WO2005105155A1 - Procédé pour fournir un produit génétique aux yeux - Google Patents

Procédé pour fournir un produit génétique aux yeux Download PDF

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Publication number
WO2005105155A1
WO2005105155A1 PCT/JP2005/008401 JP2005008401W WO2005105155A1 WO 2005105155 A1 WO2005105155 A1 WO 2005105155A1 JP 2005008401 W JP2005008401 W JP 2005008401W WO 2005105155 A1 WO2005105155 A1 WO 2005105155A1
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Prior art keywords
eye
expression
nucleic acid
expression vector
acid sequence
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PCT/JP2005/008401
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English (en)
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Keisuke Mori
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Saitama Medical School
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Publication of WO2005105155A1 publication Critical patent/WO2005105155A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to a method of delivering a gene product to the eye.
  • Vision impairment can result from most all ocular disorders, including diabetic retinopathies, proliferative retinopathies, retinal detachment, toxic retinopathies, retinal vascular diseases, retinal degenerations, vascular anomalies, age-related macular degeneration and other acquired disorders, infectious diseases, inflammatory diseases, ocular ischemia, pregnancy-related disorders, retinal tumors, choroidal tumors, choroidal disorders, vitreous disorders, trauma, cataract complications, dry eye, and inflammatory optic neuropathies.
  • Leading causes of severe vision loss and blindness are ocular-related disorders wherein the vasculature of the eye is damaged or insufficiently regulated.
  • Ocular-related diseases comprising a neovascularization aspect are many and include, for example, exudative age-related macular degeneration, diabetic retinopathy, corneal neovascularization, choroidal neovascularization, neovascular glaucoma, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, drug-related toxicities, central and branched retinal vein occlusions, and the like.
  • Damage of the retina i.e., retinal detachment, retinal tears, or retinal degeneration
  • retinal detachment i.e., retinal detachment, retinal tears, or retinal degeneration
  • vision loss While a common cause of retinal detachment, retinal tears, and retinal degeneration is abnormal, i.e., uncontrolled, vascularization of various ocular tissues, this is not always the case.
  • Atrophic complications associated with age-related macular degeneration, nonproliferative diabetic retinopathy, and inflammatory ocular damage are not associated with neovascularization, but can result in severe vision loss if not treated.
  • neovascular and atrophic components are particularly difficult to treat due to the emergence of a wide variety of complications.
  • Laser photocoagulation involves administering laser burns to various areas of the eye and is used in the treatment of many neovascularization-linked disorders.
  • focal macular photocoagulation is used to treat areas of vascular leakage outside the macula (Murphy, Amer. Family Physician, 51(A), 785-796 (1995)).
  • neovascularization in particular, advanced proliferative retinopathy, is commonly treated with scatter or panretinal photocoagulation.
  • the invention provides materials and methods for delivering a gene product, such as a therapeutic gene product, to the eye. This and other advantages of the invention will become apparent from the detailed description provided herein.
  • the invention is directed to a method of delivering a gene product to an eye.
  • the method comprises (a) inducing a stress response in the eye, and (b) subsequently administering to the eye an expression vector comprising a nucleic acid sequence encoding a gene product, wherein the expression vector transduces a host cell and the nucleic acid sequence is expressed to produce the gene product.
  • the invention further provides a method of prophylactically or therapeutically treating an animal for an ocular-related disorder.
  • the method comprises (a) inducing a stress response in an eye, and (b) subsequently administering to the eye an expression vector comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and/or a neurotrophic agent such that the expression vector transduces a host cell and the nucleic acid sequence is expressed to produce the inhibitor of angiogenesis and/or neurotrophic agent to treat prophylactically or therapeutically the ocular-related disorder.
  • inducing a stress response in the eye comprises applying photodynamic therapy or photocoagulation therapy to the eye.
  • the expression vector is an adenoviral vector.
  • the invention is directed to a method of delivering a gene product, such as a therapeutic gene product, to the eye.
  • the method comprises (a) inducing a stress response in the eye, and (b) subsequently administering to the eye an expression vector comprising a nucleic acid sequence encoding a gene product, wherein the expression vector transduces a host cell and the nucleic acid sequence is expressed to produce the gene product.
  • Any gene product can be delivered to the eye in accordance with the inventive method, including therapeutic gene products, diagnostic gene products, gene products used for research purposes (e.g., marker proteins or antigenic proteins), and the like.
  • the gene product delivered to the eye preferably is a therapeutic gene product for the prophylactic or therapeutic treatment of an animal, preferably a human, for at least one ocular-related disorder.
  • Ocular-related disorders appropriate for treatment using the inventive method include, but are not limited to, diabetic retinopathies, proliferative retinopathies, retinopathy of prematurity, retinal vascular diseases, vascular anomalies, age-related macular degeneration and other acquired disorders, endophthalmitis, infectious diseases, inflammatory diseases, AIDS-related disorders, ocular ischemia syndrome, pregnancy-related disorders, peripheral retinal degenerations, retinal degenerations, toxic retinopathies, cataracts, retinal tumors, corneal neovascularization, choroidal tumors, choroidal disorders, choroidal neovascularization, neovascular glaucoma, vitreous disorders, retinal detachment and proliferative vitreoretinopathy, cyclitis, non-penetrating trauma,
  • the invention further provides a method of prophylactically or therapeutically treating an animal for at least one ocular-related disorder, such as ocular neovascularization.
  • the method comprises (a) inducing a stress response in an eye, and (b) subsequently administering to the eye an expression vector comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and/or a neurotrophic agent such that the expression vector transduces a host cell and the nucleic acid sequence is expressed to produce the inhibitor of angiogenesis and/or neurotrophic agent to treat prophylactically or therapeutically the ocular-related disorder.
  • the expression vector comprises a nucleic acid sequence encoding an inhibitor of angiogenesis and the same or different nucleic acid sequence encoding a neurotrophic agent.
  • the nucleic acid sequence encodes pigment epithelium-derived factor (PEDF).
  • PEDF pigment epithelium-derived factor
  • the ocular disorder preferably is ocular neovascularization, such as neovascularization of the choroid.
  • the choroid is a thin, vascular membrane located under the retina.
  • Abnormal neovascularization of the choroid results from, for example, photocoagulation, anterior ischemic optic neuropathy, Best's disease, choroidal hemangioma, metallic intraocular foreign body, choroidal nonperfusion, choroidal osteomas, choroidal rupture, bacterial endocarditis, choroideremia, chronic retinal detachment, drusen, deposit of metabolic waste material, endogenous Candida endophthalmitis, neovascularization at ora serrata, operating microscope burn, punctate inner choroidopathy, radiation retinopathy, retinal cryoinjury, retinitis pigmentosa, retinochoroidal coloboma, rubella, subretinal fluid drainage, tilted disc syndrome, Taxoplasma retinochoroiditis, tuberculosis, and the like.
  • Neovascularization of the cornea also is appropriate for treatment by the method of the invention.
  • the cornea is a projecting, transparent section of the fibrous tunic, the outer most layer of the eye.
  • the outermost layer of the cornea contacts the conjunctiva, while the innermost layer comprises the endothelium of the anterior chamber.
  • Corneal neovascularization stems from, for example, ocular injury, surgery, infection, improper wearing of contact lenses, and diseases such as, for example, corneal dystrophies.
  • the ocular neovascularization is neovascularization of the retina. Retinal neovascularization is an indication associated with numerous ocular diseases and disorders, many of which are named above.
  • the neovascularization of the retina treated in accordance with the inventive method is associated with diabetic retinopathy.
  • Common causes of retinal neovascularization include ischemia, viral infection, and retinal damage.
  • Neovascularization of the retina can lead to macular edema, subretinal discoloration, scarring, hemorrhaging, and the like.
  • Complications associated with retina neovascularization stem from growth, breakage, and leakage of newly formed blood vessels. Vision is impaired as blood fills the vitreous cavity and is not efficiently removed. Not only is the passage of light impeded, but an inflammatory response to the excess blood and metabolites can cause further damage to ocular tissue.
  • the ocular disorder can be age-related macular degeneration, which can involve both exudative (neovascular) and atrophic complications.
  • Exudative complications include, for example, disciform scars (i.e., scarring involving fibrous elements) and neovascularization.
  • Atrophic complications include, for instance, the formation of drusen and basal laminar deposits, irregularity of retinal pigmentation, and accumulation of lipofuscin granules.
  • the ocular disorder also can be ocular edema (e.g., retinal edema or macular edema).
  • prophylactic is meant the protection, in whole or in part, against ocular-related disorders, in particular ocular neovascularization or age-related macular degeneration.
  • therapeutic is meant the amelioration of the ocular-related disorder, itself, and the protection, in whole or in part, against further ocular-related disease, in particular ocular neovascularization or age-related macular degeneration.
  • inventive method can be used to treat both acute and persistent, progressive ocular-related disorders.
  • the expression vector can be administered using a single or multiple applications within a short time period.
