WO2000054813A2 - Utilisation de vecteurs d'administration d'un gene recombinant pour le traitement ou la prévention des maladies de l'oeil - Google Patents

Utilisation de vecteurs d'administration d'un gene recombinant pour le traitement ou la prévention des maladies de l'oeil Download PDF

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WO2000054813A2
WO2000054813A2 PCT/US2000/007062 US0007062W WO0054813A2 WO 2000054813 A2 WO2000054813 A2 WO 2000054813A2 US 0007062 W US0007062 W US 0007062W WO 0054813 A2 WO0054813 A2 WO 0054813A2
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fgf
eye
gene delivery
vector
cells
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PCT/US2000/007062
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WO2000054813A3 (fr
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William C. Manning, Jr.
Varavani J. Dwarki
Katherine Rendahl
Shang-Zhen Zhou
Laura H. Mcgee
Dana Lau
John G. Flannery
Sheldon Miller
Fei Wang
Adriana Di Polo
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Chiron Corporation
The Regents Of The University Of California
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Priority to AU37559/00A priority Critical patent/AU3755900A/en
Priority to CA002367375A priority patent/CA2367375A1/fr
Priority to EP00916458A priority patent/EP1183051A2/fr
Priority to JP2000604885A priority patent/JP2002539176A/ja
Publication of WO2000054813A2 publication Critical patent/WO2000054813A2/fr
Publication of WO2000054813A3 publication Critical patent/WO2000054813A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • 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
    • 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
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • the present invention relates generally to compositions and methods for treating diseases of the eye, and more specifically, to the use of various gene delivery vectors which direct the expression of selected gene products suitable for treating or preventing diseases of the eye.
  • Eye diseases represent a significant health problem in the United States and world-wide. Presently, over 80 million Americans are affected with potentially blinding eye disease, with 1.1 million being legally blind. Twelve million individuals suffer from some degree of visual impairment that cannot be corrected. The total economic impact of eye disease is also very significant. In 1981, the estimated economic impact of visual impairment on the U.S. economy was 14 billion per year. By 1995, this impact had grown to an estimated 38.4 billion (National Eye Institute NIH).
  • eye diseases can cause visual impairment, including for example, macular degeneration, diabetic retinopathies, inherited retinal degeneration such as retinitis pigmentosa, glaucoma, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically).
  • macular degeneration diabetic retinopathies
  • inherited retinal degeneration such as retinitis pigmentosa, glaucoma, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically).
  • the retina which is found at the back of the eye, is a specialized light- sensitive tissue that contains photoreceptor cells (rods and cones) and neurons connected to a neural network for the processing of visual information (see Figure 10). This information is sent to the brain for decoding into a visual image.
  • the retina depends on cells of the adjacent retinal pigment epithelium (RPE) for support of its metabolic functions.
  • RPE retinal pigment epithelium
  • Photoreceptors in the retina perhaps because of their huge energy requirements and highly differentiated state, are sensitive to a variety of genetic and environmental insults.
  • the retina is thus susceptible to an array of diseases that result in visual loss or complete blindness.
  • Retinitis pigmentosa which results in the destruction of photoreceptor cells, the RPE, and choroid typify inherited retinal degenerations. This group of debilitating conditions affects approximately 100,000 people in the United States.
  • AMD AMD Degeneration
  • the social and economic impact of this disease in the United States is increasing.
  • the macula is a structure near the center of the retina that contains the fovea. This specialized portion of the retina is responsible for the high-resolution vision that permits activities such as reading.
  • the loss of central vision in AMD is devastating.
  • Degenerative changes to the macula can occur at almost any time in life but are much more prevalent with advancing age. With growth in the aged population, AMD will become a more prevalent cause of blindness than both diabetic retinopathy and glaucoma combined.
  • Laser treatment has been shown to reduce the risk of extensive macular scarring from the "wet" or neovascular form of the disease. The effects of this treatment are short-lived, however, due to recurrent choroidal neovascularization. Thus, there are presently no effective treatments in clinical use for the vast majority of AMD patients.
  • glaucoma Other diseases of the eye, such as glaucoma, are also major public health problems in the United States. In particular, blindness from glaucoma is believed to impose significant costs annually on the U.S. Government in Social Security benefits, lost tax revenues, and healthcare expenditures.
  • glaucoma is not a uniform disease but rather a heterogeneous group of disorders that share a distinct type of optic nerve damage that leads to loss of visual function.
  • the disease is manifest as a progressive optic neuropathy that, if left untreated, leads to blindness. It is estimated that as many as 3 million Americans have glaucoma and, of these, as many as 120,000 are blind as a result. Furthermore, it is the number one cause of blindness in African- Americans.
  • Its most prevalent form, primary open-angle glaucoma can be insidious. This form usually begins in midlife and progresses slowly but relentlessly. If detected early, disease progression can frequently be arrested or slowed with medical and surgical treatment. Glaucoma can involve several tissues in the front and back of the eye.
  • glaucoma begins with a defect in the front of the eye. Fluid in the anterior portion of the eye, the aqueous humor, forms a circulatory system that brings nutrients and supplies to various tissues. Aqueous humor enters the anterior chamber via the ciliary body epithelium (inflow), flows through the anterior segment bathing the lens, iris, and cornea, and then leaves the eye via specialized tissues known as the trabecular meshwork and Schlemm's canal to flow into the venous system. Intraocular pressure is maintained vis-a-vis a balance between fluid secretion and fluid outflow. Almost all glaucomas are associated with defects that interfere with aqueous humor outflow and, hence, lead to a rise in intraocular pressure.
  • optic nerve function is compromised.
  • the result is a distinctive optic nerve atrophy, which clinically is characterized by excavation and cupping of the optic nerve, indicative of loss of optic nerve axons.
  • Primary open-angle glaucoma is, by convention, characterized by relatively high intraocular pressures believed to arise from a blockage of the outflow drainage channel or trabecular meshwork in the front of the eye.
  • normal-tension glaucoma is characterized by a severe optic neuropathy in the absence of abnormally high intraocular pressure. Patients with normal-tension glaucoma have pressures within the normal range, albeit often in the high normal range.
  • Both these forms of primary open-angle glaucoma are considered to be late-onset diseases in that, clinically, the disease first presents itself around midlife or later. However, among African-Americans, ihe disease may begin earlier than middle age. In contrast, juvenile open-angle glaucoma is a primary glaucoma that affects children and young adults. Clinically, this rare form of glaucoma is distinguished from primary open-angle glaucoma not only by its earlier onset but also by the very high intraocular pressure associated with this disease. Angle-closure glaucoma is a mechanical form of the disease caused by contact of the iris with the trabecular meshwork, resulting in blockage of the drainage channels that allow fluid to escape from the eye.
  • glaucoma This form of glaucoma can be treated effectively in the very early stages with laser surgery. Congenital and other developmental glaucomas in children tend to be severe and can be very challenging to treat successfully. Secondary glaucomas result from other ocular diseases that impair the outflow of aqueous humor from the eye and include pigmentary glaucoma, pseudoexfoliative glaucoma, and glaucomas resulting from trauma and inflammatory diseases. Blockage of the outflow channels by new blood vessels (neovascular glaucoma) can occur in people with retinal vascular disease, particularly diabetic retinopathy.
  • Primary open-angle glaucoma can be insidious. It usually begins in midlife and progresses slowly but relentlessly. If detected, disease progression can frequently be arrested or slowed with medical and surgical treatment. However, without treatment, the disease can result in absolute irreversible blindness. In many cases, even when patients have received adequate treatment (e.g., drugs to lower intraocular pressure), optic nerve degeneration and loss of vision continues relentlessly.
  • adequate treatment e.g., drugs to lower intraocular pressure
  • the present invention provides compositions and methods for treating and preventing a number of diseases of the eye, including for example, retinal diseases and degenerations such as RP and AMD, as well as other diseases such as neovascular disease.
  • the present invention also provides other, related advantages.
  • the present invention provides compositions and methods for treating, preventing, or, inhibiting diseases of the eye.
  • methods are provided for treating or preventing diseases of the eye comprising the step of intraocularly administering a gene delivery vector which directs the expression of one or more neurotrophic factors, or, anti-angiogenic factors, such that the disease of the eye is treated or prevented.
  • gene delivery vectors are provided which direct the expression of one or more neurotrophic factors such as FGF, as well as gene therapy vectors which direct the expression of one or more anti-angiogenic factors.
