AAV-mediated gene transfer for retinopathy

Abstract

The present invention provides methods and pharmaceutical compositions for treating diseases that may affect vision, such as retinal degeneration, retinal dystrophy, macular degeneration, macular dystrophy, ischemic retinopathy, and glaucoma. [Solution] Embodiments include systems and procedures that use AAV-mediated gene therapy or non-AAV-mediated DNA, mRNA, or protein therapy to target all retinal cells. AAV virions can be introduced into the eye or body of an individual (e.g., via intravitreal or subretinal injection) to express heterologous gene products such as the BMI1 protein (a homolog of B lymphoma Mo-MLV insertion region 1).

Description

[Technical Field]

[0001] This disclosure relates, in general, to gene therapy systems and methods for treating ocular conditions, more specifically, retinal and macular degeneration, dystrophy, and optic nerve diseases. [Background technology]

[0002] In a healthy eye, photoreceptors form the outermost layer of the retina. These convert light into electrical signals, which are then sent to neurons in the central layer of the retina known as bipolar cells. The bipolar cells transmit visual information to the inner layer, which consists of ganglion cells, and these then connect to the brain via the optic nerve.

[0003] Retinopathy is any damage to the retina of the eye that can cause visual impairment. Often, retinopathy refers to damage to the retina caused by retinal vascular disease, or abnormal blood flow. Often, retinopathy is secondary to conditions such as diabetes or hypertension. Other conditions that affect the retina and can affect vision include retinal degeneration, retinal dystrophy, macular degeneration, and macular dystrophy.

[0004] Retinal degeneration Retinal degeneration is a retinopathy that involves deterioration of the retina caused by the progressive death of its cells. There are several causes of retinal degeneration, including arterial or vein occlusion, diabetic retinopathy, RLF / ROP (post-lens fibrous proliferative disorder / retinopathy of prematurity), or disease (usually hereditary). Retinal degeneration can result in visual impairment, night blindness, retinal detachment, photosensitivity, tunneling vision, and loss of central or peripheral vision, or even complete loss of vision.

[0005] Progressive retinal degeneration can lead to the death of photoreceptor cells. If photoreceptor cells do not function properly, vision is impossible. The irreversible loss of these cells has been the cause of blindness in many retinal degenerative disorders, including retinitis pigmentosa (RP).

[0006] Retinal dystrophy Retinal dystrophy is a chronic and progressive impairment of visual function. "Dystrophy" refers to a condition that a person is born with; "retinal" refers to the retina. Retinal dystrophy is a clinically and genetically heterogeneous group of eye disorders characterized by degeneration of different cell types within the retina. Retinal cell types involved in retinal dystrophy include rods and cones. Generally, retinal dystrophy is classified according to the cell type in the retina primarily affected, the age at which the first symptoms appear, the progression of visual impairment over time, and the presence or absence of other medical features. Specific subtypes of retinal dystrophy include rod-cone dystrophy, e.g., retinitis pigmentosa (RT); cone-rod dystrophy or cone dystrophy, e.g., color blindness; and macular dystrophy, e.g., Stargardt disease.

[0007] Macular degeneration Macular degeneration is usually classified as atrophic or exudative. Age-related macular degeneration (AMD) is the leading cause of vision loss in people over 50. It develops when a part of the retina called the macula is damaged, which can lead to loss of central vision.

[0008] Exudative macular degeneration (AMD) is less common but more serious. Exudative AMD occurs when new, abnormal blood vessels grow beneath the retina. These vessels can leak blood or other fluids, potentially leading to macular scarring. Vision loss occurs earlier in exudative AMD, and patients may not realize they have AMD until their vision becomes very blurry. If detected early, exudative AMD can be treated with intravitreal injections of anti-VEGF drugs.

[0009] Macular dystrophy Macular dystrophy is a relatively rare eye condition. It is associated more with hereditary gene mutations than with age. Macular dystrophy causes deterioration of the macula, the most sensitive part of the retina's center, which has the highest concentration of photosensitive cells (photoreceptors). This is caused by the accumulation of pigment in the macular cells. Over time, this substance can damage cells that play a crucial role in clear central vision.

[0010] Conventional treatment options for patients with degenerative eye conditions are limited. There are currently no available treatments for retinal degeneration and dystrophy. Intravitreal anti-angiogenic therapy can temporarily inactivate the rapidly growing abnormal blood vessels originating from AMD. There are also no known cures or treatments to halt the progression of macular degeneration or dystrophy. Management typically involves regular eye examinations to monitor disease progression and complications such as choroidal neovascularization (CNV). Patients may lose their entire central visual field due to progressive atrophic AMD when geographic atrophy occurs. There is no treatment or cure for this condition. Exudative AMD can be treated with regular injections into the eye. Patients may partially recover vision if blood vessels constrict, subretinal fluid is absorbed, and some retinal cell function is restored. Improved treatment and care are needed due to the limited options for treating retinopathy.

[0011] Gene therapy has come to be recognized as a promising tool for treating diseases at both the cellular and molecular levels. In recent years, there has been considerable attention on applying gene therapy to treat human diseases that are either hereditary (e.g., adenosine deaminase (ADA) deficiency) or acquired (e.g., cancer or infectious diseases). With the emergence of improved gene transfer techniques and the identification of expanding libraries of diseases associated with deficiency genes, gene therapy is rapidly progressing from a theoretical treatment to a practical reality.

[0012] Viral vectors are commonly used tools by molecular biologists to deliver genetic material to cells. This process can take place in living organisms (in vivo) or in cell cultures (in vitro). Viruses have developed special molecular mechanisms to efficiently transport their genomes into the cells they infect. The delivery of genes or other genetic material via a vector is called transduction, and the infected cell is described as transduced.

[0013] This invention discloses systems and treatments using AAV-mediated gene therapy targeting retinal pigment epithelial (RPE) cells and photoreceptor cells. For example, AAV1, AAV2, AAV4, AAV5, and AAV8 serotypes are known to be specific to RPE cells, and AAV2, AAV5, and AAV8 are specific to ocular photoreceptor cells. AAV virions can be introduced into the eye of an individual (e.g., via intravitreous, subretinal, subinternal limiting membrane, or suprachoroidal injection) to express heterologous gene products such as the BMI1 protein (a Mo-MLV insertion region 1 homolog of B lymphoma). Treatment methods, pharmaceutical compositions thereof, and manufactured articles are also provided. [Overview of the Initiative]

[0014] The following summary is provided to facilitate understanding of some of the innovative features inherent in the disclosed embodiments and is not intended to be a complete description. A complete understanding of the various aspects of the embodiments disclosed herein can be obtained by considering the entire specification, claims, drawings, and abstract together.

[0015] The present invention includes compositions and methods for treating eye diseases in a target eye (e.g., retinal degeneration, retinal dystrophy, macular degeneration, macular dystrophy, glaucoma) using adeno-associated virus (AAV) virus particles.

[0016] In one embodiment, a pharmaceutical composition for preventing, halting the progression of, or improving retinopathy is provided, comprising a viral vector and a pharmaceutically acceptable carrier. The viral vector particle may be of type AAV8 and may contain a nucleic acid sequence encoding the BMI1 protein. Other AAVs may include type AAV2.

[0017] In another embodiment, a method is provided for targeting retinal pigment epithelial (RPE) cells for gene modification therapy in a subject requiring such treatment. The method comprises administering to a subject an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0018] In yet another embodiment, a method is provided for preventing, halting the progression of, or improving vision loss associated with a target retinal degeneration. The method comprises administering to a target an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0019] In yet another embodiment, a method is provided for preventing, halting the progression of, or improving vision loss associated with a retinal dystrophy in question. The method comprises administering to a subject an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0020] In yet another embodiment, a method is provided for preventing, halting the progression of, or improving vision loss associated with a target macular degeneration. The method comprises administering to a target an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0021] In yet another embodiment, a method is provided for preventing, halting the progression of, or improving vision loss associated with a target macular dystrophy. The method comprises administering to a target an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0022] In a further embodiment, an AAV containing the BMI1 gene in its genome is administered intravitreously, intravenously, subretinally, or posteriorly to the patient. In a further embodiment, administration of this BMI1-containing AAV increases the expression of BMI1 in retinal ganglion cells. This increased protection reduces the severity of glaucoma, ischemic optic neuropathy, and / or retinopathy.

[0023] This method includes the step of administering an effective concentration of a composition comprising one of the recombinant adeno-associated viruses (AAVs) described herein and a pharmaceutically acceptable carrier.

[0024] In another embodiment, a host or target cell transfected with an AAV or nucleic acid molecule as described herein is provided.

[0025] definition In this specification, any reference to “one embodiment / aspect” or “an embodiment / aspect” means that any particular feature, structure, or characteristic described in relation to that embodiment / subject is included in at least one embodiment / subject of this disclosure. The use of the phrases “in one embodiment / aspect” or “in another embodiment / aspect” in various places in this specification does not necessarily refer to the same embodiment / subject, nor does it refer to another or alternative embodiment that is mutually exclusive with the other embodiments / aspects. Furthermore, various features are described that may be presented by some embodiments / aspects but not by others. Similarly, various requirements are described that may be necessary in some embodiments / aspects but not in others. Embodiments and aspects may be used interchangeably in certain examples.

[0026] The terms used herein generally have their common meanings in the art within the context of the disclosure and in the specific context in which each term is used. To provide practitioners with further guidance regarding the description of this disclosure, specific terms used to describe this disclosure are considered below or elsewhere in this specification. It is recognized that the same thing can be expressed in more than one way.

[0027] Where used herein, the transitional phrase "consisting essentially of" should be interpreted as encompassing the specified materials or steps described in the claims and any other elements that do not substantially affect the essential and novel features of the claimed invention. See re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original author); also see MPEP § 2111.03. Thus, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present invention. Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination.

[0028] Furthermore, the present invention intends that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0029] The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of naive terminal repeats (TRs), Rep proteins, and capsid subunits, are publicly known in the field. Such sequences can be found in the literature or in public databases such as GenBank. For example, GenBank deposit numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF See 043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, and AY530579. These disclosures are incorporated herein by reference to teach the nucleic acid and amino acid sequences of parvovirus and AAV. Similarly, for example Srivistava et al., (1983) J Virology 45:555;Chiarini et al., (1998) J. Virology 71 :6823;Chiarini et al., (1999) J. Virology 73:1309;Bantel-Schaal et al., (1999) J. Virology 73:939;Xiao et al., (1999) J. Virology 73:3994;Muramatsu et al., (1996) Virology 221 :208;Shade et al., (1986) J. Viral. 58:921;Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854;Moris et al., (2004) Virology See 33-:375-383; International Patent Publications No. 00 / 28061, 99 / 61601, 98 / 11244; and U.S. Patent Publication No. 6,156,303.These disclosures are incorporated herein by reference to teach the nucleic acid and amino acid sequences of parvovirus and AAV.

[0030] As used herein, the term "directivity" refers to the preferential entry of a virus into a particular cell or tissue, followed optionally by the expression (e.g., transcription and optionally translation) of sequences of the viral genome in the cell, for example, recombinant viruses, of the desired heterologous nucleic acid.

[0031] As used herein, “systemic directivity” and “systemic transduction” (and equivalent terms) indicate that the viral capsid or viral vector of the present invention is directive to and / or transduces tissues throughout the body (e.g., brain, eyes, lungs, skeletal muscle, heart, liver, kidneys, and / or pancreas). In embodiments of the present invention, systemic transduction of the eye or visual system is observed. In other embodiments, systemic transduction of myocardial tissue is achieved.

[0032] As used herein, “selective directivity” or “specific directivity” means the delivery of a viral vector to specific target cells and / or specific tissues and / or the specific transduction of specific target cells and / or specific tissues.

[0033] Vectors for use in gene therapy may contain viruses. In one embodiment, the virus is a retrovirus, herpes simplex virus, or adenovirus.

[0034] Physical methods for introducing the nucleotide sequence encoding the BMI1 gene may include intraocular injection of naked DNA. Further methods include electroporation, sonoporation, and the use of a gene gun that fires DNA-coated gold particles into cells using compressed gas. Other methods include magnetofection and hydrodynamic delivery.

[0035] DNA delivery can be improved through the use of lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-permeable peptides.

[0036] Unless otherwise stated, “efficient transduction” or “efficient directivity” or similar terms may be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500%, or more transduction or directivity of the control, respectively). In certain embodiments, the viral vector efficiently transduces or has efficient directivity to nerve cells and cardiomyocytes. A suitable control depends on a variety of factors, including the desired directivity and / or transduction profile.

[0037] Similarly, whether a virus “does not efficiently transduce” or “does not have efficient targeting” or similar wording to target tissues can be determined by referring to appropriate controls. In certain embodiments, the viral vector does not efficiently transduce (i.e., does not have efficient targeting) the liver, kidneys, gonads and / or germ cells. In certain embodiments, the transduction of a tissue (e.g., liver) (e.g., undesirable transduction) is less than or equal to 25%, less than or equal to 20%, less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, or less than or equal to 0.1% of the transduction level of a desired target tissue (e.g., skeletal muscle, diaphragmatic muscle, cardiac muscle, and / or central nervous system cells).

[0038] As a result, alternative languages ​​and synonyms may be used for one or more of the terms discussed herein. Whether a term is detailed or discussed herein is not of particular importance. Synonyms are provided for specific terms. Detailed explanation of one or more synonyms does not preclude the use of other synonyms. Any use of examples in any part of this specification, including examples of any of the terms discussed herein, is merely illustrative and is not intended to further limit the scope and meaning of this disclosure or any illustrated term. Similarly, this disclosure is not limited to the various embodiments provided herein.

[0039] The terms “subject” or “patient” refer to any single animal, more preferably a mammal (including non-human animals, e.g., dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates) to which treatment is desired. Most preferably, as herein, patient is human. In one embodiment, the “subject” of a diagnosis or treatment is a prokaryotic or eukaryotic cell, a tissue culture, a tissue, or an animal, a mammal, e.g., a human.

[0040] The term "AAV" refers to adeno-associated viruses and can be used to refer to the virus itself or its derivatives. Unless otherwise required, the term encompasses all subtypes, as well as naturally occurring and recombinant forms. Adeno-associated viruses (AACs) are members of the Parvoviridae family, non-enveloped, icosahedral viruses with a single-stranded linear DNA genome ranging from 4.7 kilobases (kb) to 6 kb. AAVs are assigned to the Dependvirus genus because they were discovered as contaminants in purified adenovirus stocks. The AAV life cycle includes a latent period in which the AAV genome is site-specifically integrated into the host chromosome after infection, and an infectious period in which, after infection with either an adenovirus or herpes simplex virus, the integrated genome is subsequently rescued, replicated, and packaged into an infectious virus. Due to their non-pathogenicity, broad host infection range, inclusion of non-dividing cells, and site-specific chromosome integration capabilities, AAVs are an attractive tool for gene transfer. There are 12 AAV serotypes, including AAV1, AAV2, AAV4, AAV5, and AAV8. Additionally, there are different variants of AAV, including chimeric, pseudotyped, haploid, polyploid, and self-complementary AAVs.

[0041] In one embodiment, AAV is a variant, derivative, modified AAV, or other AAV distinct from the wild-type AAV strain of the same serotype. One example of a variant of AAV2 is AAV2.7m8, which is an engineered capsid having a 10-amino acid insertion (7m8 peptide) into the adeno-associated virus (AAV) surface variable region VIII (VR=VIII), resulting in alteration of the antigenic region of AAV2 and the ability to efficiently transduce into retinal cells or other cells such as inner or outer ear cells after intravitreous administration. Another example is AAV8BP2 and AAV9-7m8. The 7m8 peptide can also be inserted into AAV5 and AAV8. A further example is AAV2.7m8-Nr2e3.

[0042] In one embodiment, "effective dose" refers to a specified amount of a component sufficient to achieve a desired therapeutic outcome, although this is not limiting. In one embodiment, this outcome may be an effective cancer treatment.

[0043] In one embodiment, as used herein, terms such as “treating” and “treatment” are used to mean, but are not limited to, obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in that it completely or partially prevents the disorder or its signs or symptoms, and / or therapeutic in that it improves the symptoms of a disease or infection, or partially or completely cures the disorder and / or adverse effects resulting from said disorder.

[0044] As used herein, “isolated” polynucleotides (e.g., “isolated DNA” or “isolated RNA”) mean polynucleotides that have been at least partially isolated from at least a portion of other naturally occurring components of an organism or virus, such as structural components of a cell or virus, or other polypeptides or nucleic acids commonly found in association with polynucleotides. In typical embodiments, the “isolated” nucleotides are concentrated at least about 10-fold, 100-fold, 1,000-fold, 10,000-fold, or more compared to the starting material. If DNA is a polynucleotide, the DNA may be B-DNA, A-DNA, D-DNA, Z-DNA, naked DNA, and cDNA. If RNA is a polynucleotide, RNA can be mRNA, rRNA, 7 SL RNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, YRNA, TERC, SL RNA, aRNA, asRNA, cis-NAT, crRNA, IncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7sK RNA, sRNA, 5S rRNA, 5.8 It can be SrRNA, SSU rRNA, LUS rRNA, NoRC RNA, 6S RNA, SsrS RNA, asmiRNA, crRNA, CRISPR RNA, diRNA, endo-siRNA, exRNA, lincRNA, IncRNA, mrpNRA, nat-siRNA, snRNA, shRNA, circRNA, cfRNA, pre-mRNA, YRNA, or eRNA.

[0045] Similarly, “isolated” polypeptide means a polypeptide that has been at least partially isolated from at least a portion of other naturally occurring components of an organism or virus, such as a cellular or viral structural component, or other polypeptides or nucleic acids commonly found in association with said polypeptide. In typical embodiments, the “isolated” polypeptide is concentrated at least about 10-fold, 100-fold, 1,000-fold, 10,000-fold, or more compared to the starting material.

[0046] "Isolated cells" refers to cells separated from other components that would normally associate with them in their natural state. For example, isolated cells may be cells in a culture medium and / or cells in a pharmaceutically acceptable carrier of the present invention. Thus, isolated cells can be delivered to and / or introduced into a subject. In some embodiments, isolated cells may be cells that are removed from the subject, manipulated ex vivo as described herein, and then returned to the subject.

[0047] As used herein, the term “recombinant” refers to a polypeptide or polynucleotide that can be produced by combining a polynucleotide or polypeptide in a configuration that does not naturally exist and does not normally exist together. The term may also refer to a polypeptide produced via a biological host selected from mammalian, insect cell, yeast, and bacterial expression systems.

[0048] As used herein, “isolate” or “purify” a viral vector or viral particle or aggregate of viral particles (or grammatical equivalent) means that the virus or viral particle or aggregate of viral particles is separated at least partially from at least some of the other components of the starting material. In typical embodiments, the “isolated” or “purified” viral vector or viral particle or aggregate of viral particles is concentrated at least about 10-fold, 100-fold, 1,000-fold, 10,000-fold, or more compared to the starting material.

[0049] A "therapeutic polypeptide" is a polypeptide that reduces, mitigates, prevents, delays, and / or stabilizes symptoms resulting from the absence or deficiency of a protein in a cell or subject, and / or confers benefits to the subject, such as anticancer activity, improved graft survival, or induction of an immune response.

[0050] A “treatment-effective” dose, as used herein, is an amount sufficient to provide some improvement or benefit to the subject. In other words, a “treatment-effective” dose is an amount that provides some reduction, alleviation, decrease, or stabilization of at least one clinical symptom of the subject. Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some benefit is provided to the subject.

[0051] The “effective preventive” amount, as used herein, is an amount sufficient to prevent and / or delay the onset of the disease, disorder, and / or clinical symptoms of the subject, and / or to reduce and / or delay the severity of the onset of the disease, disorder, and / or clinical symptoms of the subject, compared to the disease, disorder, and / or clinical symptoms of the subject that would develop in the absence of the method of the present invention. Those skilled in the art will understand that the level of prevention does not need to be complete, as long as some preventive benefit is provided to the subject.

[0052] The terms “heterogeneous nucleotide sequence” and “heterogeneous nucleic acid molecule” are used interchangeably herein and refer to nucleic acid sequences that do not naturally exist in viruses. Generally, a heterogeneous nucleic acid molecule or heterogeneous nucleotide sequence includes an open reading frame encoding the polypeptide of interest and / or the uncoding RNA of interest (for example, for delivery to cells and / or targets).

[0053] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes AAV1 (AAV-1), AAV2 (AAV-2), AAV3 (AAV-3), AAV4 (AAV-4), AAV5 (AAV-5), AAV6 (AAV-6), AAV7 (AAV-7), AAV8 (AAV-8), AAV9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primate mammals, and "bovine AAV" refers to AAVs that infect bovine mammals.

[0054] As used herein, "rAAV vector" refers to an AAV vector containing a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically the sequence of interest for genetic transformation of cells. Generally, the heterologous polynucleotide is flanked by at least one, and usually two, AAV terminal inversion sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0055] An "AAV virus," "AAV virus particle," or "rAAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein (usually all of the capsid proteins of wild-type AAV) and an encapsulated polynucleotide rAAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is usually called an "rAAV vector particle" or simply an "rAAV vector." Therefore, the production of an rAAV particle necessarily includes the production of an rAAV vector, since the vector is contained within the rAAV particle.