  • the inventive method comprises inducing a stress response in the eye prior to administering an expression vector. It has surprisingly been determined that treating an eye with, for example, photocoagulation or photodynamic therapy increases in ocular cells the expression of cell surface molecules which facilitate transduction by expression vectors and cell adhesion.
  • the cell surface molecules most recognized as mediating adenoviral infection, coxsackievirus and adeno virus receptor (CAR) and integrins (e.g., integrins ⁇ 3 and ⁇ 5), are upregulated in the retina and choroid following, for example, photocoagulation therapy.
  • Upregulation of CAR and integrins allows transduction of a greater number of host cells with an adenoviral vector encoding a gene product as compared to adenoviral transduction efficiency without induction of the stress response (e.g., laser therapy). Accordingly, the level of transduction of host cells by the expression vector is enhanced as compared to the level of transduction of host cells by the expression vector in the absence of inducing a stress response in the eye. In some instances, the invention allows delivery of a smaller dose of expression vector than previously thought possible to achieve a desired biological response.
  • inducing a stress response in the eye prior to administering the expression vector enhances and prolongs expression of the nucleic acid sequence of the expression vector compared to expression of the nucleic acid sequence in the absence of the stress response.
  • expression of the nucleic acid sequence in the context of the inventive method is enhanced compared to expression of the nucleic acid sequence in the absence of inducing the stress response in the eye (but under otherwise similar conditions) for at least one day (preferably at least 3 days (e.g., 1, 2, or 3 days) or at least five days) following administration of the expression vector.
  • expression of the nucleic acid sequence is enhanced for at least 7 days (e.g., at least 14 days or at least 21 days) post-administration of the expression vector as compared to expression of the nucleic acid sequence in the absence of the stress response (e.g., in the absence of photocoagulation therapy) at the same timepoint.
  • expression of the nucleic acid sequence is enhanced as compared to expression of the nucleic acid sequence in the absence of inducing a stress response for at least 28 days (e.g., at least 60 days or at least 90 days) post-administration of the expression vector to the eye.
  • enhanced expression is meant any increase in transcription compared to transcription (i.e., gene or nucleic acid sequence expression) which occurs in the absence of inducing the stress response.
  • Any increase in expression of the nucleic acid sequence is appropriate in the context of the invention.
  • enhanced expression of the nucleic acid sequence can be a 2-, 3-, 5-, 10-, 20-, or 50-fold increase in expression as compared to the level of gene expression which occurs under similar conditions but in the absence of inducing the stress response in the eye.
  • the enhanced expression (transcription) can result in increased levels of RNA transcript, increased protein production, and/or an enhancement in detectable gene product activity, all of which can be detected using routine laboratory techniques.
  • a stress response can be induced in the eye by exposure to heat using, for example, lasers in photodynamic therapy, exposure to cold, exposure to light, exposure to radiation (e.g., X-rays), exposure to microwaves, exposure to ultrasound, or physical trauma, all of which can alter the ocular cellular environment to enhance transcription.
  • inducing a stress response in the eye comprises applying photodynamic therapy or photocoagulation therapy to the eye.
  • suitable expression vectors include, for instance, plasmids, plasmid-liposome complexes, and viral vectors, e.g., parvoviral-based vectors (i.e., adeno-associated virus (AAV)-based vectors), retroviral vectors, herpes simplex virus (HSV)-based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • AAV-adenoviral chimeric vectors e.g., AAV-adenoviral chimeric vectors
  • adenovirus-based vectors e.g., adenovirus-based vectors.
  • Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor,
  • Plasmids genetically engineered circular double-stranded DNA molecules, can be designed to contain an expression cassette for delivery of the nucleic acid sequence encoding the gene product to a host cell, such as an ocular cell.
  • plasmids were the first vector described for administration of therapeutic nucleic acids, the level of transfection efficiency is poor compared with other techniques. By complexing the plasmid with liposomes, the efficiency of gene transfer in general is improved. While the liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids.
  • plasmid-liposome complexes include their ability to transfer large pieces of DNA encoding a therapeutic nucleic acid and their relatively low immunogenicity. Plasmids are often used for short-term expression. However, a plasmid construct can be modified to obtain prolonged expression. It has recently been discovered that the inverted terminal repeats (ITR) of parvovirus, in particular adeno-associated virus (AAV), are responsible for the high-level persistent nucleic acid expression often associated with AAV (see, for example, U.S. Patent 6,165,754).
  • ITR inverted terminal repeats
  • AAV adeno-associated virus
  • the expression vector can be a plasmid comprising native parvovirus ITRs to obtain prolonged and substantial expression of a nucleic acid sequence encoding a gene product, e.g., at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • plasmids are suitable for use in the inventive method, preferably the expression vector is a viral vector.
  • AAV vectors are viral vectors of particular interest for use in gene therapy protocols.
  • AAV is a DNA virus, which is not known to cause human disease.
  • AAV requires co-infection with a helper virus (i.e., an adenovirus or a herpes virus), or expression of helper genes, for efficient replication.
  • AAV vectors used for administration of a therapeutic nucleic acid have approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes.
  • delivering the AAV rep protein enables integration of the AAV vector comprising AAV ITRs into a specific region of genome, if desired.
  • Host cells comprising an integrated AAV genome show no change in cell growth or morphology (see, for example, U.S. Patent 4,797,368).
  • Retrovirus is an RNA virus capable of infecting a wide variety of host cells.
  • retroviral genome Upon infection, the retroviral genome integrates into the genome of its host cell and is replicated along with host cell DNA, thereby constantly producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome.
  • pathogenic retroviruses e.g., human immunodeficiency virus (HIV) or human T-cell lymphotrophic viruses (HTLV)
  • HIV human immunodeficiency virus
  • HTLV human T-cell lymphotrophic viruses
  • a retroviral vector can additionally be manipulated to render the virus replication-incompetent.
  • retroviral vectors are thought to be particularly useful for stable gene transfer in vivo.
  • Lentiviral vectors such as HIV-based vectors, are exemplary of retroviral vectors used for gene delivery.
  • HSV-based viral vectors are suitable for use as an expression vector to introduce nucleic acids into host cells (e.g., ocular cells).
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication.
  • herpes vector has its ability to enter a latent stage that can result in long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA up to 25 kb. Of course, this ability is also a disadvantage in terms of short-term treatment regimens.
  • HSV-based vectors appropriate for use in the inventive method, see, for example, U.S. Patents 5,837,532; 5,846,782; 5,849,572; and 5,804,413; and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.
  • Adenovirus is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types.
  • the virus is preferably made replication deficient by deleting select genes required for viral replication.
  • the expendable E3 region is also frequently deleted to allow additional room for a larger DNA insert.
  • the vector can be produced in high titers and can efficiently transfer DNA to replicating and non-replicating cells.
  • the newly transferred genetic information remains epi-chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
  • the integrative properties of AAV can be conferred to adenovirus by constructing an AAV- Ad chimeric vector.
  • the AAV ITRs and nucleic acid encoding the Rep protein incorporated into an adenoviral vector enables the adenoviral vector to integrate into a mammalian cell genome. Therefore, AAV- Ad chimeric vectors are an interesting option for use in the invention.
  • the expression vector of the inventive method is a viral vector; more preferably, the expression vector is an adenoviral vector, e.g., a human adenoviral vector.
  • the adenoviral vector can be derived from any serotype of adenovirus.
  • Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassas, VA), or from any other serotype of adenovirus available from any other source.
  • ATCC American Type Culture Collection
  • VA Manassas
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype.
  • an adenovirus is of serotype 2, 5, or 9.
  • non-group C adenoviruses can be used to prepare replication-deficient adenoviral gene transfer vectors for delivery of gene products to host cells, such as ocular cells.
  • Preferred adenoviruses used in the construction of non-group C adenoviral gene transfer vectors include Ad 12 (group A), Ad7 and Ad35 (group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41 (group F).
  • Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U.S.
  • the adenoviral vector is preferably deficient in at least one gene function required for viral replication, thereby resulting in a "replication-deficient" adenoviral vector.
  • replication-deficient is meant that the adenoviral vector comprises an adenoviral genome that lacks at least one replication-essential gene function (i.e., such that the adenoviral vector does not replicate in typical host cells, especially those in the human patient that could be infected by the adenoviral vector in the course of treatment in accordance with the invention).
  • a deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part. Deletion of an entire gene region often is not required for disruption of a replication-essential gene function. However, for the purpose of providing sufficient space in the adenoviral genome for one or more transgenes, removal of a majority of a gene region may be desirable.
  • Replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA-1 and/or VA-RNA-2). More preferably, the replication-deficient adenoviral vector comprises an adenoviral genome deficient in at least one replication-essential gene function of one or more regions of the adenoviral genome.
  • the adenoviral vector is deficient in at least one essential gene function of the El region of the adenoviral genome required for viral replication.
  • the recombinant adenovirus can also have a mutation in the major late promoter (MLP).
  • MLP major late promoter
  • the mutation in the MLP can be in any of the MLP control elements such that it alters the responsiveness of the promoter, as discussed in International Patent Application WO 00/00628.
