Following intravitreal injection, significant EYFP fluorescence is also observed in RPE cells, as shown in Fig. 3B.
  • EYFP signal was observed in multiple retinal quadrants. Similar data were observed 21 days post injection. These data provide evidence that intravitreal injection of compacted DNA nanoparticles can result in deep retinal penetration and transfection of RPE cells.
  • pVMD_Y2 that contains a CMV enhancer to increase promoter activity, pVMD_Y4
  • pVMD_Y4 was compacted and dosed intravitreally, and cross-sections of retinas were evaluated at day 21 for EYFP expression by immunohistochemistry using a rabbit polyclonal anti-EYFP antibody (Santa Cruz 32897). Animals dosed intravitreally with phosphate-buffered saline served as negative controls.
  • mice were injected either intravitreally or subretinally with 4.5 ⁇ g DNA nanoparticles encoding a hVMD2 promoter transcriptionally-controlling expression of the firefly luciferase transgene (pVMD_luc4).
  • Non-dosed eyes served as a negative control.
  • eyes were harvested, lysates prepared, and luciferase activity measured using a chemiluminescent assay (Promega).
  • Fig. 5 shows these results.
  • RPE transfection following intravitreal dosing requires DNA nanoparticles to diffuse through various retinal cell layers to reach RPE cells (Fig. 1). Since the size and shape of DNA nanoparticles might influence this process, compacted DNA nanoparticles containing pVMD_luc4 were compacted as rods or ellipsoids (Fig. 6). The rods were prepared utilizing CK30PEG10k polycation having acetate as the lysine counterion prior to DNA mixing (Fig. 6B), whereas to form ellipsoids the lysine counterion was trifluoroacetate (TFA) (Fig. 6A).
  • Fig. 7 shows a dose response luciferase activity analysis comparing intravitreal dosing of rod and ellipsoidal DNA nanoparticle formulations at approximately 1, 3, or 8 ⁇ g of DNA at day 3 post-injection.
  • Lyophilizable and Enhanced Compacted Nucleic Acids US2004/0048787), use of positively charged polymers that do not constitute amino acids, the percentage of PEG substitution in the polymer, the size of PEG used in the polycation, and whether the PEG moieties are added to the nanoparticles pre- or post-compaction.
  • Other factors that may influence gene transfer include whether the nanoparticles incorporate cell targeting ligands, use of various excipients that may influence nanoparticle stability and/or diffusion, dose escalation in the vitreous humor (including removal of vitreous humor fluid prior to and/or during nanoparticle dosing), and the specific administration procedure used to deliver compacted DNA to the vitreous humor.
  • Example 4 ⁇ Intravitreal dosing of compacted DNA can improve the phenotype of retinitis pigmentosa mice.
  • RP mice retinitis pigmentosa mice having knock-out of the lecithin retinol acyltransferase gene ⁇ Lrat -I- mice).
  • the LRAT enzyme converts all-trans retinol into all-trans retinyl esters, a key step in the vitamin A regeneration cycle, which is required for generating the active form of vitamin A, 11-cis retinal, which is required for vision.
  • Lrat -I- mice have depleted vitamin A chromophore intermediates, including depleted of 11 cis-retinal, and experience significant visual impairment at an early age.
  • Lrat -I- mice at 6 weeks of age were dosed either intravitreally or subretinally with compacted DNA nanoparticles encoding a RPE-restricted wild-type LRAT gene, pVMD lrat.
  • lrat -I- mice were evaluated for various parameters related to phenotypic correction, including retinal retinoid levels and electroretinograms (ERGs).
  • the electroretinogram measures electrical activity in the retina following light exposure, and is a surrogate marker for vision.
  • Figure 8 shows ocular retinyl ester levels in lrat -I- mice 14 days after dosing with compacted pVMD_lrat DNA nanoparticles. In contrast to PBS dosed lrat -I- mice, which have no detectable retinyl esters, these mice have increased levels of retinyl esters following either subretinal or intravitreal dosing. These data demonstrate that intravitreal dosing of compacted DNA in RP mice can improve their ocular phenotype.
  • lrat -I- mice received subretinal or intravitreal doses of compacted DNA containing a derivative of pVMD_lrat incorporating a CMV enhancer, pVMD_lrat3.
  • Figure 9 are ERG a and b wave tracings 1 week following subretinal dosing and 1 month after intravitreal dosing of compacted pVMD_lrat3 nanoparticles.
  • Lrat -I- mice dosed with saline showed essentially no retinal electrical activity after light exposure
  • intravitreal dosing of DNA nanoparticles can improve the visual phenotype of lrat -I- mice, and suggest that intravitreal dosing of compacted DNA may be effective in other genotypes of RP.
  • AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs 1 -

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Abstract

Intravitreal administration of nucleic acid vectors leads to transfection and expression of the nucleic acid vectors in retinal pigment epithelium. The nucleic acid is non-viral and may be compacted to form a unimolecular nanoparticle with a polycationic polymer. The nucleic acid vectors can be used to supply a normal copy of a gene which is defective in the recipient, to inhibit a deleterious cellular product, or to supply a beneficial product to improve the symptoms or natural history of the disease.

Description

METHOD OF ADMINISTERING NON-VIRAL NUCLEIC ACID VECTORS TO THE EYE
[01] This application claims the benefit of priority to U.S. 61/231,179 filed August 4, 2009, the disclosure of which is expressly incorporated herein.
Technical Field of the Invention
[02] The invention relates to the field of gene therapy. In particular, it relates to the field of ocular gene therapy.