[0056] "Helper virus function" refers to the function encoded in the genome of a helper virus that enables the replication and packaging of AAV (in conjunction with other requirements for replication and packaging as described herein). Where described herein, "Helper virus function" can be provided in many ways, including providing a helper virus or, for example, providing a polypeptide sequence encoding the desired function in trans-producing cells.

[0057] An “infectious” virus or viral particle is a virus or viral particle that contains polynucleotide components that can be delivered into cells to which the viral species is directional. This term does not necessarily imply the ability of the virus to replicate. As used herein, an “infectious” virus or viral particle is a virus or viral particle that can access target cells, infect target cells, and express heterologous nucleic acids in target cells. Thus, “infectivity” refers to the ability of a viral particle to access target cells, infect target cells, and express heterologous nucleic acids in target cells. Infectivity may refer to in vitro or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the relevant art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of copies of the viral genome. The ability of a viral particle to express heterologous nucleic acids in cells may be called “transduction.” The ability of viral particles to express heterologous nucleic acids in cells can be assayed using a number of techniques, including the evaluation of marker genes, such as a green fluorescent protein (GFP) assay in which GFP is produced, detected, and / or measured in cells infected with viral particles (e.g., viruses contain nucleotide sequences encoding GFP), or the measurement of the produced protein by enzyme-linked immunosorbent assay (ELISA).

[0058] A “replicating competent” virus (e.g., replicating competent AVV) refers to a phenotypic wild-type virus that is infectious and capable of replicating in infected cells (i.e., in the presence of a helper virus or the function of a helper virus). In the case of AAV, replication competence generally requires the presence of a functional AAV packaging gene. Generally, the rAAV vectors described herein cannot replicate in mammalian cells (particularly human cells) because they lack one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of replicating competent AAV being produced through recombination between the AAV packaging gene and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain little to no replicating competent AAV (rcAAV, also called RCA), if any (e.g., less than 1 rcAAV / 10). 2 The number of rAAV particles is less than approximately 1 rcAAV / 10 4 The number of rAAV particles is less than approximately 1 rcAAV / 10 8 The number of rAAV particles is less than approximately 1 rcAAV / 10 12 (Individual rAAV particles, or no rcAAV).

[0059] The viral vectors of the present invention may further be “targeted” viral vectors (e.g., having directional directivity) and / or “hybrid” parvoviruses (i.e., the viral TR and viral capsid are derived from different parvoviruses), as described in International Patent Publication No. 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0060] The viral vector of the present invention may further be a double-stranded parvovirus particle, as described in International Patent Publication No. 01 / 92551 (the entire disclosure is incorporated herein by reference). Thus, in some embodiments, a double-stranded (dual) genome may be packaged in the viral capsid of the present invention.

[0061] Furthermore, viral particles or genomic elements may include other modifications, including insertions, deletions, and / or substitutions.

[0062] When used herein, "chimeric" capsid protein means an AAV capsid protein that has been modified by substitution of one or more amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) in the amino acid sequence of the capsid protein compared to the wild type, as well as by insertion and / or deletion of one or more amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) in the amino acid sequence compared to the wild type. In some embodiments, a complete or partial domain, functional region, epitope, etc. from one AAV serotype can be replaced in any combination with the corresponding wild-type domain, functional region, epitope, etc. from different AAV serotypes to produce the chimeric capsid protein of the present invention. Production of chimeric capsid proteins can be carried out according to protocols well known in the art, and numerous chimeric capsid proteins have been described in the literature and herein, which may be included in the capsids of the present invention.

[0063] Where used herein, the term “variant” includes modifications or chemical equivalents of the amino acid and nucleotide sequences disclosed herein that perform substantially the same function as the protein or nucleic acid molecule disclosed herein in substantially the same manner. For example, variants of the proteins disclosed herein include, but are not limited to, conserved amino acid substitutions. Variants of the proteins disclosed herein also include additions and deletions to the proteins disclosed herein. Furthermore, variant peptide and variant nucleotide sequences include their analogues and chemical derivatives.

[0064] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the gene code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at all positions where alanine is specified by the codon, the codon can be changed to any of the corresponding codons listed without altering the coded polypeptide. Such nucleic acid variations are "silent variations" and are a type of conservatively modified variation. Furthermore, all nucleic acid sequences in this specification that code for polypeptides describe all possible silent variations of nucleic acids. Those skilled in the art will recognize that by modifying each codon of a nucleic acid (except for AUG, usually the sole codon for methionine, and TGG, usually the sole codon for tryptophan), a functionally identical molecule can be obtained. Therefore, each silent variation of the nucleic acid encoding the polypeptide is implied in the sequences described for the expression product, rather than in the actual probe sequences.

[0065] The protein encoded by the BMI1 gene may have amino acid additions, deletions, or substitutions. The modified amino acid sequence is different from the native amino acid sequence due to the deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues. In one embodiment, the modification is a point mutation. In one embodiment, the modified therapeutic peptide does not have a naturally occurring sequence.

[0066] Amino acid substitutions can be conserved or non-conservative. “Conservative amino acid substitution,” as used herein, is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. A family of amino acid residues having similar side chains is defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The most commonly observed exchanges are bidirectional: Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly. Generally, amino acid exchanges in proteins and peptides that do not alter the activity of the protein or peptide are well known in the field (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979).

[0067] The term "protein derivative" refers to a protein having one or more residues that have been chemically derivatized by the reaction of functional side chain groups. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, or formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters, or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-imu-benzylhistidine. Peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids are also included as derivatives. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0068] In one embodiment, the modified therapeutic protein disclosed herein may have any amino acid substitutions, deletions, or deletions in the amino acid sequence of the protein. In another embodiment, the modified therapeutic protein disclosed herein may have 1 to 13 amino acid additions, deletions, or substitutions. In one embodiment, the therapeutic protein has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acid additions, substitutions, or deletions. The substitutions may be conserved or non-conserved. In another embodiment, the therapeutic protein may have up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid additions, substitutions, or deletions. In yet another embodiment, the therapeutic protein is 1-13, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-4, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-4, 3-13, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-10, 4-9, 4 It may have ~8, 4~7, 4~6, 4~5, 5~12, 5~10, 5~9, 5~8, 5~7, 5~6, 5~5, 6~12, 6~10, 6~9, 6~8, 6~7, 7~13, 7~12, 7~10, 7~9, 7~8, 8~13, 8~12, 8~10, 8~9, 9~13, 9~12, 9~10, 10~12, 11~13, 11~12, or 12~13 amino acid additions, substitutions, or deletions.

[0069] For sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, the coordinates of the subsequences are specified as needed, and the parameters of the sequence algorithm program are specified. Preferably, default program parameters can be used, or different parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence compared to the reference sequence based on the program parameters.

[0070] Where used herein, "comparison window" refers to any one segment of a number of consecutive positions in a reference sequence, selected from the group consisting of 20 to full-length, typically about 25 to 100, or 50 to about 150, more typically about 100 to 150, in which the sequence can be compared to the same number of consecutive positions in the reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved by, for example, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0071] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST2.0, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST2.0 are used with the parameters described herein to determine the percent sequence identity of nucleic acids and proteins of the present invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with words of the same length in a database sequence. T is called the neighbor word score threshold (see Altschul et al., above). These initial adjacent word hits act as seeds to initiate the search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated using parameters M (reward score for pairs of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0) for nucleotide sequences. For amino acid sequences, the cumulative score is calculated using a score matrix. Extension of word hits in each direction stops if the cumulative alignment score falls by amount X from the maximum value achieved; if the cumulative score becomes 0 or less due to the accumulation of alignments of one or more negative scoring residues; or if the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses 11 word length (W) and 10 expected value (E), M=5, N=-4, and a comparison of both strands as defaults.For amino acid sequences, the BLASTP program uses a word length of 3, an expected value of 10 (E), and a BLOSUM62 score matrix of 50 (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment (B), an expected value of 10 (E), M=5, N=-4, and a comparison of both strands by default.

[0072] The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may also include non-nucleotide components. Where present, modifications to the nucleotide structure may be provided before or after the assembly of the polymer. As used herein, the term polynucleotide is interchangeable between double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both a double-stranded form and two complementary single-stranded forms that are known or expected to constitute the double-stranded form.

[0073] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein after transcription or translation.

[0074] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, and other techniques that result in a construct different from naturally occurring polynucleotides. A recombinant virus is a viral particle containing recombinant polynucleotides. These terms include, respectively, a replica of the original polynucleotide construct and a progeny of the original viral construct.

[0075] The term “regulatory element” or “regulatory sequence” refers to a nucleotide sequence involved in molecular interactions that contribute to the functional control of polynucleotides, including replication, duplication, transcription, splicing, translation, or degradation. This control can affect the frequency, rate, or specificity of a process and may be inherently enhancing or inhibiting. Examples of regulatory elements known in this field include transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that can bind to RNA polymerase under certain conditions and initiate transcription of a coding region typically located downstream (3' direction) of the promoter.

[0076] The terms "operatively ligated" or "operably ligated" refer to the juxtaposition of gene elements in a relationship that allows the elements to operate in a predictable manner. For example, a promoter is operatively ligated to a codon region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the codon region, as long as this functional relationship is maintained.

[0077] The term "expression vector" refers to a vector containing a region that codes for a target polypeptide and is used to influence protein expression in intended target cells. Expression vectors also include regulatory elements operably ligated to the coding region to facilitate protein expression in the target. The combination of regulatory elements and the gene(s) operably ligated for expression is sometimes called an "expression cassette," many of which are publicly known and available in the field, or can be readily constructed from components available in the field.

[0078] "Heterogeneous" means that the entity originates from an entity whose genotype differs from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector from a different species through genetic engineering is a heterogeneous polynucleotide. A promoter that has been removed from its natural coding sequence and operably ligated with a coding sequence not found ligated in nature is a heterogeneous promoter. Therefore, for example, an rAAV containing heterogeneous nucleic acids encoding a heterogeneous gene product is an rAAV containing nucleic acids not normally found in naturally occurring wild-type AAV, and the encoded heterogeneous gene product is a gene product not normally encoded by naturally occurring wild-type AAV.

[0079] As used herein, the term “homologous recombination” refers to a type of genetic modification in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also results in novel recombinations of DNA sequences. These novel combinations of DNA represent variations in genes. Homologous recombination is also used for horizontal gene transfer to exchange genetic material between different strains or species of viruses.

[0080] The terms “genetic alteration” and “genetic modification” (and their grammatical variants) refer to the process by which a genetic element (e.g., polynucleotide) is introduced into a cell outside of mitosis or meiosis. This element may be heterologous to the cell, or it may be a further copy or improved version of an element already present in the cell. Genetic alteration can be achieved by transfecting a cell with recombinant plasmids or other polynucleotides via any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with polynucleotide-liposome complexes. Genetic alteration can also be achieved by transduction or infection with DNA or RNA viruses or viral vectors, for example. Generally, genetic elements are introduced into the cell’s chromosomes or minichromosomes, but any alteration that alters the phenotype and / or genotype of the cell and its offspring is included in this term.

[0081] Certain nucleic acid sequences implicitly include “splice variants.” Similarly, certain proteins encoded by nucleic acids implicitly include any proteins encoded by splice variants of that nucleic acid. “Splice variants,” as the name suggests, are the products of alternative splicing of genes. After transcription, the initial nucleic acid transcript may be spliced ​​so that different (alternative) nucleic acid splice products encode different polypeptides. The mechanisms for splice variant production vary but include alternative splicing of exons. Another nucleic acid derived from the same nucleic acid by read-through transcription is also included by this definition. All products of splicing reactions, including recombinant splice products, are included in this definition. An example of a potassium channel splice variant is discussed in Leicher et al., J. Biol. Chem. 273(52):35095-35101 (1998).

[0082] As used herein, the terms “gene editing,” “genome editing,” or “genomic manipulation” refer to a type of genetic manipulation in which DNA is inserted, deleted, or replaced in the genome of an organism using a manipulated nuclease or “molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.

[0083] As used herein, the term “gene delivery” means the process by which foreign DNA is introduced into host cells for the application of gene therapy.

[0084] Cells are said to be "stable" altered, transduced, genetically modified, or transformed by their gene sequence if the sequence is available to perform its function during long-term cell culture in vitro. Generally, such cells are "genetically" altered (genetically modified) in the sense that the genetic alteration is introduced which is similarly inherited by the offspring of the altered cell.

[0085] "Isolated" plasmids, nucleic acids, vectors, viruses, virions, host cells, or other substances refer to preparations of a substance that lack at least some of other components that would similarly be present if the substance or a similar substance were naturally occurring or initially prepared from it. Thus, for example, an isolated substance may be prepared by using purification techniques to concentrate it from a mixture of sources. Concentration can be measured in absolute terms, such as weight per volume of solution, or it can be measured in relation to a second potentially interfering substance present in the mixture of sources. Increasing the concentrate of embodiments of the present invention leads to increasing isolation. Isolated plasmids, nucleic acids, vectors, viruses, host cells, or other substances are purified in some embodiments to be, for example, about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% or more pure.

[0086] The term "retinal pigment epithelium," "RPE," or "Müller" cells refers to a single layer of epithelial cells lining the inner surface of the posterior segment of the eye. This layer is located between the photoreceptor cells that detect light and the choroid capillaries. Like other epithelial cell types, RPE cells are polarized.

[0087] As used herein, the terms “treatment,” “treating,” etc., refer to obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses all treatments of diseases in mammals, particularly humans, and includes (a) preventing the onset of a disease in subjects who may be predisposed to or at risk of acquiring the disease but have not yet been diagnosed; (b) inhibiting the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing a regression of the disease.

[0088] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to humans and non-primates, including monkeys and humans; mammals used for sport (e.g., horses); mammals used for domestication (e.g., sheep, goats, etc.); mammals used for pets (e.g., dogs, cats, etc.); and mammals, including rodents (e.g., mice, rats, etc.).

[0089] As used herein, “gene replacement therapy” refers to the administration of exogenous genetic material encoding a therapeutic agent to a recipient and the subsequent in situ expression of the administered genetic material. This includes injection into the vitreous cavity of the eye. Thus, “conditions suitable for gene replacement therapy” encompass conditions such as hereditary diseases (i.e., disease conditions resulting from the deletion of one or more genes), acquired conditions (i.e., conditions not resulting from congenital defects), cancer, and prophylactic processes (i.e., prevention of disease or undesirable medical conditions). Thus, as used herein, the term “therapeutic agent” refers to any agent or material having a useful effect on a mammalian recipient. Thus, “therapeutic agent” encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0090] The term "ailment" refers to a disease, illness, or medical condition. An ailment could be a disease of the eye, such as retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy.

[0091] Many hereditary eye diseases are known. Furthermore, effective therapeutic agents for these diseases are also known, as they are proteins / enzymes that are known to be deficient in these diseases. In specific embodiments, the disease or condition is retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy.

[0092] The terms "BMIG," "Bmi1," "Bmi-1," or "BMI-1 gene" refer to the "Mo-MLV insertion region 1 homolog of B lymphoma." Studies suggest that BMI1 overexpression plays an important role in several types of cancer.

[0093] The terms "Polycomb complex protein BMI1," "Polycomb group ring finger protein 4," "PCGF4," "Ring finger protein 51," or "RNF51" refer to proteins encoded by the Bmi1 gene in humans (Moloney mouse leukemia virus integration site 1, specific to B cells). Polycomb group (PcG) proteins are proteins that modify chromatin, which plays a crucial role in development. They also regulate cell proliferation, aging, and tumorigenesis. Polycomb group (PcG) protein BMI1 is an important regulator of cell proliferation.

[0094] The terms "Mel-18," "Polycomb group RING finger 2," or "PCGF2" refer to closely related PcG proteins. The Mel-18 gene product is structurally similar to Bmi1. Studies suggest that Bmi1 and Mel-18 control a unique, overlapping set of genes.

[0095] The terms “substantial homology” or “substantial similarity,” when referring to a nucleic acid or a fragment thereof, mean that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, the aligned sequence has nucleotide sequence identity in at least about 95–99% of the aligned sequence. Preferably, the homology extends over a full-length sequence, its open reading frame, or another suitable fragment of at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0096] The terms “identical” or “percent “identical” refer to two or more nucleic acid or polypeptide sequences that, in terms of two or more nucleic acid or polypeptide sequences, are identical or have a specific percentage of identical amino acid residues or nucleotides (i.e., approximately 60% identity across a specific region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity when compared and aligned to maximize correspondence across a comparison window or specified region) when measured using the BLAST or BLAST2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection. Thus, such sequences are said to be “substantially identical.” This definition may also be applied to complements of test sequences. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Preferred algorithms may take gaps, etc., as described below. Preferably, the identity exists over a region of at least about 25 amino acids or 25 nucleotides, or more preferably, over a region of 50 to 100 amino acids or 50 to 100 nucleotides.

[0097] In the context of nucleic acid sequences, the terms “sequence identity,” “percent identity,” or “percent identical” refer to residues in two sequences that are identical when aligned to their maximum corresponding extent. The length over which sequence identity is compared may preferably span a full-length genome, a full-length genetic coding sequence, or a fragment of at least 500–5000 nucleotides. However, identity between smaller fragments, e.g., at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, and at least about 36 or more nucleotides, may also be desirable. Similarly, “percent sequence identity” can be readily determined with respect to amino acid sequences across a full-length protein or a fragment thereof. Appropriately, a fragment may be at least about 8 amino acids long and up to about 700 amino acids. Examples of suitable fragments are described herein.

[0098] The terms “substantial homology” or “substantial similarity,” when referring to an amino acid or a fragment thereof, mean that, with appropriate amino acid insertions or deletions, when optimally aligned with another amino acid (or its complementary chain), the aligned sequence has sequence identity in at least about 95–99% of the aligned sequence. Preferably, the homology extends over the full-length sequence or its protein, e.g., a cap protein, a rep protein, or a fragment thereof that is at least 8 amino acids long, or more preferably at least 15 amino acids long. Examples of suitable fragments are described herein.

[0099] The term "highly preserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art by relying on algorithms and computer programs known to those skilled in the art.

[0100] In general, when referring to “identity,” “homology,” or “similarity” between two different adeno-associated viruses, “identity,” “homology,” or “similarity” is determined by referring to “aligned” sequences. An “aligned” sequence, or “alignment,” often refers to multiple nucleic acid sequences or protein (amino acid) sequences that include deletions or additional bases or amino acid complements compared to a reference sequence. For example, AAV alignment is performed using a publicly available AAV2 or AAV1 sequence as a reference point. However, those skilled in the art can easily select a different AAV sequence as the reference. Alignment is performed using one of a variety of publicly available or commercially available Multiple Sequence Alignment Programs. Examples of such programs include “Clustal W,” “CAP Sequence Assembly,” “MAP,” and “MEME,” which are available via web servers on the Internet.

[0101] The term "serotype" refers to differences in AAVs that have serologically distinct capsids from other AAV serotypes. Serological differences are determined based on the absence of cross-reactivity between antibodies against AAVs when compared to other AAVs.

[0102] The term “exogenous genetic material” refers to natural or synthetic nucleic acids or oligonucleotides that are not found naturally in cells; or, if found naturally in cells, are not transcribed or expressed at a biologically significant level by those cells. Thus, “exogenous genetic material” includes, for example, nucleic acids that do not exist naturally and can be transcribed into antisense RNA, and “heterogenes” (i.e., genes that encode proteins that are not expressed or are not expressed at a biologically significant level in naturally occurring cells of the same species).

[0103] Alternatively, an amino acid may be a modified amino acid residue, and / or an amino acid modified by post-translational modifications (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). Amino acids that do not exist in nature may be “unnatural” amino acids that can be used to chemically link the desired molecule to the AAV capsid protein or other types of viral vectors.

[0104] As used herein, the term “homologous recombination” means a type of genetic modification in which the nucleotide sequences of two similar or identical molecules, which are DNA, are exchanged. Homologous recombination also results in novel combinations of DNA sequences that may represent genetic variations. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.

[0105] As used herein, the terms “gene editing,” “genome editing,” or “genomic manipulation” refer to a type of genetic manipulation in which DNA is inserted, deleted, or replaced in the genome of an organism using a manipulated nuclease or “molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.

[0106] As used herein, the term “gene delivery” means the process by which foreign DNA is introduced into host cells for the application of gene therapy. As used herein, the term “CRISPR” means Clustered Regularly Interspaced Short Palindromic Repeats, a feature of the bacterial defense system that forms the basis of CRISPR-Cas9 genome editing technology.

[0107] In some embodiments, the AAV particles of the present invention may be synthetic viral vectors designed to exhibit a range of desirable phenotypes suitable for different in vivo and in vivo applications. Thus, in one embodiment, the present invention provides AAV particles comprising adeno-associated virus (AAV).