  • a viral promoter e.g., CMV
  • an inducible promoter e.g., tet
  • gene delivery vectors suitable for use within the present invention may be generated from viruses such as retroviruses (e.g. , FIV or HIV), herpesviruses, adenoviruses, adeno-associated viruses, and alphaviruses, or from non- viral vectors.
  • a wide variety of diseases of the eye may be readily treated or prevented, including for example, glaucoma, macular degeneration, diabetic retinopathies, inherited retinal degeneration such as retinitis pigmentosa, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically).
  • a wide variety of neurotrophic factors may be utilized (either alone or in combination) within the context of the present invention, including for example, NGF, BDNF, CNTF, NT-3, NT-4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF- 21.
  • the gene delivery vector be utilized to deliver and express an anti-angiogenic factor for the treatment, prevention, or, inhibition of diabetic retinopathy, wet ARMD, and other neovascular diseases of the eye (e.g., ROP).
  • an anti-angiogenic factor for the treatment, prevention, or, inhibition of diabetic retinopathy, wet ARMD, and other neovascular diseases of the eye (e.g., ROP).
  • ROP neovascular diseases of the eye
  • the gene delivery vector be utilized to deliver and express a neurotrophic growth factor to treat, prevent, or, inhibit diseases of the eye, such as, for example, glaucoma, retinitis pigmentosa, and dry ARMD.
  • a gene delivery vector which expresses both an anti- angiogenic molecule and a neurotrophic growth factor, or two separate vectors which independently express such factors, in the treatment, prevention, or inhibition of an eye disease (e.g., for diabetic retinopathy).
  • the above-mentioned methods utilizing gene delivery vectors may be administered along with other methods or therapeutic regimens, including for example, photodynamic therapy (e.g., for wet ARMD), laser photocoagulation (e.g., for diabetic retinopathy and wet ARMD), and intraocular pressure reducing drugs (e.g., for glaucoma).
  • photodynamic therapy e.g., for wet ARMD
  • laser photocoagulation e.g., for diabetic retinopathy and wet ARMD
  • intraocular pressure reducing drugs e.g., for glaucoma
  • isolated nucleic acid molecules comprising the sequence of Figure 2, vectors which contain, and/or express this sequence, and host cells which contain such vectors.
  • gene delivery vectors which direct the expression of a neurotrophic factor, or, an anti-angiogenic factor.
  • neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT-4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21.
  • anti-angiogenic factors include soluble Fit- 1 , soluble Tie-2 receptor, and PEDF.
  • suitable gene delivery vectors include adenovirus, retroviruses (e.g., HIV or FIV-based vectors), alphaviruses, AAV vectors, and naked DNA vectors.
  • Figure 1 is a schematic illustration of pKm201bFGF-2.
  • Figure 2 is the nucleic acid sequence of pKm201bFGF-2 (SEQ I.D.
  • Figure 3 is a schematic illustration of pD10-CMV-FGF-5.
  • Figure 4 is a western analysis of FGF-5 rAAV infected 293 cells.
  • Figure 5 is a schematic illustration of pD10-CMV-FGF-5 (sig-).
  • Figure 6 is a western analysis of pD10-CMV-FGF-5 (sig-) transfected 293 cells.
  • Figure 7 is a schematic illustration of pD 10-CMV-FGF- 18.
  • Figure 8 western analysis of 293 cells transfected pD10-CMV-FGF-18.
  • Figures 9A and 9B are photographs which show that bluo-gal staining is visible across 40% of a retina transfected with AAV-CMV-lacZ. All photoreceptors appear to express lacZ at the injection site, except at the edge where individual cells are visible.
  • Figure 10 is a schematic illustration which shows the retina within the eye, and the organization of cells within the retina.
  • FIGS 11A and 11B are photographs of wild-type and degenerated S334ter rat retinas.
  • S334ter is a rat model for retinitis pigmentosa.
  • Figures 12A, 12B and 12C are photographs of degenerated S334ter,
  • FGF-2 injected S334ter and PBS injected S334ter rat retinas substantially slows the progression of disease.
  • Figure 13 is a graph which plots Outer Nuclear Layer (ONL) thickness for FGF-2 subretinally injected, PBS subretinally injected, and an uninjected control.
  • ONL Outer Nuclear Layer
  • Figure 14 is a bar graph which plots ONL thickness at p60.
  • Figures 15 A, 15B and 15C are photographs of FGF-2 expressing cells stained with an anti-FGF-2 antibody.
  • Figures 16A and 16B are photographs which show gene delivery to cells in the ganglion cell layer following intraocular injection of recombinant rAAV-CMV- lacZ.
  • RGCs Retinal radial section showing the AAV -mediated LacZ gene product in cells of the ganglion cell layer.
  • a large number of these cells can be identified as RGCs because of the intense LacZ staining in axons projecting to the optic nerve head (asterisk).
  • RPE retinal pigment epithelium
  • PS photoreceptor segments
  • ONL outer nuclear layer
  • OPL outer plexiform layer
  • INL inner nuclear layer
  • IPL inner plexiform layer
  • GCL ganglion cell layer.
  • Scale bars a) 0.5 mm, b) 50 ⁇ m.
  • Figure 17 is a graph which shows the time-course of AAV-mediated transgene expression in the ganglion cell layer of the adult rat retina.
  • a recombinant AAV vector (rAAV-CMV-lacZ) was injected into the vitreous chamber of adult rats and 2, 4 and 8 weeks later, Lac-Z positive neurons in the ganglion cell layer (GCL) were counted in retinal flat-mounts.
  • the values are the mean of 3-4 retinas per time point ⁇ standard deviation (pO.OOl).
  • FIGS 18A and 18B are photographs which show the localization of the AAV-mediated LacZ gene product in retrogradely labeled RGCs.
  • FIG. 19 is a graph which quantifies Fluorogold- and LacZ-positive cells in the ganglion cell layer following intravitreal injection of rAAV-CMV-lacZ.
  • FIG 20 is a photograph which shows the localization of the heparan sulfate (HS) proteoglycan receptor, the cellular receptor for AAV, in the intact adult rat retina.
  • Retinal cryosection immunostained with a polyclonal antibody against the heparan sulfate (HepSS-1; diluted 1:200) followed by biotinylated anti-rabbit Fab fragment, avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame, CA). The section was reacted in a solution containing 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.06% hydrogen peroxide in PB (pH 7.4) for 5 min.
  • DABC Elite Vector Labs Burlingame, CA
  • FIG. 21 is a schematic illustration of pDlO-VEGFuc
  • Figure 22 is the nucleotide sequence of pDlO-VEGFuc
  • Figure 23 is a bar graph which shows pDlO-VEGFuc rAAV virus infection of 293 cells.
  • Figure 24 is a three dimensional bar-graph which shows VEGF secretion by hfRPE after infection with VEGF AAV.
  • Figure 25 is a three dimensional bar-graph which shows VEGF secretion by hfRPE after infection with VEGF AV
  • Figure 26 is a three dimensional bar-graph which shows resistance of hfRPE after infection with VEGF AV.
  • Figure 27 is a schematic illustration of pD 10-sFlt- 1.
  • Figure 28 is the nucleotide sequence of pDlO-sFlt-1.
  • Figure 29 is the nucleotide sequence of FGF-20.
  • Figure 30 is the nucleotide sequence of FGF-21.
  • Figure 31 is a schematic illustration of pD10K-FGF-2Sc.
  • Figure 32 is the nucleotide sequence of pD 10K-FGF-2Sc.
  • Figure 33 is a graph which compares ONL thickness (um) after injection of various vectors into the eye.
  • Gene delivery vector refers to a construct which is capable of delivering, and, within preferred embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • Representative examples of such vectors include viral vectors, nucleic acid expression vectors, naked DNA, and certain eukaryotic cells
  • producer cells e.g., producer cells
  • Recombinant adeno-associated virus vector refers to a gene delivery vector based upon an adeno-associated virus.
  • the rAAV vectors should contain 5' and 3' adeno-associated virus inverted terminal repeats (ITRs), and a transgene or gene of interest operatively linked to sequences which regulate its expression in a target cell.
  • the transgene may be operably linked to a heterologous promoter (such as CMV), or, an inducible promoter such as (tet).
  • the rAAV vector may have a polyadenylation sequence.