Background of the Invention
[03] Previously, subretinal injections have been used with both viral vectors and with non- viral vectors, such as nanoparticles. Subretinal injection is a technique whereby a needle is directly placed in the deep retina in a potential space between the photoreceptor and retinal pigment epithelial (RPE) cell layers (Fig. 1). However, subretinal injections are invasive, operative procedures in humans that cause retinal detachment, which usually reattaches within several days to a week. Moreover, the retinal detachment process can induce apoptosis in retinal cells from subjects having retinal diseases, such as retinitis pigmentosa. Thus, even though these injections are successful in accessing the deep retina, including the photoreceptor cells and retinal pigment epithelium, their negative effects are too severe to disregard.
[04] In contrast, intravitreal injection is a less invasive procedure that can be performed in a physician's office, and is far less likely to cause retinal detachment. In this technique, the ophthalmologist introduces a needle into the vitreous humor, which is contained in the posterior chamber between the lens and the retina (Fig. 1). Although intravitreal injection of antibodies, such as the anti-VEGF antibody Lucentis™ (Genentech), is effective in penetrating into the deep retina, nucleic acid vectors evaluated to date, including viral vectors such as adeno associated virus (AAV), do not transduce RPE cells following intravitreal injection, and photoreceptor transduction is limited (Hellstrόm, Petrs-Silva). In addition, various studies have documented that introduction of non- viral vectors into the vitreous humor has limited effectiveness and that the vitreous humor fluid can destabilize these vectors (Pitkanen L). [05] There is a continuing need in the art for safe and efficient means of delivering nucleic acids to the retinal pigment epithelium.
Summary of the Invention
[06] An aspect of the invention is a method of delivering nucleic acids to retinal pigment epithelium. An effective dose of a non-viral nucleic acid is administered by intravitreal injection to an eye. The non-viral nucleic acid encodes an RNA and/or protein product.
Retinal pigment epithelial cells are thereby transfected and express the RNA and/or protein product.
[07] Another aspect of the invention is a single dose vial for delivery of the dose intravitreally to an eye. The single dose is 0.05 to 0.5 ml of a non-viral nucleic acid which encodes an RNA and/or protein product. The nucleic acid is compacted to achieve a minor dimension of less than or equal to 30 nm. Transcription of the product is controlled by a tissue- or cell type- specific promoter. The promoter is operably linked to the nucleic acid which encodes the product.
[08] Another aspect of the invention is a kit. The kit comprises a single dose vial for delivery of the dose intravitreally to an eye. The single dose is 0.05 to 0.5 ml of a non-viral nucleic acid which encodes an RNA and/or protein product. The nucleic acid is compacted to achieve a minor dimension of less than 30 nm. Transcription of the product is controlled by a retinal pigment epithelium-specific promoter. The promoter is operably linked to the nucleic acid which encodes the product. The kit further comprises a filter needle and an injection needle. The kit optionally comprises a product insert which details methods of administration, and contraindications.
[09] Another aspect of the invention is a method of delivering nucleic acid to retinal pigment epithelium or retinal photoreceptor cells. An effective dose of a non-viral nucleic acid is administered by intravitreal injection to an eye. The non-viral nucleic acid is an siRNA. Retinal pigment epithelium cells or retinal photoreceptor cells are transfected by the siRNA.
[10] Yet another aspect of the invention is a method of delivering nucleic acid to retinal photoreceptor cells. An effective dose of a non- viral nucleic acid is administered by intravitreal injection to an eye. The non-viral nucleic acid encodes an RNA and/or protein product. Retinal photoreceptor cells are trans fected and express a detectable amount of the RNA and/or protein product.
[11] Still another aspect of the invention is a method of treating an ocular disease. An effective dose of a non-viral nucleic acid is administered to an eye by intravitreal injection. The non-viral nucleic acid encodes or is a therapeutic product. Vision by the eye is improved due to the administration.
Brief Description of the Drawings
[12] Fig. 1. Diagram of retinal architecture, including deep (posterior) cell layers of retinal pigment epithelial (RPE) cells, and rod and cone photoreceptor cells. Light passes through superficial (anterior) retinal layers, including ganglion, amacrine, bipolar, and horizontal cells, and is absorbed by various opsins in rods and cones, with subsequent electrical signals conveyed to ganglion cells, whose axonal processes constitute the optic nerve. The posterior eye chamber, between the lens and retina, contains vitreous humor fluid. The sites of subretinal and intravitreal injections are noted.
[13] Fig. 2. Transmission electron micrograph of unimolecularly compacted pVMD_Y2 DNA nanoparticles formulated with CK3 OPEG 10k having an acetate counterion at the time of DNA mixing. Nanoparticles were concentrated to approximately 4 mg/ml of DNA with solvent exchange to normal saline. Bar indicates 200 nm.
[14] Fig. 3A-3B. Detection of EYFP protein in deep retinal RPE cells following intravitreal dosing. Shown are EYFP direct fluorescent images of retinal flat mounts from Balb/c mice dosed with approximately 1 μl volumes (~4 μg of pVMD_Y2 DNA nanoparticles) administered either subretinally (Fig. 3A) or intravitreally (Fig. 3B).
