BR112017017867B1 - MODIFIED ADENOASSOCIATED VIRUS (AAV) CAPSID PROTEIN, NUCLEIC ACID SEQUENCE, VIRAL VECTOR PARTICLE, PHARMACEUTICAL COMPOSITION, AS WELL AS USES OF THE VIRAL VECTOR - Google Patents

Abstract

METHODS AND COMPOSITIONS FOR TREATMENT OF GENETIC EYE DISEASES. The present disclosure provides targeting peptides and vectors that contain a sequence encoding targeting peptides that deliver agents to the eyes. The present inventors have discovered peptides that function to target agents, such as viral vectors, to ocular cells. The present description describes a method of using these new peptides to target, for example, viral capsids to the cell type of interest. In this case, ocular cells (such as retinal cells) are targeted by the identified peptides. The capsid proteins carrying the vectors, modified to include these peptides, can be used to deliver therapeutic agents to the eyes.

Description

RELATED REQUEST

[001] This application claims priority to United States Provisional Application No. 62/118,934, filed on February 20, 2015, the entirety of which is incorporated herein by reference.

INTRODUCTION

[002] Gene transfer is now widely recognized as a powerful tool for analyzing biological events and disease processes, both at the cellular and molecular levels. Recently, the application of gene therapy for the treatment of human diseases, whether hereditary (e.g., adenosine deaminase (ADA) deficiency) or acquired (e.g., cancer or infectious disease), has received considerable attention. With the advent of improved gene transfer techniques and the identification of an ever-growing library of diseases related to defective genes, gene therapy has rapidly evolved from a theory for treatment to a practical reality.

[003] Traditionally, gene therapy has been defined as a procedure in which an exogenous gene is introduced into a patient's cells in order to correct an innate genetic error. Although more than 4500 human diseases are currently classified as genetic, specific mutations in the human genome have been identified for relatively few of these diseases. Until recently, these rare genetic diseases represented the exclusive targets of gene therapy efforts. Therefore, most gene therapy protocols approved by the NIH to date have been directed toward introducing a functional copy of a defective gene into the somatic cells of an individual who has a known innate genetic error. Only recently have researchers and clinicians begun to realize that most human cancers, certain forms of cardiovascular disease, and many degenerative diseases also have important genetic components, and for the purposes of designing new gene therapies, should be considered "genetic diseases." . Therefore, gene therapy has more recently been broadly defined as the correction of a disease phenotype by introducing new genetic information into the affected organism.

[004] In in vivo gene therapy, a transferred gene is introduced into the cells of the recipient organism in situ, that is, inside the recipient. In vivo gene therapy has been examined in several animal models. Several recent publications have reported the feasibility of direct in situ gene transfer in tissues and organs such as muscle, hematopoietic stem cells, the arterial wall, the nervous system and lungs. Direct injection of DNA into skeletal muscle, cardiac muscle and injection of DNA-lipid complexes into the vasculature have also been reported to obtain a detectable expression level of the gene product(s) inserted in alive.

[005] Treatment of genetic eye diseases, for example, hereditary genetic eye diseases, such as diseases that cause blindness, remains a problem. Examples of such diseases are retinitis pigmentosa, maculopathies, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, oculocutaneous albinism, glaucoma, Stargardt's disease, choroideremia, macular degeneration related to age, Spinocerebellar Ataxia Type 7 (SCAT), color blindness, and lysosomal storage diseases that affect the cornea, such as Mucopolysaccharidosis (MPS) IV and MPS VII. Therefore, therapies for genetic eye diseases need to be developed.

SUMMARY

[006] The present inventors have discovered peptides that function for targeting agents, such as viral vectors for ocular cells. The present disclosure describes a method for using these new peptides to target, for example, viral capsids to the cell type of interest. In this case, ocular cells (such as retinal cells) are targeted by the identified peptides. Vectors harboring capsid proteins modified to include such peptides can be used to deliver therapeutic agents to the eye.

[007] As used herein, the term "targets" means that the capsid protein of a virus, such as an adeno-associated virus (AAV), preferentially binds to a tissue type (e.g., retinal cells ) over another type of tissue (e.g., brain tissue). In certain embodiments, the genetically modified capsid protein can "target" ocular cells by binding at a level 10% to 1000% higher than a comparable unmodified capsid protein. For example, an AAV with a genetically modified capsid protein can bind to eye cells at a level 50% to 100% higher than an unmodified AAV virus.