  • Neurotrophic Factor refers to proteins which are responsible for the development and maintenance of the nervous system.
  • Representative examples of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT-4, and Fibroblast Growth Factors.
  • FGF Fibroblast Growth Factor
  • Anti-angiogenic Factor refers to a factor or molecule which is able to inhibit the proliferation of vascular growth.
  • assays may be utilized to assess the anti-angiogenic activity of a given molecule, including for example, the assay provided in Example 15, which measures HUVEC (human umbilical vein endothelial cell) proliferation.
  • anti-angiogenic factors include for example, Angiostatin, 1,25-Di-hydroxy-vitamn D 3 , Endostatin, IGF-1 receptor antagonists, Interferons alpha, beta and gamma, Interferon gamma-inducible protein IP- 10, Interleukin 1 alpha and beta, Interleukin 12, 2-Methoxyestradiol, PEDF, Platelet factor 4, Prolactin (16kd fragment), Protamin, Retinoic acid, Thrombospondin-1 and 2, Tissue inhibitor of metalloproteinase-1 and -2, Transforming growth factor beta, soluble Tie-2 receptor, soluble Tie-2 receptor, soluble Flt-1 and Tumor necrosis factor - alpha.
  • Diseases of the Eve refers to a broad class of diseases wherein the functioning of the eye is affected due to damage or degeneration of the photoreceptors; ganglia or optic nerve; or neovascularization.
  • Representative examples of such diseases include macular degeneration, diabetic retinopathies, inherited retinal degeneration such as retinitis pigmentosa, glaucoma, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically).
  • the present invention provides compositions and methods for treating, preventing, or, inhibiting diseases of the eye, comprising the general step of administering intraocularly a recombinant adeno-associated viral vector which directs the expression of one or more neurotrophic factors, such that the disease of the eye is treated or prevented.
  • a more detailed discussion is provided below regarding (A) gene delivery vectors; (B) Neurotrophic Factors; (C) Anti-angiogenic factors; and (D) methods of administering the rAAVs in the treatment or prevention of diseases of the eye.
  • retroviral gene delivery vectors are provided which are constructed to carry or express a selected gene(s) or sequence(s) of interest.
  • retroviral gene delivery vectors of the present invention may be readily constructed from a wide variety of retrovimses, including for example,
  • retrovimses as well as spumavimses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • retrovimses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC”; Rockville, Maryland), or isolated from known sources using commonly available techniques.
  • ATCC American Type Culture Collection
  • retroviral gene delivery vectors may be readily utilized in order to assemble or construct retroviral gene delivery vectors given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkel, RN4S ⁇ 2:488, 1985).
  • portions of the retroviral gene delivery vectors may be derived from different retrovimses.
  • retrovims LTRs may be derived from a Murine Sarcoma Vims, a tR ⁇ N binding site from a Rous Sarcoma Vims, a packaging signal from a Murine Leukemia Vims, and an origin of second strand synthesis from an Avian Leukosis Vims.
  • retrovector constmcts comprising a 5' LTR, a tR ⁇ A binding site, a packaging signal, one or more heterologous sequences, an origin of second strand D ⁇ A synthesis and a 3' LTR, wherein the vector constmct lacks gaglpol or env coding sequences.
  • retroviral gene delivery vectors may likewise be utilized within the context of the present invention, including for example EP 0,415,731; WO 90/07936; WO 91/0285, WO 9403622; WO 9325698; WO 9325234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-961, 1993; Ram et al., Cancer Res. 5 :83-88, 1993; Takamiya et al, J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg.
  • Packaging cell lines suitable for use with the above described retrovector constmcts may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also U.S. Serial No. 07/800,921, filed November 27, 1991), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.
  • VCLs vector cell lines
  • the rAAV should be comprised of, in order, a 5' adeno-associated vims inverted terminal repeat, a transgene or gene of interest operatively linked to a sequence which regulates its expression in a target cell, and a 3' adeno-associated vims inverted terminal repeat.
  • the rAAV vector may preferably have a polyadenylation sequence.
  • rAAV vectors should have one copy of the AAV ITR at each end of the transgene or gene of interest, in order to allow replication, packaging, and efficient integration into cell chromosomes.
  • the ITR consists of nucleotides 1 to 145 at the 5' end of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same sequence) at the 3' end of the AAV DNA genome.
  • the rAAV vector may also include at least 10 nucleotides following the end of the ITR (i.e., a portion of the "D region").
  • the transgene sequence will be of about 2 to 5 kb in length (or alternatively, the transgene may additionally contain a "stuffer” or "filler” sequence to bring the total st. c of the nucleic acid sequence between the two ITRs to between 2 and 5 kb).
  • the transgene may be composed of same heterologous sequence several times (e.g., two nucleic acid molecules which encode FGF-2 separated by a ribosome readthrough, or alternatively, by an Internal Ribosome Entry Site or "IRES"), or several different heterologous sequences (e.g., FGF-2 and FGF-5, separated by a ribosome readthrough or an IRES).
  • Recombinant AW vectors of the present invention may be generated from a variety of adeno-associated vimses, including for example, serotypes 1 through 6.
  • ITRs from any AAV serotype are expected to have similar stmctures and functions with regard to replication, integration, excision and transcriptional mechanisms.
  • expression of the transgene may be accomplished by a separate promoter (e.g., a viral promoter).
  • suitable promoters include a CMV promoter, RSV promoter, SV40 promoter, or MoMLV promoter.
  • Other promoters that may similarly be utilized within the context of the present invention include cell or tissue specific promoters (e.g., a rod, cone, or ganglia derived promoter), or inducible promoters.
  • suitable inducible promoters include tetracycline-response promoters ("Tet", see, e.g., Gossen and Bujard, Proc. Natl. Acad. Sci. USA.
  • the rAAV vector may also contain additional sequences, for example from an adenovims, which assist in effecting a desired function for the vector.
  • additional sequences include, for example, those which assist in packaging the rAAV vector in adenovims-associated vims particles.
  • Packaging cell lines suitable for producing adeno-associated viral vectors may be readily accomplished given readily available techniques (see e.g., U.S. Patent No. 5,872,005).
  • the present invention also provides a variety of Alphavims vectors which may function as gene delivery vectors.
  • Sindbis vims is the prototype member of the alphavims genus of the togavims family.
  • the unsegmented genomic RNA (49S RNA) of Smdbis vims is approximately 11,703 nucleotides in length, contains a 5' cap and a 3' poly-adenylated tail, and displays positive polarity.
  • Infectious enveloped Sindbis vims is produced by assembly of the viral nucleocapsid proteins onto the viral genomic RNA in the cytoplasm and budding through the cell membrane embedded with viral encoded glycoproteins.
  • the packaging sequence resides within the nonstmctural coding region, therefore only the genomic 49S RNA is packaged into virions.
  • Sindbis vector systems may be constmcted and utilized within the present invention. Representative examples of such systems include those described within U.S. Patent Nos. 5,091,309 and 5,217,879, and PCT Publication No. WO 95/07994.
  • viral gene delivery vectors In addition to retroviral vectors and alphavims vectors, numerous other viral vectors systems may also be utilized as a gene delivery vector. Representative examples of such gene delivery vectors include vimses such as pox viruses, such as canary pox vims or vaccinia vims (Fisher-Hoch et al., RN4S 86:311-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 5 9:86-103, 1989; Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent ⁇ os.
  • vimses such as pox viruses, such as canary pox vims or vaccinia vims (Fisher-Hoch et al., RN4S 86:311-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 5 9:86-103, 1989; Flexner et al., Vaccine 5:17
  • viral carriers may be homologous, non-pathogenic(defective), replication competent vims (e.g., Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular immune responses, including CTL.
  • non-viral gene delivery vectors may likewise be utilized within the context of the present invention.
  • Representative examples of such gene delivery vectors include direct delivery of nucleic acid expression vectors, naked DNA alone (WO 90/11092), polycation condensed DNA linked or unlinked to killed adenovims (Curiel et al., Hum. Gene Ther. 3:147-154, 1992), DNA ligand linked to a ligand with or without one of the high affinity pairs described above (Wu et al, J. of Biol.
  • nucleic acid containing liposomes e.g., WO 95/24929 and WO 95/12387
  • certain eukaryotic cells e.g., producer cells - see U.S. Serial No. 08/240,030, filed May 9, 1994, and U.S. Serial No. 07/800,921).