[15] Fig. 4. Immunohistochemical localization of EYFP protein in RPE cells. Shown are retinal cross sections of Balb/c mouse eyes dosed intravitreally with ~4 μg pVMD_Y4 DNA nanoparticles or phosphate-buffered saline. Eyes were harvested 21 days post-injection. Primary antibody was a rabbit polyclonal anti-EYFP antibody (Santa Cruz 32897). Arrow indicates RPE cell layer in nanoparticle dosed eyes. Detection of EYFP protein was localized specifically in RPE cells. [16] Fig. 5. Quantitative comparison of RPE-based luciferase transgene activity following subretinal or intravitreal dosing of Balb/c mouse eyes with approximately 4.5 μg of compacted pVMD_Luc4 DNA nanoparticles. Eyes were harvest 159 days post injection and then evaluated for luciferase activity in eye lysates (expressed as log RLU/mg protein). There was a non-significant (NS) 4.5 -fold difference between animals dosed subretinally or intravitreally (t- test, p2=0.066). Both DNA nanoparticle dosed eye groups were significantly different than the non-dosed groups.
[17] Fig. 6A-6B. Transmission electron micrographs of unimolecularly compacted pVMD_luc4 DNA nanoparticles formulated with CK3 OPEG 10k having either a trifluoroacetate (Fig. 6A) or an acetate (Fig. 6B) counterion at the time of DNA mixing. Nanoparticles were concentrated to approximately 4 mg/ml of DNA with solvent exchange to normal saline. The nanoparticles in (Fig. 6A) are ellipsoids and some short rod forms, whereas the nanoparticles in (Fig. 6B) are longer rods. Bar indicates 200 nm.
[18] Fig. 7. Intravitreal dose response curves comparing ellipsoidal and rod-like DNA nanoparticles. The pVMD_luc4 DNA nanoparticles shown in Figures 6A and 6B were administered intravitreally at a dose of approximately 1, 3, or 8 μg. Eyes were harvested 3 days post injection and eye lysates were assayed for luciferase activity (expressed as RLU/mg protein). The 7.1 μg dose of ellipsoids was significantly different than each of the other DNA nanoparticle groups (1 way ANOVA with Bonferroni's multiple comparison test, *).
[19] Fig. 8. Ocular retinyl ester levels in lrat -I- mice 14 days after either subretinal or intravitreal dosing with compacted pVMD_lrat DNA nanoparticles.
[20] Fig. 9. Electroretinogram (ERG) analysis of 6 week old lrat -I- mice dosed with pVMD_lrat3 DNA nanoparticles. Shown are representative ERGs in lrat -I- mice dosed 1 week after a subretinal injection or 1 month after an intravitreal injection of compacted DNA. For comparison is an ERG from an lrat -I- mouse 1 week after a subretinal dose of saline, which demonstrates minor ERG signal. In contrast, substantial electrical activity is noted in the lrat -I- mouse eyes dosed with pVMD Irat3 DNA nanoparticles. As a positive control, an age- matched wild type eye ERG (no injection) is also shown. Detailed Description of the Invention
[21] It is a discovery of the inventor that intravitreal injection is effective for transfection of retinal pigment epithelium (RPE) and photoreceptor cells by non- viral vectors. One suitable form for delivery of non- viral vectors is as nanoparticles. The nanoparticles may be formed by condensation between the nucleic acids and polycations. One suitable form which can be used is a unimolecularly-compacted DNA nanoparticle. The effective transfection and expression of non-viral vectors is unexpected because intravitreal injection of viral vectors has been shown to be ineffective for transfection of RPE.
[22] Intravitreal injections of non- viral vectors can be used to treat retinitis pigmentosa and other diseases of RPE cells and photoreceptors. In experimental treatments, improved vision has resulted in as little as 1 week.
[23] Additionally, such methods can be used to also transfect and obtain expression in retinal photoreceptor cells. Moreover, such methods are also useful for delivering nucleic acids which are non-coding but are themselves therapeutic, such as siRNA or antisense RNA. When therapeutic products are produced in the eye, improvement in the function of diseased eyes has been observed.
[24] Doses per eye or per injection may be at least O.Olug, at least 0.1 ug, at least 1 ug, at least 10 ug, at least 102 ug, at least 103 ug, at least 2 x 103 ug, at least 4 x 103 ug, at least ό x lO3 ug, at least 8 x 103 ug, at least 104 ug, and up to about 105 ug of nucleic acid or nanoparticle. Dosings may be divided into multiple injections, or injections may be repeated, for example, if expression of the delivered gene declines over time. Volume that can be added to the vitreous humor is limited. In some cases some of the natural volume can be reduced by withdrawing some of the vitreous humor prior to or during injection. Typical injection volumes may be from 0.05, from 0.06, from 0.07, from 0.08, or from 0.09 up to 0.1, to 0.2, to 0.3, to 0.4, to 0.5 ml, to 1 ml, or to 5 ml. As is known in the art a fine gauge needle can be used such as a 30 gauge, Vi inch for intravitreal injections. Other fine gauge needles can be used. The volume of vitreous humor in a mouse eye is believed to be between about 3 and 5 ul. The volume of vitreous humor in a human eye is believed to be between about 3 and 5 ml. Other species may be treated as well, and the volumes of vitreous humor in their eyes may vary. If the additional volume necessary to treat is too great, then endogenous vitreous humor may be withdrawn and replaced with the volume of the dosing.
[25] Posterior ocular conditions which can be treated according to the invention include acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; retinal
neovascularization; diabetic uveitis; histoplasmosis; infections, such as bacterial, fungal or viral-caused infections; macular degeneration, such as acute macular degeneration, non- exudative age related macular degeneration and exudative age related macular degeneration; edema, such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma which affects a posterior ocular site or location; ocular tumors;
retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic opthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy, photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; other forms of optic neuropathy and optic neuritis; non-retinopathy diabetic retinal dysfunction; retinitis pigmentosa; and glaucoma. Glaucoma can be considered a posterior ocular condition because the therapeutic goal is to prevent the loss of or reduce the occurrence of loss of vision due to damage to or loss of retinal cells or optic nerve ganglion cells (i.e., neuroprotection).