[0108] In certain embodiments, the mammalian recipient has a condition suitable for gene replacement therapy. As used herein, “gene replacement therapy” refers to the administration of exogenous genetic material encoding a therapeutic agent to the recipient and the subsequent in situ expression of the administered genetic material. Thus, the phrase “condition suitable for gene replacement therapy” encompasses conditions such as hereditary diseases (i.e., disease conditions resulting from the deletion of one or more genes), acquired conditions (i.e., conditions not resulting from congenital deletions), cancer, and prophylactic processes (i.e., prevention of disease or undesirable medical conditions). Thus, as used herein, the term “therapeutic agent” refers to any agent or material having a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” encompasses both therapeutic and prophylactic molecules having nucleic acid (e.g., antisense RNA) and / or protein components. [Brief explanation of the drawing]

[0109] [Figure 1A-C] Figures 1A-C show photographs of a healthy retina in a young person (Figure 1A), a photograph of an eye with moderate atrophic macular degeneration (Figure 1B), and a photograph of advanced atrophic macular degeneration with subfoveal geographic atrophy (Figure 1C). [Figure 2A-C] Figures 2A-2C show photographs of normal retinal structures (Figure 2A), images of normal retinal structures from OCT (optical coherence tomography) retinal imaging (Figure 2C), and stained cross-sections showing normal retinal structures (Figure 2C), respectively, all of which exhibit retinal structures free from toxicity and histopathology. [Figure 3A] Figure 3A shows mRNA expression in different regions of a human eye derived from donor eye tissue. [Figure 3B]Figure 3B shows protein expression in specific regions of the retina and RPE-choroidal layer of a human donor eye. [Figure 3C] Figure 3C shows different regions of the eye, each identified by a specific number. [Figure 4A] Figure 4A shows mRNA expression from the cornea, lens, iris, vitreous humor, retina, and RPE-choroid of human donor eyes based on qRT-PCR performed for the human BMI1 gene, with human GAPDH gene expression used as a control. Values ​​are expressed as mean ± SD. Values ​​are normalized to corneal expression of BMI1 in human eyes. [Figure 4B] Figure 4B shows protein expression using indirect ELISA for proteins extracted from the cornea, lens, iris, vitreous humor, retina, and RPE of human donor eyes. ELISA was performed using a commercially available anti-rabbit antibody against BMI1. Human recombinant BMI1 was used for the standard curve. Values ​​are expressed as mean ± SD. [Figure 5A] Figure 5A shows total mRNA extracted from the cornea, lens, vitreous humor, retina, and RPE-choroid of pig eyes by qRT-PCR using primers for porcine BMI1. GAPDH was used as a control gene. Values ​​are expressed as mean ± SD. Values ​​are normalized to corneal expression of BMI1 in pig eyes. [Figure 5B] Figure 5B shows Western blot analysis of BMI1 protein extracted from the cornea, lens, vitreous humor, retina, and RPE-choroid of a pig eye. A rabbit antibody against BMI1 was used to detect BMI1, and β-actin was used as a loading control. The numbers refer to the KDa of the standard substance. [Figure 5C] Figure 5C shows the results of indirect ELISA performed using proteins extracted from the cornea, lens, iris, vitreous humor, retina, and RPE of pig eyes. ELISA was performed using a commercially available anti-rabbit antibody against BMI1. Human recombinant BMI1 was used for the standard curve. Values ​​are expressed as mean ± SD. [Figure 6A]Figure 6A shows representative images of the retina obtained from young and aged C57BL6J mice using H&E staining. Different layers are shown on the right side of the image. [Figure 6B] Figure 6B shows the quantitative analysis of retinal thickness and ONL thickness from the H&E images shown in Figure 6A. Values ​​are expressed as mean ± SD. ***P-value < 0.001, ****P-value < 0.0001 (one-way ANOVA). [Figure 6C] Figure 6C shows total BMI1 mRNA extracted from the cornea, lens, vitreous humor, retina, and RPE of a pig eye, after qRT-PCR using primers for the porcine BMI1 gene. GAPDH was used as a control gene. Values ​​are expressed as mean ± SD. Values ​​are normalized to corneal expression of human BMI1. *P value < 0.05 (one-way ANOVA) [Figure 6D] Figure 6D shows Western blot analysis of proteins extracted from the cornea, lens, vitreous humor, retina, and RPE-choroid of a pig eye. BMI1 anti-rabbit antibody was used to detect the BMI1 protein, and β-actin was used as a loading control. The numbers refer to the KDa of the standard substance. [Figure 6E] Figure 6E shows the quantification of bands from the Western blot derived from Figure 6D by concentration measurement, where the BMI1 protein level values ​​are normalized to the β-actin control. The values ​​represent the sum of retina and RPE and are expressed as mean ± SD. **P value < 0.01 (one-way ANOVA). [Figure 7A] Figure 7A shows the amount of total mRNA extracted from the retina of control mice and mice transduced with AAV5.BMI1, after qRT-PCR using primers for the mouse BMI1 gene. GAPDH was used as the control gene. Values ​​are expressed as mean ± SD. Values ​​are normalized to the control level of BMI1 in control mice. *P value < 0.5 **P value < 0.01, ****P value < 0.0001 (one-way ANOVA). [Figure 7B]Figure 7B shows the total mRNA levels after qRT-PCR performed using primers for the mouse BMI1 gene, extracted from the RPE-choroid of the eyes of control mice and mice transduced with AAV5.BMI1. GAPDH was used as the control gene. Values ​​are expressed as mean ± SD. Values ​​are normalized to the control level of BMI1 in control mice. **P value < 0.01 (one-way ANOVA). [Figure 7C] Figure 7C shows the results from indirect ELISA performed using proteins extracted from the RPE-choroid of the eyes of control mice and mice transduced with AAV5.BMI1. ELISA was performed using a commercially available anti-rabbit antibody against BMI1. Human recombinant BMI1 was used for the standard curve. Values ​​are expressed as mean ± SD. *P-value < 0.05 (Student's t-test). [Figures 7D-7F] Figures 7D-7F show representative images of eye sections obtained from control mice (Figure 1C) and mice transduced with AAV5.BMI1 (1 × 10⁹ (Figure 7D) and 1 × 10¹⁰ vg / eye (Figure 7E)) after H&E staining. [Figure 8A] Figure 8A shows the relative expression levels of BMI1 mRNA in control retinas treated with NaLO3-saline for 4 weeks, and in retinas treated with NaLO3 and transduced with AAV5.BMI1. mRNA was amplified using qRT-PCR with primers for the mouse BMI1 gene. GAPDH was used as a control. Values ​​are expressed as mean ± SD. Values ​​are normalized to the control level of BMI1 levels in eyes treated with NaLO3-saline. **P-value < 0.01 (independent t-test). [Figures 8B-8D] Figures 8B–8D show the results of indirect immunofluorescence using rhodopsin-stained eye sections obtained from eyes that did not receive injection (Figure 8B), eyes administered with NaLO3-physiological saline (Figure 8C), and eyes administered with NaLO3-AA5.BMI1 (Figure 8D). The nuclei are stained with DAPI. [Figure 8E-8G]Figures 8E-8G show representative images of H&E-stained eye sections obtained from the control group (Figure 8E), the group treated with NaLO3-physiological saline (Figure 8F), and the group treated with NaLO3-AA5.BMI1 (Figure 8G). [Figure 8H] Figure 8H shows the quantified retinal thickness (upper graph) and ONL (lower graph) thickness measurements using the H&E images shown in Figures 8E-8G. Values ​​are expressed as mean ± SD. *P-value < 0.05, ****P-value < 0.0001 (one-way ANOVA). [Figure 9] Figure 9 shows the results of quantified ONL thickness measurements using OCT scans in control mice, NaLO3 mice injected with physiological saline, and NaLO3 mice injected with AAV5.BMI1. Values ​​are expressed as mean ± SD. *P-value < 0.05, **P-value < 0.01, ****P-value < 0.0001 (one-way ANOVA). [Figure 10] Figure 10 shows the results of quantified ONL thickness measurements using OCT scans in control mice, LIR mice injected with physiological saline, and LIR mice injected with AAV5.BMI1. Values ​​are expressed as mean ± SD. *P-value < 0.05, **P-value < 0.01, ****P-value < 0.0001 (one-way ANOVA). [Figure 11] Figure 11 shows the results after extraction of total mRNA from the retina of human donor eyes treated with AAVrh10.BMI1 for 5 days. mRNA was subjected to qRT-PCR using primers for the human BMI1 gene. Human GAPDH was used as a control. Values ​​are expressed as mean ± SD. Values ​​are normalized to retinal expression of BMI1 without AAV transduction. [Figure 12A] Figure 12A shows that ARPE-19 cells with overexpressed BMI1 significantly increased cell viability upon NlO3 exposure compared to shRNA-mediated knockdown of BMI1 expression (P<0.001). Values ​​refer to the mean ± SD. [Figure 12B]Figure 12B shows that LDH levels, a measure of cytotoxicity, are significantly higher in BMI1 shRNA cells compared to cells treated with BMI1 overexpression. The values ​​represent the mean + standard deviation. [Figure 13A] Figure 13A is a graph showing the quantitative changes in mRNA expression of the Bcl2 and Bax genes, which are involved in cellular apoptosis, via qRT-PCR analysis after transduction with AAV5.BMI1 (Bmi1) in ARPE-19 cells. The values ​​are normalized to the mRNA levels of ARPE-19 cells without transduction (control). *P-value < 0.05, **P-value < 0.01 (independent t-test). [Figure 13B] Figure 13B is a quantitative graph of changes in mRNA expression of Gpx1, Gpx3, Sod1, and Sod2, which are involved in cellular resistance to oxidative stress, as analyzed by qRT-PCR after transduction with AAV5.BMI1 (Bmi1) in ARPE-19 cells. Values ​​are normalized to mRNA levels in ARPE-19 cells without transduction (control). *P-value < 0.05, **P-value < 0.01 (independent t-test). [Figure 13C] Figure 13C is a quantitative graph of changes in mRNA expression of P21 and p53, which are involved in cellular senescence in response to oxidative stress, as analyzed by qRT-PCR after transduction with AAV5.BMI1 (Bmi1) in ARPE-19 cells. The values ​​are normalized to mRNA levels in ARPE-19 cells without transduction (control). *P value < 0.05 (independent t-test). [Figure 13D] Figure 13D is a quantitative graph of changes in vegfa mRNA expression, which is involved in the inflammatory response, as analyzed by qRT-PCR after transduction with AAV5.BMI1 (Bmi1) in ARPE-19 cells. The values ​​are normalized to the mRNA levels of ARPE-19 cells without transduction (control). *P-value < 0.05 (independent t-test). [Figure 14A]Figure 14A is a quantitative graph of changes in mRNA expression of Gpx1, Sod1, and Sod2, which are involved in cellular resistance to oxidative stress, via qRT-PCR analysis after subretinal injection of AAV5.BMI1 (Bmi1) into Balb / c mice. Values ​​are normalized to mRNA levels of ARPE-19 cells without transduction (control). *P-value < 0.05 (independent t-test). [Figure 14B] Figure 14B is a quantitative graph of P21 and p53 mRNA expression, which are involved in cellular senescence in response to oxidative stress, via qRT-PCR analysis after subretinal injection of AAV5.BMI1 (Bmi1) into Balb / c mice. Values ​​are normalized to mRNA levels in untransduced ARPE-19 cells (control). *P-value < 0.05 (independent t-test). [Figure 14C] Figure 14C is a quantitative graph of changes in Vegfa mRNA expression, which is involved in the inflammatory response of RPE cells, as analyzed by qRT-PCR after subretinal injection of AAV5.BMI1 (Bmi1) into Balb / c mice. Values ​​are normalized to mRNA levels of ARPE-19 cells without transduction (control). *P-value < 0.05 (independent t-test). [Modes for carrying out the invention]

[0110] The specific configurations discussed in the following description are non-limiting examples that may vary and are cited merely to illustrate at least one embodiment, and are not intended to limit their scope.

[0111] The present invention provides a method for treating diseases such as eye / ocular diseases in mammals by administering a viral vector described herein to the mammal. In certain embodiments, the mammal is a human. In certain embodiments, the disease is retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy.

[0112] Polycomb group proteins form large macromolecular complexes that silence specific target genes by modifying chromatin structure. The Polycomb group protein BMI1 is a component of the Polycomb repressive complex 1 (PCR1), which promotes chromatin condensation and gene repression through its monoubiquitin ligase activity at lysine 119 on histone H2A. In one embodiment of the present invention, the BMI1 protein is transduced into the eye cells of patients suffering from diseases (such as retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy). The applicants have found that the expression of the BMI1 protein in retinal pigment epithelial (RPE) cells can reduce retinopathy. Specifically, this expression can prevent, arrest, and / or improve the signs and symptoms of retinal degeneration and / or macular degeneration. In some cases, vision loss associated with retinopathy can be prevented or restored.

[0113] The BMI1 protein includes either the native or naturally occurring BMI1 protein and any derivatives or variants of the BMI1 protein. This includes BMI1 proteins with amino acid additions, deletions, and substitutions. It also includes BMI1 proteins with one or more nucleotide additions, deletions, and substitutions in the nucleic acid sequence encoding the BMI1 protein.

[0114] Embodiments include the introduction and expression of the BMI1 protein in retinal pigment epithelial (RPE) cells. Transduction can be achieved by vectors such as adeno-associated virus (AAV) vectors, adenovirus vectors, retroviruses, or lentiviral vectors based on human immunodeficiency virus or feline immunodeficiency virus. Examples of such AAVs can be found, for example, in Davidson et al., PNAS (2000) 97:3428-3432. AAV and lentiviruses can confer persistent expression, while adenoviruses can provide transient expression.

[0115] An expression vector may include a promoter for controlling the transcription of a heterologous gene. This promoter may be an inducible or constitutive promoter. The expression system is suitable for administration to a mammalian recipient. The expression system may include a plurality of non-immortalized genetically modified cells, where each cell contains at least one recombinant gene encoding at least one therapeutic agent.

[0116] A cell expression system can be formed in vivo. According to another aspect, a method for treating a mammalian recipient in vivo is provided. The method includes, for example, introducing in situ into the patient's cells an expression vector for expressing a heterologous gene product, via intravitreal administration. To form an expression system in vivo, an expression vector for expressing a therapeutic agent is introduced into a mammalian recipient in vivo.

[0117] AAV vector Adeno-associated virus (AAV) is a small non-pathogenic virus (20 nm) that may be useful for treating human diseases. A construct is made around a promoter linked to a target gene having an AAV inverted terminal repeat (ITR) sequence.

[0118] In one embodiment, the viral vector of this disclosure is an AAV vector. The term “AAV” vector represents an adeno-associated virus and may be used to refer to the naturally occurring wild-type virus itself or its derivatives. The term encompasses all subtypes, serotypes, and pseudotypes, as well as both naturally occurring and recombinant forms, unless otherwise required. As used herein, the term “serotype” refers to an AAV that is identified by the reactivity of a specified antiserum and capsid protein and distinguished from other AAVs, for example, primate AAVs have many known serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRhl0, as well as modified capsids of these serotypes). The abbreviation “rAAV” refers to a recombinant adeno-associated virus, also called a recombinant AAV vector (or “rAAV vector”).

[0119] An "AAV virus" or "AAV virus particle" refers to a viral particle consisting of at least one AAV capsid protein (preferably all of the wild-type capsid proteins) and an encapsulated polynucleotide. If the particle is a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is usually called "rAAV".

[0120] In one embodiment, the AAV expression vector is constructed using known techniques to provide at least a regulatory element containing a transcription start region, the DNA of interest, and a transcription termination region as components operably linked in the direction of transcription. The regulatory element is selected to be functional in mammalian cells. The resulting construct, including the operably linked components, is facilitated (at 5' and 3') with a functional AAV ITR sequence.

[0121] An "adeno-associated virus terminal inversion sequence" or "AAV ITR" refers to a region recognized in the field found at each end of the AAV genome that functions together in cis as the DNA replication origin and as a packaging signal for the virus. AAV ITRs, along with AAV rep coding regions, provide efficient excision and rescue of nucleotide sequences inserted between two adjacent ITRs, as well as integration into the mammalian cell genome.

[0122] Other vectors The present invention also includes other non-AAV vectors for use in treating eye diseases with the BMI1 protein. These include retroviruses, parvoviruses, lentiviruses, adenoviruses, and herpes simplex viruses. These non-AAV vectors enable the introduction of BMI1-like genes into cells, respectively, either through the insertion of the gene's nucleic acid into the host cell genome or through episomal expression of nucleic acid molecules such as plasmids that are not integrated into the host cell genome. With respect to each of the non-AAV vectors identified below, it should be understood that those skilled in the art know how to construct vectors for the expression of BMI1-like genes in host cells.

[0123] Parvovirus vector The non-AAV virus vectors that can be used in the present invention are parvovirus vectors. Parvovirus vectors are animal viruses with relatively small DNA containing a linear, single-stranded DNA genome. Parvoviruses include self-replicating parvoviruses and dependviruses. Self-replicating parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Coronavirus. Examples of self-replicating parvoviruses, but not limited to, include mouse microvirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other self-replicating parvoviruses are known to those skilled in the art.

[0124] The parvovirus vector of the present invention may be a “hybrid” parvovirus. The parvovirus may have a “chimeric capsid” containing sequences derived from different parvoviruses or other viruses or a “targeted” capsid (e.g., directionally oriented). The parvovirus may also have a polyploid capsid (different capsid proteins derived from different serotypes of parvoviruses or even different viruses). The genome of a double-stranded parvovirus vector may contain sufficient packaging sequences for capsid formation within a selected parvovirus capsid.

[0125] Adenovirus vector Another non-AAV viral vector is the adenovirus vector. Adenovirus vectors are effective in transduction of many mammalian cell types, but not necessarily all. Furthermore, adenovirus vectors have the advantage that the vector DNA remains in the episome without being integrated into the host genome.

[0126] lentiviral vectors Another non-AAV viral vector is the lentivirus, a type of retrovirus that inserts its genetic material into dividing and non-dividing cells, resulting in the integration of the genetic material into the host genome and enabling continuous expression. Lentiviral vectors have the ability to infect non-dividing cells and insert their genetic material into the host cell's genome. Lentiviral vectors are used to treat diseases such as diabetes mellitus, mouse hemophilia A, prostate cancer, chronic granulomatous disease, rheumatoid arthritis, and vascular diseases.

[0127] Listeria monocytogenes Listeria monocytogenes is an intracellular bacterium that can be used to deliver protein-coding genes into cells.

[0128] Poxvirus vector A further non-AAV virus vector of the present invention is a poxvirus vector. Examples of poxvirus vectors include vaccinia virus (VACV) and myxomavirus (MYXV), both of which can be used as viral vectors for the transfer of genes into cells. Vaccinia virus (VV) is known to transduce a wide range of cells, resulting in transient expression. Poxvirus vectors have a substantial genome that can be largely replaced and allow for the insertion of large DNA fragments up to 25 kb in size. Other poxvirus vectors, such as modified vaccinia ankara (MVA) or fowlpox virus, do not fully replicate in mammalian cells but can similarly be used to transduce cells.

[0129] Herpes simplex virus Another non-AAV virus vector is the herpes simplex virus vector, or HSV vector. These have a 150kb double-stranded linear genome. HSV exhibits directionality to a wide variety of cell types and is highly infectious to both dividing and non-dividing cells. HSV expresses more than 80 different genes, many of which are not essential for its replication cycle. Therefore, HSV has the potential to carry a substantial payload, allowing for the insertion of multiple or very many transgenes into the highly deficient vectors described below. The HSV genome in latent is not integrated into the cell's DNA but maintains an episomal structure as a closed circular molecule, thus avoiding the risk of insertional mutations.

[0130] Nonviral / nonbacterial gene delivery methods Nonviral / nonbacterial gene delivery methods use synthetic or natural compounds or physical forces to deliver DNA or RNA fragments to cells. Cell or tissue specificity with these methods can be achieved by utilizing cell-specific functionality in their design. Methods include needle and jet injection for gene delivery to cells. Jet injection includes the use of gene guns, also known as ballistic DNA delivery or irradiation of DNA-coated particles. Another method for gene delivery to cells is sonoporation, which uses ultrasound to create defects in the cell membrane through acoustic cavitation. Sonoporation can be performed in combination with contrast agents or microbubbles.

[0131] Furthermore, gene transfer into cells can be achieved using cationic lipids. Cationic lipids have a structure consisting of a positively charged hydrophilic head and a hydrophobic tail linked via a linker structure. The most commonly found hydrophilic head groups are primary, secondary, tertiary amines, or quaternary ammonium salts. However, guanidino, imidazole, pyridinium, phosphorus, and arsenic groups have also been developed. Cationic polymers can also be used for gene transfer into cells. After mixing with DNA or RNA, these polymers often form nano-sized complexes called polyplexes. Polyplexes are generally more stable than lipoplexes.