  • the vector is deficient in at least one essential gene function of the El region and at least part of the E3 region (e.g., an Xba I deletion of the E3 region).
  • the adenoviral vector can be deficient in at least part of the El a region and at least part of the El b region.
  • the adenoviral vector can comprise a deletion of the entire El region and part of the E3 region of the adenoviral genome (i.e., nucleotides 355 to 3,511 and 28,593 to 30,470).
  • a singly-deficient adenoviral vector can be deleted of approximately nucleotides 356 to 3,329 and 28,594 to 30,469 (based on the adenovirus serotype 5 genome).
  • the adenoviral vector genome can be deleted of approximately nucleotides 356 to 3,510 and 28,593 to 30,470 (based on the adenovirus serotype 5 genome).
  • the endpoints defining the deleted nucleotide portions can be difficult to precisely determine and typically will not significantly affect the nature of the adenoviral vector, i.e., each of the aforementioned nucleotide numbers can be +/- 1, 2, 3, 4, 5, or even 10 or 20 nucleotides.
  • the adenoviral vector is "multiply deficient," meaning that the adenoviral vector is deficient in one or more essential gene functions required for viral replication in each of two or more regions.
  • the aforementioned El -deficient or E1-, E3 -deficient adenoviral vectors can be further deficient in at least one essential gene function of the E4 region.
  • Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses.
  • the adenoviral vector genome can comprise a deletion of, for example, nucleotides 32,826 to 35,561 (based on the adenovirus serotype 5 genome), optionally in addition to deletions in the El region (e.g., nucleotides 356 to 3,329 or nucleotides 356 to 3,510) and/or deletions in the E3 region (e.g., nucleotides 28,594 to 30,469 or nucleotides 28,593 to 30,470).
  • the adenoviral vector lacks all or part of the El region and all or part of the E2 region (e.g., the E2A region).
  • adenoviral vectors lacking all or part of the El region, all or part of the E2 region, and all or part of the E3 region also are contemplated herein.
  • the adenoviral vector lacks all or part of the El region, all or part of the E2 region, all or part of the E3 region, and all or part of the E4 region.
  • Suitable replication-deficient adenoviral vectors are disclosed in LT.S. Patents 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826.
  • suitable replication-deficient adenoviral vectors include those with at least a partial deletion of the El a region, at least a partial deletion of the Elb region, at least a partial deletion of the E2a region, and at least a partial deletion of the E3 region.
  • the replication-deficient adenoviral vector can have at least a partial deletion of the El region, at least a partial deletion of the E3 region, and at least a partial deletion of the E4 region.
  • other regions of the adenoviral genome also can be deleted such as the VAI gene and VAII gene as described in International Patent Application No. PCT/US02/29111.
  • Multiply-deficient viral vectors are particularly useful in that such vectors can accept large inserts of exogenous DNA.
  • adenoviral amplicons an example of a multiply-deficient adenoviral vector which comprises only those genomic sequences required for packaging and replication of the viral genome, can accept inserts of approximately 36 kb. Therefore, in a preferred embodiment, the expression vector of the inventive method is a multiply-deficient adenoviral vector lacking all or part of the El region, all or part of the E3 region, all or part of the E4 region, and, optionally, all or part of the E2 region.
  • an at least E4-deficient adenoviral vector expresses a transgene at high levels for a limited amount of time in vivo and that persistence of expression of a transgene in an at least E4-def ⁇ cient adenoviral vector can be modulated through the action of a trans-acting factor, such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205 osteosarcoma cell line that functions like HSV ICPO, or the cellular factor in PC 12 cells that is induced by nerve growth factor, among others.
  • a trans-acting factor such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205 osteosarcoma cell line that functions like HSV ICPO, or the
  • a nucleic acid sequence encoding a trans-acting factor that modulates the persistence of expression of the nucleic acid sequence encoding the gene product can be administered.
  • Use of trans-acting factors in combination with replication deficient adenoviral vectors is further described in U.S. Patents 6,225,113; 6,660,521; and 6,649,373; and International Patent Application WO 00/34496. It should be appreciated that the deletion of different regions of the adenoviral vector can alter the immune response of the mammal. In particular, deletion of different regions can reduce the inflammatory response generated by the adenoviral vector.
  • the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509. Such modifications are useful for long-term treatment of persistent ocular disorders.
  • the adenoviral vector when multiply replication-deficient, especially in replication-essential gene functions of the El and E4 regions, preferably includes a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication-deficient adenoviral vectors, particularly an adenoviral vector comprising a deficiency in the El region.
  • the spacer is desirably located between the L5 fiber region and the right-side ITR. More preferably in such an adenoviral vector, the E4 polyadenylation sequence alone or, most preferably, in combination with another sequence exists between the L5 fiber region and the right-side ITR, so as to sufficiently separate the retained L5 fiber region from the right-side ITR, such that viral production of such a vector approaches that of a singly replication deficient adenoviral vector, particularly a singly replication deficient El deficient adenoviral vector.
  • the spacer element can contain any sequence or sequences which are of a desired length, such as sequences at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs in length.
  • the spacer element sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region.
  • the spacer can also contain a promoter-variable expression cassette. More preferably, the spacer comprises an additional polyadenylation sequence and/or a passenger gene.
  • both the E4 polyadenylation sequence and the E4 promoter of the adenoviral genome or any other (cellular or viral) promoter remain in the vector.
  • the spacer is located between the E4 polyadenylation site and the E4 promoter, or, if the E4 promoter is not present in the vector, the spacer is proximal to the right-side ITR.
  • the spacer can comprise any suitable polyadenylation sequence.
  • polyadenylation sequences include synthetic optimized sequences, BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus) and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus).
  • BGH Bovine Growth Hormone
  • polyoma virus TK (Thymidine Kinase)
  • EBV Epstein Barr Virus
  • the spacer includes an SV40 polyadenylation sequence.
  • the SV40 polyadenylation sequence allows for higher virus production levels of multiply replication deficient adenoviral vectors.
  • the spacer is composed of the glucuronidase gene.
  • the adenoviral vector requires, at most, complementation of replication-essential gene functions of the El, E2A, and/or E4 regions of the adenoviral genome for replication (i.e., propagation).
  • the adenoviral genome can be modified to disrupt one or more replication-essential gene functions as desired by the practitioner, so long as the adenoviral vector remains deficient and can be propagated using, for example, complementing cells and/or exogenous DNA (e.g., helper adenovirus) encoding the disrupted replication-essential gene functions.
  • the adenoviral vector can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome.
  • the adenoviral vector also can have essentially the entire adenoviral genome removed, in which case it is preferred that at least the viral inverted terminal repeats (ITRs) and a packaging signal are left intact (i.e., an adenoviral amplicon).
  • ITRs viral inverted terminal repeats
  • Suitable replication-deficient adenoviral vectors including multiply replication-deficient adenoviral vectors, are disclosed in U.S.
  • the pharmaceutical composition is virtually free of replication-competent adenovirus (RCA) contamination (e.g., the pharmaceutical composition comprises less than about 1% of RCA contamination).
  • the pharmaceutical composition is RCA-free.
  • Adenoviral vector compositions and stocks that are RCA-free are described in U.S. Patents 5,944,106 and 6,482,616, U.S.
  • the pharmaceutical composition also is free of El-revertants when the adenoviral vector is El -deficient in combination with deficiencies in other replication-essential gene functions of another region of the adenoviral genome, as further described in International Patent Application WO 03/040314.
  • the adenoviral genome can contain benign or non-lethal modifications, i.e., modifications which do not render the adenovirus replication-deficient, or, desirably, do not adversely affect viral functioning and/or production of viral proteins, even if such modifications are in regions of the adenoviral genome that otherwise contain replication-essential gene functions.
  • benign or non-lethal modifications i.e., modifications which do not render the adenovirus replication-deficient, or, desirably, do not adversely affect viral functioning and/or production of viral proteins, even if such modifications are in regions of the adenoviral genome that otherwise contain replication-essential gene functions.
  • modifications commonly result from DNA manipulation or serve to facilitate expression vector construction.
  • benign mutations often have no detectable adverse effect on viral functioning.
  • the adenoviral vector can comprise a deletion of nucleotides 10,594 and 10,595 (based on the adenoviral serotype 5 genome), which are associated with VA-RNA-1 transcription, but the deletion of which does not prohibit production of VA-RNA- 1.
  • the coat protein of a viral vector preferably an adenoviral vector, can be manipulated to alter the binding specificity or recognition of a virus for a viral receptor on a potential host cell.
  • such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • the expression vector is an adenoviral vector comprising a chimeric coat protein (e.g., a fiber, hexon pIX, pllla, or penton protein), which differs from the wild-type (i.e., native) coat protein by the introduction of a nonnative amino acid sequence, preferably at or near the carboxyl terminus.
  • a nonnative amino acid sequence is inserted into or in place of an internal coat protein sequence.