[008] The present invention provides a modified adeno-associated virus (AAV) capsid protein that contains a targeting peptide, wherein the targeting peptide is 3 to 10 amino acids (i.e., 3, 4, 5, 6, 7 , 8, 9 or 10 amino acids) in length and wherein the targeting peptide directs an AAV to an eye cell. In certain embodiments, the targeting peptide is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[009] The present invention provides an AAV virus containing capsid protein genetically modified to encode the peptides described above.

[0010] The present invention provides a method for treating diseases (for example, an eye disease) in a mammal by administering the viral vector described above or the cell described above to the mammal. In certain embodiments, the mammal is human. In certain embodiments, the disease is retinitis pigmentosa, maculopathies, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, oculocutaneous albinism, glaucoma, Stargardt's disease, choroideremia, macular degeneration age-related, SCAT, color blindness and lysosomal storage diseases that affect the cornea, such as MPS IV and MPS VII.

[0011] In certain embodiments, the disease is a lysosomal storage disease (LSD). In certain embodiments, the disease is a deficiency or defect in TPP1 (tripeptidyl peptidase I), CLN3 (Batenin), PPT1 (palmitoyl thioesterase I protein), CLN6 (neuronal ceroid lipofuscinosis protein 6) or CLN8 expression or activity. In certain embodiments, the disease is a neurodegenerative disease, such as neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late infantile NCL, juvenile NCL (Batten disease), and adult NCL.

[0012] The present invention provides a method for administering an agent to the eye of an individual by transducing ocular cells with a viral vector described above, such that the transduced ocular cells express the therapeutic agent and administer the agent to the eye of the individual. . In certain embodiments, the agent is Ataxin 7 mirRNA (e.g., to treat SCA 7). In certain embodiments, the agent is the 65 kDa protein specific for retinal pigment epithelium (RPE 65) (e.g., to treat Leber congenital amaurosis). In certain embodiments, the agent is an inhibitor of Vascular Endothelial Growth Factor (VEGF) or soluble VEGF receptor 1 (sFifl) (e.g., to treat age-related macular degeneration). In certain embodiments, the agent is REP1 (e.g., to treat choroideremia). In certain embodiments, the agent is L-opsin (e.g., to treat color blindness). Table 1 sets out exemplary diseases, target genes, and corresponding encoded proteins. TABLE 1

BRIEF DESCRIPTION OF FIGURES

[0013] Figure 1. Peptide-modified AAV (PMAAV)-GST sequence. AAV2/capsid sequence 2 (AAV2 ITR/AAV2 capsid) (in italics) and retinal glutathione S-transferase (GST) target peptide (bold and underlined).

[0014] Figure 2. Fluorescent graphic representation in live animals showed relative infection efficiencies. Spotty regions of green fluorescent protein (GFP) positivity were observed in AAV2/2-injected eyes, whereas there were broad and uniform GFP signals in PMAAV-GST eyes.

[0015] Figure 3. PMAAV-GST.CMV.eGFP targeted the retinal pigment epithelium (RPE) and Outer Nuclear Layer (ONL) layers and some cells in the ganglion cell layer (GCL). AAV2/2 primarily targeted OPL. CMV - Cytomegalovirus; eGFP - enhanced GFP.

[0016] Figure 4. Subretinal injection.

[0017] Figure 5. Unmodified AAV2 - subretinal injection.

[0018] Figure 6. Unmodified AAV2 - subretinal injection.

[0019] 20X images of PM-modified AAV2 - subretinal injection.

[0020] Figure 8. Intravitreal injection.

[0021] Figure 9. Intravitreal injection of AAV2.

[0022] Figure 10. Intravitreal injection of AAV2.

[0023] Figure 11. Intravitreal injection of AAV2 PM GST.

[0024] Figure 12. Intravitreal injection of AAV2 PM GST.

DETAILED DESCRIPTION

[0025] Certain embodiments of the present disclosure provide a viral vector comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that directs the viral vector to ocular cells. In certain embodiments, the eye cell is a retinal cell.