[0132] Gene transfer can also be performed using inorganic nanoparticles typically prepared from metals (e.g., iron, gold, silver), inorganic salts, or ceramics (e.g., phosphates or carbonates of calcium, magnesium, or silicon). Metal ion-based salts typically result in complexes with sizes in the range of 10 to 100 nm in diameter. The surface of these nanoparticles can be coated to facilitate DNA binding or targeted gene delivery. This small particle size offers several advantages, including typically bypassing most physiological and cellular barriers and resulting in high gene expression. Nanoparticles have the ability to efficiently transfect post-mitotic cells. Furthermore, they tend to be non-toxic or exhibit low toxicity and are inert to the immune response. Superparamagnetic iron oxide-based nanoparticles can also provide a magnetic response to a magnetic field and enable magnetic field-induced targeted DNA delivery.

[0133] Method for introducing genetic material into cells The present invention also provides a method for delivering an agent to an eye of a subject by transducing a viral vector into an eye cell such that the transduced eye cell expresses a therapeutic agent and delivers the agent to the eye of the subject. In certain embodiments, the agent is the BMI1 protein. In certain embodiments, the eye cell is a retinal pigment epithelium (RPE) cell.

[0134] Foreign genetic material (e.g., cDNA encoding more than one therapeutic protein) is introduced into cells ex vivo or in vivo by gene transfer methods such as transfection or transduction, providing genetically modified cells. Various expression vectors (i.e., vehicles for facilitating delivery of foreign genetic material into target cells) are known to those skilled in the art.

[0135] As used herein, “cellular transfection” refers to the acquisition of novel genetic material by a cell through the incorporation of added DNA. Thus, transfection refers to the insertion of nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those skilled in the art, including calcium phosphate DNA coprecipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; and microparticle gun methods facilitated by tungsten particles. Strontium phosphate DNA coprecipitation is another promising transfection method.

[0136] In contrast, "transduction of cells" refers to the process of introducing nucleic acids into cells using DNA or RNA viruses. RNA viruses (i.e., retroviruses) used to introduce nucleic acids into cells are referred to herein as transduction chimeric retroviruses. The exogenous genetic material contained within the retrovirus is integrated into the genome of the transduced cell. Cells transduced with a chimeric DNA virus (for example, an adenovirus carrying cDNA encoding a therapeutic agent) do not have the exogenous genetic material integrated into their genome, but are capable of expressing the exogenous genetic material that is retained outside the chromosomes within the cell.

[0137] Examples of expression systems that can be used for the purposes of the present invention include, but are not limited to, mammalian cell lines (e.g., COS, CHO, BHK, 293, 3T3).

[0138] Typically, exogenous genetic material contains a heterologous gene (usually in the form of a cDNA containing exons encoding a therapeutic protein) along with a promoter to control the transcription of the novel gene. Characteristically, this promoter has a specific nucleotide sequence necessary to initiate transcription. Optionally, exogenous genetic material further contains additional sequences (i.e., enhancers) necessary to obtain the desired gene transcriptional activity. For the purposes of this discussion, “enhancer” is simply any non-coding DNA sequence that acts adjacent to the coding sequence (cis) to modify the basal transcriptional level directed by the promoter. Exogenous genetic material can be introduced into the cellular genome immediately downstream from the promoter so that the promoter and coding sequence are operably linked to allow transcription of the coding sequence. Retroviral expression vectors may contain exogenous promoter elements to control the transcription of the inserted exogenous gene. Such exogenous promoters may include both constitutive and inductive promoters.

[0139] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes under the control of constitutive promoters are expressed under all conditions of cell growth. Exemplary constitutive promoters include those for the following genes encoding specific constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, and other constitutive promoters known to those skilled in the art. Furthermore, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40; long-chain terminal repeats (LTRs) of Moloney's leukemia virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus. Thus, any of the constitutive promoters mentioned above can be used to control the transcription of heterologous gene inserts.

[0140] Genes under the control of an inductive promoter are expressed only in the presence of an inducer, or to a greater degree (for example, transcription under the control of a metallothionein promoter is greatly increased in the presence of a specific metal ion). Inductive promoters contain a response element (RE) that stimulates transcription when bound to their inducer. REs exist for serum factors, steroid hormones, retinoic acid, and cyclic AMP, for example. To obtain an inductive response, a promoter containing a specific RE can be selected, and in some cases, the RE itself may bind to a different promoter, thereby conferring inductive ability to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive vs. inductive; strong vs. weak), it is possible to control both the presence and expression level of therapeutic agents in genetically modified cells. When a gene encoding a therapeutic agent is under the control of an inductive promoter, in situ delivery of the therapeutic agent is induced by exposing the genetically modified cells in situ to conditions that allow transcription of the therapeutic agent, for example, by intraperitoneal injection of a specific inducer of the inductive promoter that controls the transcription of the agonist. For example, the in situ expression of therapeutic agents encoded by genes under the control of the metallothionein promoter in genetically modified cells can be enhanced by contacting the genetically modified cells in situ with a solution containing appropriate (i.e., inducible) metal ions.

[0141] Therefore, the amount of therapeutic agent delivered in situ is regulated by controlling factors such as (1) the nature of the promoter used to direct the transcription of the inserted gene (i.e., whether the promoter is constitutive or inductive, strong or weak); (2) the copy number of the exogenous gene inserted into the cell; (3) the number of transduced / transfected cells administered to the patient (e.g., transplanted); (4) the size of the implant (e.g., graft or encapsulated expression system); (5) the number of implants; (6) the duration that the transduced / transfected cells or implants remain in situ; and (7) the rate of therapeutic agent production by the genetically modified cells. The selection and optimization of these factors for the delivery of a therapeutically effective dose of a particular therapeutic agent should be within the scope of the art, without excessive experimentation, by considering the factors disclosed above and the patient's clinical profile.

[0142] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may include a selection gene, such as a neomycin resistance gene, to facilitate the selection of cells being transfected or transduced with the expression vector. Alternatively, cells may be transfected with two or more expression vectors, one of which contains a gene encoding a therapeutic agent and the other containing a selection gene. The selection of appropriate promoters, enhancers, selection genes, and / or signal sequences (described below) should be within the scope of those skilled in the art without excessive experimentation.

[0143] Therapeutic agents can be targeted for delivery to extracellular, intracellular, or membrane locations. If it is desirable for the gene product to be secreted from the cell, the expression vector is designed to include an appropriate secretion "signal" sequence for secreting the therapeutic gene product from the cell into the extracellular environment. If it is desirable for the gene product to be retained within the cell, this secretion signal sequence is omitted. Similarly, expression vectors can be constructed to include a "retention" signal sequence for immobilizing the therapeutic agent within the cell membrane. For example, all membrane proteins have a hydrophobic transmembrane region that prevents protein translocation across the membrane and thus prevents protein secretion. Constructing expression vectors containing signal sequences for targeting gene products to specific locations is considered within the scope of the art and does not require excessive experimentation.

[0144] The selection and optimization of a specific expression vector for expressing a specific gene product in isolated cells is achieved by the steps of: obtaining a gene containing one or more suitable regulatory regions (e.g., promoter, insertion sequence); preparing a vector construct containing the vector to be inserted into the gene; transfecting or transducing the vector construct into in vitro cultured cells; and determining whether the gene product is present in the cultured cells. In certain embodiments, viruses of the Adeno-Associated Viridae family are used. In certain embodiments, expression vectors for AAV8-based gene therapy are used.

[0145] Therefore, as will be apparent to those skilled in the art, a variety of suitable viral expression vectors are available for transmitting exogenous genetic material into cells. The selection of a suitable expression vector for expressing a therapeutic agent under specific conditions suitable for gene replacement therapy and the optimization of conditions for insertion of the selected expression vector into cells is within the scope of those skilled in the art and does not require excessive experimentation.

[0146] In alternative embodiments, the expression vector is in plasmid form and is introduced into target cells by one of several methods: physical (e.g., microinjection, electroporation, scrape loading, microparticle gun) or by chemical complex (e.g., calcium or strontium coprecipitation, complexation with lipids, complexation with ligands). Several commercially available products for cationic liposome complexation are available, including Lipofectin® (Gibco-BRL, Gaithersburg, Md.) and Transfectam® (ProMega, Madison, Wis.). However, the efficiency of transfection by these methods is highly dependent on the properties of the target cells, and therefore, the conditions for optimal transfection of nucleic acids into cells using the methods described above must be optimized. Such optimization is within the scope of those skilled in the art and does not require excessive experimentation.

[0147] For simplicity, this specification refers to AAV1, AAV2, AAV4, AAV5, or AAV8, but it should be understood that mutations in the homologous regions of the capsid of other AAV serotypes are also included in the present invention. Where used herein, the term “wild-type” refers to a naturally occurring AAV sequence without mutations in amino acids 587–595 (using the numbering for AAV8) of the capsid protein. However, it is not intended that naturally occurring AAVs are the only source of wild-type sequences. In this specification, non-naturally occurring AAVs, including but not limited to recombinant AAVs, modified or altered AAVs, chimeric AAVs, hybrid AAVs, synthetic AAVs, and artificial AAVs, are useful. This includes AAVs with mutations in capsid regions other than amino acids 587–595, insofar as they are used as “start sequences” for constructing the mutant capsids described herein.

[0148] This specification discloses systems and methods for delivering gene products to target retinal epithelial cells. The method includes the step of administering the rAAV virion of the present invention to an individual. The method generally includes the step of introducing the rAAV virion of the present invention into the eye of an individual, wherein the rAAV virion enters the retinal cells of the eye and the gene product encoded by the heterologous polynucleotide present in the rAAV virion is produced in the retinal cells. The eye may be an eye with visual impairment and / or an eye disease (e.g., retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy). Alternatively, the eye may be an eye at high risk of developing visual impairment and / or an eye disease. The introduction of the rAAV virion of the present invention into the eye of an individual may be carried out by intravitreal injection, intravitreal injection, intravitreal implantation, subretinal injection, choroidal administration, intravenous administration, or any other convenient form or route of administration known in the art.

[0149] In retinal gene therapy, AAV can enter cells and transduce RPE cells by expressing therapeutic DNA sequences. Because retinal cells do not divide, AAV can persist and provide expression of therapeutic DNA sequences for extended periods, potentially lasting several years.

[0150] A further aspect of the present invention relates to a method for administering a viral vector and / or viral capsid to a subject. Administration of a viral vector and / or capsid according to the present invention to a human subject or animal requiring such administration may be by any means known in the art. Optionally, the viral vector and / or capsid is delivered in a therapeutic or preventive dose on a pharmaceutically acceptable carrier.

[0151] Depending on the circumstances, when the AAV virion of the present invention is introduced into the eye of an individual (for example via intravitreal injection), it provides high levels of production of heterologous gene products encoded by AAV in the eye. For example, heterologous polypeptides encoded by AAV may be produced in the eye at levels of about 1 μg to about 50 μg, or more than 50 μg. As another example, heterologous polypeptides encoded by AAV may be produced in the vitreous fluid of the eye at levels of about 100 pg / mL to about 5000 pg / mL, for example, about 100 pg / mL to about 500 pg / mL, about 500 pg / mL to about 1000 pg / mL, about 1000 pg / mL to about 2000 pg / mL, about 2000 pg / mL to about 3000 pg / mL, about 3000 pg / mL to about 4000 pg / mL, or about 4000 pg / mL to about 5000 pg / mL. In some cases, polypeptides encoded by AAV may be produced in the vitreous fluid of the eye at levels exceeding 5000 pg / mL of vitreous fluid.

[0152] Depending on the circumstances, when the AAV virion of the present invention is introduced into the eye of an individual (for example via intravitreal injection), it provides the production of heterologous gene products encoded by AAV in at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80% of the Müller cells of the eye.

[0153] In some embodiments, when the AAV virion in question is introduced into the eye of an individual (for example via intravitreal injection), it provides the production of heterogeneous gene products encoded by AAV for a period of approximately 2 days to approximately 6 months, for example, approximately 2 days to approximately 7 days, approximately 1 week to approximately 4 weeks, approximately 1 month to approximately 2 months, or approximately 2 months to approximately 6 months. In some embodiments, when the AAV virion in question is introduced into the eye of an individual (for example via intravitreal injection), it provides the production of heterogeneous gene products encoded by AAV for a period of more than 6 months, for example, approximately 6 months to 20 years or more, or more than 1 year, for example, approximately 6 months to approximately 1 year, approximately 1 year to approximately 2 years, approximately 2 years to approximately 5 years, approximately 5 years to approximately 10 years, approximately 10 years to approximately 15 years, approximately 15 years to approximately 20 years or more, or more than 20 years.

[0154] Gene products can be polypeptides or nucleic acids. Examples of nucleic acid gene products include the aforementioned interfering RNAs (e.g., shRNA, siRNA, etc.), ribozymes, antisense RNAs, and aptamers.

[0155] Pharmaceutical composition Another embodiment is a pharmaceutical composition. This pharmaceutical composition may comprise the polypeptide described above and a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to treat a pathological condition suitable for gene replacement therapy. The exogenous genetic material may comprise a heterologous gene encoding a therapeutic agent for treating the disease or condition. The pharmaceutical composition may also comprise a sufficient amount of polypeptide to deliver a therapeutically effective dose of the therapeutic agent to the patient.

[0156] In one embodiment, recombinant AAV (rAAV) containing a transgene and cell-specific promoter desirable for use in the target ocular cells described above is optionally evaluated for impurities by conventional methods and subsequently formulated into a pharmaceutical composition intended for subretinal or intravitreal injection. Such formulations include the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly suitable for administration to the eye, for example, by subretinal injection, such as buffered saline or other buffers, such as HEPES to maintain pH at an appropriate physiological level, and optionally other medical agents, pharmaceutically active agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. In the case of injection, the carrier is usually a liquid. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate-buffered saline. Various such known carriers are provided in U.S. Patent Publication No. 7,629,322, which is incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is an equilibrium salt solution. In one embodiment, the carrier is Tween. When the virus is to be stored for a long period of time, it can be frozen in the presence of glycerol or Tween20.

[0157] In certain embodiments of the methods described herein, the pharmaceutical compositions described above are administered to the target by subretinal injection. In other embodiments, the pharmaceutical compositions are administered by intravitreous injection, suprachorionic injection, or orbital injection. Other forms of administration that may be useful in the methods described herein include, but are not limited to, direct delivery to the desired organ (e.g., the eye), oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. The administration routes may be combined as needed.

[0158] Furthermore, in certain embodiments, it is desirable to perform non-invasive retinal imaging and functional testing to identify specific areas of ocular cells to be targeted for treatment. In these embodiments, clinical diagnostic tests are used to determine the precise location for one or more subretinal injections. These tests may include fundus examination, electroretinography (ERG) (particularly measurement of b-waves), visual field measurement, topological mapping of retinal layers and measurement of the thickness of those layers by confocal scanning laser ophthalmoscope (cSLO) and optical coherence tomography (OCT), topological mapping of cone density via adaptive optics (AO), and functional testing of the eye. These and other desirable tests are described in International Patent Publication No. 2013 / 022628. From the viewpoint of imaging and functional testing, in some embodiments, one or more injections are given in the same eye to target different areas of retained bipolar cells. The volume and viral titer of each injection are determined individually, as further described later, or may be the same as or different from other injections given in the same or opposite eye. In other embodiments, a single, larger volume injection is given to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition are selected so that only a specific region of eye cells is affected. In another embodiment, the volume and / or concentration of the rAAV composition is greater so that it reaches a larger portion of the eye containing undamaged eye cells.

[0159] The composition can be delivered in a volume of from about 0.1 μL to about 1 mL (including all numbers within this range), depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL.

[0160] The effective concentration of recombinant adeno-associated virus having a nucleic acid sequence encoding a desired transgene under the control of a cell-specific promoter sequence is preferably in the range of about 10 7 ~10 13 vector genomes / milliliter (vg / mL) (also referred to as genome copies / mL (GC / mL)). rAAV infectious units are measured as described in S.K. McLaughlin et al, 1988 J. Virol., 62:1963, which is incorporated herein by reference. Preferably, the concentration in the retina is from about 1.5×10 9 vg / mL to about 1.5×10 12 vg / mL, more preferably from about 1.5×10 9 vg / mL to about 1.5×10 11 vg / mL. In one embodiment, the effective concentration is about 1.4×10 8 vg / mL. In one embodiment, the effective concentration is about 3.5×10 10 vg / mL. In another embodiment, the effective concentration is about 5.6×10 11 vg / mL. In another embodiment, the effective concentration is about 5.3×10 12The concentration is vg / mL. In yet another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 12 The effective concentration is vg / mL. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 13 The concentration is vg / mL. In one embodiment, the effective dose (total genome copies delivered) is approximately 10 7 ~10 13 This is a vector genome. It is desirable to utilize the lowest possible concentrations of the virus to reduce the risk of undesirable effects such as toxicity, retinal dysplasia, and detachment. Further other doses and volumes within this range may be selected by the attending physician, taking into account the subject being treated, preferably the human physical condition, the subject's age, specific eye disorders, and, if progressive, the degree of progression of the disorder. For extraocular delivery, the dose is increased in proportion to the scale-up from the retina. For intravenous delivery, for example, 1.5 × 10⁻⁶ doses are used. 13 Doses in the order of vg / kg may be required.

[0161] The "therapeutic effective dose" is within a relatively wide range, which can be determined through experiments and / or clinical trials. The dose of viral vector and / or capsid administered to a subject may vary depending on the mode of administration, the disease or condition being treated and / or prevented, the individual subject's condition, the specific viral vector or capsid, and the nucleic acid being delivered, and may be determined in the usual manner. For example, in in vivo injection, e.g., direct injection into the eye, the therapeutically effective dose is approximately 10 6 ~about 10 15 billion rAAVs, for example, about 10 8 ~10 12 It is in the order of rAAV virions. For example, in in vivo injection, such as direct injection into the eye, the therapeutically effective dose is approximately 10 6 ~about 10 12 Infection units, for example, about 10 8 ~about 10 12 It is in the order of the infectious unit. Other effective doses can be readily established by those skilled in the art through conventional tests to establish dose-response curves. In one embodiment, an exemplary dose to achieve a therapeutic effect is at least about 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 The trait introduction unit is selected at random, resulting in approximately 10 8 ~about 10 13 This is the potency of the trait-introducing unit.

[0162] In some cases, the therapeutically effective dose of rAAV virion of the present invention is effective in slowing the progression of retinal degeneration in an individual when administered to the individual in one or more doses (for example, via intravitreal injection into the individual's eye (e.g., an eye with visual impairment; an eye with eye disease; an eye at risk of developing eye disease)). For example, the therapeutically effective dose of rAAV virion of the present invention is effective in slowing the progression of retinal degeneration in an individual when administered to the individual in one or more doses (for example, via intravitreal injection into the individual's eye), The amount of rAAV virion may be effective in slowing the progression of retinal degeneration by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80% compared to the progression of retinal degeneration without treatment.

[0163] Depending on the circumstances, the therapeutically effective dose of rAAV virion of the present invention is an effective amount to improve the visual acuity of an individual when administered to the individual in one or more doses (for example, by intravitreal injection into the individual's eye). For example, the therapeutically effective dose of rAAV virion of the present invention, when administered to an individual in one or more doses (for example, by intravitreal injection into the individual's eye), is effective in improving visual acuity by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80% compared to the visual acuity of an individual who has not been treated with rAAV virion.

[0164] In another embodiment, the therapeutically effective dose of rAAV virion of the present invention, when administered to an individual in one or more doses (e.g., administered to the individual's eye via intravitreal injection), is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12% of the individual's visual acuity compared to the individual's visual acuity without treatment with rAAV virion. %, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 4 3%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately It is effective in improving vision by approximately 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0165] In another embodiment, the therapeutically effective dose of rAAV virion of the present invention, when administered to an individual in one or more doses (for example, administered to the individual's eye via intravitreal injection), is 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, and 14% or less compared to the individual's visual acuity without treatment with rAAV virion. , 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less Lower, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less Lower, 45% or less, 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% Below, 60% or less, 61% or less, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% The following are effective for improving vision: 75% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0166] In another embodiment, the therapeutically effective dose of rAAV virion of the present invention, when administered to an individual in one or more doses (e.g., administered to the individual's eye via intravitreal injection), is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, less than the visual acuity of the individual not treated with rAAV virion. At least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45% , at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 7 3%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%,It is effective in improving eyesight.

[0167] In some cases, the therapeutically effective dose of rAAV virion of the present invention, when administered to an individual in one or more doses (for example, to the individual's eye via intravitreal injection), is effective in reducing the rate of vision loss in eyes with visual impairment.

[0168] Improvement in clinical symptoms is monitored by one or more methods known to those skilled in the art, such as tests of functional vision including visual acuity, visual field, contrast sensitivity, color vision, mobility, and light sensitivity. Clinical symptoms may also be monitored by anatomical or physiological means, such as indirect fundus examination, fundus photography, fluorescein angiopathy, optical coherence tomography, electroretinography (whole field, multifocal, or other), external eye examination, slit-lamp biomicroscopy, applanation tonometry, corneal thickness measurement, autorefraction, or other measurements of functional vision.