  • neurotrophic factor refers to proteins which are responsible for the development and maintenance of the nervous system.
  • Representative examples of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT-4, and Fibroblast Growth Factors.
  • Fibroblast Growth Factor refers to a family of related proteins, the first of which was isolated from the pituitary gland (see Gospodarowicz, D., Nature, 249:123-127, 1974). From this original FGF (designated basic FGF) a family of related proteins, protein muteins, and protein analogs have been identified (see, e.g., U.S. Patent Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226; see generally Baird and Gospodarowicz, D. Ann NY. Acad. Sci.
  • the first two members of the family to be identified were acidic fibroblast growth factor (aFGF/FGF-1) and basic fibroblast growth factor (bFGF/FGF-2). Additional members of the FGF family include: i-nt-2/FGF-3, (Smith et al., EMBO J. 1: 1013, 1988); FGF-4 (Delli-Bovi et al., Cell 50: 729, 1987); FGF-6 (Maries et al., Oncogene 4: 335 (1989); keratinocyte growth factor/FGF-7, (Finch et al, Science 245: 752, 1989); FGF-8 (Tanaka et al, Proc. Natl. Acad Sci. USA 89: 8928, 1992); and FGF-9 (Miyamoto et al., Mol. Cell Biol. 13: 4251, 1993).
  • FGF-5 was originally isolated as an oncogene. See Goldfarb et al. US Patent Nos. 5,155,217 and 5,238,916, Zhan et al. "Human Oncogene Detected by a Defined Medium Culture Assay” (Oncogene 1:369-316, 1987), Zhan et al. "The Human FGF-5 Oncogene Encodes a Novel Protein Related to Fibroblastic Growth Factors" (Molecular and Cellular Biology 5:3487-3495, 1988), and Bates et al. "Biosynthesis of Human Fibroblast Growth Factor 5": (Molecular and Cellular Biology 11: 1840-1845, 1991).
  • FGFs include those disclosed in U.S. Patent Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226. 5,852,177, and 5,872,226, as well as FGF-20 (U.S. Provisional Application No. 60/161,162) and FGF-21 (U.S. Provisional Application No. 60/166,540).
  • FGF-20 U.S. Provisional Application No. 60/161,162
  • FGF-21 U.S. Provisional Application No. 60/166,540.
  • anti-angiogenic factors may also be expressed from the gene delivery vectors of the present invention, including for example, Angiostatin (O'Reilly et al, Cell 79:315-328, 1994; O'Reilly et al., Nat. Med. 2:689-92, 1996; Sim et al, Cancer Res. 57:1329-34, 1997), 1,25-Di-hydroxy-vitamn D 3 (Shibuya et al., Oncogene 5:519-24, 1990; Oikawa et al, Eur. J. Pharmacol.
  • VEGF antagonists such as soluble Flt-1 (Kendall and Thomas, PNAS 90: 10705, 1993), and Ang-1 antagonists such as soluble Tie-2 receptor (Thurston et al., Science 255:2511, 1999; see also, generally Aiello et al, RN4S 92:10457, 1995; Robinson et al., RN4S 93:4851, 1996; Seo et al., Am. J. Pathol. 754:1743, 1999).
  • VEGF antagonists such as soluble Flt-1 (Kendall and Thomas, PNAS 90: 10705, 1993)
  • Ang-1 antagonists such as soluble Tie-2 receptor (Thurston et al., Science 255:2511, 1999; see also, generally Aiello et al, RN4S 92:10457, 1995; Robinson et al., RN4S 93:4851, 1996; Seo et al., Am. J. Pathol. 754:174
  • the present invention provides methods which generally comprise the step of intraocularly administering a gene delivery vector which directs the expression of one or more neurotrophic factor to the eye, or an anti-angiogenic factor to the eye in order to treat, prevent, or inhibit the progression of an eye disease.
  • a gene delivery vector which directs the expression of one or more neurotrophic factor to the eye, or an anti-angiogenic factor to the eye in order to treat, prevent, or inhibit the progression of an eye disease.
  • the terms "treated, prevented, or, inhibited” refers to the alteration of a disease course or progress in a statistically significant manner. Determination of whether a disease course has been altered may be readily assessed in a variety of model systems, discussed in more detail below, which analyze the ability of a gene delivery vector to delay, prevent or rescue photoreceptors, as well as other retinal cells, from cell death.
  • a wide variety of diseases of the eye may be treated given the teachings provided herein.
  • gene delivery vectors are administered to a patient intraocularly in order to treat or prevent macular degeneration.
  • MD macular degeneration
  • AMD age related macular degeneration
  • laser treatment has been shown to reduce the risk of extensive macular scarring from the "wet" or neovascular form of the disease, there are currently no effective treatments for the vast majority of patients with MD.
  • gene delivery vectors can be administered to a patient intraocularly in order to treat or prevent an inherited retinal degeneration.
  • a patient intraocularly in order to treat or prevent an inherited retinal degeneration.
  • RP retinitis pigmentosa
  • photoreceptor cells and the RPE.
  • gene delivery vectors which direct the expression of a neurotrophic growth factor can be administered to a patient intraocularly in order to treat or prevent glaucoma.
  • glaucoma is not a uniform disease but rather a heterogeneous group of disorders that share a distinct type of optic nerve damage that leads to loss of visual function.
  • the disease is manifest as a progressive optic neuropathy that, if left untreated leads to blindness. It is estimated that as many as 3 million Americans have glaucoma and, of these, as many as 120,000 are blind as a result. Furthermore, it is the number one cause of blindness in African- Americans. Its most prevalent form, primary open-angle glaucoma, can be insidious.
  • neurotrophic growth factors such as FGF-2, 5, 18, 20, and, 21.
  • gene delivery vectors can be administered to a patient intraocularly in order to treat or prevent injuries to the retina, including retinal detachment, photic retinopathies, surgery-induced retinopathies, toxic retinopathies, retinopathies due to trauma or penetrating lesions of the eye.
  • the present invention also provides methods of treating, preventing, or, inhibiting neovascular disease of the eye, comprising the step of administering to a patient a gene delivery vector which directs the expression of an anti- angiogenic factor.
  • neovascular diseases include diabetic retinopathy, ARMD (wet form), and retinopathy of prematurity.
  • AMD Age-related Macular Degeneration
  • Retinal neovascularization occurs in diseases such as diabetic retinapathy and retinopathy of prematurity (ROP), the most common cause of blindness in the young.
  • Particularly preferred vectors for the treatment, prevention, or, inhibition of neovascular diseases of the eye direct the expression of an anti-angiogenic factor such as, for example, soluble tie-2 receptor or soluble Fit- 1.
  • a novel model for neovascularization (either choroidal or subretinal) can be generated by subretinal injection of a recombinant vims (rAV or rAAV) containing an angiogenic transgene such as VEGF and/or angiopoietin.
  • rAV or rAAV recombinant vims
  • the extent and duration of neovascularization induced by rAAV and rAV vectors containing an angiogenic transgene such as VEGF can be determined using fundus photography, fluorescein angiography and histochemistry.
  • a D10- sFlt-1 rAAV (or other gene delivery vector which directs the expression of an anti-angiogenic factor) is intraocularly injected, either by subretinal or intravitreal routes of injection.
  • subretinal injection of the gene delivery vector may be utilized to achieve delivery to both the choroidal and inner retinal vasculature.
  • Intravitreal injection can be utilized to infect Muller cells and retinal ganglion cells (RGCs), which deliver anti-angiogenic protein to the retinal vasculature. Muller cells span the retina and would secrete the therapeutic protein into the subretinal space.
  • Such injections may be accomplished either prior to, simultaneous with, or subsequent to administration of an angiogenic factor or gene delivery vector which expresses an angiogenic factor.
  • inhibition of neovascularization can be determined using fundus photography, fluorescein angiography and/or histochemistry.
  • VEGF transgenic mouse While there are many animal models of retinal neovascularization such as oxygen-induced ischemic retinopathy (Aiello et al., PNAS 93: 4881, 1996.) and the VEGF transgenic mouse (Okamoto et al., Am. J. Pathol. 151 : 281, 1997), there are fewer models of choroidal neovascularization (e.g., laser photocoagulation as described by Murata et al., IOVS 39: 2474, 1998). Subretinal neovascularization from the retinal rather than choroidal blood supply is also observed in VEGF transgenic animals (Okamoto et al., Am. J. Pathol. 151: 281, 1997). Hypoxic stimulation of VEGF expression is known to correlate with neovascularization in human ocular disease.