  • the nonnative amino acid sequence can be inserted within the internal coat protein sequence or at the end of the internal coat protein sequence.
  • the resultant chimeric viral coat protein is able to direct entry into cells of the viral, i.e., adenoviral, vector comprising the coat protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type viral coat protein rather than the chimeric viral coat protein.
  • the chimeric virus coat protein binds a novel endogenous binding site present on the cell surface that is not recognized, or is poorly recognized by a vector comprising a wild-type coat protein.
  • the virus preferably, the adenovirus
  • the expression vector is a viral vector comprising a chimeric virus coat protein not selective for a specific type of eukaryotic cell.
  • the chimeric coat protein differs from the wild-type coat protein by an insertion of a nonnative amino acid sequence into or in place of an internal coat protein sequence.
  • the chimeric virus coat protein efficiently binds to a broader range of eukaryotic cells than a wild-type virus coat, such as described in International Patent Application WO 97/20051.
  • Specificity of binding of an adenovirus to a given cell can also be adjusted by use of an adenovirus comprising a short-shafted adenoviral fiber gene, as discussed in U.S. Patent 5,962,311.
  • Use of an adenovirus comprising a short-shafted adenoviral fiber gene reduces the level or efficiency of adenoviral fiber binding to its cell-surface receptor and increases adenoviral penton base binding to its cell-surface receptor, thereby increasing the specificity of binding of the adenovirus to a given cell.
  • an adenovirus comprising a short-shafted fiber enables targeting of the adenovirus to a desired cell-surface receptor by the introduction of a nonnative amino acid sequence either into the penton base or the fiber knob.
  • the ability of a viral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein.
  • complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables one of ordinary skill in the art to target the vector to a particular cell type. Suitable modifications to a viral vector, specifically an adenoviral vector, are described in U.S.
  • Adenoviral vectors can be constructed and/or purified using methods known in the art (e.g., using complementing cell lines, such as the 293 cell line, Per.C6 cell line, or 293-ORF6 cell line) and methods set forth, for example, in U.S. Patents 5,965,358; 5,994,128; 6,033,908; 6,168,941; 6,329,200; 6,383,795; 6,440,728; 6,447,995; and 6,475,757; U.S.
  • Adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).
  • the selection of expression vector for use in the inventive method will depend on a variety of factors such as, for example, the host, immunogenicity of the vector, the desired duration of protein production, and the like.
  • the nucleic acid sequence is desirably present as part of an expression cassette, i.e., a particular nucleotide sequence that possesses functions which facilitate subcloning and recovery of a nucleic acid sequence (e.g., one or more restriction sites) or expression of a nucleic acid sequence (e.g., polyadenylation or splice sites).
  • the nucleic acid sequence is preferably located in the El region (e.g., replaces the El region in whole or in part) of the adenoviral genome.
  • the El region can be replaced by a promoter- variable expression cassette comprising the nucleic acid sequence(s).
  • the expression cassette is preferably inserted in a 3'-5' orientation, e.g., oriented such that the direction of transcription of the expression cassette is opposite that of the surrounding adjacent adenoviral genome.
  • the adenoviral vector can comprise other expression cassettes containing nucleic acid sequences encoding other products, which cassettes can replace any of the deleted regions of the adenoviral genome.
  • an expression cassette into the adenoviral genome can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.
  • a unique restriction site at a given position of the adenoviral genome.
  • the nucleic acid sequence is operably linked to regulatory sequences necessary for expression, i.e., a promoter.
  • a "promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a nucleic acid sequence is "operably linked" to a promoter when the promoter is capable of directing transcription of that nucleic acid sequence.
  • a promoter can be native or non-native to the nucleic acid sequence to which it is operably linked. Any promoter (i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques) can be used in connection with the invention to provide for transcription of the nucleic acid sequence.
  • the promoter preferably is capable of directing transcription in a eukaryotic (desirably mammalian) cell. The functioning of the promoter can be altered by the presence of one or more enhancers and/or silencers present on the vector.
  • Enhanccers are cis-acting elements of DNA that stimulate or inhibit transcription of adjacent genes.
  • Enhancers differ from DNA-bindin ' g sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements") in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.
  • Promoter regions can vary in length and sequence and can further encompass one or more DNA binding sites for sequence-specific DNA binding proteins and/or an enhancer or silencer. Enhancers and/or silencers can similarly be present on a nucleic acid sequence outside of the promoter per se.
  • a cellular or viral enhancer such as the cytomegalovirus (CMV) immediate-early enhancer, is positioned in the proximity of the promoter to enhance promoter activity.
  • CMV cytomegalovirus
  • splice acceptor and donor sites can be present on a nucleic acid sequence to enhance transcription.
  • the invention preferentially employs a viral promoter.
  • Suitable viral promoters include, for instance, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter, promoters derived from human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.
  • CMV cytomegalovirus
  • HMV human immunodeficiency virus
  • RSV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • HSV promoters such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.
  • promoters derived from SV40 or Epstein Barr virus an adeno-associated viral promoter, such as the p5 promoter, and the like.
  • the viral promoter is an adenoviral promoter, such as the Ad2 or Ad5 major late promoter and tripartite leader, a CMV promoter, or an RSV promoter.
  • the invention employs a cellular promoter, i.e., a promoter that drives expression of a cellular protein.
  • Preferred cellular promoters for use in the invention will depend on the desired expression profile to produce the therapeutic agent(s).
  • the cellular promoter is preferably a constitutive promoter that works in a variety of cell types, such as cells associated with the eye.
  • Suitable constitutive promoters can drive expression of genes encoding transcription factors, housekeeping genes, or structural genes common to eukaryotic cells.
  • YY1 Ying Yang 1
  • NMP-1, NF-E1, and UCRBP Ying Yang 1
  • UCRBP ubiquitous nuclear transcription factor that is an intrinsic component of the nuclear matrix
  • JEM-1 also known as HGMW and BLZF-1
  • JEM-1 is a ubiquitous nuclear transcription factor identified in normal and tumorous tissues (Tong et al., Leukemia, 12(11), 1733-1740 (1998), and Tong et al., Genomics, 69(3), 380-390 (2000)). JEM-1 is involved in cellular growth control and maturation, and can be upregulated by retinoic acids.
  • the JEM-1 promoter does not comprise a TATA box.
  • the ubiquitin promoter specifically UbC, is a strong constitutively active promoter functional in several species.
  • the UbC promoter is further characterized in Marinovic et al., J. Biol. Chem., 277(19), 16673-16681 (2002). Many of the above-described promoters are constitutive promoters.
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals.
  • the regulatory sequences can comprise a hypoxia driven promoter, which is active when an ocular disorder is associated with hypoxia.
  • suitable inducible promoter systems include, but are not limited to, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system.
  • promoters that are selectively activated at different developmental stages can be employed.
  • the promoter sequence that regulates expression of the nucleic acid sequence can contain at least one heterologous regulatory sequence responsive to regulation by an exogenous agent.
  • the regulatory sequences are preferably responsive to exogenous agents such as, but not limited to, drugs, hormones, or other gene products (ideally gene products produced in the eye).
  • the regulatory sequences, e.g., promoter preferably are responsive to glucocorticoid receptor-hormone complexes, which, in turn, enhance the level of transcription of a therapeutic gene or a therapeutic fragment thereof.
  • the regulatory sequences can comprise a tissue-specific promoter, i.e., a promoter that is preferentially activated in a given tissue and results in expression of a gene product in the tissue where activated.
  • tissue-specific promoter suitable for use in the invention can be chosen by the ordinarily skilled artisan based upon the target tissue or cell-type.
  • Preferred tissue-specific promoters for use in the inventive method are specific to ocular tissue, such as a rhodopsin promoter. Examples of rhodopsin promoters include, but are not limited to, a GNAT cone- transducing alpha-subunit gene promoter or an interphotoreceptor retinoid binding protein promoter.
  • the expression vector comprises a nucleic acid encoding a cis-acting factor, wherein the cis-acting factor modulates the expression of the nucleic acid sequence.
  • the cis-acting factor comprises matrix attachment region (MAR) sequences (e.g., immunoglobulin heavy chain (Jenunwin et al., Nature, 3S5(16), 269 (1997)), apolipoprotein B, or locus control region (LCR) sequences, among others.
  • MAR sequences have been characterized as DNA sequences that associate with the nuclear matrix after a combination of nuclease digestion and extraction (Bode et al., Science, 255(5041), 195-197 (1992)).
  • MAR sequences are often associated with enhancer-type regulatory regions and, when integrated into genomic DNA, MAR sequences augment transcriptional activity of adjacent nucleotide sequences. It has been postulated that MAR sequences play a role in controlling the topological state of chromatin structures, thereby facilitating the formation of transcriptionally-active complexes. Similarly, it is believed LCR sequences function to establish and/or maintain domains permissive for transcription. Many LCR sequences give tissue specific expression of associated nucleic acid sequences. Addition of MAR or LCR sequences to the expression vector can further enhance expression of the nucleic acid sequence. To optimize protein production, preferably the nucleic acid sequence further comprises a polyadenylation site following the coding region of the nucleic acid sequence.