[0169] In certain embodiments, more than one dose (e.g., two, three, four, five, six, seven, eight, nine, ten, or more doses) may be used over periods of varying intervals, for example, every hour, every day, every week, every month, or every year, to achieve a desired level of gene expression. Dosage may be single doses or cumulative (sequential), which can be readily determined by those skilled in the art. For example, treatment of a disease or disorder may involve a single dose of an effective amount of the pharmaceutical composition viral vector disclosed herein. Alternatively, treatment of a disease or disorder may involve multiple doses of an effective dose of the viral vector administered over periods such as once a day, twice a day, three times a day, once every few days, or once a week. The timing of administration may vary between individuals depending on factors such as the severity of the individual's symptoms. For example, an effective dose of the viral vector disclosed herein may be administered to an individual once every six months for an unspecified period or until the individual no longer requires treatment. Those skilled in the art will recognize that the condition of an individual can be monitored throughout the treatment process and that the effective dose of the viral vector disclosed herein can be adjusted accordingly.

[0170] In one embodiment, the duration of viral vector administration is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In further embodiments, the period of discontinuation of administration may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0171] Multiple doses of the rAAV virion of the present invention may be administered to individuals requiring it. When multiple doses are administered over a period of time, the effective agent may be administered at intervals of approximately once a month to approximately once a year, approximately once a year to approximately once every two years, approximately once every two years to approximately once every five years, approximately once every five years to approximately once every ten years. For example, the rAAV virion of the present invention may be administered over periods of approximately three months to approximately two years, approximately two years to approximately five years, approximately five years to approximately ten years, approximately ten years to approximately twenty years, or more than twenty years. Actual administration frequency and actual treatment duration will vary depending on various factors.

[0172] As an example, a method for treating an eye disorder may include the steps of administering an initial dose of rAAV virion according to the present invention and administering at least a second dose (subsequent dose) of rAAV virion. If two or more subsequent doses are administered, the subsequent doses may be spaced at intervals of at least one month, at least three to six months, at least six months to one year, at least one to five years, at least five to ten years, at least ten to twenty years, or more than 20 years from each other.

[0173] Other eye diseases that can be prevented, improved, or treated using the methods described herein include: acute macular neuroretinopathy; macular telangiectasia; Behçet's disease; choroidal neovascularization; diabetic eye disease; uveitis; histoplasmosis; macular degeneration, e.g., age-related macular degeneration, Sausby's macular dystrophy, early or moderate (atrophic) macular degeneration, or progressive macular degeneration, e.g., exudative macular degeneration or geographic atrophy; edema, e.g., macular edema, cystic macular edema, and diabetic macular edema; multiple choroiditis; ocular trauma affecting the site or position of the posterior eye; ocular tumors; retinal disorders, e.g., central retinal vein occlusion, glucose These include urea retinopathy (including proliferative and nonproliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal detachment, uveal retinal disease; sympathetic ophthalmitis; Vogt-Koyanagi-Harada (VKH) syndrome; uveal exudation; posterior eye conditions caused or affected by laser treatment of the eye; eye conditions caused or affected by photodynamic therapy or photocoagulation, radiation retinopathy; epiretinal membrane disorders; central retinal vein occlusion or branch retinal vein occlusion; central artery occlusion or branch artery occlusion, anterior ischemic optic neuropathy, diabetic retinal dysfunction; retinitis pigmentosa; retinal schizophrenia; and glaucoma.

[0174] In one embodiment, the patient is administered an AAV containing the BMI1 gene in its genome via intravitreous, intravenous, subretinal, or retroocular administration. In a further embodiment, the BMI1-containing AAV increases the expression of BMI1 in retinal ganglion cells. This increased protection results in a reduction in the severity of glaucoma, ischemic optic neuropathy, and / or retinopathy.

[0175] In another embodiment, the subject exhibits clinical signs of ocular impairment. Clinical signs of ocular impairment may include decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color perception, decreased visual acuity, decreased photoreceptor function, and pigment changes. In one embodiment, the subject exhibits degeneration of the outer nucleus layer (ONL). In another embodiment, the subject has been diagnosed with ocular impairment. In yet another embodiment, the subject has never exhibited clinical signs of ocular impairment.

[0176] In one embodiment, the subject is symptomatic with respect to ocular impairment. In another embodiment, the subject has 10% or more photoreceptor damage / loss. In another embodiment, the subject has 20% or more photoreceptor damage / loss. In another embodiment, the subject has 30% or more photoreceptor damage / loss. In another embodiment, the subject has 40% or more photoreceptor damage / loss. In another embodiment, the subject has 50% or more photoreceptor damage / loss. In another embodiment, the subject has 60% or more photoreceptor damage / loss. In another embodiment, the subject has 70% or more photoreceptor damage / loss. In another embodiment, the subject has 80% or more photoreceptor damage / loss. In another embodiment, the subject has 90% or more photoreceptor damage / loss. In another embodiment, the bipolar cell network to the ganglion cells and optic nerve of the subject remains intact.

[0177] Optionally, the compositions of the present invention may include, in addition to rAAV and carriers, other conventional pharmaceutical components, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0178] Virus delivery In another aspect, the present invention provides a method for delivering a transgene to a host, comprising transfecting or infecting selected host cells with a recombinant viral vector constructed with the AAV9 / HU.14 sequence (or a functional fragment thereof) of the present invention. Methods for delivery are well known to those skilled in the art and do not limit the present invention.

[0179] In one preferred embodiment, the present invention provides a method for AAV-mediated delivery of a transgene to a host. The method comprises the step of transfecting or infecting selected host cells with a recombinant viral vector containing a selected transgene and an AAV9 capsid protein under the control of a sequence that directs its expression.

[0180] Optionally, a host sample may first be assayed for the presence of antibodies against a selected AV source (e.g., serotype). Various assay forms for detecting neutralizing antibodies are well known to those skilled in the art. The selection of such assays is not limiting to the present invention. See, for example, Fisher et al, Nature Med., 3(3):306-312 (March 1997) and WC Manning et al, Human Gene Therapy, 9:477-485 (Mar. 1, 1998). The results of this assay can be used to determine which AAV vector containing a particular source's capsid protein is preferred for delivery, for example, by the absence of neutralizing antibodies specific to that capsid source.

[0181] In one aspect of this method, delivery of a vector containing the AAV capsid protein of the present invention may occur before or after delivery of a gene via a vector containing a different AAV capsid protein. Thus, gene delivery via an rAAV vector may be used for repeated gene delivery to a selected host. Preferably, the subsequently administered rAAV vector carries the same transgene as the first rAAV vector, but the subsequently administered vector contains a capsid protein (and preferably a different serotype) from a different source than the first vector. For example, if the first vector contains the AAV9 / HU.14 capsid protein, the subsequently administered vector may optionally contain a capsid protein selected from another AAV of a different serotype or another clade.

[0182] Multiple rAAV vectors, which can be optionally used to deliver a large or multiple transgene, can be concatenated in vivo by co-administration of rAAV vectors to form a single vector genome. In such embodiments, the first AAV may have an expression cassette expressing a single transgene (or its subunits), and the second AAV may have an expression cassette expressing a second transgene (or a different subunit) for co-expression in host cells. The first AAV may have an expression cassette which is a first fragment of a polycistronic construct (e.g., a promoter and a transgene, or a subunit), and the second AAV may have an expression cassette which is a second fragment of a polycistronic construct (e.g., a transgene or subunit and a polyA sequence). These two fragments of the polycistronic construct form a concatemer in vivo to form a single vector genome that co-expresses the transgenes delivered by the first and second AAVs. In such embodiments, an rAAV vector carrying a first expression cassette and an rAAV vector having a second expression cassette may be delivered in a single pharmaceutical composition. In other embodiments, two or more rAAV vectors may be administered as separate pharmaceutical compositions, which may be administered substantially simultaneously, or immediately before or after each other.

[0183] The injectable formulation may be prepared in conventional forms, such as a liquid solution or suspension, a solid form suitable for dissolution or suspension in a liquid before injection, or an emulsion. Alternatively, the viral vector and / or viral capsid of the present invention may be administered topically rather than systemically, for example in a depot or sustained-release formulation. Furthermore, the viral vector and / or viral capsid may be delivered attached to a surgically implantable matrix (for example, as described in U.S. Patent Publication No. 2004-0013645). The viral vector and / or viral capsid disclosed herein may be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of breathable particles consisting of the viral vector and / or viral capsid, which the subject inhales. The breathable particles may be liquid or solid. The aerosol of liquid particles containing the viral vector and / or viral capsid may be delivered by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, which are known to those skilled in the art. Similarly, aerosols of solid particles containing viral vectors and / or viral capsids can be produced using any solid particulate drug aerosol generator by techniques known in the field of pharmaceuticals.

[0184] The recombinant vectors described above can be delivered to host cells according to the published methods. Preferably, rAAV suspended in a physiologically compatible carrier can be administered to human or non-human mammalian patients. A suitable carrier can be readily selected by those skilled in the art, taking into account the adaptation to which the introduced virus is directed. For example, one suitable carrier is saline, which can be formulated with various buffers (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not limiting to the present invention.

[0185] The vector can be administered in sufficient quantities to transfect cells and to provide sufficient levels of gene introduction and expression to deliver therapeutic benefits with medically acceptable physiological effects or without excessive adverse effects, and this quantity can be determined by those skilled in the art of medicine. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., the eye (optionally via the hepatic artery) or the lung), ocular, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes. Routes of administration may be combined as desired.

[0186] The dose of viral vectors depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore can vary among patients. For example, the therapeutically effective human dose of viral vectors is generally about 1 × 10⁶ of the viral vector. 9 ~1 × 10 16 The concentration of the genome is in the range of approximately 0.1 mL to approximately 100 mL of solution. The preferred human dose for delivery to large organs (e.g., liver, muscle, heart, and lungs) is approximately 5 × 10¹⁶ in a volume of approximately 1 to 100 mL. 16 ~5×10 13 This can be AAV genomes / kg. The preferred dose for delivery to the eye is generally about 5 × 10 in a volume of about 0.1 mL to 1 mL. 9 ~5×10 12 This is a single genome copy. The dose is adjusted to balance the therapeutic benefits with all side effects, and such doses may vary depending on the therapeutic application in which the recombinant vector is used. The expression level of the transgene may be monitored to determine the frequency of administration resulting in a viral vector, preferably an AAV vector containing a minigene. Optionally, dosing regimens similar to those described for therapeutic purposes may be used for immunization using the compositions of the present invention.

[0187] Examples of therapeutic and immunogenic products for delivery by the AAV-containing vectors of the present invention are provided below. These vectors can be used in a variety of therapeutic or vaccine regimens as described herein. Furthermore, these vectors can be delivered in combination with one or more other vectors or active ingredients in the desired therapeutic and / or vaccine regimen.

[0188] For example, diabetic retinopathy is characterized by neovascularization. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors intraocularly (e.g., into the vitreous humor) or periorbitally (e.g., in the sub-Tenon's region). Alternatively, one or more neurotrophic factors may be delivered simultaneously intraocularly (e.g., into the vitreous humor) or periorbitally.

[0189] For comparison, retinitis pigmentosa is characterized by retinal degeneration. In typical embodiments, retinitis pigmentosa can be treated by intraocular (e.g., intravitreal) administration or other delivery methods of delivery vectors encoding one or more neurotrophic factors.

[0190] Age-related macular degeneration includes both neovascularization and retinal degeneration. In one embodiment, this disorder may be treated by administering the present invention's delivery vectors encoding one or more neurotrophic factors intraocularly (e.g., into the vitreous humor) and / or one or more anti-angiogenic factors intraocularly or periocularly (e.g., into the subtenon region).

[0191] Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment of glaucoma involves administering one or more neuroprotective agents that protect cells from excitotoxic damage using the delivery vectors of the present invention. Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic delivery factors delivered intraocularly, or optionally into the vitreous humor.

[0192] In other embodiments of this embodiment, the viral vector containing AAV reduces the severity of eye disease or impairment by, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, and at least 22%. , at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, less At least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least Reduce by 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0193] In other embodiments of this embodiment, the viral vector containing AAV reduces the severity of eye disease or impairment by, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, and about 2%. 0%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47% Approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately Reduce by 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0194] In other embodiments of this embodiment, the viral vector containing AAV reduces the severity of eye disease or impairment to, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, 21% or less. % or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less , 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48 % or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less, 61% or less , 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less, 75 Reduce to % or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0195] In yet another embodiment of this embodiment, the viral vector reduces the severity of eye disease or impairment to, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, 21% Below, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48% Below, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less, 61% or less , 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less, 75 Reduce to % or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0196] In further other embodiments of this embodiment, the viral vector is used to measure the severity of the disease or disorder, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, Approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 7 Reduce by 5%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0197] In yet another embodiment of this embodiment, the viral vector reduces the severity of the disease or disorder by, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, and less At least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, and at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 7 Reduce by 5%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0198] In further other embodiments of this embodiment, the viral vector reduces the severity of the disease or disorder to, for example, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, and about 30% Reduces by approximately 90%, 40%–90%, 50%–90%, 60%–90%, 70%–90%, 10%–80%, 20%–80%, 30%–80%, 40%–80%, 50%–80%, or 60%–80%, 10%–70%, 20%–70%, 30%–70%, 40%–70%, or 50%–70%.

[0199] The viral vectors containing AAV disclosed herein may contain a sufficient amount of solvent, emulsion, or other diluent to dissolve the viral vectors disclosed herein. In other embodiments of this specification, the viral vector containing AAV disclosed herein may contain, for example, less than about 90(v / v)%, less than about 80(v / v)%, less than about 70(v / v)%, less than about 65(v / v)%, less than about 60(v / v)%, less than about 55(v / v)%, less than about 50(v / v)%, less than about 45(v / v)%, less than about 40(v / v)%, less than about 35(v / v)%, less than about 30(v / v)%, less than about 25(v / v)%, less than about 20(v / v)%, less than about 15(v / v)%, less than about 10(v / v)%, less than about 5(v / v)%, or less than about 1(v / v)%, a solvent, emulsion, or diluent. In other embodiments of this specification, the viral vectors containing AAV disclosed herein are, for example, about 1(v / v)%~90(v / v), about 1(v / v)%~70(v / v), about 1(v / v)%~60(v / v), about 1(v / v)%~50(v / v), about 1(v / v)%~40(v / v), about 1(v / v)%~30(v / v)%, approximately 1(v / v)%~20(v / v)%, approximately 1(v / v)%~10(v / v)%, approximately 2(v / v)%~50(v / v)%, approximately 2(v / v)%~40(v / v)% , approximately 2(v / v)%~30(v / v)%, approximately 2(v / v)%~20(v / v)%, approximately 2(v / v)%~10(v / v)%, approximately 4(v / v)%~50(v / v)%, approximately 4 (v / v)%~40(v / v)%, approximately 4(v / v)%~30(v / v)%, approximately 4(v / v)%~20(v / v)%, approximately 4(v / v)%~10(v / v)%, approximately 6(v / v)%~50(v / v)%, approx. 6(v / v)%~40(v / v)%, approx. 6(v / v)%~30(v / v)%, approx. 6(v / v)%~20(v / v)%, approx. 6(v / v)% It may contain amounts of solvent, emulsion, or other diluents ranging from ~10(v / v)%, approximately 8(v / v)%~50(v / v)%, approximately 8(v / v)%~40(v / v)%, approximately 8(v / v)%~30(v / v)%, approximately 8(v / v)%~20(v / v)%, approximately 8(v / v)%~15(v / v)%, or approximately 8(v / v)%~12(v / v)%.

[0200] Aspects of this specification disclose partial treatment of an individual suffering from a disease or disorder. As used herein, the term “treating” means reducing or eliminating the clinical symptoms of a disease or disorder in an individual, or delaying or preventing the onset of the clinical symptoms of a disease or disorder in an individual.For example, the term "treating" means treating the symptoms of a condition characterized by a disease or disorder, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, and 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, and less At least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, This could mean a reduction of at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.Actual symptoms associated with a particular disease or disorder may be determined by a person skilled in the art by considering factors including, but not limited to, the location of the disease or disorder, the cause of the disease or disorder, the severity of the disease or disorder, and / or the tissue or organ affected by the disease or disorder. A person skilled in the art is familiar with appropriate symptoms or indicators associated with a particular type of disease or disorder and is familiar with methods for determining whether an individual is a candidate for the treatment disclosed herein.

[0201] In aspects of this embodiment, a therapeutically effective dose of a viral vector containing AAV disclosed herein is, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, and at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, less 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, little Reduce by at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0202] In other embodiments of this embodiment, the therapeutically effective dose of the viral vector containing AAV disclosed herein is, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, Approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately Reduces by 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0203] In other embodiments of this model, the therapeutically effective dose of the viral vector containing AAV disclosed herein is, for example, less than or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or less, 20% or less, 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less , 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less , 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less Lower, 61% or less, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% The following percentages should be reduced to 75% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0204] In other embodiments of this embodiment, a therapeutically effective dose of a viral vector containing AAV disclosed herein reduces symptoms associated with a disease or disorder by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In further other embodiments of this embodiment, a therapeutically effective dose of a viral vector containing AAV disclosed herein reduces symptoms associated with a disease or disorder by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0205] In one embodiment, a viral vector containing AAV disclosed herein reduces the level and / or amount of protein encoded in the viral vector administered to a patient compared to a patient who did not receive the same treatment, for example, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%. , at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, less At least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,Or it is possible to increase it by at least 100%.

[0206] In another embodiment, a viral vector containing AAV disclosed herein is administered to a patient at levels and / or amounts of the protein encoded in the viral vector, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, Approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74% It is possible to increase it by approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0207] In further embodiments, the viral vectors containing AAV disclosed herein may have levels and / or amounts of the protein encoded in the viral vector administered to a patient such that they are, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less. , 17% or less, 18% or less, 19% or less, 20% or less, 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31 % or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less , 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 6 0% or less, 61% or less, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less It is possible to increase it to 75% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0208] In other embodiments of this embodiment, the viral vector containing AAV reduces the severity of the disease or disorder in individuals with the disease or disorder compared to patients who did not receive the same treatment, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, and about 20% It is possible to reduce the levels by approximately 90%, 30%–90%, 40%–90%, 50%–90%, 60%–90%, 70%–90%, 10%–80%, 20%–80%, 30%–80%, 40%–80%, 50%–80%, or 60%–80%, 10%–70%, 20%–70%, 30%–70%, 40%–70%, or 50%–70%.

[0209] In aspects of this embodiment, the therapeutically effective dose of a viral vector containing AAV disclosed herein is, compared to an individual that did not receive the same treatment, the amount of protein encoded in the viral vector in the individual is, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 1 8%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, and 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, and less Increase by at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0210] In further embodiments of this embodiment, the therapeutically effective amount of a viral vector containing AAV disclosed herein is, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 7 Increase by 3%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0211] In other embodiments of this embodiment, the therapeutically effective amount of a viral vector containing AAV disclosed herein is, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 1 7% or less, 18% or less, 19% or less, 20% or less, 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31 % or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less Lower, 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less , 60% or less, 61% or less, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, Increase by 74% or less, 75% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0212] In other embodiments of this embodiment, a therapeutically effective dose of a viral vector containing AAV disclosed herein reduces or maintains the severity of disease or impairment in an individual by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In further other embodiments of this embodiment, a therapeutically effective dose of a viral vector containing AAV disclosed herein reduces or maintains the severity of disease or impairment in an individual by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0213] The viral vector is administered to an individual or patient. The individual or patient is usually a human, but may be an animal, including, but is not limited to, dogs, cats, birds, cattle, horses, sheep, goats, reptiles, and other animals, whether or not they are livestock.

[0214] In one embodiment, diseases of the eye and RPE cells, including the retina, can be treated using AAV, where AAV comprises a recipient AAV which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10. In one embodiment, the recipient AAV is AAV2, and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10. In another embodiment, the recipient AAV is AAV3, and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10.

[0215] In another embodiment, a method for forming the above-described pharmaceutical composition is provided. This method includes the steps of introducing an expression vector for expressing a heterologous gene product into cells to form genetically modified cells, and placing the genetically modified cells in a pharmaceutically acceptable carrier.

[0216] Although this invention is primarily described for treating eye diseases such as retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy, it is understood that the invention is not so limited and may be used to treat other diseases.

[0217] Aspects of this specification disclose, in part, the treatment of an individual affected by retinopathy. As used herein, “treating” means reducing or eliminating the clinical symptoms of retinopathy in an individual; or delaying or preventing the onset of the clinical symptoms of retinopathy in an individual.For example, the term "treating" does not limit the symptoms of a condition characterized by retinopathy, including visual impairment, to, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, and less 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, and at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least This could mean a reduction of 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.Actual symptoms associated with cancer are well known and can be determined by those skilled in the art. Those skilled in the art are aware of appropriate symptoms or indicators associated with certain types of retinopathy and are aware of methods for determining whether an individual is a candidate for the treatment disclosed herein.