  • RRCs retinal ganglion cells
  • the loss of RGCs caused by ON transection in adult mammals varies from 50% to more than 90% depending on the techniques used to identify RGCs, the proximity of the lesion to the eye, and the age and species of the animal. For example, in a study in adult rats, in which retrogradely transported tracers were used to distinguish RGCs from displaced amacrine cells (Villegas-Perez, M.P., Vidal-Sanz, M., Bray, G.M. and Aguayo, A.J., J. Neurosci. 5:265-280, 1988). ON transection near the eye (0.5-1 mm) leads to the loss of more than 90% of the RGCs by 2 weeks.
  • the posterior pole of the left eye and the origin of the optic nerve are exposed through a superior temporal intraorbital approach.
  • a longitudinal excision of the ON dural sheath is performed.
  • the ON is then gently separated from the dorsal aspect of this sheath and completely transected within the orbit, within 1 mm of the optic disc. Care is taken to avoid damage to the ophthalmic artery, which is located on the inferomedial dural sheath of the ON.
  • RGC survival and death following gene delivery can also be examined using two alternative models of ON injury: 1) ON cmsh; and (2) increased intraocular pressure.
  • ON cmsh ON cmsh
  • intraocular pressure In the first model the ON is exposed, and then clamped at a distance of about one millimeter from the posterior pole using a pair of calibrated forceps as previously described (Li et al., Invest. Ophthalmol. Vis. Sci. 40:1004, 1999).
  • chronic moderately elevated intraocular pressure can be produced unilaterally by cauterization of three episcleral vessels as described by Neufeld et al. in PNAS 17:9944, 1999).
  • a variety of animal models can be utilized for photoreceptor degeneration, including the RCS rat model, P23H transgenic rat model, the rd mouse, and the S334ter transgenic rat model.
  • the S334ter transgenic rat model a mutation occurs resulting in the tmncation of the C-terminal 15 amino acid residues of rhodopsin (a seven- transmembrane protein found in photoreceptor outer segments, which acts as a photopigment).
  • the S334ter mutation is similar to rhodopsin mutations found in a subset of patients with retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • RP is a heterogeneous group of inherited retinal disorders in which individuals experience varying rates of vision loss due to photoreceptor degeneration. IN many RP patients, photoreceptor cell death progresses to blindness.
  • Transgenic S334ter rats are bom with normal number of photoreceptors. The mutant rhodopsin gene begins expression at postnatal day 5 in the rat, and photoreceptor cell death begins at postnatal day 10-15.
  • transgenic line S334ter-3 approximately 70% of the outer nuclear layer has degenerated by day 60 in the absence of any therapeutic intervention. The retinal degeneration in this model is consistent from animal to animal and follows a predictable and reproducible rate.
  • S334ter rats are utilized as a model for RP as follows. Briefly, S334ter transgenic rats are euthanized by overdose of carbon dioxide inhalation and immediately perfused intracardially with a mixture of mixed aldehydes (2% formaldehyde and 2.5 % glutaraldehyde). Eyes are removed and embedded in epoxy resin, and 1 ⁇ m thick histological sections are made along the vertical meridian.
  • Tissue sections are aligned so that the ROS and Muller cell processes crossing the inner plexiform layer are continuous throughout the plane of section to assure that the sections are not oblique, and the thickness of the ONL and lengths of RIS and ROS are measured. These retinal thickness measurements are plotted and establish the baseline retinal degeneration rates for the animal model.
  • the assessment of retinal thickness is as follows: briefly, 54 measurements of each layer or stmcture were made at set points around the entire retinal section. These data were either averaged to provide a single value for the retina, or plotted as a distribution of thickness or length across the retina.
  • TgN(s334ter) line 4 (abbreviated s334ter 4) can be utilized for in vivo experiments. Briefly, in this rat model expression of the mutated opsin transgene begins at postnatal day P5 in these rats, leading to a gradual death of photoreceptor cells. These rats develop an anatomically normal retina up to P15, with the exception of a slightly increased number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) than in non- transgenic control rats. In this animal model , the rate of photoreceptor cell death is approximately linear until P60, resulting in loss of 40-60% of the photoreceptors. After P60, the rate of cell loss decreases, until by one year the retinas have less than a single row of photoreceptor nuclei remaining.
  • ONL outer nuclear layer
  • Gene delivery vectors of the present invention may be administered intraocularly to a variety of locations depending on the type of disease to be treated, prevented, or, inhibited, and the extent of disease. Examples of suitable locations include the retina (e.g., for retinal diseases), the vitreous, or other locations in or adjacent to the eye.
  • the human retina is organized in a fairly exact mosaic.
  • the mosaic is a hexagonal packing of cones.
  • the rods break up the close hexagonal packing of the cones but still allow an organized architecture with cones rather evenly spaced surrounded by rings of rods.
  • the cone density is highest in the foveal pit and falls rapidly outside the fovea to a fairly even density into the peripheral retina (see Osterberg, G. (1935) Topography of the layer of rods and cones in the human retina.
  • the amount of the specific viral vector applied to the retina is uniformly quite small as the eye is a relatively contained stmcture and the agent is injected directly into it.
  • the amount of vector that needs to be injected is determined by the intraocular location of the chosen cells targeted for treatment.
  • the cell type to be transduced will be determined by the particular disease entity that is to be treated.
  • a single 20-micro liter volume (of 10" physical particle titer/ml rAAV) may be used in a subretinal injection to treat the macula and fovea.
  • a larger injection of 50 to 100 microliters may be used to deliver the rAAV to a substantial fraction of the retinal area, perhaps to the entire retina depending upon the extent of lateral spread of the particles.
  • a 100-ul injection will provide several million active rAAV particles into the subretinal space. This calculation is based upon a titer of 10' 3 physical particles per milliliter. Of this titer, it is estimated that 1/1000 to 1/10,000 of the AAV particles are infectious.
  • the retinal anatomy constrains the injection volume possible in the subretinal space (SRS). Assuming an injection maximum of 100 microliters, this would provide an infectious titer of 10 8 to 10 9 rAAV in the SRS. This would have the potential of infecting all of the -150 x 10 6 photoreceptors in the entire human retina with a single injection.
  • SRS subretinal space
  • Smaller injection volumes focally applied to the fovea or macula may adequately transfect the entire region affected by the disease in the case of macular degeneration or other regional retinopathies.
  • Gene delivery vectors can alternately be delivered to the eye by intraocular injection into the vitreous.
  • the primary target cells to be transduced are the retinal ganglion cells, which are the retinal cells primarily affected in glaucoma.
  • the injection volume of the gene delivery vector could be substantially larger, as the volume is not constrained by the anatomy of the subretinal space. Acceptable dosages in this instance can range from 25 ul to 1000 ul.
  • assays may be utilized in order to determine appropriate dosages for administration, or to assess the ability of a gene delivery vector to treat or prevent a particular disease. Certain of these assays are discussed in more detail below.
  • Electroretinographic analysis can be utilized to assess the effect of gene delivery administration into the retina. Briefly, rats are dark adapted overnight and then in dim red light, then anesthetized with intramuscular injections of xylazine (13 mg/kg) and ketamine (87 mg/kg). Full-field scotopic ERGs are elicited with 10- ⁇ sec flashes of white light and responses were recorded using a UTAS-E 2000 Visual Electrodiagnostic System (LKC Technologies, Inc., Gaithersburg, MD). The corneas of the rats are the anesthetized with a drop of 0.5% proparacaine hydrochloride, and the pupils dilated with 1% atropine and 2.5% phenylephrine hydrochloride.
  • Small contact lenses with gold wire loops are placed on both corneas with a drop of 2.5% methylcellulose to maintain corneal hydration.
  • a silver wire reference electrode is placed subcutaneously between the eyes and a ground electrode is placed subcutaneously in the hind leg.
  • Stimuli are presented at intensities of -1.1, 0.9 and 1.9 log cd m ⁇ 2 at 10-second, 30- second and 1 -minute intervals, respectively.
  • Responses are amplified at a gain of 4,000, filtered between 0.3 to 500 Hz and digitized at a rate of 2,000 Hz on 2 channels. Three responses are averaged at each intensity.