  • nucleic acid sequence preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the nucleic acid sequence will be properly expressed in the cells into which it is introduced.
  • the nucleic acid sequence also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production.
  • the nucleic acid sequence encodes a protein or peptide, which is a processed or secreted protein or acts intracellularly, preferably the nucleic acid sequence further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like. In certain embodiments, it may be advantageous to modulate expression of the nucleic acid sequence encoding the gene product.
  • An especially preferred method of modulating expression of a nucleic acid sequence comprises addition of site-specific recombination sites on the expression vector. Contacting an expression vector comprising site-specific recombination sites with a recombinase will either up- or down-regulate transcription of a coding sequence, or simultaneously up-regulate transcription one coding sequence and down-regulate transcription of another, through the recombination event.
  • site-specific recombination to modulate transcription of a nucleic acid sequence is described in, for example, U.S. Patents 5,801,030 and 6,063,627 and International Patent Application WO 97/09439.
  • the expression vector can comprise a nucleic acid sequence that encodes any gene product.
  • the gene product can be a protein or RNA useful in methods of treatment, diagnostic methods, or useful in ocular-related research.
  • the gene product is a therapeutic gene product (i.e., a gene product that achieves a beneficial biological effect in a patient).
  • Suitable gene products include, but are not limited to, cytokines, enzymes, inhibitors of angiogenesis, neurotrophic factors, antibodies, and biologically-active fragments of any of the foregoing.
  • the expression vector of the inventive method comprises a nucleic acid encoding an inhibitor of angiogenesis.
  • the nucleic acid sequence can encode multiple inhibitors of angiogenesis.
  • inhibitor of angiogenesis is meant any factor that prevents or ameliorates neovascularization.
  • an inhibitor of angiogenesis includes, for instance, an anti-angiogenic factor, an anti-sense molecule specific for an angiogenic factor, a ribozyme, a small interfering RNA (siRNA, an RNA interfering molecule), a receptor for an angiogenic factor, and an antibody that binds a receptor for an angiogenic factor.
  • the anti-angiogenic factors contemplated for use in the invention include, for example, pigment epithelium-derived factor, angiostatin, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., INF ⁇ ), tissue inhibitor of metalloproteinase 3 (TIMP3), and the like.
  • pigment epithelium-derived factor angiostatin, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., INF ⁇ ), tissue inhibitor of metalloproteinase 3 (TIMP3), and the like.
  • INF ⁇ interferons
  • TRIP3 tissue inhibitor of metalloproteinase 3
  • anti-angiogenic factors are described in International Patent Application WO 02/22176.
  • any anti-angiogenic factor can be modified or truncated and retain anti-angiogenic activity.
  • an anti-sense molecule specific for an angiogenic factor should generally be substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the nucleic acid encoding the angiogenic factor to be inhibited, but need not be identical.
  • the anti-sense nucleic acid molecule can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the nucleic acid.
  • the introduced anti-sense nucleic acid molecule also need not be full-length relative to either the primary transcription product or fully processed mRNA.
  • anti-sense molecule need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.
  • Antisense phosphorothiotac oligodeoxynucleotides is exemplary of an anti-sense molecule specific for an angiogenic factor.
  • Other RNA interfering agents such as siRNA (see, e.g., Chui et al., Mol. Cell, 10(3), 549-61 (2002)).
  • Ribozymes can be designed that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme.
  • the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature, 334, 585-591 (1988).
  • the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.
  • Receptors specific for angiogenic factors inhibit neovascularization by sequestering growth factors away from functional receptors capable of promoting a cellular response.
  • Fit and Flk receptors e.g., soluble fit (sflt)
  • VEGF-receptor chimeric proteins compete with VEGF receptors on vascular endothelial cells to inhibit endothelial cell growth (Aiello, PNAS, 92, 10457 (1995)).
  • growth factor-specific antibodies and fragments thereof e.g., Fab, F(ab') 2 , and Fv
  • the expression vector can comprise a nucleic acid sequence encoding a vessel maturation factor.
  • vessel maturation factors reduce the amount of vascular leakage and, therefore, are useful in treating, for example, exudative ocular disorders.
  • Vessel maturation factors include, but are not limited to, angiopoietins (Ang, e.g., Ang-1 and Ang-2), tumor necrosis factor-alpha (TNF- ⁇ ), midkine (MK), COUP-TFII, hepatic growth factor (HGF), and heparin-binding neurotrophic factor (HBNF, also known as heparin binding growth factor).
  • Ang angiopoietins
  • TNF- ⁇ tumor necrosis factor-alpha
  • MK midkine
  • COUP-TFII hepatic growth factor
  • HGF hepatic growth factor
  • HBNF heparin-binding neurotrophic factor
  • the invention also contemplates delivery of a nucleic acid sequence encoding at least one neurotrophic agent (or neurotrophic factor) to ocular cells. Neurotrophic factors are thought to be responsible for the maturation of developing neurons and for maintaining adult neurons.
  • the method of the invention can be used to inhibit or reverse neural cell degeneration and death not associated with neovascular diseases.
  • Neurotrophic factors are divided into three subclasses: neuropoietic cytokines; neurotrophins; and the fibroblast growth factors.
  • Ciliary neurotrophic factor (CNTF) is exemplary of neuropoietic cytokines. CNTF promotes the survival of ciliary ganglionic neurons and supports certain neurons that are NGF-responsive.
  • Neurotrophins include, for example, brain-derived neurotrophic factor and nerve growth factor, perhaps the best characterized neurotrophic factor.
  • neurotrophic factors suitable for being encoded by the nucleic acid sequence of the inventive method include, for example, transforming growth factors, glial cell-line derived neurotrophic factor, neurotrophin 3, neurotrophin 4/5, and interleukin 1- ⁇ .
  • Neurotrophic factors associated with angiogenesis such as aFGF and bFGF, are less preferred.
  • the neurotrophic factor can also be a neuronotrophic factor, e.g., a factor that enhances neuronal survival. It has been postulated that neurotrophic factors can actually reverse degradation of neurons. Such factors, conceivably, are useful in treating the degeneration of neurons associated with vision loss. Neurotrophic factors function in both paracrine and autocrine fashions, making them ideal therapeutic agents.
  • the nucleic acid sequence of the invention encodes both an inhibitor of angiogenesis and a neurotrophic factor. More preferably, the nucleic acid sequence encodes at least one factor comprising both anti-angiogenic and neurotrophic properties. Most preferably, the factor comprising both anti-angiogenic and neurotrophic properties is PEDF.
  • PEDF also named early population doubling factor- 1 (EPC-1), is a secreted protein having homology to a family of serine protease inhibitors named serpins. PEDF is made predominantly by retinal pigment epithelial cells and is detectable in most tissues and cell types of the body.
  • PEDF has been observed to induce differentiation in retinoblastoma cells and enhance survival of neuronal populations (Chader, Cell Different., 20, 209-216 (1987)). Factors that enhance neuronal survival under adverse conditions, such as PEDF, are termed "neuronotrophic," as described herein. PEDF further has gliastatic activity, or has the ability to inhibit glial cell growth. As discussed above, PEDF also has anti-angiogenic activity. Anti-angiogenic derivatives of PEDF include SLED proteins, discussed in WO 99/04806. It has also been postulated that PEDF is involved with cell senescence (Pignolo et al., J. Biol. Chem., 268(12), 8949-8957 (1998)).
  • PEDF for use in the inventive method can be derived from any source, and is further characterized in U.S. Patent 5,840,686 and International Patent Applications WO 93/24529 and WO 99/04806.
  • the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 1.
  • the expression vector e.g., the adenoviral or the adeno-associated viral vector, also can comprise a nucleic acid sequence encoding a protein fragment, such as a therapeutic fragment of at least one inhibitor of angiogenesis or at least one neurotrophic factor.
  • any inhibitor of angiogenesis or neurotrophic factor e.g., PEDF
  • coding sequences for therapeutic fragments i.e., those fragments having biological activity sufficient to, for example, inhibit angiogenesis or promote neuron survival
  • nucleic acid sequences comprising substitutions, deletions, or additions, but which encode a functioning inhibitor of angiogenesis or neurotrophic factor or a therapeutic fragment of any of the foregoing.
  • a functioning inhibitor of angiogenesis or a therapeutic fragment thereof prevents or ameliorates neovascularization.
  • a functioning neurotrophic factor or a therapeutic fragment thereof desirably promotes neuronal cell differentiation, inhibits glial cell proliferation, and/or promotes neuronal cell survival.
  • a functioning neurotrophic factor or a therapeutic fragment thereof desirably promotes neuronal cell differentiation, inhibits glial cell proliferation, and/or promotes neuronal cell survival.
  • complete prevention or amelioration of neovascularization is not required in order to realize a therapeutic effect.
  • complete induction of neuron survival or differentiation is not required in order to realize a benefit. Therefore, both partial and complete prevention and amelioration of angiogenesis or promotion of neuron survival is appropriate.