[26] Other ocular disorders, conditions, and diseases that can be treated using the methods of the present invention are severe visual impairment (i.e., blindness), including diseases related to degeneration of cells of the retina and macula, including, but not limited to, Usher syndrome, Stargardt disease, Bardet-Biedl syndrome, Best disease, choroideremia, gyrate-atrophy, retinitis pigmentosa, macular degeneration, Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), Blue-cone monochromacy, retinoschisis, Malattia Leventinese, Oguchi Disease, or Refsum disease, or other diseases related to impairment of the function of the retina or macula. [27] Macular degeneration disorders may include but are not limited to any of a number of conditions in which the retinal macula degenerates or becomes dysfunctional, e.g., as a consequence of decreased growth of cells of the macula, increased death or rearrangement of the cells of the macula (e.g., RPE cells), loss of normal biological function, or a combination of these events such as North Carolina macular dystrophy, Sorsby's fundus dystrophy, pattern dystrophy, dominant drusen, and any condition which alters or damages the integrity or function of the macula (e.g., damage to the RPE or Bruch's membrane). For example, macular degeneration may involve retinal detachment, chorioretinal degenerations, retinal
degenerations, photoreceptor degenerations, RPE degenerations, mucopolysaccharidoses, rod- cone dystrophies, cone-rod dystrophies, and cone degenerations.
[28] Furthermore, the methods disclosed herein for delivering nucleic acids to the eye via non- viral nanoparticles may be used to treat or prevent ocular diseases or conditions, such as the following: maculopathies and retinal degeneration; macular degeneration, including age related macular degeneration (AMD), such as non-exudative age related macular degeneration and exudative age related macular degeneration; choroidal neovascularization; retinal
neovascularization; retinopathy, including diabetic retinopathy, acute and chronic macular neuroretinopathy; central serous chorioretinopathy, and macular edema, including cystoid macular edema, and diabetic macular edema; uveitis; retinitis; choroiditis; acute multifocal placoid pigment epitheliopathy; Behcet's disease; birdshot retinochoroidopathy; infectious (syphilis, lyme, tuberculosis, toxoplasmosis) uveitis; intermediate uveitis (pars planitis) and anterior uveitis; multifocal choroiditis; multiple evanescent white dot syndrome (MEWDS); ocular sarcoidosis; posterior scleritis; serpignous choroiditis; subretinal fibrosis; uveitis syndrome; Vogt-Koyanagi-Harada syndrome; vascular diseases/exudative diseases including retinal arterial occlusive disease, central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal arterial microaneurysms, Coat's disease, parafoveal telangiectasis, hemi- retinal vein occlusion, papillophlebitis, central retinal artery occlusion, branch retinal artery occlusion, frosted branch angitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, familial exudative vitreoretinopathy, and Eales disease; traumatic/surgical conditions including sympathetic ophthalmia, uveitic retinal disease, retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusion during surgery, radiation retinopathy, and bone marrow transplant retinopathy; proliferative disorders including proliferative vitreal retinopathy and epiretinal membranes, and proliferative diabetic retinopathy; infectious disorders including ocular histoplasmosis, ocular toxocariasis, presumed ocular histoplasmosis syndrome (POHS), endophthalmitis, toxoplasmosis, retinal diseases associated with HIV infection, choroidal disease associated with HIV infection, uveitic disease associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal diseases, bacterial diseases, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, and myiasis; genetic disorders including retinitis pigmentosa, systemic disorders with associated retinal dystrophies, congenital stationary night blindness, cone dystrophies,
Stargardt's disease and fundus flavimaculatus, Bests disease, pattern dystrophy of the retinal pigmented epithelium, X-lmked retinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti's crystalline dystrophy, and pseudoxanthoma elasticum; retinal tears/holes including retinal detachment, macular hole, and giant retinal tear; tumors including retinal disease associated with tumors, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined hamartoma of the retina and retinal pigmented epithelium, retinoblastoma, vasoproliferative tumors of the ocular fundus, retinal astrocytoma, intraocular lymphoid tumors; and miscellaneous conditions including punctate inner choroidopathy, acute posterior multifocal placoid pigment
epitheliopathy, myopic retinal degeneration, and acute retinal pigment epithelitis.
[29] The invention may employ nucleic acid nanoparticles comprising one or more of the genes CA4, CRX, FSCN2, GUCAlB, IMPDHl, NR2E3, NRL, PRPF3, PRPF8, PRPF31, PRPH2, RHO, ROMl, RPl, RP9, SEMA4A, TOPORS, ABCA4, CERKL, CNGAl, CNGBl, CRBl, LRAT, MERTK, NRL, PDE6A, PDE6B, PRCD, PROMl, RGR, RLBPl, RPl, RPE65, SAG, TULPl, USH2A, RP2, and RPGR for use in treating autosomal dominant, autosomal recessive, or X-linked forms of retinitis pigmentosa. These nucleic acids may be used to express other genes in other genotypes of retinitis pigmentosa not listed above. The nucleic acids can also be used in other forms and formulations as well.
[30] Other nucleic acids which may be used in nanoparticles are one or more of genes
ABCA4, ARMS2, C2, C3, CFB, CFH, ERCC6, FBLN5, HMCNl, HTRAl, RAX2 and TLR4 for use in treating age-related macular degeneration (AMD) and one or more of genes BESTl, C1QTNF5, EFEMPl, EL0VL4, FSCN2, GUCAlB, PRPH2, TIMP3, and RPGR for use in treating autosomal dominant macular degeneration, autosomal recessive macular degeneration, or X-linked macular degeneration. [31] In addition to gene replacement therapy using wild-type genes to offset the defects of mutant genes, other forms of gene therapy may be practiced using the present technique.