  • a-waves are measured from the baseline to the peak in the comea-negative direction, and b-waves are measured from the comea- negative peak to the major comea-positive peak.
  • the values from all the stimulus intensities are averaged for a given animal.
  • retinal tissue analysis can also be utilized to assess the effect of gene delivery administration into the retina.
  • Gene delivery vectors may be prepared as a pharmaceutical product suitable for direct administration.
  • the vector should be admixed with a pharmaceutically acceptable carrier for intraocular administration.
  • suitable carriers are saline or phosphate buffered saline.
  • nucleic acid sequence of this deposit as well as the amino acid sequence of the polypeptide encoded thereby, are incorporated herein by reference and should be referred to in the event of an error in the sequence described herein.
  • a license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.
  • pKm201CMV is an AAV cloning vector in which an expression cassette, consisting of a CMV immediate early promoter/enhancer and a bovine growth hormone (BGH) polyadenylation site, is flanked by inverted terminal repeat (ITR) sequences from AAV-2.
  • ITR inverted terminal repeat
  • pCMVlink a derivative of pCMV ⁇ c (see Chapman, Nucleic Acids Res. 79:193-198 (1991)) in which the BGH poly A site has been substituted for the SV40 terminator, was inserted between the ITRs of pKm201 to generate pKm201CMV.
  • pKm201bFGF-2 was constmcted by cloning the following, in order, into the multiple cloning site of pKm201CMV: the encephalomyocarditis vims (EMCV) internal ribosome entry site (IRES), the bovine FGF-2 cDNA, and the human growth hormone polyadenylation sequence.
  • the cDNA for FGF-2 has two mutations that change amino acid 121 from serine to threonine and amino acid 137 from pro line to serine.
  • the DNA sequence of pKm201bFGF-2 is shown in Figure 2 and the plasmid has been deposited with the American Type Culture Collection (ATCC). rAAV vector particles were produced by a triple transfection protocol (Nucleic Acids Res. 24:596-601, 1996; J. Exp. Med. 779:1867-1875, 1994).
  • human embryonic kidney 293 cells grown to 50% confluence in a 10 layer Nunclon cell factory (Nalge Nunc, Int., NaperviUe, IL), were co-transfected with 400 ⁇ g of helper plasmid pKSrep/cap (Hum. Gene Ther. 9:477-485, 1998) 400 ⁇ g of vector plasmid, and 800 ⁇ g of adenovims plasmid pBHGlO (Microbix Biosystems, Inc., Toronto, Ontario) using the calcium phosphate co-precipitation method.
  • MOI multiplicity of infection
  • Packaged rAAV was purified from adenovims by two rounds of cesium chloride equilibrium density gradient centrifugation. Residual adenovims contamination was inactivated by heating at 56°C for 45 min.
  • rAAV vector construct and the AAV helper gene constmct on one plasmid. This would allow rAAV to be produced by transfecting 293 cells with two plasmids. Alternatively, one could add the adenovims helper genes to this plasmid to make a single plasmid containing all that is required to make rAAV particles.
  • the vims stock was treated with DNAse I, and encapsidated DNA was extracted with phenol-chloroform, and precipitated with ethanol. DNA dot blot analysis against a known standard was used to determine titer (Blood 75:1997-2000, 1990).
  • DNA dot blot analysis against a known standard was used to determine titer (Blood 75:1997-2000, 1990).
  • 293 cells were infected with 10 ⁇ l of purified rAAV stock and followed for any signs of cytopathic effect. All stocks were negative for adenovims contamination (level of detection greater than or equal to lOO PFU/ml).
  • 293 cells were co-infected with serial dilutions of rAAV stocks and adenovims dl312 at a MOI of 2. Three days later the cells were harvested, lysed by three cycles of freezing/thawing, and centrifuged to remove cell debris. The supernatant was heat inactivated (56°C for 10 min) and fresh plates of 293 cells (6 x 10 6 ) were infected in the presence of adenovims dl312 at a MOI of 2. Forty-eight hours after infection, low molecular-weight DNA was isolated (J Mol. Biol.
  • the wild type AAV titer was defined as the highest dilution of rAAV vector stock demonstrating a positive hybridization signal.
  • the rAAV preparations contained less than 1 wild type AAV genome per 10 9 rAAV genomes.
  • Etoposide is a topoisomerase inhibitor which has been shown to increase transduction efficiency of rAAV vectors (Proc. Natl. Acad. Sci. USA, 92:5719- 5723, 1995).
  • the pDIO AAV vector is constmcted by replacing the AAV gene encoding sequences of pD-10 (see Wang, X. et al. J. Virol. 71 :3077 (1997), with the
  • oligonucleotides 5'-ggtatttaaa acttgcggcc gcggaatttc gactctaggc c-3' SEQ I.D. No.
  • the FGF-5 coding region (see U.S. Serial No. 08,602,147) was cloned into the rAAV pDlO-CMV vector by digestion with the enzymes SacII and Xmnl, resulting in an 814 bp fragment. This removed an ORF (ORF-1) upstream of and overlapping with the FGF-5 coding region. The ends of the FGF-5 fragment were then blunted with T4 DNA polymerase, and it was cloned into the rAAV pDlO-CMV vector linearized with Stul. This vector contains a 1353 bp insertion of a bacteriophage Phi X174 HaeIII fragment.
  • the pD10-CMV-FGF-5 vector is illustrated schematically in Figure 3.
  • this plasmid contains the CMV immediate/early enhancer + promoter, the CMV intron A, an FGF-5 coding region, the bovine growth hormone polyA site, and AAV ITR sequences.
  • PhiX 174 bacteriophage DNA cloned into the Notl site between one ITR and the CMV immediate early enhancer + promoter region.
  • rAAV vims was packaged using a triple transfection method as described in Example 1. However, rather than cesium chloride equilibrium density gradient centrifugation, heparin sulfate column chromatography is utilized. More specifically, a cell pellet is resuspended in TNM buffer: 20mM Tris pH 8.0, 150mM NaCl, 2mM MgCl 2 . Deoxycholic Acid is added to 0.5% to lyse the cells. 50 U/ml Benzonase is added and the lysed cells are incubated at 37 degrees to digest any nucleic acids.
  • the cell debris is pelleted and the supernatant is filtered through a 0.45um filter and then a 0.22um filter.
  • the vims is then loaded onto a 1.5ml Heparin sulfate column using the Biocad HPLC.
  • the column is then washed with 20mM Tris pH 8.0, lOOmM NaCl.
  • the rAAV particles are eluted with a gradient formed with increasing concentrations of NaCl.
  • the fractions under the peak are pooled and filtered through a 0.22um filter before overnight precipitation with 8% PEG 8000.
  • CaCl 2 is added to 25mM and the purified particles are pelleted and then resuspended in HBS#2: 150mM NaCl, 50mM Hepes pH7.4.
  • tissue culture media and cell lysates were harvested and analysed by Western blotting. Briefly, protein samples were ran on a 4-20% tris-glycine gradient gel, and transferred to nitrocellulose by standard procedures. After blocking with 5% milk in PBS, the membrane was incubated with an anti-human FGF-5 antibody (R and D systems, made in goat) at a dilution of 1 : 1 ,000 for one hour at room temperature.
  • an anti-human FGF-5 antibody R and D systems, made in goat
  • the membrane was washed 3 times in PBS + 0.05% Tween-20, it was incubated with an anti-goat secondary antibody conjugated to peroxidase (1 :5,000 dilution). The membrane was then washed and the FGF-5 protein detected by chemiluminescence.
  • lane 1 represents 50 ng of the 29.5 Kd recombinant FGF-5 protein (R and D systems).
  • Lane 2 media from cells infected with 8 x 10 9 viral particles and treated with etoposide, shows no FGF-5 expression.
  • Lane 3 is an uninfected cell lysate control.
  • Lane 4 and 5 are lysates from cells infected with 8 x 10 8 or 8 x 10 9 viral particles, respectively, and
  • Lanes 6 and 7 are lysates from cells infected with 8 x 10 8 or 8 x 10 9 viral particles and treated with 3 uM etoposide.
  • Lanes 4-7 all show positive FGF-5 expression.
  • Lane 8 is a negative control of lysate from uninfected cells. In summary, although the FGF-5 signal sequence was intact, FGF-5 protein was detected in the cell lysate only.
  • Oncogenic activity is associated with the wild-type FGF-5 molecule
  • the 5' primer contains point mutations which destabilize G/C rich hairpin stmctures of the FGF-5 mRNA, and should increase levels of gene expression.