  • the ordinarily skilled artisan has the ability to determine whether a modified therapeutic factor or a fragment thereof has neurotrophic and anti-angiogenic therapeutic activity using, for example, neuronal cell differentiation and survival assays (see, for example, U.S.
  • the inhibitor of angiogenesis and/or the neurotrophic factor can be a factor that acts upon a receptor for an anti-angiogenic factor or a receptor for a neurotrophic factor, thereby resulting in the desired biological effect.
  • the expression vector can comprise a nucleic acid sequence encoding an antibody or peptide agonist that binds and activates the PEDF receptor, which signals a series of intracellular events responsible for the biological activity of PEDF.
  • the expression vector can comprise a nucleic acid sequence encoding a peptide that interacts with a PEDF receptor to achieve a biological effect.
  • a dominant positive protein can be constructed which constitutively activates cell-signaling via the PEDF receptor.
  • the invention also contemplates the use of nucleic acid sequences encoding chimeric or fusion peptides. Through recombinant DNA technology, scientists have been able to generate fusion proteins that contain the combined amino acid sequence of two or more proteins.
  • a fusion protein such as a fusion protein comprising an anti-angiogenic factor or neurotrophic factor or, a therapeutic fragment thereof and for example, a moiety that stabilizes peptide conformation, also can be present in the expression vector.
  • the chimeric peptide can comprise the entire amino acid sequences of two or more peptides or, alternatively, can be constructed to comprise portions of two or more peptides (e.g., 10, 20, 50, 75, 100, 400, 500, or more amino acid residues).
  • the chimeric peptide comprises anti-angiogenic and neurotrophic activity, which can be determined using routine methods.
  • the method of the invention can be part of a treatment regimen involving other therapeutic modalities. It is appropriate, therefore, if the ocular-related disorder, namely ocular neovascularization or age-related macular degeneration, has been treated, is being treated, or will be treated with any of a number of additional ocular therapies, such as drug therapy, panretinal therapy, thermotherapy, radiation therapy, or surgery.
  • the surgery can comprise, for instance, macular translocation, removal of subretinal blood, or removal of subretinal choroidal neovascular membrane.
  • the expression vector is preferably administered intraocularly for the prophylactic or therapeutic treatment of an ocular-related disorder, e.g., age-related macular degeneration or persistent or recurrent ocular neovascularization, which is also treated with drugs, surgery, laser photocoagulation, and/or photodynamic therapies.
  • an ocular-related disorder e.g., age-related macular degeneration or persistent or recurrent ocular neovascularization
  • the expression vector of the inventive method is delivered to the eye, wherein the expression vector transduces host cells. Delivery to the eye can be achieved by administering the expression vector to any component of the ocular apparatus (e.g., eye globe, layers of the eye globe, muscles or connective tissue associated with the eye, etc.), such that the gene product is produced and delivered to target ocular cells.
  • any component of the ocular apparatus e.g., eye globe, layers of the eye globe, muscles or connective tissue associated with the eye, etc.
  • Ocular cells include, but are not limited to, cells of neural origin, cells of all layers of the retina, especially retinal pigment epithelial cells, glial cells, pericytes, endothelial cells, iris epithelial cells, corneal cells, ciliary epithelial cells, Mueller cells, astrocytes, muscle cells surrounding and attached to the eye (e.g., cells of the lateral rectus muscle), fibroblasts (e.g., fibroblasts associated with the episclera), orbital fat cells, cells of the sclera and episclera, connective tissue cells, muscle cells, cells of the trabecular meshwork, fibroblasts, and vascular endothelial cells.
  • retinal pigment epithelial cells e.g., glial cells, pericytes, endothelial cells, iris epithelial cells, corneal cells, ciliary epithelial cells, Mueller cells, astrocytes, muscle cells surrounding and attached to the eye (e.g.,
  • the expression vector can be administered to an area of vascular leakage.
  • the expression vector desirably is administered in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the expression vector(s).
  • a pharmaceutical composition which comprises a pharmaceutically acceptable carrier and the expression vector(s).
  • Any suitable pharmaceutically acceptable carrier can be used within the context of the invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
  • Suitable formulations include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti- ⁇ xjdants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood or intraocular fluid of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • the expression vector is administered in a pharmaceutical composition formulated to protect and/or stabilize the expression vector from damage prior to administration.
  • the pharmaceutical composition can be formulated to reduce loss of the expression vector on devices used to prepare, store, or administer the expression vector, such as glassware, syringes, pellets, slow-release devices, pumps, or needles.
  • the pharmaceutical composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the expression vector.
  • the pharmaceutical composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • the formulation comprises Tris base (10 mM), NaCl (75 mM), MgCl-6H 2 O (1 mM), polysorbate 80 (0.0025%) and trehalose dehydrate (5%).
  • Use of such a pharmaceutical composition will extend the shelf life of the vector, facilitate administration, and increase the efficiency of the inventive methods.
  • a pharmaceutical composition also can be formulated to enhance transduction efficiency. Suitable compositions are further described in U.S.
  • the expression vector, e.g., viral vector, of the invention can be present in a composition with other therapeutic or biologically-active agents.
  • therapeutic factors useful in the treatment of a particular indication can be present.
  • hyaluronidase can be added to a composition to, for example, affect the break down of blood and blood proteins in the vitreous of the eye.
  • Factors that control inflammation such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the viral vector and ocular distress.
  • Inflammation also can be controlled by down-regulating the effects of cytokines involved in the inflammation process (e.g., TNF ⁇ ).
  • cytokines involved in the inflammation process e.g., TNF ⁇
  • agonists for chemokines which control inflammation e.g., TGF ⁇
  • Immune system suppressors can be administered in combination with the inventive method to reduce any immune response to the vector itself or associated with an ocular disorder.
  • Anti-angiogenic factors such as soluble growth factor receptors (sflt), growth factor antagonists (e.g., angiotensin), an anti-growth factor antibody (e.g., Lucentis ), Squalamine (an aminosterol), and the like also can be part of the composition, as well as additional neurotrophic factors.
  • vitamins and minerals, anti-oxidants, and micronutrients can be co-administered.
  • Antibiotics i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.
  • Ligands for nuclear receptors such as thyroid hormones, retinoids, specific prostaglandins, estrogen hormone, glucocorticoids or their analogues can be part of the composition.
  • Small molecule agonists for the PEDF receptor also can be included in the formulation. Such small molecule agonists can amplify the therapeutic effect of the inventive method.
  • Suitable drugs for inclusion in the formulation include, but are not limited to, a prostaglandin analogue, a beta-blocker (as commonly used for glaucoma treatment), hyaluronidase (e.g., VitraseTM available from Allergan), pegaptanib sodium (e.g., MacugenTM), tetrahydrozoline hydrochloride (e.g., VisineTM), dorzolamide hydrochloride (CosoptTM and TruspotTM), and an alpha-2-adrenergic agonist (e.g., AlphaganTM).
  • these compounds can be administered separately to the animal.
  • suitable methods i.e., invasive and noninvasive methods, of administering an expression vector to the eye are available.
  • a particular route can be used to administer a particular expression vector to an eye, such as a human eye, a particular route can provide a more immediate and more effective reaction than another route.
  • the described routes of administration are merely exemplary and are in no way limiting. Any route of administration is appropriate so long as the expression vector transduces a host cell.
  • the expression vector can be appropriately formulated and administered in the form of an injection, eye lotion, ointment, implant and the like.
  • An expression vector can be applied, for example, topically, intracamerally, subconjunctivally, intraocularly, retrobulbarly, periocularly (e.g., subtenon delivery), subretinally, intravitreously, or suprachoroidally for direct administration to the eye.
  • Multiple applications of the expression vector may also be required to achieve the desired effect.
  • Topical administration of the expression vector may be most appropriate.
  • Topical formulations are well known to those of skill in the art. Such formulations are suitable in the context of the invention for application to the skin.
  • patches, corneal shields (see, e.g., U.S. Patent 5,185,152), and ophthalmic solutions (see, e.g., U.S. Patent 5,710,182) and ointments, e.g., eye drops, is also within the skill in the art.
  • the expression vector can also be administered non-invasively using a needleless injection device, such as the Biojector 2000 Needle-Free Injection Management System® available from Bioject, Inc.
  • the expression vector can be present in or on a device that allows controlled or sustained release of the expression vector, such as an ocular sponge, meshwork, mechanical reservoir, or mechanical implant.
  • Implants see, e.g., U.S. Patents 4,853,224; 4,997,652; and 5,443,505
  • devices see, e.g., U.S.
  • Patents 4,863,457; 5,098,443; 5,554,187; and 5,725,493) such as an implantable device, e.g., a mechanical reservoir, an intraocular device or an extraocular device with an intraocular conduit, or an implant or a device comprised of a polymeric composition are particularly useful for ocular administration of the expression vector.
  • An expression vector also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent 5,378,475) comprising, for example, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), or a polylactic-glycolic acid.