Genes which provide a supportive function, but do not replace a defective gene, may be used. Genes whose protein products inhibit neovascularization or anti-sense nucleic acids that inhibit expression of genes that stimulate neovascularization may be used. Anti-sense
oligonucleotides, anti-sense plasmid constructs, siRNA, and shRNA plasmids can be used to provide a desired therapeutic effect. Thus the nucleic acids may be coding or non-coding. The therapeutic product may be a nucleic acid or a protein.
[32] Expression of the therapeutic product or persistence of the therapeutic product in the eye can be monitored once or multiple times. The monitoring may be used to determine, e.g., if a sufficient dosing has been achieved or to determine if an additional dosing is required after passage of time.
[33] A patient may be treated therapeutically or prophylactically by administering the compacted nucleic acid nanoparticles to the patient by at least one of intravitreal placement, subretinal placement, subconjuctival placement, conjuctival placement, anterior chamber placement, episcleral placement, sub-tenon placement, retrobulbar placement, suprachoroidal placement, and systemic injection via intravenous and/or intraarterial administration.
Placement methods may include injection and/or surgical insertion. The compacted nucleic acid nanoparticle is administered via intravitreal injection. In an embodiment of the invention, the amount of nucleic acid per dosage is provided to the subject's eye at a concentration of 0.01 ug/ul to 15 ug/ul, depending on the desired level of expression in the ocular cells. Individual dosage volumes may range (in non-limiting examples) for example from 1 ul to 5000 ul, or from 10 ul to 1000 ul. The total amount of DNA administered per eye may be at least 1 ug, at least 3 ug, at least 8 ug, at least 10 ug, at least 12 ug, at least 15 ug, at least 20 ug, at least 50 ug, at least 100 ug, at least 500 ug, at least 1,000 ug, at least 5,000 ug, at least 10.000 ug, at least 50,000 ug. The nucleic acid nanoparticles may be provided in a composition comprising any pharmaceutically acceptable carrier, such as a saline solution (e.g., PBS). Depending on the dose administered, a volume of vitreous humor may be removed to improve tolerance of the injected volume containing the nanoparticles. As other beneficial genes are identified, they too can be administered in this manner. [34] In one embodiment a subject having an ocular disorder is treated by providing a compacted nucleic acid nanoparticle having a minor dimension equal to or below 30 nm or below 25 nm and a nucleic acid non-covalently linked to a cationic polymeric material. The minor dimension may even be below 20, 15, or 11 nm as measured by transmission electron microscopy. The compacted nanoparticle is administered to a tissue of the eye of the patient for treating the ocular disorder. In some embodiments, the ocular condition or disorder to be treated is related to retinal and macular degeneration, Usher syndrome, Stargardt disease, Bardet-Biedl syndrome, Best disease, choroideremia, gyrate-atrophy, retinitis pigmentosa, Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), various types of optic neuropathy and optic neuritis, Blue-cone monochromacy, retinoschisis, Malattia Leventinese, Oguchi Disease, and Refsum disease, retinal detachment, chorioretinal degenerations, retinal degenerations, photoreceptor degenerations, degeneration of the retinal pigment epithelium, mucopolysaccharidoses, rod-cone dystrophies, cone -rod dystrophies, cone degenerations, conditions involving decreased growth of cells of the macula, increased death or rearrangement of the retinal pigment epithelial cells of the macula, North Carolina macular dystrophy, Sorsby's fundus dystrophy, pattern dystrophy, dominant drusen, or any condition which alters or damages the integrity or function of the macula.
[35] Any gene may be used that will be useful in the retina. These may encode at least one of opsin protein of rhodopsin (RHO), cyclic GMP phosophodiesterase α-subunit (PDE6A) or β- subunit (PDE6B), the α subunit of the rod cyclic nucleotide gated channel (CNGAl), RPE65, RLBPl, ABCR, ABCA4, CRBl, LRAT, CRX, IPl, EFEMPl, peripherin/RDS, ROMl, arrestin (SAG), α-transducin (GNATl), rhodopsin kinase (RHOK), guanylate cyclase activator IA (GUCAlA), retina specific guanylate cyclase (GUCY2D), the α subunit of the cone cyclic nucleotide gated cation channel (CNGA3), and cone opsins BCP, GCP, and RCP. Other genes may also be used including those encoding ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), pigment epithelium-derived factor (PEDF), and ORE 15 variant of Retinitis Pigmentosa GTPase Regulator (RPGR). Examples of genes which are related to macular degeneration include CFH (Complement Factor H), CFB (Complement Factor B), ABCR and ACBA4, C2 (Complement Component 2), C3 (Complement Component 3), HTRAl, T2-TrpRS, and RdCVF; any of these alone or in combination may be used in nanoparticles to treat, mitigate or prevent macular degeneration conditions. [36] Nucleic acid nanoparticles can be made, for example, according to the methods disclosed in Hanson, Perales, and/or Cooper. See U.S. 6506890, 5877302, 5844107, the disclosures of which are expressly incorporated here. Nanoparticles may be 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 11 nm or less, as measured by transmission electron microscopy.