[0218] In another embodiment, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of retinopathy-related disorders. In this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of retinopathy-related disorders by, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, and at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, At least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, and at least Reduce by 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0219] In aspects of this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of retinopathy-related disorders by, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, and about 19%. Approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 4 7%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, Reduces by approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0220] In this embodiment, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of retinopathy-related disorders to, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, and 20% or less. , 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34 % or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less, 61% Below, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less, 7 Reduce by 5% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0221] In other embodiments of this specification, the pharmaceutical compositions disclosed herein reduce the severity of symptoms of retinopathy-related disorders to, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, and about 3% Reduces from 0% to approximately 90%, 40% to approximately 90%, 50% to approximately 90%, 60% to approximately 90%, 70% to approximately 90%, 10% to approximately 80%, 20% to approximately 80%, 30% to approximately 80%, 40% to approximately 80%, 50% to approximately 80%, or 60% to approximately 80%, 10% to approximately 70%, 20% to approximately 70%, 30% to approximately 70%, 40% to approximately 70%, or approximately 50% to approximately 50%.

[0222] In aspects of this embodiment, the therapeutically effective amount of the pharmaceutical composition disclosed herein is, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, and at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, At least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, and at least Reduce by 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0223] In aspects of this embodiment, the therapeutically effective amount of the pharmaceutical composition disclosed herein is, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% Approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 4 7%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, Reduces by approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0224] In this embodiment, the therapeutically effective amount of the pharmaceutical composition disclosed herein is, for example, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, to alleviate symptoms associated with retinopathy. , 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34 % or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less, 61% Below, 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less, 7 Reduce by 5% or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0225] In other embodiments of this embodiment, a therapeutically effective amount of the pharmaceutical composition disclosed herein reduces symptoms associated with retinopathy by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In further other embodiments of this embodiment, a therapeutically effective amount of the pharmaceutical composition disclosed herein reduces symptoms associated with retinopathy by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0226] In further other embodiments of this embodiment, the therapeutically effective dose of the pharmaceutical composition disclosed herein is generally in the range of about 0.001 mg / kg to about 100 mg / kg, administered, for example, every 3, 5, 7, 10, or 14 days.

[0227] In aspects of this embodiment, the effective amount of the pharmaceutical composition disclosed herein may be, for example, at least 0.001 mg / kg, at least 0.01 mg / kg, at least 0.1 mg / kg, at least 1.0 mg / kg, at least 5.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, or at least 50 mg / kg, and may be administered, for example, every 3, 5, 7, 10, or 14 days. In other embodiments of this specification, the effective amount of the pharmaceutical composition disclosed herein may be, for example, about 0.001 mg / kg to about 10 mg / kg, about 0.001 mg / kg / day to about 15 mg / kg, about 0.001 mg / kg to about 20 mg / kg, about 0.001 mg / kg to about 25 mg / kg, about 0.001 mg / kg to about 30 mg / kg, about 0.001 mg / kg to about 35 mg / kg, about 0.001 mg / kg to about 40 mg / kg, about 0.001 mg / kg to about 45 mg / kg, about 0.001 mg / kg to about 50 mg / kg, about 0.001 mg / kg to about 75 mg / kg, or about 0.001 mg / kg to about 100 mg / kg, and may be administered, for example, every 3, 5, 7, 10, or 14 days. In further other embodiments of this embodiment, the effective amount of the pharmaceutical composition disclosed herein may be, for example, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 35 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 45 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.01 mg / kg to about 75 mg / kg, or about 0.01 mg / kg to about 100 mg / kg, and may be administered, for example, every 3, 5, 7, 10, or 14 days.In further other embodiments of this embodiment, the effective amount of the pharmaceutical composition disclosed herein may be, for example, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 15 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 35 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 45 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 75 mg / kg, or about 0.1 mg / kg to about 100 mg / kg, and may be administered, for example, every 3, 5, 7, 10, or 14 days.

[0228] Administration may be a single dose or cumulative (sequential), and this can be readily determined by those skilled in the art. For example, treatment for retinopathy may involve a single dose of an effective amount of the pharmaceutical composition disclosed herein. Alternatively, treatment for retinopathy may involve multiple doses of an effective dose of a viral vector administered over a range of periods, such as once daily, twice daily, three times daily, once every few days, or once a week. The timing of administration may vary between individuals depending on factors such as the severity of the individual's symptoms. For example, an effective dose of the pharmaceutical composition disclosed herein may be administered to an individual once daily for an indefinite period or until the individual no longer requires treatment. Those skilled in the art will recognize that the individual's condition can be monitored throughout the treatment process and that the effective dose of the pharmaceutical composition disclosed herein can be adjusted accordingly.

[0229] A pharmaceutical composition or retinopathy treatment is administered to an individual. The individual is usually a human, but may be an animal, including, but is not limited to, dogs, cats, birds, cattle, horses, sheep, goats, reptiles, and other animals, whether or not they are livestock. Typically, any individual that is a candidate for treatment is a candidate that has some form of retinopathy.

[0230] In one embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy. In this embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy in amounts of, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22% , at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, less At least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least Reduce by 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0231] In another embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy. In this embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy in amounts of, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 2% 0%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47% Approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately Reduce by 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0232] In another embodiment, the compositions disclosed herein reduce the symptoms of disorders associated with retinopathy. In this embodiment, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy, for example, at concentrations of 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, 21% or less % or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less , 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48 % or less, 49% or less, 50% or less, 51% or less, 52% or less, 53% or less, 54% or less, 55% or less, 56% or less, 57% or less, 58% or less, 59% or less, 60% or less, 61% or less , 62% or less, 63% or less, 64% or less, 65% or less, 66% or less, 67% or less, 68% or less, 69% or less, 70% or less, 71% or less, 72% or less, 73% or less, 74% or less, 75 Reduce to % or less, 76% or less, 77% or less, 78% or less, 79% or less, 80% or less, 81% or less, 82% or less, 83% or less, 84% or less, 85% or less, 86% or less, 87% or less, 88% or less, 89% or less, 90% or less, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97% or less, 98% or less, 99% or less, or 100% or less.

[0233] In other embodiments of this specification, the pharmaceutical compositions disclosed herein reduce the symptoms of disorders associated with retinopathy, for example, in concentrations of about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to Reduces by approximately 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, or 60% to 80%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, or 50% to 70%.

[0234] Figure 1 provides photographs comparing a retina taken from a young person with one taken from an elderly person. Figure 1A is a photograph of a healthy young person's retina. A healthy young person's retina contains high levels of antioxidants, which also replace dead or dying cells in a timely manner. A healthy young person's retina does not contain drusen and can repair DNA damage. Figure 1B shows a photograph of an elderly person's retina, which contains low levels of antioxidants and is hardly able to replace damaged cells. The elderly retina also has a reduced ability to remove drusen. This image shows moderate atrophic retinal degeneration. Figure 1C is a photograph of an elderly person's retina, which suffers from progressive atrophic macular degeneration or geographic atrophy. Macular degeneration results in inflammation and retinal death, which is visible in the large "hole" in the retina near the center of the photograph in Figure 1C.

[0235] Figure 2A shows normal retinal structure as seen through images of the eye of a mouse treated with AAV5.BMI1. Figures 2B and 2C show normal histopathology after treatment with our therapy, which is identical to that of a normal mouse eye. As can be seen in Figure 2C, the cellular structure is consistent and fluid throughout the image, and no significant cellular damage or holes are observed in the retina.

[0236] Examples The following non-limiting examples are provided solely for illustrative purposes to facilitate a more complete understanding of the representative embodiments intended herein. These examples are intended to be merely a subset of all possible combinations of the components of the formulation. Therefore, these examples should not be construed as limiting any of the embodiments described herein, including those relating to the types and amounts of the components of the formulation, and / or the methods and uses thereof.

[0237] Example 1 Treatment method for retinal degeneration using AAV In retinal gene therapy, AAV can "transduce" RPE cells by entering the cells and expressing therapeutic DNA sequences. Because retinal cells do not divide, AAV can persist and continue to provide expression of therapeutic DNA sequences for a long period, potentially lasting several years.

[0238] According to one embodiment, target cells are transformed or otherwise genetically modified in vivo. Parental cells are transformed (i.e., transduced) in vivo with a vector containing exogenous genetic material to express a heterologous (e.g., recombinant) gene encoding a therapeutic agent, and this therapeutic agent is delivered in situ.

[0239] AAV can transduce multiple cell types within the retina, including RPE cells. AAV serotype 8 can be administered via one of two routes: intravitreous or subretinal. When using the intravitreous route, AAV is injected into the vitreous humor of the eye. When using the subretinal route, AAV is injected under the retina, utilizing the potential space between the photoreceptors and the RPE layer. This is more invasive than the intravitreous route, but the humor is absorbed by the RPE and photoreceptors, and the retina flattens within about 14 days without significant complications. Subretinal AAV is preferred because this treatment is less likely to induce an immune response. Other serotypes (e.g., 1, 2, 5, 7, 8, and 9) can also be used to transduce RPE cells.

[0240] In this embodiment, the patient presents with signs and / or symptoms of age-related macular degeneration (AMD). For example, the patient may experience a gradual loss of the ability to see objects clearly, distorted shapes of objects, and / or loss of clear color vision.

[0241] AAV-mediated gene therapy is used to target affected retinal pigment epithelial (RPE) cells, photoreceptors, other cells in the inner and outer retina, and ganglion cells. AAV virions can be introduced into the patient's eye via intravitreal injection. Subsequently, the expression of the BMI1 protein in RPE cells is monitored. The patient's vision is also monitored. One or more subsequent injections may be administered after the initial injection.

[0242] For example, the therapeutically effective amount of the pharmaceutical compositions disclosed herein is generally about 1 × 10⁶ per dose. 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , about 10×10 8 , about 1×10 9 , about 2×10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 10×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , 1 about 10×10 10 , about 1×10 11, about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 10×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 10×10 12 , about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 10×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 6×10 14 , about 7×10 14 , about 8×10 14 , about 9×10 14 , or about 10×10 14 and are in the range of individual AAV virions.

[0243] For example, the therapeutically effective amount of the pharmaceutical composition disclosed herein is generally about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , about 10×108 , about 1×10 9 , about 2×10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 10×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , 1 about 10×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 10×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 10×10 12 , about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 10×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×1014 , about 5×10 14 , about 6×10 14 , about 7×10 14 , about 8×10 14 , about 9×10 14 , or approximately 10 x 10 14 It is within the range of AAVvg / eye.

[0244] For example, the therapeutically effective amount of the pharmaceutical compositions disclosed herein is generally at least 1 × 10⁶ per dose. 8 , at least 2 × 10 8 , at least 3 × 10 8 , at least 4 × 10 8 , at least 5 × 10 8 , at least 6 × 10 8 , at least 7 × 10 8 , at least 8 × 10 8 , at least 9 × 10 8 , at least 10 × 10 8 , at least 1 × 10⁹, at least 2 × 10 9 , about 4×10 9 , at least 5 × 10 9 , at least 6 × 10 9 , at least 7 × 10 9 , at least 8 × 10 9 , at least 9 × 10 9 , at least 10 × 10 9 , at least 1 × 10 10 , at least 2 × 10 10 , at least 3 × 10 10 , at least 4 × 10 10 , at least 5 × 10 10 , at least 6 × 10 10 , at least 7 × 10 10 , at least 8 × 10 10 , at least 9 × 10 10 , 1 at least 10 × 10 10 , at least 1 × 10 11 , at least 2 × 10 11 , at least 3 × 10 11 , at least 4 × 10 11 , at least 5 × 10 11 , at least 6 × 1011 , at least 7 × 10 11 , at least 8 × 10 11 , at least 9 × 10 11 , at least 10 × 10 11 , at least 1 × 10 12 , at least 2 × 10 12 , at least 3 × 10 12 , at least 4 × 10 12 , at least 5 × 10 12 , at least 6 × 10 12 , at least 7 × 10 12 , at least 8 × 10 12 , at least 9 × 10 12 , at least 10 × 10 12 , at least 1 × 10 13 , at least 2 × 10 13 , at least 3 × 10 13 , at least 4 × 10 13 , at least 5 × 10 13 , about 7×10 13 , at least 8 × 10 13 , at least 9 × 10 13 , at least 10 × 10 13 , at least 1 × 10 14 , at least 2 × 10 14 , at least 3 × 10 14 , at least 4 × 10 14 , at least 5 × 10 14 , at least 6 × 10 14 , at least 7 × 10 14 , at least 8 × 10 14 , at least 9 × 10 14 , or at least 10 × 10 14 It is within the range of one AAV billion.

[0245] For example, the therapeutically effective amount of the pharmaceutical compositions disclosed herein is generally at least 1 × 10⁻⁶ 8 , at least 2 × 10 8 , at least 3 × 10 8 , at least 4 × 10 8 , at least 5 × 10 8 , at least 6 × 10 8, at least 7 × 10 8 , at least 8 × 10 8 , at least 9 × 10 8 , at least 10 × 10 8 , at least 1 × 10 9 , at least 2 × 10 9 , about 4×10 9 , at least 5 × 10 9 , at least 6 × 10 9 , at least 7 × 10 9 , at least 8 × 10 9 , at least 9 × 10 9 , at least 10 × 10 9 , at least 1 × 10 10 , at least 2 × 10 10 , at least 3 × 10 10 , at least 4 × 10 10 , at least 5 × 10 10 , at least 6 × 10 10 , at least 7 × 10 10 , at least 8 × 10 10 , at least 9 × 10 10 , 1, at least 10 × 10 10 , at least 1 × 10 11 , at least 2 × 10 11 , at least 3 × 10 11 , at least 4 × 10 11 , at least 5 × 10 11 , at least 6 × 10 11 , at least 7 × 10 11 , at least 8 × 10 11 , at least 9 × 10 11 , at least 10 × 10 11 , at least 1 × 10 12 , at least 2 × 10 12 , at least 3 × 10 12 , at least 4 × 10 12 , at least 5 × 10 12 , at least 6 × 10 12 , at least 7 × 10 12 , at least 8 × 10 12 , at least 9 × 10 12 2. At least 10 × 10 12, at least 1 × 10 13 , at least 2 × 10 13 , at least 3 × 10 13 , at least 4 × 10 13 , at least 5 × 10 13 , about 7×10 13 , at least 8 × 10 13 , at least 9 × 10 13 , at least 10 × 10 13 , at least 1 × 10 14 , at least 2 × 10 14 , at least 3 × 10 14 , at least 4 × 10 14 , at least 5 × 10 14 , at least 6 × 10 14 , at least 7 × 10 14 , at least 8 × 10 14 , at least 9 × 10 14 , or at least 10 × 10 14 It is within the range of AAVvg / eye.

[0246] For example, the therapeutically effective amount of the pharmaceutical compositions disclosed herein is generally 1 × 10⁶ per dose. 8 Below, 2 x 10 8 Below, 3 x 10 8 Below, 4 x 10 8 Below, 5 x 10 8 Below, 6 x 10 8 Below, 7 x 10 8 Below, 8 x 10 8 Below, 9 x 10 8 Below, 10 x 10 8 Below, 1 x 10 9 Below, 2 x 10 9 Below, approximately 4×10 9 , 5×10 9 Below, 6 x 10 9 Below, 7 x 10 9 Below, 8 x 10 9 Below, 9 x 10 9 Below, 10 x 10 9 Below, 1 x 10 10 Below, 2 x 10 10 Below, 3 x 10 10 Below, 4 x 10 10 Below, 5 x 1010 Below, 6 x 10 10 Below, 7 x 10 10 Below, 8 x 10 10 Below, 9 x 10 10 Below, 1 10×10 10 Below, 1 x 10 11 Below, 2 x 10 11 Below, 3 x 10 11 Below, 4 x 10 11 Below, 5 x 10 11 Below, 6 x 10 11 Below, 7 x 10 11 Below, 8 x 10 11 Below, 9 x 10 11 Below, 10 x 10 11 Below, 1 x 10 12 Below, 2 x 10 12 Below, 3 x 10 12 Below, 4 x 10 12 Below, 5 x 10 12 Below, 6 x 10 12 Below, 7 x 10 12 Below, 8 x 10 12 Below, 9 x 10 12 Below, 10 x 10 12 Below, 1 x 10 13 Below, 2 x 10 13 Below, 3 x 10 13 Below, 4 x 10 13 Below, 5 x 10 13 Below, about 7×10 13 , 8×10 13 Below, 9 x 10 13 Below, 10 x 10 13 Below, 1 x 10 14 Below, 2 x 10 14 Below, 3 x 10 14 Below, 4 x 10 14 Below, 5 x 10 14 Below, 6 x 10 14 Below, 7 x 10 14 Below, 8 x 10 14 Below, 9 x 10 14 The following, or 10 x 10 14 It falls within the following AAV billion range.

[0247] For example, the therapeutically effective amount of the pharmaceutical compositions disclosed herein is generally 1 × 10⁻⁶8 Below, 2×10 8 Below, 3×10 8 Below, 4×10 8 Below, 5×10 8 Below, 6×10 8 Below, 7×10 8 Below, 8×10 8 Below, 9×10 8 Below, 10×10 8 Below, 1×10 9 Below, 2×10 9 The following, approximately 4×10 9 5×10 9 Below, 6×10 9 Below, 7×10 9 Below, 8×10 9 Below, 9×10 9 Below, 10×10 9 Below, 1×10 10 Below, 2×10 10 Below, 3×10 10 Below, 4×10 10 Below, 5×10 10 Below, 6×10 10 Below, 7×10 10 Below, 8×10 10 Below, 9×10 10 Below, 1 10×10 10 Below, 1×10 11 Below, 2×10 11 Below, 3×10 11 Below, 4×10 11 Below, 5×10 11 Below, 6×10 11 Below, 7×10 11 Below, 8×10 11 Below, 9×10 11 Below, 10×10 11 Below, 1×10 12 Below, 2×10 12 Below, 3×10 12 Below, 4×10 12 Below, 5×10 12 Below, 6×10 12 Below, 7×10 12 Below, 8×10 12 Below, 9×10 12 Below, 10×10 12 Below, 1×10 13Below, 2 x 10 13 Below, 3 x 10 13 Below, 4 x 10 13 Below, 5 x 10 13 Below, about 7×10 13 , 8×10 13 Below, 9 x 10 13 Below, 10 x 10 13 Below, 1 x 10 14 Below, 2 x 10 14 Below, 3 x 10 14 Below, 4 x 10 14 Below, 5 x 10 14 Below, 6 x 10 14 Below, 7 x 10 14 Below, 8 x 10 14 Below, 9 x 10 14 The following, or 10 x 10 14 It is within the following AAVvg / eye range.

[0248] In gene therapy methods, those skilled in molecular biology and gene therapy can determine appropriate doses and routes of administration of the expression vector used in the novel methods of this disclosure without performing excessive experiments.

[0249] Example 2 Treatment methods for retinal dystrophy using AAV In this embodiment, the patient presents with signs and / or symptoms of retinal dystrophy. For example, retinitis pigmentosa, the most common retinal dystrophy, is an example of such a hereditary disease. The patient may experience symptoms such as difficulty seeing at night and loss of peripheral vision.

[0250] As described above, AAV-mediated gene therapy is used to target affected retinal pigment epithelial (RPE) cells. AAV virion is introduced into the patient's eye via intravitreal injection. Subsequently, the expression of the BMI1 protein in RPE cells is monitored. The patient's vision is also monitored. One or more subsequent injections may be given after the initial injection.

[0251] Example 3 Treatment methods for macular degeneration using AAV In this embodiment, the patient presents with signs and / or symptoms of macular degeneration. Patients experiencing age-related macular degeneration may have a blind spot in the center of their vision. As the condition worsens, central vision may be lost.

[0252] As described above, AAV-mediated gene therapy is used to target affected retinal pigment epithelial (RPE) cells. AAV virion is introduced into the patient's eye by intravitreal, subretinal, and / or choroidal injection. Subsequently, the expression of the BMI1 protein in RPE cells is monitored. The patient's vision is also monitored. One or more subsequent injections may be given after the initial injection.

[0253] Example 4 Treatment methods for macular dystrophy using AAV In this example, the patient presents with signs and / or symptoms of macular dystrophy. Patients experiencing vitiligo macular degeneration may experience progressive vision loss. This condition causes an increase in fatty xanthochromic pigment (lipofuscin) in the cells of the lower layer of the macula. Over time, the abnormal accumulation of this substance can damage cells crucial for clear central vision. As a result, the patient may lose central vision, and their vision may become blurred or distorted.

[0254] As described above, AAV-mediated gene therapy is used to target affected retinal pigment epithelial (RPE) cells. AAV virion is introduced into the patient's eye via intravitreal injection. Subsequently, the expression of the BMI1 protein in RPE cells is monitored. The patient's vision is also monitored. One or more subsequent injections may be given after the initial injection.

[0255] Example 5 Evaluation of cellular senescence in cells overexpressing BMI1 compared to wild-type cells in vitro.

[0256] In this example, RPE cell lines, such as the ARPE-19 cell line or iPS cell lines, are cultured in vitro, and these are used to identify differences in cellular senescence between RPE cells into which BMI1 / AAV virions were introduced to overexpress BMI1, and RPE cells into which BMI1 / AAV virions were not introduced. After virion introduction, RPE cells treated with BMI1 / AAV virions are found to express BMI1 at levels higher than untreated RPE cells. Furthermore, increased BMI1 production is found to result in a loss of senescence compared to RPE cells into which BMI1I AAV virions were not introduced. Since senescence is associated with retinal cell death, this loss of senescence may be associated with a reduction in retinal cell death and a reduction in macular degeneration.