  • the expression vector can be administered using invasive procedures, such as, for instance, intravitreal injection or subretinal injection, optionally preceded by a vitrectomy, or periocular (e.g., subtenon) delivery.
  • the expression vector can be injected into different compartments of the eye, e.g., the vitreal cavity or anterior chamber.
  • Pharmaceutically acceptable carriers for injectable compositions are well-known to those of ordinary skill in the art (see Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4 ed., pages 622-630 (1986)).
  • the expression vector can also be administered in vivo by particle bombardment, i.e., a gene gun.
  • the expression vector is administered via an ophthalmologic instrument for delivery to a specific region of an eye.
  • a specialized ophthalmologic instrument ensures precise administration of the expression vector while minimizing damage to adjacent ocular tissue.
  • a preferred ophthalmologic instrument is a combination of forceps and subretinal needle or sharp bent cannula.
  • injectable compositions also can be administered intramuscularly, intravenously, intraarterially, intraperitoneally, parenterally, systemically, or subcutaneously.
  • Expression vectors also can be administered intratracheally, orally, trans-dermally, or intranasally.
  • any expression vector administered to a patient using these routes of administration in the context of the invention is specifically targeted to ocular cells.
  • an expression vector can be modified to alter the binding specificity or recognition of an expression vector for a receptor on a potential host cell. With respect to adenovirus, such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • parenteral administration can require large doses or multiple administrations to effectively deliver the expression vector to the appropriate host cells.
  • dosage and routes of administration can be selected to minimize loss of expression vector due to a host's immune system.
  • a null expression vector i.e., an expression vector not comprising the nucleic acid sequence encoding the gene product
  • Prior administration of null expression vectors can serve to create an immunity (e.g., tolerance) in the host to the expression vector, thereby decreasing the amount of vector cleared by the immune system.
  • the dose of expression vector administered to an animal, particularly a human, in accordance with the invention should be sufficient to affect the desired response in the animal over a reasonable time frame.
  • dosage will depend upon a variety of factors, including the age, species, the pathology in question, and condition or disease state. Dosage also depends on the gene product, e.g., inhibitor of angiogenesis and/or neurotrophic factor, to be expressed, as well as the amount of ocular tissue to be transduced and/or about to be affected or actually affected by the ocular-related disease. The size of the dose also will be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular expression vector and the desired physiological effect. It will be appreciated by one of ordinary skill in the art that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.
  • Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art.
  • about 10 6 viral particles to 19 • about 10 viral particles are delivered to the patient.
  • a pharmaceutical composition can be administered that comprises an expression vector concentration of from about particles/ml (including all integers within the range of about particles/ml), preferably from about 10 10 particles/ml to about ically involve the intraocular administration of from about 0.1 ⁇ l to about 100 ⁇ l of such a pharmaceutical composition per eye.
  • an injection can comprise from about 0.5 mL to about 1 mL of pharmaceutical composition.
  • a dose of about 1 x 10 6 , about 1 x 10 65 , about 1 x 10 7 , about 1 x 10 75 , about 1 x 10 8 , about 1 x 10 8,5 , about 1 x 10 9 , or about 1 x 10 9 ' 5 particles of adenoviral vector (e.g., about 3 x 10 , 3 x 10 , or 3 x 10 particles of adenoviral vector) is administered per eye to a patient via intravitreal injection.
  • the adenoviral vector of the inventive method is administered subretinally in a dose of about 1 x 10 5 , about 1 x 10 5'5 , about 1 x 10 6 , about 1 x 10 6 ' 5 , about 1 x 10 7 , about 1 x 10 7,5 , about 1 x 10 8 , or about 1 x 10 8 ' 5 particles per eye.
  • the dose of adenoviral vector administered preferably is about 1 x 10 7 , about 1 x 10 7 - 5 , about 1 x 10 8 , about 1 x 10 85 , about 1 x 10 9 , about 1 x 10 95 , about 1 x 10 10 , about 1 x 10 10 - 5 , about 1 x 10 11 , about 1 x 10 1 5 , or about 1 x 10 12 particles per eye.
  • the expression vector is a plasmid, preferably about 0.5 ng to about 1000 ⁇ g of DNA is administered. More preferably, about 0.1 ⁇ g to about 500 ⁇ g is administered, even more preferably about 1 ⁇ g to about 100 ⁇ g of DNA is administered.
  • the expression vector can be administered any time after inducing a stress response in the eye. It is desirable to administer the expression vector after inducing the stress response such that transduction of host cells is enhanced and expression of the nucleic acid sequence (i.e., transcription) is increased as compared to an expression vector administered in the absence of inducing a stress response in the eye.
  • the expression vector is administered within 3 months (e.g., within 2 months) of inducing a stress response in the eye (e.g., exposing the eye to photocoagulation or photodynamic therapy). More preferably, the expression vector is administered within 28 days (e.g., within 21 days or within 14 days) of inducing a stress response in the eye. Also preferably, the expression vector is administered to the eye within 7 days of (e.g., 1, 2, 3, 4, 5, 6, or 7 days after) inducing a stress response in the eye. Also preferably, the expression vector is administered within 3 days (e.g., 1, 2, or 3 days) or within 1 day of inducing the stress response in the eye.
  • 3 days e.g., 1, 2, or 3 days
  • the inventive method provides for multiple applications of the expression vector.
  • at least two applications of an expression vector comprising an exogenous nucleic acid encoding a gene product e.g., a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic agent, can be administered to the same eye.
  • the multiple doses are administered while retaining gene expression above background levels.
  • two applications or more of the expression vector is administered within about 30 days or more.
  • the expression vector of the inventive method comprises a nucleic acid sequence that encodes a gene product (e.g., at least one inhibitor of angiogenesis and/or at least one neurotrophic factor).
  • the nucleic acid sequence can encode multiple, i.e., two, three, or more, gene products, or comprise additional transgenes.
  • expression of one or more additional gene products is beneficial, e.g., prophylactically or therapeutically beneficial, to the ocular cell or eye.
  • the gene product confers a prophylactic or therapeutic benefit to the cell
  • the transgene can exert its effect at the level of RNA or protein.
  • a peptide other than an inhibitor of angiogenesis or neurotrophic factor that can be employed in the treatment or study of a disorder, e.g., an ocular-related disorder, can be encoded by the expression vector.
  • nucleic acid sequence encoding an antisense molecule, a ribozyme, siRNA, a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the level of expression of another gene within the cell i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a process protein), such as by mediating an altered rate of mRNA accumulation or transport or an alteration in post-transcriptional regulation, can be included in the expression vector.
  • the nucleic acid sequence encodes PEDF and ciliary neurotrophic factor (CNTF) or sflt.
  • Multiple inhibitors of angiogenesis and/or multiple neurotrophic factors can be operably linked to different promoters.
  • Multiple gene products can be encoded by multiple expression vectors, which are administered to the eye and produced within a target cell.
  • the transgene can encode a chimeric peptide for combination treatment of an ocular-related disorder.
  • a nucleotide sequence encoding an immunosuppressor also can be incorporated into the expression vector to reduce any inappropriate immune response within the eye as a result of an ocular-related disorder or the administration of the expression vector.
  • a transgene encoding a marker protein, such as green fluorescent protein or luciferase can be incorporated into the expression vector. Such marker proteins are useful in vector construction and determining vector migration.
  • Marker proteins also can be used to determine points of injection or treated ocular tissues in order to efficiently space injections of the expression vector to provide a widespread area of treatment, if desired.
  • the inventive method also can involve the co-administration of other pharmaceutically active compounds.
  • co-administration is meant administration before, concurrently with, e.g., in combination with the expression vector in the same formulation or in separate formulations, or after administration of the expression vector as described above.
  • Any of the exogenous materials, drugs, proteins, and the like described herein can be co-administered with the expression vector as adjuvant therapy.
  • factors that control inflammation such as ibuprofen or steroids, can be co-administered to reduce swelling and inflammation associated with intraocular administration of the expression vector.
  • Immunosuppressive agents can be co-administered to reduce inappropriate immune responses related to an ocular disorder or the practice of the inventive method.
  • Anti-angiogenic factors such as soluble growth factor receptors, growth factor antagonists, i.e., angiotensin, and the like can also be co-administered, as well as neurotrophic factors.
  • the expression vector of the inventive method can be administered with anti-proliferative agents such as siRNA, aptamers, or antibodies which sequester or inactivate angiogenic factors such as, for example, VEGF.
  • vitamins and minerals, anti-oxidants, and micronutrients can be co-administered.
  • Antibiotics i.e., microbicides and fungicides
  • Other therapeutics for ocular disorders can be administered in conjunction with the inventive method.
  • Visudyne® Novartis
  • MacugenTM Pfizer
  • RetaaneTM Alcon
  • LucentisTM Genentech/Novartis
  • Squalamine Genaera
  • Cosopt and Alphagan
  • Alphagan can be formulated with the expression vector or can be administered separately before, during, or after administration of the expression vector to the animal.