[37] Nucleic acids may optionally contain a plasmid origin of replication. Nucleic acids may optionally contain coding sequences for RNA and/or protein products that are transcribed under the control of promoters that are cell-type or tissue-type specific. One embodiment of the invention utilizes the promoter from the VMD gene, which is specifically expressed in the retinal pigment epithelium. Promoters that are specific for rods or cones may be used as well. Types of RNA molecules which may be used include siRNA. Antisense oligonucleotides or antisense-producing constructs may be used as well, including DNA plasmids transcribing shRNA. shRNA (small hairpin RNA) is a transcript that is processed by the cell to siRNA. The siRNA inhibits expression of a target mRNA.
[38] Nucleic acids of the invention may be packaged in any suitable manner known in the art. They may be packaged in single dose vials, which are typically aseptic. The vials may be provided in a kit with additional components to facilitate the successful delivery to the eye. Additional components include an injection needle, a filter needle, a 1-cc tuberculin syringe, and a sterile, disposable eyelid speculum. Instructions and other information may be provided on a package insert, for example. Suitable types of needles for injection and for preparation include 30 gauge, Vi inch for injection and 19 gauge, 5 micron for preparation and withdrawal from a vial.
[39] Improvement in the symptoms or phenotype of an ocular disease can be monitored in any way that the disease is usually assessed. These may involve neurological assessments, behavioral assessments, electronic assessments, reading or object recognition, imaging assessments, etc. Assessments may be combined. Assessments may be carried out once or many times to determine the trajectory of the disease process and/or the therapeutic process.
[40] Those of skill in the art will readily recognize that aspects of the invention may be combined in different ways to achieve desirable results. Examples
Example l~Intravitreal dosing of compacted DNA nanoparticles transfects the deep retina, including retinal pigment epithelial (RPE) cells.
[41] Figure 1 presents a diagram of the retina, including deep retinal layers (retinal pigment epithelial cells, rod and cone photoreceptor cells), and location of injection sites for subretinal and intravitreal delivery. Subretinal dosing of viral vectors, such as AAV, transduces RPE and photoreceptor cells, whereas intravitreal dosing of AAV fails to express well in photoreceptors and not at all in RPE cells (Hellstrόm, Petrs-Silva) in wild-type mice and most forms of retinitis pigmentosa evaluated to date. One exception to this observation is the ability of AAV vectors to have improved transduction of photoreceptors in one type of retinitis pigmentosa, X- linked juvenile retinoschisis, where microcysts and internal retinal dissection disturbs the normal lamination of various neuronal and plexiform layers (Park). Additionally, use of proteolytic enzymes, such as pronase, delivered in the vitreous humor can facilitate deep retina transduction (Dalkara), although the use of such proteolytic enzymes in subjects with retinitis pigmentosa and other ocular diseases has unknown and potential deleterious side effects. In view of toxicities associated with subretinal dosing, including retinal detachment and associated retina cell apoptosis, as has been documented in various forms of retinitis pigmentosa, there is a significant unmet medical need to develop nucleic acid delivery technologies that can safely express transgenes in photoreceptor and RPE cells following the less-invasive intravitreal dosing procedure, and without the need for proteolytic enzymes.
[42] To determine if compacted DNA nanoparticles can transfect the deep retina following intravitreal delivery, we prepared an expression plasmid (pVMD_Y2) in which enhanced yellow fluorescent protein (EYFP) cDNA is transcriptionally-controlled by the promoter from the hVMD2 gene, which encodes for bestrophin-1. The hVMD2 promoter is well-established to be restricted in activity to RPE cells, which parallels the natural tissue-restricted expression pattern of bestrophin-1 protein. In tissue culture studies using RPE and photoreceptor cell lines, the pVMD_Y2 plasmid showed increased relative activity in RPE cells (data not shown). Plasmid pVMD_Y2 was unimolecularly compacted with CK3 OPEG 10k polycation, a 30-mer lysine peptide with an N-terminal cysteine that is covalently modified via a S-C bond with 10 kDa polyethylene glycol (PEGlOk) having a maleimide reactive group. Polycation preparation and the DNA compaction protocols were conducted essentially as previously published (Liu, Fink, Method of Nucleic Acid Compaction US 6,506,890). After compaction, the compacted DNA was processed with tangential flow filtration to remove excess uncompacted polycation, exchange water solvent with normal saline, and to concentrate the DNA nanoparticles to approximately 4 mg/ml of DNA. The final compacted DNA preparation was then evaluated with a series of quality control assays, including transmission electron microscopy, turbidity parameter analysis, gel analysis, sedimentation analysis, serum stability assays, and other studies as previously described (Liu, Fink, Ziady, Konstan). The compacted DNA met or exceeded all quality control assay end-release specifications, indicating compaction of the DNA, colloidal stability of the DNA nanoparticles in normal saline, and stability of the compacted DNA in DNAse-rich solutions.
[43] Fig. 2 shows an image of an electron micrograph of the compacted pVMD_Y2 nanoparticles in saline, which are compacted into rod-like shapes by using CK30PEG10k with an acetate lysine counterion prior to mixing of the polycation with DNA (see Fink et al. for discussion of rod-like nanoparticle dimensions).