  • the PCR product was digested with Hindlll and Xhol (restriction sites introduced by the primers), and cloned by standard methods, into the pDIO vector digested with the same enzymes.
  • the pD10-CMV-FGF-5 (sig-) vector is illustrated schematically in Figure 5.
  • the pD10-CMV-FGF-5 (sig-) plasmid contains the CMV immediate/early enhancer + promoter, the CMV intron A, the FGF-5 coding region with the modifications described in Example C above, the bovine growth hormone polyA site, and the AAV ITR sequences.
  • FGF-5 protein was demonstrated by transient transfection of 293 cells with the plasmid pD10-CMV-FGF-5 (sig-), by standard methods. After 48 hours, tissue culture media and cell lysates were harvested. Western analysis was performed with an anti-human FGF-5 antibody (R and D systems) as described above.
  • lane 1 represents 50 ng of the 29.5 Kd recombinant FGF-5 prot? ⁇ n (R and D systems).
  • Lanes 2 and 3 showing FGF-5 expression, are cell lysates from 293 cells transfected with two different clones of the pD10-CMV-FGF-5 sig- plasmid.
  • Lane 4 is lysate from cells transfected with a negative control plasmid CMV-Epo.
  • Lanes 5, 6 and 7 represent media from cells transfected with different clones of the pD10-CMV-FGF-5 sig- plasmid, respectively, and the CMV-Epo plasmid.
  • FGF- 5 protein was detected in the cell lysate only.
  • FGF-5 signal - rAAV.
  • FGF-5 (sig-) rAAV vims is packaged using the triple transfection method described in more detail above.
  • the vector contains a 1353 bp insertion of PhiX 174 bacteriophage DNA
  • FIG. 7 A schematic illustration of pD10-CMV-FGF-18 is provided in Figure 7. Briefly, this plasmid contains the CMV immediate/early enhancer + promoter, the CMV intron A, the FGF-18 coding region, the bovine growth hormone polyA site, and the AAV ITR sequences. There is a 1353 bp insertion of PhiX 174 bacteriophage DNA cloned into the Notl site between one ITR and the CMV immediate early enhancer + promoter region.
  • FGF-18 protein was assessed by transient transfection of 293 cells followed by Western analysis, using standard methods. Cell lysates and tissue culture media were harvested at 48 hours post transfection. An anti-peptide FGF-18 rabbit polyclonal antibody, generated against a selected polypeptide from recombinant FGF-18, was used at a dilution of 1 :2,500 for one hour at room temperature. The secondary antibody, an anti-rabbit IgG conjugated to peroxidase, was used at a dilution of 1 :25,000.
  • lanes 1-3 represent 1, 2 and 10 ul of tissue culture media from cells transfected with the pD10-CMV-FGF-18 plasmid.
  • Lane 4 is blank.
  • Lanes 5, 6 and 7 contain 2, 10 and 20 ul of lysate from the transfected cells.
  • Lanes 8 and 9 are negative controls; 20 ul of tissue culture media and cell lysate, respectively, from uninfected cells.
  • MBP FGF-18-maltose binding protein fusion
  • FGF-18 rAAV vims was generated by the triple transfection method.
  • EXAMPLE 5 AAV - LACZ INJECTED RETINA
  • Albino Sprague-Dawley rats were injected at 14 -15 days postnatal (PI 4- PI 5). Animals were anesthetized by ketamine/xylazine injection, and a local anesthetic (proparacain HC1) was applied topically to the cornea. An aperture was made through the inferior cornea of the eye with a 28 gauge needle. Subretinal injections of 2-3 ⁇ l of AAV-CMV-Lac-Z were then made by inserting a blunt 32 gauge needle through the opening and delivering the rAAV suspension into the subretinal space in the posterior retina. The contralateral eye was either uninjected, injected subretinally with PBS, or with a control rAAV containing a reporter gene.
  • Line 3 albino transgenic rats (P23H-3) on an albino Sprague-Dawley background (produced by Chrysalis DNX Transgenic Sciences, Princeton, NJ) were injected at the ages of P14 or PI 5.
  • Animals were anesthetized by ketamine/xylazine injection, and a local anesthetic (proparacain HC1) was applied topically to the cornea.
  • An aperture was made through the inferior cornea of the eye with a 28 gauge needle.
  • the subretinal injections of 2 ⁇ l were then made by inserting a blunt 32 gauge needle through the opening and delivering the rAAV suspension into the subretinal space in the posterior retina.
  • the intent was to inject into the subretinal space of the posterior superior hemisphere, but we sometimes found histologically that the injection site was located just inferior to the optic nerve head.
  • the opposite eye was either uninjected, injected subretinally with PBS, with control rAAV containing no neurotrophin or containing proteins not known to possess neurotrophic properties.
  • the rats were euthanized by overdose of carbon dioxide inhalation and immediately perfused intracardially with a mixture of mixed aldehydes (2% formaldehyde and 2.5 % glutaraldehyde). Eyes were removed and embedded in epoxy resin, and 1 ⁇ m thick histological sections were made along the vertical meridian. Tissue sections were aligned so that the ROS and Muller cell processes crossing the inner plexiform layer were continuous throughout the plane of section to assure that the sections were not oblique, and the thickness of the ONL and lengths of RIS and ROS were measured as described (LaVail, et al) . Briefly, 54 measurements of each layer or structure were made at set points around the entire retinal section.
  • S334ter rats after day P15 (the left eye was not injected).
  • S334(4) transgenic animals were used to assess the rescue effect of rAAV-CMV- FGF-2 on degenerating photoreceptor cells when delivered by intravitreal injection.
  • the rats were all sacrificed at age p60 and the embedded in plastic and sectioned to assess morphology and therapeutic effect as assayed by the preservation of thickness of outer nuclear layer. Superior and inferior regions of eyecup are quantitated by measuring the ONL thickness using a BioQuant morphometric measuring system (BioQuant). Injected eyes were evaluated along with uninjected control eyes.
  • BioQuant BioQuant morphometric measuring system
  • FGF-2 FGF-2
  • A. Injection Protocol Albino Sprague-Dawley rats were injected with rAAV-CMV-FGF-2 at the ages of P14 or PI 5 essentially as follows. Briefly, wild-type animals were anesthetized by ketamine/xylazine injection, and a local anesthetic (proparacain HC1) was applied topically to the cornea. An aperture was made through the inferior cornea of the eye with a 28 gauge needle. The subretinal injections of 2-3 ul of rAAV-CMV- FGF-2 were then made by inserting a blunt 32 gauge needle through the opening and delivering the rAAV suspension into the subretinal space in the posterior retina. The contralateral eye was either uninjected, injected subretinally with PBS, wild-type rAAV, or with rAAV-CMV-lacZ. B. Staining Protocol
  • Immunohistochemistry was used to look for expression of FGF-2 in the eye. Two rats were examined every week starting at 3 weeks post-injection. Retinas were examined for expression of FGF-2 and also examined histopathologically for signs of inflammation or neovascularization.
  • results are shown in Figure 15. Briefly, expression of FGF-2 was found in retinal photoreceptor cells as well as RPE cells at 35 days following inoculation with 2-3 ul of rAAV-CMV-FGF-2. Less significant expression was noted in retinal bipolar intemeurons and retinal ganglion cells (RGCs) following injection into the subretinal space (SRS). No significant staining above background was observed in sections injected with PBS or rAAV-CMV-lacZ vectors.
  • the rats were euthanized by overdose of carbon dioxide inhalation and immediately perfused intracardially with a mixture of mixed aldehydes (2% formaldehyde and 2.5 % glutaraldehyde). Eyes were removed and embedded in epoxy resin, and 1 ⁇ m thick histological sections were made along the vertical meridian. Tissue sections were aligned so that the ROS and Muller cell processes crossing the inner plexiform layer were continuous throughout the plane of section to assure that the sections were not oblique, and the thickness of the ONL was measured as described (LaVail, et al 19XX). Briefly, 54 measurements of each layer or stmcture were made at set points around the entire retinal section. The 27 measurements from the inferior region and the superior region of the retina were averaged separately to give two values for each eye. This separation was made because the retina degenerates at different rates in these two regions of the S334ter-4 animal model.