[0257] Example 6 Evaluation of cell death in BMI1-overexpressing cells compared to wild-type cells in vitro.

[0258] In this example, RPE cell lines, such as the ARPE-19 cell line or iPS cell lines, are cultured in vitro, and these are used to identify differences in the rate of cell death between RPE cells into which BMI1 / AAV virions were introduced to overexpress BMI1, and RPE cells into which BMI1 / AAV virions were not introduced. After virion introduction, RPE cells treated with BMI1 / AAV virions are found to express BMI1 at a higher level than untreated RPE cells. Furthermore, the increased production of BMI1 is found to result in reduced cell death compared to RPE cells into which BMI1I AAV virions were not introduced. The reduction in cell death is associated with a reduction in cases of macular degeneration and loss of retinal function. In addition, it is determined that cells into which BMI1I AAV virions were introduced proliferate at a greater rate than cells into which virions were not introduced.

[0259] Example 7 Evaluation of cell recovery from oxidative stress death in BMI1-overexpressing cells compared to wild-type cells in vitro.

[0260] In this example, RPE cell lines, such as the ARPE-19 cell line or iPS cell lines, are cultured in vitro and used to identify differences in cell recovery from cellular stress, including oxidative stress, which results in an increased rate of apoptosis. Cellular stress is induced in one experiment in which RPE cells are introduced with BMI1 / AAV virions to overexpress BMI1, compared to RPE cells that are not introduced with BMI1 / AAV virions. Cellular stress is induced by introducing lactate into the culture medium in which cells proliferate. The inventors measured the rate of cell death between RPE cells introduced with BMI1 / AAV virions to overexpress BMI1 and RPE cells that are not introduced with BMI1 / AAV virions. It is found that RPE cells treated with BMI1 / AAV virions, which express BMI1 at levels higher than untreated RPE cells after introduction of lactate, recover faster from lactate-induced oxidative stress compared to RPE cells that are not introduced with virions. Furthermore, increased BMI1 production is found to result in reduced cell death due to oxidative stress compared to RPE cells that were not introduced with BMI1 / AAV virions. This reduction in cell death is associated with a decrease in cases of macular degeneration and loss of retinal function. In addition, cells introduced with BMI1 / AAV virions are found to proliferate at a greater rate than cells that were not introduced with virions. It is understood that stress is induced by means other than the introduction of lactate into the cell culture medium.

[0261] Example 8 Evaluation of macular degeneration in mouse animal models after introduction of BMI1 / AAV virion, which leads to increased BMI1 expression.

[0262] In this example, a mouse model of macular degeneration is used. BMI1 / AAV virion is introduced subretinally into the eyes of mice. Mice are evaluated over time for retinal degeneration. The eyes of the mice are examined to determine whether the photoreceptors and outer granule cells of mice introduced with BMI1 / AAV virion are healthier and less dead (and therefore not macular degeneration) than the same cells of mice that were not introduced with BMI1 / AAV virion. After examining the eyes of the mice, it was found that the photoreceptors and outer granule cells of mice introduced with BMI1 / AAV virion were healthier and less likely to die than those that were not introduced with BMI1 / AAV virion.

[0263] Example 9 In this example, BMI1 mRNA and protein expression were measured in different parts of the eye. As seen in Figure 3A, BMI1 mRNA expression was found primarily in the retina and lens, and less so in the retinal periapical area (RPE). BMI1 mRNA expression was lowest in the cornea and vitreous solution. In Figure 3B, the location of BMI1 expression in the retina was found to be relatively uniform throughout the retina, as represented by the different retinal regions identified in Figure 3C. Similar results are shown in the RPE in Figure 3B, based on results from samples taken from the center, central, and peripheral parts of the eye.

[0264] Figure 3C provides a schematic diagram of the eye, showing different regions indicated by multiple presentations. In Figure 3C, the region represented by number 9 constitutes the macula, numbers 2, 4, 6, and 8 refer to the mid-periphery of the retina, and numbers 1, 3, 5, and 7 constitute the far-periphery of the retina. For the purposes of this Example 9, samples were taken from the eye regions shown in Figure 3C to determine relative expression in Figure 3B.

[0265] Example 10: In vivo distribution of BMI1 in the human eye Background: The following describes the determination of the amount of BMI1 protein in the human eye. It also describes the determination of the expression level of the BMI1 gene in the eye. In both cases, the amounts of BMI1 protein and gene expression levels were determined based on their in vivo distribution within the eye.

[0266] Methods: Human donor eyes were obtained from the One Legacy Foundation. Human donor eyes (average age 64 years) were maintained under optimal conditions until receipt. Upon arrival, the eyes were dissected and separated into different tissue fractions, including the cornea, lens, vitreous humor, iris, retina, and RPE-choroidal layer. Each tissue fraction was rapidly frozen in liquid nitrogen and then treated for RNA and protein extraction. Total mRNA was extracted from the tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using the Maxima Reverse Transcriptase Kit (Thermo Fisher Scientific) by reaction of 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in 96-well plates using a QuantStudio 6 Pro system (Thermo Fisher Scientific) with a total of 20 μl of mixed solution. Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primer, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized to GAPDH levels. The ploidy of the BMI1 gene was normalized to corneal gene expression. The sequences of the primers used are listed in Table 1. [Table 1] TIFF2026108759000003.tif96162

[0267] Eye samples were analyzed, and BMI1 protein levels were determined using standard indirect enzyme-linked immunosorbent assay (ELISA). Protein extracts were prepared in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Halt® phosphatase inhibitor and Halt® protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using the Micro BCATM protein assay kit (Thermo Fisher Scientific). This was done by first loading a 96-well plate with a triple sample of 2 μg / ml protein extract. A standard curve for protein levels was prepared using human recombinant BMI1 (Origene) as a control. Polyclonal anti-BMI1 antibody (Bethyl Laboratories) was used as the primary antibody for ELISA. The actual detection reaction was performed using the ELISA Buffer kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0268] Results: Figure 4A shows the results from the determination of BMI1 gene mRNA levels in the human eye. It is observed that BMI1 gene expression is found to be primarily detected in the lens and retina. As shown in Figure 4B, mRNA levels correlate with the location of highest BMI1 protein expression.

[0269] Example 11: In vivo distribution of BMI1 in pig eyes Background: The following describes the determination of the dose, gene expression level, and in vivo distribution of BMI protein in the eyes of young pigs in our investigation.

[0270] Methods: Young pig eyes were obtained from Fist Visiontech, Inc. Pig eye specimens were maintained at our facility to ensure optimal recovery of biomolecules after receiving the pig eyes. Upon arrival, the eyes were dissected to obtain the cornea, lens, vitreous humor, iris, retina, and RPE-choroidal layer. The tissues were rapidly frozen in liquid nitrogen and then processed for RNA and protein extraction.

[0271] Total mRNA was extracted from tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reacting 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed using a QuantStudio 6 Pro system (Thermo Fisher Scientific) in a total of 20 μl of mixed solution in a 96-well plate. Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primers, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized relative to GAPDH levels. The ploidy of the BMI1 gene was normalized relative to corneal gene expression. The sequences of the primers used are listed in Table 1. BMI1 mRNA quantification was performed as described.

[0272] In Western blot analysis of BMI1, samples were analyzed for BMI1 and β-actin expression levels using standard immunoblotting techniques. Briefly, aliquots of cell extracts were subjected to SDS gel electrophoresis on 4%–12% polyacrylamide gels, transferred to nitrocellulose membranes by electrophoresis using the iBLOT system (Thermo Fisher Scientific), and probed with rabbit anti-BMI1 (1:1,000 Bethyl Laboratories) or mouse anti-β-actin (1:5,000; R&D Systems) antibodies. Anti-rabbit and anti-mouse horseradish peroxidase (Thermo Fisher Scientific) were used for detection, and all immunoreactions were visualized by enhanced chemiluminescence using Super Signal West Pico Plus (Thermo Fisher Scientific). In particular, protein extracts were prepared in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Halt® phosphatase inhibitor and Halt® protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using the Micro BCA® Protein Assay Kit (Thermo Fisher Scientific). This was done by first loading a 96-well plate with a triple sample of 2 μg / ml protein extract. A standard curve for protein levels was prepared using human recombinant BMI1 (Origene) as a control. Polyclonal anti-BMI1 antibody (Bethyl Laboratories) was used as the primary antibody for the ELISA. The actual detection reaction was performed using the ELISA Buffer Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0273] Results: As shown in Figure 5A, BMI1 mRNA levels were detected at high levels in the retina of young pig eyes and at lower levels in the cornea. A similar expression pattern was observed for the BMI1 protein in the eyes of young pigs. As shown in the Western blot (Figure 5B), BMI1 protein expression was prominent in the retina and less pronounced in the RPE-choroid and cornea. This is also shown based on the concentration of BMI1 in each histological type of young pig eye, with the highest levels of BMI1 expression found in the retina (Figure 5C).

[0274] Example 12: BMI1 is downregulated in aged mice. Background: In this example, the inventors evaluated whether BMI1 mRNA expression is downregulated in aged retinas. To determine this, the inventors evaluated the levels of BMI1 mRNA and protein in young and aged mice.

[0275] Methods: C57BL6 / J mice at 9 weeks and 90 weeks of age were obtained from the Jackson Laboratory. Eyes were extracted from these mice, and the retina of the right eye was dissected. This tissue was rapidly frozen in liquid nitrogen and then processed for RNA and protein extraction.

[0276] Total mRNA was extracted from tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reacting 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in a total of 20 μl of mixed solution in a 96-well plate using a QuantStudio 6 Pro system (Thermo Fisher Scientific). Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primers, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized relative to GAPDH levels. The ploidy of the BMI1 gene was normalized relative to corneal gene expression. The primer sequences used are listed in Table 1.

[0277] Standard indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze eye samples and determine BMI1 protein levels. Protein extracts were prepared in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Halt® phosphatase inhibitor and Halt® protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using the Micro BCA® protein assay kit (Thermo Fisher Scientific). This was done by first loading a 96-well plate with a triple sample of 2 μg / ml protein extract. Human recombinant BMI1 (Origene) was used as a control to prepare a standard curve for protein levels. Polyclonal anti-BMI1 antibody (Bethyl Laboratories) was used as the primary antibody for the ELISA. The actual detection reaction was performed using the ELISA Buffer kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0278] For histological analysis, the left eye was embedded in an OCT scanner and frozen in isopentane cooled with liquid nitrogen. The tissue was then sectioned into 10 μm sections using a cryostat (Leica), and hematoxylin and eosin (H&E) staining was performed for morphological observation of the retinal layer. ONL and total retinal thickness were measured using ImageJ software.

[0279] Results: As shown in Figures 6A and 6B, retinal and ONL layer thicknesses decreased with age in mice. As shown in Figures 6A and 6B, the retinal and ONL layers of aged mice (90 weeks old) were thinner than those found in juvenile mice (9 weeks old). Similar differences were found from the analysis of BMI1 mRNA expression levels, where the retinal and ONL layers of juvenile mice had higher BMI1 mRNA levels than those of aged mice (Figure 6C). Tracking BMI1 protein expression in the retinal and ONL layers by BMI1 mRNA expression levels revealed that the retinal and ONL layers of juvenile mice showed higher levels of BMI1 protein compared to those of aged mice (Figure 6D). Figure 6E shows the quantification of the concentration measurements of the bands shown in Figure 6D, with values ​​normalized by β-actin in the loading control. As shown in Figure 6E, BMI1 protein expression is higher in young mice compared to aged mice.

[0280] Example 13: Administration study of AAV5.BMI1 in Balb / c mice Background: The optimal dose of AAV5.BMI1 was determined by subretinal delivery in Balb / c mice.

[0281] Methods: Six-week-old Balb / c mice were obtained from Jackson Laboratory. For all experiments, the mice were anesthetized with isoflurane. After anesthesia, the pupils of all animals were dilated using topical 1% tropicamide and 2.5% phenylepherine. AAV5.BMI1 was injected into the subretinal space of the eye using a 10 mM 33 gauge subcutaneous needle attached to a 10 μl syringe (Hamilton). Next, 1 μl of the vector suspension was injected into 1 × 10⁶ cells. 7 or 5 x 10 10Subretinal injections were administered within the dose range of vg / eye. Control animals were injected with 1 μl of physiological saline (0.9% NaCl). 1% chloramphenicol ophthalmic ointment was administered to the cornea of ​​all animals. Mice were euthanized 4 weeks after injection, their eyes were removed, and the retina of the right eye was dissected. This tissue was rapidly frozen in liquid nitrogen and then processed for mRNA and protein extraction.

[0282] Total mRNA was extracted from tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reacting 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed using a QuantStudio 6 Pro system (Thermo Fisher Scientific) in a total of 20 μl of mixed solution in a 96-well plate. Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primers, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, and the mean value was determined. PCR products were quantified using QuantStudio 6 Pro software. Expression levels were normalized relative to GAPDH levels. The ploidy of the BMI1 gene was normalized relative to corneal gene expression. The primer sequences used are listed in Table 1.

[0283] Eye samples were analyzed, and BMI1 protein levels were determined using standard indirect enzyme-linked immunosorbent assay (ELISA). Protein extracts were prepared in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with Halt® phosphatase inhibitor and Halt® protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using the Micro BCA® protein assay kit (Thermo Fisher Scientific). This was done by first loading a 96-well plate with a triple sample of 2 μg / ml protein extract. Human recombinant BMI1 (Origene) was used as a control to prepare a standard curve for protein levels. Polyclonal anti-BMI1 antibody (Bethyl Laboratories) was used as the primary antibody for ELISA. The actual detection reaction was performed using the ELISA Buffer kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0284] For histological analysis, the left eye was embedded in an OCT scanner and frozen in isopentane cooled with liquid nitrogen. The tissue was then sectioned into 10 μm sections using a cryostat (Leica), and stained with hematoxylin and eosin (H&E) for morphological observation of the retinal layer. ONL and total retinal thickness were measured using ImageJ software.

[0285] Results: As shown in Figures 7A (retina) and 7B (RPE-cochloride), BMI1 mRNA levels increased with increasing dose of AAV5. In particular, BMM mRNA levels increased with increasing dose of AAV5.BMI1. 8 From 1 x 10 9 , ultimately 5 x 10 9 mRNA levels increased as the dose of AAV5.BMI1 increased to vg / eye. mRNA levels were higher than the control at all doses after administration of AAV5.BMI1 (Figures 7A and 7B). 9When administered at a dose of vg / ocular, BMI1 protein in the RPE-choroid was statistically significantly increased compared to the control (Figure 7C). Figures 7D, 7E, and 7F show changes in ocular tissue compared to the control (Figure 7) and 1 × 10⁻⁶, respectively. 9 Eyes treated with vg / eye AAV5.BMI1 (Figure 7E), or 5×10 9 This indicates that it was not found in eyes administered with vg / eye AAV5.BMI1 (Figure 7F).

[0286] Example 14: Effect of BMI1 in a sodium iodate-induced retinal degeneration (AMD) model. Background: A preliminary study evaluated the ability of BMI1 to protect the retina from NaLO3-induced damage in vivo.

[0287] Methods: In this study, 6-week-old Balb / c mice were obtained from the Jackson Laboratory. One mouse per group was fed 1 × 10⁶ of saturation solution. 9 AAV5.BMI1 and saline solution were injected subretinically into each eye. In all experiments, mice were anesthetized with isoflurane. After anesthesia, the pupils of each mouse were dilated using topical 1% tropicamide and 2.5% phenylephrine. AAV5.BMI1 was injected into the subretinal space of the eye using a 10 mM 33 gauge subcutaneous needle attached to a 10 μl syringe (Hamilton). Next, 1 μl of the vector suspension was injected into 1 × 10⁶ mice. 7 or 5 x 10 10Subretinal injection was administered within the dose range of vg / eye. Control mice were injected with 1 μl of physiological saline (NaCl 0.9%). The corneas of all mice were coated with 1% chloramphenicol ophthalmic ointment. Four weeks after injection, the mice were euthanized, their eyes were removed, and the retina of the right eye was dissected. This tissue was rapidly frozen in liquid nitrogen and then processed for mRNA extraction. After subretinal injection of physiological saline and AAV5.BMI1, mice were intraperitoneally injected with NaLO3 (50 mg / kg). Mice that did not receive NaLO3 injection were used as the control group. The control mice were also euthanized four weeks after injection, their eyes were removed, and the retina of the right eye was dissected.

[0288] Total mRNA was extracted from tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reacting 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in a total of 20 μl of mixed solution in a 96-well plate using a QuantStudio 6 Pro system (Thermo Fisher Scientific). Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primers, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized relative to GAPDH levels. The ploidy of the BMI1 gene was normalized relative to corneal gene expression. The primer sequences used are listed in Table 1.

[0289] For histological analysis, the left eye was embedded in an OCT scanner and frozen in isopentane cooled with liquid nitrogen. This tissue was then sectioned into 10 μm sections using a cryostat (Leica), and stained with hematoxylin and eosin (H&E) for morphological observation of the retinal layer. ONL and total retinal thickness were measured using ImageJ software. For rhodopsin staining, 10 μm frozen eye sections were fixed in 4% paraformaldehyde (PFA). They were rinsed three times for 5 minutes each with phosphate-buffered saline (PBS), permeabilized in PBS-0.2% Triton X-100 for 15 minutes, incubated in IHC / ICC Blocking Buffer-High Protein (Thermo Fisher Scientific) for 60 minutes, and then incubated overnight at 4°C in a humidified chamber with blocking buffer containing mouse B630 anti-rhodopsin antibody 1:500 (Novus Biologicals). Retinal sections were washed four times with PBS and incubated with Alexa-conjugated goat anti-mouse antibody (Thermo Fisher Scientific) at room temperature for 1 hour. The nuclei were stained with DAPI (1:10,000 dilution) for 5 minutes. The sections were mounted using Fluoromount G mounting solution (Thermo Fisher Scientific). Immunofluorescence images were then acquired using the EVOS cell imaging system (Thermo Fisher Scientific).

[0290] Results: Figure 8A shows increased mRNA expression in NalO3-AAV5.BMI1 mice compared to control (NalO3-saline) mice. As can be seen in Figure 8A, efficient transduction of AAV in the mouse retina resulted in increased expression in eyes damaged by NalO3. Figures 8B, 8C, and 8D show photoreceptors (rods) in control mice (no NalO3) (Figure 8B), mice administered NalO3-saline (Figure 8C), and mice administered NalO3 and AAV5.BMI1 (Figure 8D). As observed, recovery occurred in NalO3-AAV5.BMI1 mice (Figure 8D) compared to NalO3-saline mice (Figures 8B and 8D). In Figures 8E–8G, eye sections of control mice, NaLO3-saline mice, and NaLO3-AAV5.BMI1 mice were immunostained with rhodopsin to visualize rod cells in the retinal photoreceptor layer. Histological analyses shown in Figures 8E–8G indicate that NaLO3 induced a decrease in retinal thickness in the two control groups (Figures 8E and 8F) compared with mice treated with NaLO3 and AAV5.BMI1 (Figure 8G). In the AAV5.BMI1 group, retinal thickness and ONL thickness were observed to have recovered when comparing Figure 8E with Figure 8G. This result is confirmed in Figure 8H, showing that the retinal thickness and ONL thickness in the AAV5.BMI1 group were similar to those of control mice that did not receive NaLO3.

[0291] Example 15: BMI1 protects against retinal degeneration in a sodium iodate-induced AMD model. Background: To validate the data obtained in previous examples, we evaluated the ability of BMI1 to protect the retina from NaLO3-induced damage in vivo in a larger group of mice.

[0292] Methods: In this study, 6-week-old Balb / c mice were obtained from Jackson Laboratory. Mice treated with AAV BMI1 were given 1 × 10⁶ doses. 9AAV5.BMI1 (n=20) and physiological saline (n=11) were injected subretinically into each eye. In all experiments, mice were anesthetized with isoflurane. After anesthesia, the pupils of all animals were dilated using topical 1% tropicamide and 2.5% phenylephrine. AAV5.BMI1 was injected into the subretinal space of the eye using a 10 mM 33 gauge subcutaneous needle attached to a 10 μl syringe (Hamilton). Then, 1 μl of the vector suspension was injected into the eye at a rate of 1 × 10⁶. 7 or 5 x 10 10 Subretinal injection was administered within the dose range of vg / eye. Control animals were injected with 1 μl of physiological saline (0.9% NaCl). 1% chloramphenicol ophthalmic ointment was administered to the cornea of ​​all animals. Four weeks after subretinal injection of physiological saline and AAV5.BMI1, mice were intraperitoneally injected with NaLO3 (50 mg / kg). Mice that did not receive NaLO3 injection were used as the control group (n=4). One week after NaLO3 injection, in vivo imaging was performed using spectral domain optical coherence tomography (OCT) adapted for use in mice (Specfraiis Multiline, Heidelberg Engineering GmbH, Heidelberg, Germany). Mice were anesthetized with isoflurane. Pupils were dilated with a mixture of tropicamide and phenylephrine. Artificial tears were used during this procedure to maintain corneal hydration and clarity. Twenty-five linear B-scans were obtained, each with an average of 9 frames, and these were used to quantify the outer retinal thickness.