[44] pVMD_Y2 nanoparticles were dosed subretinally and intravitreally into wild-type Balb/c mice. Approximately 4 ug was administered to the mice dosed intravitreally. The DNA concentration in the vitreous humor would be about 0.8 ug/ul, assuming a typical 5 ul volume of vitreous humer per mouse eye. Two days post injection, eyes were harvested, retinal flat mounts were prepared, and eyes were imaged for EYFP direct fluorescence. Fig. 3 A shows robust EYFP expression in RPE cells at the site of subretinal injection, which is an expected result for these nanoparticles. Following intravitreal injection, significant EYFP fluorescence is also observed in RPE cells, as shown in Fig. 3B. In some animals, EYFP signal was observed in multiple retinal quadrants. Similar data were observed 21 days post injection. These data provide evidence that intravitreal injection of compacted DNA nanoparticles can result in deep retinal penetration and transfection of RPE cells. To further evaluate that RPE cells were transfected, a derivative of pVMD_Y2 that contains a CMV enhancer to increase promoter activity, pVMD_Y4, was compacted and dosed intravitreally, and cross-sections of retinas were evaluated at day 21 for EYFP expression by immunohistochemistry using a rabbit polyclonal anti-EYFP antibody (Santa Cruz 32897). Animals dosed intravitreally with phosphate-buffered saline served as negative controls. Detection of EYFP protein was localized specifically in RPE cells (Figure 4). The PBS-dosed animals had no significant signal. These data demonstrate RPE-specific transgene expression following intravitreal dosing of compacted DNA nanoparticles. Example 2~Quantitative comparison of luciferase activity in RPE cells following intravitreal or subretinal dosing of compacted DNA encoding a VMD promoter luciferase transgene.
[45] To quantitate gene transfer into RPE cells following intravitreal delivery better, Balb/c mice were injected either intravitreally or subretinally with 4.5 μg DNA nanoparticles encoding a hVMD2 promoter transcriptionally-controlling expression of the firefly luciferase transgene (pVMD_luc4). Non-dosed eyes served as a negative control. On post-injection day 159, eyes were harvested, lysates prepared, and luciferase activity measured using a chemiluminescent assay (Promega). Fig. 5 shows these results. Compared to non-dosed eyes, there was statistically significant luciferase activity in eyes injected either subretinally or intravitreally, and the 4.5 fold difference between the subretinal and intravitreal groups was not significant (t- test, p2=0.066). These data further demonstrate the ability of compacted DNA nanoparticles to transfect the deep retina RPE cells following intravitreal injection.
Example 3--E valuation of nanoparticle dose, size, and shape on RPE gene transfer following intravitreal delivery.
[46] RPE transfection following intravitreal dosing requires DNA nanoparticles to diffuse through various retinal cell layers to reach RPE cells (Fig. 1). Since the size and shape of DNA nanoparticles might influence this process, compacted DNA nanoparticles containing pVMD_luc4 were compacted as rods or ellipsoids (Fig. 6). The rods were prepared utilizing CK30PEG10k polycation having acetate as the lysine counterion prior to DNA mixing (Fig. 6B), whereas to form ellipsoids the lysine counterion was trifluoroacetate (TFA) (Fig. 6A). For a given plasmid size typically used for therapeutic intent, the minimum cross-sectional diameter of the rods (8-11 nm) will be smaller than the ellipsoid minor diameter (Fink), which may improve diffusion. However, the longer length of the rod compared to the ellipsoidal major diameter may adversely affect diffusion properties, so the optimized gene transfer nanoparticle requires empiric testing. Fig. 7 shows a dose response luciferase activity analysis comparing intravitreal dosing of rod and ellipsoidal DNA nanoparticle formulations at approximately 1, 3, or 8 μg of DNA at day 3 post-injection. There was statistically improved luciferase activity in eyes dosed intravitreally with the highest dose of ellipsoids compared to the other groups (1 way ANOVA with Bonferroni's multiple comparison test, *). These data indicate that the shape and dose of the DNA nanoparticle are important parameters to consider when optimizing deep retinal gene transfer. Other formulation parameters also may influence RPE gene transfer, and these findings are not meant to restrict the matrix of nanoparticle properties that may influence RPE transfection. Other such nanoparticle formulation parameters are the polycation design, including type of positively charged amino acids used, use of polycations having other types of counterions prior to mixing with DNA (see
Lyophilizable and Enhanced Compacted Nucleic Acids, US2004/0048787), use of positively charged polymers that do not constitute amino acids, the percentage of PEG substitution in the polymer, the size of PEG used in the polycation, and whether the PEG moieties are added to the nanoparticles pre- or post-compaction. Other factors that may influence gene transfer include whether the nanoparticles incorporate cell targeting ligands, use of various excipients that may influence nanoparticle stability and/or diffusion, dose escalation in the vitreous humor (including removal of vitreous humor fluid prior to and/or during nanoparticle dosing), and the specific administration procedure used to deliver compacted DNA to the vitreous humor.
Example 4~Intravitreal dosing of compacted DNA can improve the phenotype of retinitis pigmentosa mice.
[47] To evaluate if intravitreal dosing of compacted DNA nanoparticles can correct the visual phenotype of retinitis pigmentosa (RP) mice caused by mutations in a gene normally expressed in RPE cells, we selected RP mice having knock-out of the lecithin retinol acyltransferase gene {Lrat -I- mice). The LRAT enzyme converts all-trans retinol into all-trans retinyl esters, a key step in the vitamin A regeneration cycle, which is required for generating the active form of vitamin A, 11-cis retinal, which is required for vision. Lrat -I- mice have depleted vitamin A chromophore intermediates, including depleted of 11 cis-retinal, and experience significant visual impairment at an early age.
[48] Lrat -I- mice at 6 weeks of age were dosed either intravitreally or subretinally with compacted DNA nanoparticles encoding a RPE-restricted wild-type LRAT gene, pVMD lrat. At multiple times post-injection, lrat -I- mice were evaluated for various parameters related to phenotypic correction, including retinal retinoid levels and electroretinograms (ERGs). The electroretinogram measures electrical activity in the retina following light exposure, and is a surrogate marker for vision.