  • EXAMPLE 1 This example demonstrates the ability of an expression vector comprising a nucleic acid sequence encoding PEDF to inhibit choroidal neovascularization (CNV).
  • CNV choroidal neovascularization
  • Replication-deficient (El -/E3 -deficient) adenoviral vectors (AdPEDF.10) comprising the coding sequence for PEDF operably linked to the CMV immediate early promoter were constructed using standard techniques.
  • Adult C57BL/6 mice were injected intravitreously with AdNull.10 or AdPEDF.10 using a Harvard pump microinjection apparatus and pulled glass micropipettes.
  • mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight). Topicamide (1%) was utilized to dilate the pupils prior to rupture of Bruch's membrane by diode laser photocoagulation. Rupture of Bruch's membrane is known to induce neovascularization of the choroid.
  • choroidal flat mounts described in Edelman et al., Invest. Ophthalmol. Vis.
  • EXAMPLE 2 This example demonstrates the ability of an expression vector comprising a nucleic acid sequence encoding PEDF to inhibit ischemia-induced retinal neovascularization.
  • Replication-deficient adenoviral vectors comprising the coding sequence for PEDF operably linked to the CMV immediate early promoter were constructed using standard techniques.
  • mice seven day old mice (P7) were exposed to an atmosphere of 75 +/- 3% oxygen for five days.
  • mice were injected intravitreously with 10 9 particles of AdPEDF.11 or AdNull.11 , returned to oxygen for two days, then returned to room atmosphere.
  • OCT optimum cutting temperature embedding compound
  • the eyes were sectioned and histochemically stained with biotinylated griffonia simplicifolia lectin B4 (GSA, Vector Laboratories, Burlingame, CA).
  • Eyes injected with AdNull.l 1 showed less neovascularization than uninjected eyes, but significantly more neovascularization of the retina than eyes injected with AdPEDF.11. Eyes injected with AdPEDF.l 1 comprised the least amount of neovascularization.
  • This example clearly demonstrates the ability of adenoviral vector-mediated delivery of PEDF to inhibit an ocular-related disorder, namely ischemia-induced retinal neovascularization, in a clinically relevant animal model.
  • EXAMPLE 3 This example demonstrates the ability of the inventive method to deliver a gene product to the eye.
  • Forty albino Lewis rats were used in the study, a portion of which were administered photocoagulation therapy or photodynamic therapy.
  • Half of the posterior fundus was laser treated, and the remaining hemisphere was untreated as an internal control.
  • the remaining rats were not pre-treated with laser therapy and served as negative controls.
  • An adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions of the El, E3, and E4 regions of the adenoviral genome and comprising the ⁇ -galactosidase gene (AdZ.l 1) was injected intravitreously using Hamilton syringes.
  • Retina pre-treated with photodynamic therapy showed less frequent enhanced gene expression than photocoagulation-treated retina.
  • rats were administered 3 x 10 9 particles of AdZ.l 1 per eye three days following laser treatment. Eyes were enucleated and examined histochemically at 5, 14, 28, and 90 days after adenovector administration. Expression of the nucleic acid sequence encoding the ⁇ -galactosidase gene product was enhanced at all timepoints examined and was sustained at 90 days after administration of AdZ.l 1 (the longest duration examined) in photocoagulation-treated animals.
  • a study of various doses in the context of the inventive method was performed.
  • EXAMPLE 4 This example demonstrates enhanced transgene expression in the eye as a result of the inventive method.
  • Female Lewis rats were obtained at 4 to 8 weeks of age, and were administered photocoagulation therapy or photodynamic therapy (PDT).
  • photocoagulation therapy involved delivery of a thermal diode laser PC (532 nm wavelength, 200 ⁇ m spot size, 0.5 second duration, 300 mW) using the slitlamp delivery system (SL130, Zeiss, Germany) and a hand-held cover slide as a contact lens. Fifty burns were confined to the left half of the posterior fundus in each eye.
  • PDT was performed using the hydrophilic photosensitizer mono-L-aspartyl chlorin e6 (NPe6, LSI 1, Light Science, Seattle, WA) (see, e.g., Mori et al., Ophthalmology, 106, 1384-1391 (1999), Peyman et al., Ophthalmology, 107, 29-35 (2000), and Mori et al., Retina, 21, 499-508 (2001)).
  • a solution of 10 mg/kg body weight LSI 1 was administered to each rat via the tail vein. Irradiation of the diode laser (664 nm wavelength, 100 ⁇ m spot size, 10 second duration, 4.5 mW) was started within five minutes after intravenous injection.
  • Rats not pre-treated with laser therapy served as negative controls.
  • One group of rats was administered a dose of 3 x 10 9 particle units (pu) of AdZ.l 1 (Example 3) per eye on day 1, 3, 7, and 28 after laser treatment.
  • the adenoviral vector was injected intravitreously using Hamilton syringes. Five days after adenoviral vector injection, eyes were enucleated and examined histochemically for LacZ staining.
  • Histochemical analysis consisted of fixing eyes in 2.5% glutaraldehyde in phosphate buffer solution (PBS) for 60 minutes and rinsing five times for 10 minutes in PBS. The anterior segments were removed and the remaining posterior segments were incubated overnight in 1 mg/mL 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal, Sigma, St. Louis, MO) in a solution containing 5mM K 3 Fe(CN) 6 , 5 mM K Fe(CN)6-3H2 ⁇ , and 1 mM MgCl 2 in PBS. Eyes were post-fixed for 15 minutes and then rinsed with PBS.
  • PBS phosphate buffer solution
  • X-gal staining in the posterior segment of eyes For quantitative analysis of X-gal staining in the posterior segment of eyes, a dissecting microscope and its camera system (MZ 8 and MPS 30, Leica, Wetzlar, Germany) were used. Images were captured, digitized and analyzed by image-analysis software (Win ROOF, Mitani Corporation, Fukui, Japan) to delineate X-gal stained areas. Area ratios (X-gal stained area to total retinal area) were calculated for each eye. Statistical analysis comparing the area ratio between treated left hemisphere and the untreated right hemisphere was performed using Wilcoxon signed-ranks test. A p- value of less than 0.05 was prospectively assigned as the level at which a finding would be considered statistically significant.
  • Eyes were enucleated and examined histochemically five days after AdZ.11 administration. Vector transduction and lacZ expression were enhanced at all dose levels (p ⁇ 0.05).
  • This example demonstrates the ability of the inventive method to deliver a gene product to the eye, and effect enhanced and prolonged gene expression in the eye.
  • EXAMPLE 5 This example demonstrates the expression of adenovirus cell surface receptors in retinal and choroidal tissues following photocoagulation therapy (PC) or photodynamic therapy (PDT).
  • PC photocoagulation therapy
  • PDT photodynamic therapy
  • RNA expression was isolated from samples composed of the retina and choroids, including retinal pigment epithelium.
  • RNA samples were homogenized in TRIzol reagents (LifeTechnologies, Greand Island, NY) and treated with RNase-free DNase (DNase I, Gibco, Paisley, UK) to remove genomic DNA contamination.
  • First-strand cDNA was synthesized by reverse transcription of total RNA using reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA) with random hexamers as primers in a total reaction volume of 20 ⁇ L.
  • Amplification of the control gene ARP acidic ribosomal phosphoprotein P0 was used for normalization (see, e.g., Simpdon et al., Mol. Vis., 6, 178-183 (2000), and hackam et al., Mol.
  • Nucleic acid sequences encoding CAR, integrins ⁇ V, ⁇ 3, and ⁇ 5, and ARP were amplified using commercially available primers and probe sets (assay IDs: Mm00438361_ml for CAR, Mm00434506_ml for integrin ⁇ V, Mm00443980_ml for integrin ⁇ 3, Mm00439825_ml for integrin ⁇ 5, and Mm00725448_sl for ARP, Applied Biosystems, Foster City, CA).

Abstract

L'invention est axé sur un procédé defourniture d'un produit génétique á un oeil. Le procédé comprend (a) l'induction d'une réponse au stress dans l'oeil et (b) l'administration subséquente à l'œil d'un vecteur d'expression comprenant une séquence d'acide nucléique encodant un produit génétique, le vecteur d'expression traduit une cellule hôte et la séquence d'acide nucléique est exprimée pour produire le produit génétique. L'invention vise en outré un procédé de traitement thérapeutique ou prophylactique d'un animal pour un trouble oculaire. Le procédé comprend (a) l'incitation d'une réponse au stress dans un oeil (b) et l'administration consécutive á un oeil d'un vecteur d'expression comprenant une séquence d'acide nucléique encodant un inhibiteur d'angiogenèse d'expression et/ou un agent neurotrophique pour traiter de manière pophylactique ou thérapeutique le trouble oculaire.
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WO2017062990A1 (fr) * 2015-10-09 2017-04-13 Case Western Reserve University Compositions et procédés de libération d'acides nucléiques
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US10792374B2 (en) 2015-10-09 2020-10-06 Case Western Reserve University Compositions and methods for the delivery of nucleic acids
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