[0293] Results: Figure 9 shows the reduction in ONL thickness in mice treated with NaLO3 and the effect of BMI1 in mice treated with AAV BMI1 on preventing this degeneration.

[0294] Example 16: BMI1 protects against retinal degeneration in a photo-induced retinopathy model. Background: To validate the data obtained in the sodium iodate model, the inventors evaluated the ability of BMI1 to protect mice from photo-induced retinopathy (LIR) in vivo.

[0295] In this study, 6-week-old Balb / c mice were obtained from the Jackson Laboratory. These mice were subjected to 1 × 10⁶ 9 AAV5.BMI1 (n=15) and physiological saline (n=7) were injected subretinically into each eye. For all experiments, mice were anesthetized with isoflurane. After anesthesia, the pupils of all animals were dilated using topical 1% tropicamide and 2.5% phenylephrine. AAV5.BMI1 was injected into the subretinal space of the eye using a 10 mM 33 gauge subcutaneous needle attached to a 10 μl syringe (Hamilton). Next, 1 μl of the vector suspension was injected into 1 × 10⁶ mice. 7 or 5 x 10 10Subretinal injections were administered within the dose range of vg / eye. Control animals were injected with 1 μl of physiological saline (NaCl 0.9%). The corneas of all animals were administered 1% chloramphenicol ophthalmic ointment. Four weeks after subretinal injection of physiological saline and AAV5.BMI1, mice were induced to develop retinal degeneration via exposure to intensive white LED light (LIR). 1% cyclopentolate hydrochloride and 2.5% phenylephrine were used in combination to dilate the pupils of the mice. After 45 minutes of adaptation to a darkroom, mice were individually placed in containers covered with aluminum foil and directly exposed to 6,000K and 12,000-15,000 lux light for 2 hours to induce photoreceptor degeneration. The temperature inside the containers was kept constant using a mini-fan. After exposure to intensive light, the mice were returned to a normal 12-hour light / dark cycle. One week after NalO3 injection, in vivo imaging was performed using spectral-domain optical coherence tomography (OCT) adapted for use in mice (Specfraiis Multiline, Heidelberg Engineering GmbH, Heidelberg, Germany). Mice were anesthetized with isoflurane. Pupils were dilated with a mixture of tropicamide and phenylephrine. Artificial tears were used during the procedure to maintain corneal hydration and clarity. Twenty-five linear B-scans, each with an average of 9 frames, were obtained and used to quantify the outer retinal thickness.

[0296] Results: Figure 10 shows a decrease in ONL thickness in control mice subjected to LIR and the effect of BMI1 administered to AAV-treated mice in preventing this degeneration.

[0297] Example 17: AAVrh10.BMI1 efficiently transduced human retinal explants. Background: Evaluation of AAVrh10.BMI1's ability to transduce human retina.

[0298] Methods: Eyes from two human donors were obtained from One Legacy Foundation (Los Angeles, CA). The specimens were kept under optimal conditions until delivery to our facility. Upon arrival, the eyes were dissected, and 5 mm diameter retinal explants were obtained using a dermal punch. These retinal explants were washed with HBSS (Hanks' Balanced Salt Solution) and then incubated in Neurobasal® (a medium supplemented with B-27 and N-2 supplements, 200 mM glutamine, and antibiotic-antifungal agents) at 37°C in a 5% CO2 incubator. AAV5.BMI1 was added to the explant medium (1 × 10⁶). 11 Explants were incubated for 5 days at a concentration of vg / ml. One retinal explant from each donor was not transduced and was used as a control.

[0299] Total mRNA was extracted from tissue using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA quantity and quality were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reacting 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in a total of 20 μl of mixed solution in a 96-well plate using a QuantStudio 6 Pro system (Thermo Fisher Scientific). Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primers, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized to GAPDH levels. The ploidy of the BMI1 gene was normalized to retinal gene expression. The primer sequences used are listed in Table 1.

[0300] Results: As shown in Figure 11, BMI1 mRNA levels increased approximately twofold in retinal explants transduced with AAVrh10.BMI1.

[0301] Example 18: Effects of BMI1 on cell proliferation, cell viability, and cytotoxicity in vitro Methods: ARPE-19 cells were grown in DMEM-F12 medium supplemented with 10% FBS at 5% CO2 / 37°C until the cells reached confluence. Afterward, the cells were placed in 50 μL of medium / well containing AAV5.BMI1 or AAV5.BMI1-shRNA at a rate of 1 × 10⁶ cells. 9Transduction was performed in vg for 72 hours. Cell viability was measured in transfected cells treated with different doses of NaLO3 (Sigma) for 15 hours. Mitochondrial damage was assessed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thermo Fisher Scientific). After stimulation, cells were washed with PBS and incubated with 20 μl of stock MTT solution for 4 hours. Subsequently, 200 μl of DMSO was added to each well. The plate was then shaken on a plate shaker at room temperature for 15 minutes. Cell viability was determined by measuring absorbance at 570 nm using an ELISA plate reader. Cytotoxicity was measured in AAV-transfected cells treated with different concentrations of tert-butyl hydroperoxide (tBH). Briefly, cells were seeded in a 96-well plate. Next, 72 hours after transduction with BMI1 and BMI1 shRNA, the culture medium was replaced with serum-free medium containing the specified concentration of tBH and incubated for 15 hours. Toxicity was determined by measuring cytoplasmic lactate dehydrogenase in the condition medium using the CyQUANT® LDH cytotoxicity assay (Thermo Fisher Scientific). Absorbance was measured using an ELISA microplate reader.

[0302] Results: Figure 12A shows that ARPE-19 cells overexpressing BMI1 significantly increased cell viability upon exposure to NaL3 compared to cells in which BMI1 expression was knocked down by shRNA. Figure 12B shows LDH levels, a measure of cytotoxicity. Figure 12B shows that cytotoxicity was significantly higher in BMI1 shRNA cells compared to cells treated with BMI1 overexpression.

[0303] Example 19: Infection with AV5.BMI1 in ARPE-19 cells altered the expression of genes involved in multiple cellular pathways. Background: Gene expression analysis was performed to determine the molecular mechanisms involved in the gene pathway of BMI1 function in ARPE-19 cells. These mechanisms include cell proliferation / apoptosis / senescence.

[0304] Methods: ARPE-19 cells were cultured and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS (Invitrogen) and 1% penicillin / streptomycin (Invitrogen). Cells were placed in 35 mm 6-well plates in 0.5-10 6Cells were seeded at a density of 100 cells / well. After 12 hours, cells were infected with AAV5.NM201 (treated group) or AAV5.GFP (control group) in the range of 2500–60000 MOI. 48 hours after viral infection, confluent monolayers were exposed to oxidative stress for 1 hour using tert-butyl hydroperoxide (tBH) (Invitrogen). Total mRNA was then extracted from the cells using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. The quantity and quality of mRNA were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reaction of 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in 96-well plates using a QuantStudio 6 Pro system (Thermo Fisher Scientific) in a total of 20 μl of mixed solution. Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primer, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized to GAPDH levels. The sequences of the primers used are listed in Table 1.

[0305] Results: Figures 13A–13D show that overexpression of BMI1 via AAV5.BMI1 increases the Bcl2 / Bax ratio (Figure 13A), which correlates with a reduction in apoptosis in ARPE-19 cells. BMI1 also upregulates the mRNA expression of Gpx1, Gpx3, Sod1, and Sod2 (Figure 13B), which correlates with increased cellular resistance to oxidative stress in the treated group of ARPE-19 cells. Furthermore, the treated group cells downregulate the mRNA expression of p21, p53 (Figure 13C), and Vegfa (Figure 13D) (Figure 13D). This correlates with a reduction in cellular senescence and inflammatory responses in ARPE-19 cells.

[0306] Example 20: Subretinal injection of AAV5.BMmi1 in Balb / c mice altered the expression of genes involved in multiple cellular pathways. Background: The study disclosed in this example was conducted to determine the molecular mechanisms involved in the gene pathway of BMI1 expression in mouse eyes. This was done through gene expression analysis, and the requirements for the cell proliferation / apoptosis / senescence network were evaluated.

[0307] Methods: Six-week-old Balb / c mice were obtained from Jackson Laboratory. For all experiments, the mice were anesthetized with isoflurane. After anesthesia, the pupils of all animals were dilated using topical 1% tropicamide and 2.5% phenylephrine. AAV5.BMI1 was injected into the subretinal space of the eye using a 10 mM 33 gauge subcutaneous needle attached to a 10 μl syringe (Hamilton). Next, 1 μl of the vector suspension was injected into 1 × 10⁶ cells. 7 or 5 x 10 10Subretinal injection was administered within the dose range of vg / eye. Control animals were injected with 1 μl of physiological saline (0.9% NaCl). The corneas of all animals were coated with 1% chloramphenicol ophthalmic ointment. Mice were euthanized 4 weeks after injection, their eyes were removed, and the retina of the right eye was dissected. This tissue was rapidly frozen in liquid nitrogen and then processed for mRNA and protein extraction. Total mRNA was then extracted from cells using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions. The quantity and quality of mRNA were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). First-strand cDNA synthesis was performed using a Maxima reverse transcriptase kit (Thermo Fisher Scientific) by reaction of 1.0 μg of total mRNA with random primers. Conventional RT-PCR (qRT-PCR) was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qPCR was performed in 96-well plates using a QuantStudio 6 Pro system (Thermo Fisher Scientific) in a total of 20 μl of mixed solution. Each 20 μl reaction mixture contained 10 μl of SYBR Green Master (Thermo Fisher Scientific), 0.5 μM primer, and diluted cDNA. Real-time PCR quantification was performed in triplicate for each sample, the mean was determined, and the PCR product was quantified using QuantStudio 6 Pro software. Expression levels were normalized to GAPDH levels. The sequences of the primers used are listed in Table 1.

[0308] Results: Figures 14A–14C show that overexpression of BMI1 induced by AAV5.NM201 administration resulted in upregulation of Gpx1, Sod1, and Sod2 mRNA expression (Figure 14A). This correlated with increased resistance to oxidative stress in the eyes of mice treated with AAV5.NM201. Furthermore, AAV5.NM201 administration correlated with downregulation of p16, p21, p53 (Figure 14B), and Vegfa (Figure 14C) mRNA expression. This downregulation correlated with reduced cellular senescence and inflammatory responses in the eyes of mice.

[0309] Finally, although aspects of this specification are emphasized by reference to specific embodiments, those skilled in the art will readily understand that these disclosed embodiments are merely examples of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is not in any way limited to specific compounds, compositions, articles, apparatus, methods, protocols, and / or reagents, etc., unless otherwise explicitly stated. Furthermore, those skilled in the art will understand that specific changes, modifications, rearrangements, alterations, additions, deletions, and sub-combinations thereof can be made in accordance with the teachings herein without departing from the spirit of this specification. Therefore, the appended claims below and the claims introduced herein herein are intended to be construed to include all such changes, modifications, rearrangements, alterations, additions, deletions, and sub-combinations, insofar as they are in their true spirit and scope.

[0310] Specific embodiments of the Invention, including the best modes known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the above description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and they intend that the Invention may be carried out in ways other than those specifically described herein. Thus, the Invention includes, to the extent permitted by applicable law, all modifications and equivalents of the subject matter described in the claims appended herein. Furthermore, all combinations of the above embodiments in all possible variations are encompassed by the Invention unless other meanings are described herein or other meanings are clearly inconsistent with the context.

[0311] A group of other embodiments, elements, or steps of the present invention should not be construed as limiting. Members of each group may be referenced and claimed individually or in combination with members of other groups disclosed herein. It is anticipated that one or more members of a group may be included in or excluded from a group for convenience and / or patentability. If any such inclusion or exclusion occurs, this specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0312] Unless otherwise stated, all numbers used herein and in the claims to refer to features, items, quantities, parameters, characteristics, terms, etc., should be understood to be modified in all examples by the term “approximately.” Where used herein, the term “approximately” means that the quantified feature, item, quantity, parameter, characteristic, or term encompasses a range of more than ±10% and less than ±10% of the value of the stated feature, item, quantity, parameter, characteristic, or term. Thus, unless otherwise stated, the mathematical parameters described herein and in the appended claims are variable approximations. For example, since mass spectrometers can vary slightly in determining the mass of a given analyte, the term “approximately” refers to ±0.50 atomic mass units in the context of the mass of an ion and / or the mass / charge ratio of an ion. At the very least, not as an attempt to limit the application of the doctrine of equivalents in the claims, each numerical description should be interpreted by applying ordinary rounding techniques, at least in terms of the number of significant figures reported.

[0313] The use of the terms “may” or “can” in relation to an embodiment or aspect of an embodiment also has the alternative meaning of “may not” or “cannot.” Therefore, where this specification discloses that an embodiment or aspect of an embodiment may be part of or may be included as part of the subject matter of the invention, it also expressly means a negative limitation or exclusive proviso meaning that the embodiment or aspect of an embodiment may not be part of or may not be included as part of the subject matter of the invention. Similarly, the use of the term “optionally” in relation to an embodiment or aspect of an embodiment means that such embodiment or aspect of an embodiment may be included as part of the subject matter of the invention, or may not be included as part of the subject matter of the invention. Whether such a negative limitation or exclusive proviso applies depends on whether the negative limitation or exclusive proviso is stated in the claimed subject matter. Furthermore, the terms “include,” “includes,” and “including” mean, without limitation, include, includes, and / or including, as well as include, includes, and including.

[0314] While the numerical ranges and values ​​described in the broad scope of this invention are approximations, the numerical ranges and values ​​described in specific examples are reported as accurately as possible. However, any numerical range or value inherently contains certain errors that inevitably arise from the standard deviation found in its respective test measurement. The descriptions of numerical ranges of values ​​herein are intended simply to function as a concise way of individually referring to each separate numerical value within that range. Unless otherwise stated herein, each individual value within a numerical range is incorporated herein as if it were individually described herein.

[0315] The terms “a,” “an,” “the,” and similar references used in the context describing this invention (particularly in the context of the following claims) should be interpreted as encompassing both singular and plural forms unless otherwise specified or explicitly contradicts the context. Furthermore, ordinal nominatives, such as “first,” “second,” “third,” etc., in identified elements, are used to distinguish between elements and do not refer to or imply a required or limited number of such elements, nor do they refer to a specific position or order of such elements, unless otherwise specifically specified. All methods described herein can be performed in any suitable order unless otherwise specified or explicitly contradicts the context. Any use of any examples or illustrative language provided herein (e.g., “such as”) is intended solely to illustrate the invention in good terms and does not impose any limitation on the scope of the invention unless otherwise specified. The language herein should not be interpreted as indicating all unspecified elements necessary for the practice of the invention.

[0316] When used in the claims, whether filed or added with each amendment, the open-ended transitional clause “comprising” (and equivalent open-ended transitional clauses such as including, containing, and having) encompasses all specified elements, limitations, steps, and / or characteristics, either alone or in combination with unspecified subject matter; while named elements, limitations, and / or characteristics are important, other unspecified elements, limitations, and / or characteristics may be added and further form a composition within the claims. Specific embodiments disclosed herein may be further limited in the claims using the closed-ended transitional clause “consisting of” or “consisting essentially of” as a substitute for or amendment to “comprising.” When used in the claims, whether filed or added with each amendment, the closed-ended transitional clause “consisting of” excludes all elements, limitations, steps, or characteristics not specified in the claims. The closed-end transitional clause "consisting essentially of" limits the claims to any other elements, limitations, steps, and / or characteristics that do not substantially affect the basic and novel features of the claimed subject matter, as well as any other elements, limitations, steps, and / or characteristics that do not substantially affect the elements, limitations, steps, and / or characteristics that are specified in the claims. Thus, the meaning of the open-end transitional clause "comprising" is defined as encompassing all the elements, limitations, steps, and / or characteristics that are specifically specified in the claims, as well as any further unspecified elements, limitations, steps, and / or characteristics of any choice. The meaning of the closed-end transitional clause "consisting of" is defined as including only the elements, limitations, steps, and / or characteristics that are specified in the claims, as well as any other elements, limitations, steps, and / or characteristics that do not substantially affect the basic and novel features of the claimed subject matter.Therefore, the open-ended transitional clause “comprising” (and equivalent open-ended transitional clauses) includes, in limited cases, the claimed subject matter as specified by the closed-ended transitional clause “consisting of” or “consisting essentially of.” Thus, the embodiments described or claimed herein with respect to the words “comprising” are clearly, or essentially, described, enabled, and supported herein with respect to the words “consisting essentially of” and “consisting of.”

[0317] All patents, patent publications, and other publications referenced and identified herein are incorporated herein individually and expressly by reference in their entirety for the purpose of describing and disclosing compositions and methods described in such publications, for example, which may be used in connection with the present invention. These publications are provided solely in relation to disclosures prior to the filing date of this application. This should not be construed as an acknowledgment that the inventors do not have prior authority to such disclosures for the sake of prior art or for any other reason. All statements regarding dates or expressions relating to the contents of these documents are based on information available to the applicants and do not constitute an acknowledgment of the accuracy of the dates or contents of these documents.

[0318] Finally, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which is defined only by the claims. Therefore, the invention is not limited to what is precisely shown and described.

[0319] While embodiments of this disclosure are described in considerable detail to encompass possible forms, those skilled in the art will understand that other versions of this disclosure are also possible.

[0320] Although the present invention is described in both a general and detailed form from the perspective of specific embodiments and applications, it should be understood that these descriptions are not intended to limit the scope in any way to such embodiments and applications, and that many substitutions, modifications, and variations in the embodiments, applications described herein, and in the details of the methods and systems and their operation exemplified herein can be made by those skilled in the art without departing from the spirit of the invention.

Claims

1. A method for preventing, halting the progression of, or improving retinopathy, comprising expressing a heterologous BMI1 protein in the patient's membrane pigment epithelial (RPE) cells.

2. The method according to claim 1, wherein the retinopathy is at least one of retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy.

3. The method according to claim 1, wherein the BMI1 protein is transduced by AAV-mediated gene therapy.

4. The method according to claim 1, wherein the BMI1 protein is constitutively expressed.

5. The method according to claim 1, wherein the BMI1 protein is expressed by an inducible promoter.

6. The method according to claim 3, wherein the AAV-mediated gene therapy uses AAV8 type.

7. The method according to claim 3, wherein the AAV-mediated gene therapy uses one of AAV types 1, 2, 5, 7, 9, or rh10.

8. The method according to claim 3, wherein adeno-associated virus (AAV) is introduced by intravitreal injection.

9. The method according to claim 3, wherein adeno-associated virus (AAV) is introduced by subretinal injection, suprachoroidal injection, and / or subinternal limiting membrane injection.

10. A pharmaceutical composition for preventing, halting the progression of, or improving retinopathy, comprising a viral vector and a pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10, wherein the retinopathy is at least one of retinal degeneration, retinal dystrophy, macular degeneration, or macular dystrophy.

12. The pharmaceutical composition according to claim 10, wherein the viral vector particle is of type AAV8.

13. The pharmaceutical composition according to claim 10, wherein the viral vector particles include a nucleic acid sequence encoding a BMI protein.

14. The pharmaceutical composition system according to claim 10, wherein the pharmaceutical composition is introduced into the eye by intravitreal injection, subretinal injection, subinternal limiting membrane injection, or suprachoroidal injection.

15. The pharmaceutical composition according to claim 10, wherein the viral vector is an AAV virion encoding the BMI1 protein.

16. An isolated host cell transduced by or containing the viral vector particles described in claim 15.

17. A method for treating an eye disease in a mammal, comprising the step of administering the viral vector described in claim 15 to the mammal.

18. A method for delivering a therapeutic agent to a target eye, comprising the step of transfecting an eye cell with a viral vector according to claim 15, thereby causing the transfected eye cell to express the therapeutic agent and deliver the agonist to the target eye.

19. A method for preventing, halting the progression of, or improving vision loss associated with retinal degeneration in a patient, comprising the step of administering to the patient a composition comprising a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the adeno-associated virus comprises an AAV virion expressing a heterologous BMI1 protein.

20. A method for preventing, halting the progression of, or improving vision loss associated with retinal dystrophy in a patient, comprising the step of administering to the patient a composition comprising recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the adeno-associated virus comprises an AAV virion expressing a heterologous BMI1 protein.

21. A method for preventing, halting the progression of, or improving vision loss associated with macular degeneration in a patient, comprising the step of administering to the patient a composition comprising a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the adeno-associated virus comprises an AAV virion expressing a heterologous BMI1 protein.

22. A method for preventing, halting the progression of, or improving vision loss associated with macular dystrophy in a patient, comprising the step of administering to the patient a composition comprising a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the adeno-associated virus comprises an AAV virion expressing a heterologous BMI1 protein.

23. A method for preventing, halting the progression of, or improving vision loss associated with diabetic retinopathy, ischemic retinopathy, retinal vein occlusion, retinal artery occlusion, ischemic optic neuropathy and glaucomatous optic neuropathy in a patient, comprising the step of administering to the patient a composition comprising recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the adeno-associated virus comprises an AAV virion expressing a heterologous BMI1 protein.

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