[49] Figure 8 shows ocular retinyl ester levels in lrat -I- mice 14 days after dosing with compacted pVMD_lrat DNA nanoparticles. In contrast to PBS dosed lrat -I- mice, which have no detectable retinyl esters, these mice have increased levels of retinyl esters following either subretinal or intravitreal dosing. These data demonstrate that intravitreal dosing of compacted DNA in RP mice can improve their ocular phenotype.
[50] To determine if therapy with DNA nanoparticles encoding the wild-type lrat gene can improve vision, lrat -I- mice received subretinal or intravitreal doses of compacted DNA containing a derivative of pVMD_lrat incorporating a CMV enhancer, pVMD_lrat3. Presented in Figure 9 are ERG a and b wave tracings 1 week following subretinal dosing and 1 month after intravitreal dosing of compacted pVMD_lrat3 nanoparticles. Lrat -I- mice dosed with saline showed essentially no retinal electrical activity after light exposure, whereas DNA nanoparticle treated mice, following either subretinal and intravitreal delivery, showed quite significant ERG signals, approximately 25% to 50% of positive control wild-type mouse a and b wave amplitudes, respectively. Together, these data demonstrate that intravitreal dosing of DNA nanoparticles can improve the visual phenotype of lrat -I- mice, and suggest that intravitreal dosing of compacted DNA may be effective in other genotypes of RP.
References
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Claims

I Claim:
1. A method of delivering nucleic acid to retinal pigment epithelium, comprising:
administering an effective dose of a non-viral nucleic acid to an eye by intravitreal injection, wherein the non- viral nucleic acid encodes an RNA and/or protein product, whereby retinal pigment epithelial cells are trans fected and express a detectable amount of the RNA and/or protein product.
2. The method of claim 1 wherein the non- viral nucleic acid is compacted to achieve a minor dimension of 30 nm or less as assessed by transmission electron microscopy.
3. The method of claim 1 wherein the non- viral nucleic acid is compacted to achieve a minor dimension of 20 nm or less as assessed by transmission electron microscopy.
4. The method of claim 1 wherein the non- viral nucleic acid is compacted to achieve a minor dimension of 15 nm or less as assessed by transmission electron microscopy.
5. The method of claim 1 wherein the non- viral nucleic acid is compacted to achieve a minor dimension 11 nm or less as assessed by transmission electron microscopy.
6. The method of claim 1 further comprising the step of assessing expression of the product in the retinal pigment epithelium.
7. The method of claim 1 further comprising the step of quantitatively assessing expression of the product in the retinal pigment epithelium.
8. The method of claim 1 further comprising the step of assessing expression of the product in the retinal pigment epithelium at a plurality of time points.
9. The method of claim 1 wherein a tissue-specific or cell-specific promoter controls
transcription of the non- viral nucleic acid.
10. The method of claim 1 wherein a retinal pigment epithelium-specific promoter controls transcription of the non- viral nucleic acid.
11. The method of claim 1 wherein a VMD (bestrophin) promoter controls transcription of the non- viral nucleic acid.
12. The method of claim 1 wherein the protein product supplements a deficiency in the eye.
13. The method of claim 1 wherein the eye is diseased and expression of the RNA or protein product reduces the severity of symptoms of the disease.
14. A single dose vial for delivery of the dose intravitreally to an eye, comprising 0.05 to 5 ml of a non-viral nucleic acid which encodes an RNA and/or protein product, wherein the nucleic acid is compacted to achieve a minor dimension of 30 nm or less, and wherein transcription of the product is controlled by a tissue-specific or cell type-specific promoter which is operably linked to the nucleic acid which encodes the product.
15. A kit comprising :
• the single dose vial of claim 14; and
• an injection needle.
16. The kit of claim 15 further comprising a filter needle.
17. The kit of claim 15 wherein the injection needle is 30 gauge, Vi inch.
18. The kit of claim 15 further comprising:
• a 1-cc tuberculin syringe.
19. The kit of claim 15 further comprising :
• a sterile, disposable eyelid speculum.
20. The kit of claim 16 wherein the filter needle is 19 gauge, 5 micron.
21. The method of claim 1 wherein the RNA is shRNA.
22. A method of delivering nucleic acid to retinal pigment epithelium or retinal photoreceptor cells, comprising:
administering an effective dose of a non-viral nucleic acid to an eye by intravitreal injection, wherein the non- viral nucleic acid is an siRNA, wherein retinal pigment epithelial cells or retinal photoreceptor cells are transfected by the siRNA.
23. A method of delivering nucleic acid to retinal photoreceptor cells, comprising:
administering an effective dose of a non- viral nucleic acid to an eye by intravitreal injection, wherein the non-viral nucleic acid encodes an RNA and/or protein product, whereby retinal photoreceptor cells are transfected and express a detectable amount of the RNA and/or protein product.
24. The method of claim 23 wherein a retinal photoreceptor-specifϊc promoter controls
transcription of the non- viral nucleic acid.
25. A method of treating an ocular disease, comprising:
administering an effective dose of a non- viral nucleic acid to an eye by intravitreal injection, wherein the non- viral nucleic acid encodes or is a therapeutic product, whereby vision by the eye is improved.
26. The method of claim 25 wherein retinal pigment epithelium cells are transfected and express a detectable amount of an RNA and/or protein product encoded by said non- viral nucleic acid.
PCT/US2010/044234 2009-08-04 2010-08-03 Method of administering non-viral nucleic acid vectors to the eye WO2011017313A1 (en)

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