  • RPCs For visualization of RGCs, neurons were retrogradely labeled with the fluorescent tracer Fluorogold (Fluorochrome, Englewood, CO) at 2% in 0.9% NaCl containing 10% dimethyl sulfoxide by application of the tracer to both superior colliculi 7 days prior to analyses as described (Vidal-Sanz et al, 1988). Anesthetized rats were then perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) and the eyes were immediately enucleated. The anterior part of the eye and the lens were removed and the remaining eye cup was immersed in the same fixative for 2 hr at 4°C.
  • Fluorogold Fluorogold
  • Eye cups were cryoprotected in graded sucrose solutions (10-30% in PB) for several hours at 4°C, embedded in O.C.T. compound (Tissue-Tek, Miles Laboratories, Elkhart, IN) and frozen in a 2-methylbutane/liquid nitrogen bath. Retinal radial cryosections (12-15 ⁇ m), obtained along the vertical meridian of the eye, were collected onto gelatin-coated slides and processed for immunocytochemistry. Alternatively, entire eyes were rinsed three times (15 min each) in PBS at room temperature with gentle shaking, to whole-mount histochemical staining as described below. C. Histochemical Analysis
  • cryosections were incubated in 10% normal goat serum (NGS) in 0.2% Triton X-100 (Sigma, St. Louis, MO) in phosphate buffer saline (PBS) for 30 min at room temperature to block non-specific binding.
  • NGS normal goat serum
  • PBS phosphate buffer saline
  • Two primary antibodies raised against the lacZ gene product were used with similar results.
  • a polyclonal anti-betagal antibody diluted 1:1000; 5 prime- 3 prime, Inc., Boulder, CO
  • a monoclonal anti- LacZ antibody diluted 1:500; Promega, Madison, Wl.
  • Primary antibodies were added in 2% NGS in 0.2% Triton X-100 and incubated overnight at 4°C.
  • Sections were subsequently processed with anti-rabbit Cy3 -conjugated IgG (diluted 1:500, Jackson Immunoresearch, West Grove, PA) or anti-mouse Cy 3 -conjugated IgG (diluted 1 :500, Jackson Immunoresearch) and mounted. Control sections were treated in the same way but with omission of the primary antibody. Sections were visualized by fluorescent microscopy (Polyvar, Reichert-Jung).
  • heparan sulfate proteoglycan receptor in the retina was examined using a monoclonal anti-heparan sulfate antibody (He ⁇ SS-1, diluted 1 :1,000, Seikagaku Corporation, Tokyo, Japan). Following overnight incubation at 4°C, sections were processed with biotinylated anti-mouse Fab fragment (Jackson Immunoresearch), avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame, CA), followed by reaction in a solution containing 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.06% hydrogen peroxide in PB (pH 7.4) for 5 min.
  • DABC Elite Vector Labs Burlingame, CA
  • Quantification of AAV-transduced cells in the ganglion cell layer of retinal flat-mounts was performed in two ways: i) by counting the entire number of Bluo-gal positive cells in each of the retinal quadrants (superior, inferior, temporal and nasal); and ii) by counting the number of cells in three standard areas (at 1, 2 and 3 mm from the optic disc) of each quadrant as previously described (Villegas-Perez et al, 1993).
  • the human VEGF- 165 cDNA was cloned from the PCR-Blunt II Topo
  • pDlO-CMV rAAV vector as an EcoRI fragment
  • the pDlO-VEGFuc vector is illustrated schematically in Figure 21, and its nucleotide sequence is shown in Figure 22.
  • the VEGFuc rAAV vims was packaged using the triple transfection method and purified by column chromatography. Briefly, a cell pellet is resuspended in TNM buffer: 20mM Tris pH 8.0,
  • fractions under the peak are pooled and filtered through a 0.22um filter before overnight precipitation with 8% PEG 8000.
  • CaCl 2 is added to 25mM and the purified particles are pelleted and then resuspended in HBS#2: 150mM NaCl, 50mM Hepes pH7.4.
  • the functionality of the viral particles was assessed by infection of 293 cells with 3 different viral multiplicities of infection (MOIs); 1 x 10e7, 1 x 10e8 and 1 x 10e9 viral particles per 4 x 10e5 293 cells, in the presence of 1.5 uM etoposide.
  • MOIs viral multiplicities of infection
  • tissue culture media (sups) and cell lysates were harvested.
  • VEGF protein levels were determined using a Quantikine human VEGF sandwich ELISA kit (R and D Systems, see Figure 23). VEGF protein concentrations are given in pg/ml.
  • the two highest MOIs gave values significantly above that of the cells infected with a negative control vims.
  • the levels of secreted VEGF were approximately 4-7 fold higher than those of the lysates.
  • EXAMPLE 12 INFECTION OF RETINAL PIGMENT EPITHELIAL (RPE) CELLS WITH D 10- VEGF RAAV
  • VEGF expression levels in a monolayer of cultured primary (or very early passage) human fetal RPE cells infected with D 10- VEGF 165 rAAV were clearly elevated relative to endogenous levels. Cells were infected with a range of rAAV particles from 0 to 1 x 10e5 per cell. VEGF expression was dose dependent, increased over time, and secretion appeared to be somewhat higher from the apical surface. In a representative experiment, RPE cells infected with 1 x 10e5 viral particles secreted > 100 ng/1 x 10e6 cells from the apical surface and 50 ng/1 x 10e6 cells from the basal surface at 8 days post infection. The polarity of VEGF secretion from human fetal RPE cells infected with 3 different MOIs is shown in Figure 24.
  • VEGF adenovims results in secretion of very high levels of VEGF from both the apical and basal surfaces of the RPE.
  • MOI's 0 to 1,000 or 0 to 10,000 particles per cell were used in two separate experiments. In both cases, expression levels increased over time, peaking at approximately 100-200 ug/1 x 10e6 cells at the highest MOI's (see Figure 25). In addition, the total transepithelial membrane resistance of the RPE monolayer decreased significantly at all MOIs, and by approximately 4-5 fold at the highest MOIs (see Figure 26).
  • the sFlt-1 cDNA was cloned from the Blunt II Topo Vector (Invitrogen) into the pDlO-CMV rAAV vector as an EcoRI fragment (pDlO-sFlt-1).
  • the pDlO- sFlt-1 vector is illustrated schematically in Figure 27, and its nucleotide sequence is shown in Figure 28.
  • the human sFlt-1 rAAV vims was packaged using the triple transfection technique and purified by column chromatography.
  • HUVEC human umbilical vein endothelial cell proliferation assay, which can be utilized to determine the anti-angiogenic activity of a molecule (see generally, Gerritsen et al, 1999. JBC 274:9116-9121). Briefly, HUVEC cells are seeded on collagen-coated 96-well plates at 6,000 cells/cm 2 in Clonetics EGM media. EGM media contains endothelial cell growth supplements, 10% fetal bovine semm, 2mM L-glutamine, and antibiotics. Cells are allowed to attach for 4 hours.
  • IX basal medium consisting of Ml 99 supplemented with 1% fetal bovine semm, IX ITS, 2 mM L-glutamine, 50 ug/ml of ascorbic acid, 26.5 mM NaHCO 3 , and appropriate concentration of antibiotics.
  • Cells are cultured in the above medium in the presence of the sample supernatant or vector for 4 hours.
  • the sample supernatant would come from 293 cells transfected with the appropriate plasmid constmct expressing the molecule we are testing for anti-angiogenic activity.

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Abstract

L'invention concerne des vecteurs d'administration de gènes, tels que, par exemple, des vecteurs de virus associés aux adénovirus recombinants, ainsi que des techniques d'utilisation de tels vecteurs pour le traitement ou la prévention des maladies de l'oeil.
PCT/US2000/007062 1999-03-15 2000-03-15 Utilisation de vecteurs d'administration d'un gene recombinant pour le traitement ou la prévention des maladies de l'oeil WO2000054813A2 (fr)

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AU37559/00A AU3755900A (en) 1999-03-15 2000-03-15 Use of recombinant gene delivery vectors for treating or preventing diseases of the eye
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EP00916458A EP1183051A2 (fr) 1999-03-15 2000-03-15 Utilisation de vecteurs d'administration d'un gene recombinant pour le traitement ou la pr vention des maladies de l'oeil
JP2000604885A JP2002539176A (ja) 1999-03-15 2000-03-15 眼の疾患を処置または予防するための組換え遺伝子送達ベクターの使用

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