WO2011017313A1 - Procédé d'administration de vecteurs d'acide nucléique non viraux à l'œil - Google Patents

Procédé d'administration de vecteurs d'acide nucléique non viraux à l'œil Download PDF

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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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nucleic acid
viral nucleic
retinal
eye
compacted
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PCT/US2010/044234
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Mark J. Cooper
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Copernicus Therapeutics Inc.
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    • 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
    • A61K48/0008—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 '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
    • A61K48/0025—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 '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
    • 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
    • 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
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • 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

  • 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 -

Abstract

L'invention porte sur une administration intravitréale de vecteurs d'acide nucléique, laquelle administration conduit à la transfection et à l'expression des vecteurs d'acide nucléique dans l'épithélium du pigment rétinien. L'acide nucléique est non viral et peut être compacté pour former une nanoparticule unimoléculaire avec un polymère polycationique. Les vecteurs d'acide nucléique peuvent être utilisés pour fournir une copie normale d'un gène qui est défectueux dans le destinataire, pour inhiber un produit cellulaire délétère ou pour fournir un produit bénéfique afin d'améliorer les symptômes ou l'historique naturelle de la maladie.
PCT/US2010/044234 2009-08-04 2010-08-03 Procédé d'administration de vecteurs d'acide nucléique non viraux à l'œil WO2011017313A1 (fr)

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US11883541B2 (en) 2017-10-02 2024-01-30 The Johns Hopkins University Nonviral gene transfer to the suprachoroidal space
WO2019108570A3 (fr) * 2017-11-29 2019-07-25 Copernicus Therapeutics, Inc. Thérapie génique pour l'amélioration des fonctions oculaires
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