[0026] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is AAV2, although the tropism is modified such that such modifications would be found to alter the tropism of any AAV.

[0027] In certain embodiments, the targeting peptide comprises a sequence of up to 10 amino acids in length having in it (or consisting of) the GSTPPPM (SEQ ID NO: 1) or GETRAPL (SEQ ID NO: 4) amino acid sequence, such as expressed in an amino to carboxy orientation or in a carboxy to amino orientation. It should be noted that the orientation of the sequence is not important. For example, the peptide may be oriented from the amino-terminal end to the carboxy-terminal end of the peptide to be GSTPPPM (SEQ ID NO: 1) or it may be from the amino-terminal end to the carboxy-terminal end of the peptide to be MPPPTSG (SEQ ID NO: 5).

[0028] In certain embodiments, the targeting peptide is inserted between AAV2 capsid residues at positions P34-A35, T138-A139, A139-P140, G453-T454, N587-R588 and/or R588-Q589. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO: 10.

[0029] In certain embodiments, the targeting peptide is inserted after AAV9 capsid residues at positions D384, G385, I560, T561, N562, E563, E564, E565, N704 and/or Y705. An exemplary wild-type reference AAV9 capsid protein sequence is provided in SEQ ID NO: 11.

[0030] In certain embodiments, the capsid protein comprises or consists of SEQ ID NO: 8 or SEQ ID NO: 9.

[0031] The present invention provides a viral vector comprising a nucleic acid encoding capsid protein as described above. In certain embodiments, the viral vector additionally contains a nucleic acid sequence that encodes a nucleic acid of interest. In certain embodiments, the nucleic acid of interest is a therapeutic agent. In certain embodiments, the therapeutic agent is an enzyme or an RNAi molecule (e.g., SiRNA, shRNA, or miRNA molecule). In certain embodiments, the therapeutic agent is Ataxin 7 mirRNA, RPE65, VEGF inhibitor or soluble VEGF receptor 1 (sFifl), REP1, L-opsin, Rho, PDE6β, ABCA4, LRAT, RDS/Peripherin, MERTK, IMPDH1, GUCY2D, RDS/Peripherin, AIPL1, ABCA4, RPGRIP1, IMPDH1, AIPL1, GUCY2D, LRAT, MERTK, RPGRIP1, RPE65, ABCA4, GNAT2, CNGB3, Rsl, OA1, (OCAI) tyrosinase, P21 WAF-1/Cipl, PDGF , Endostatin, Angiostatin, arylsulfatase B or β-glucuronidase.

[0032] Certain embodiments of the present disclosure provide a nucleic acid sequence that encodes a viral vector as described herein.

[0033] Certain embodiments of the present disclosure provide a nucleic acid sequence that encodes a modified capsid as described herein. Certain embodiments of the present disclosure provide a nucleic acid sequence comprising or consisting of SEQ ID NO: 2 (GGGTCGACGCCGCCTCCTATG). Certain embodiments of the present disclosure provide a modified capsid encoded by a nucleic acid sequence described herein.

[0034] Certain embodiments of the present disclosure provide a cell comprising a viral vector as described herein.

[0035] Certain embodiments of the present disclosure provide a cell transduced by a viral vector as described herein.

[0036] In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human cell. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in vivo. In certain embodiments, the cell is an eye cell, such as a retinal cell.

[0037] Certain embodiments of the present disclosure provide a method of treating a disease in a mammal, comprising administering a viral vector or cell as described herein to the mammal. In certain embodiments, the targeting peptide targets a diseased ocular cell. In certain embodiments, the targeting peptide targets an ocular cell in an individual who has retinitis pigmentosa, maculopathies, Leber congenital amaurosis, Leber hereditary optic neuropathy, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, albinism oculocutaneous, glaucoma, Stargardt disease, choroideremia, age-related macular degeneration, SCAT, color blindness, and lysosomal storage diseases that affect the cornea, such as MPS IV and MPS VII.

[0038] In certain embodiments, the individual has a lysosomal storage disease or disorder. In certain embodiments, the disease is a deficiency or defect in TPP1 (tripeptidyl peptidase I), CLN3 (Batenin), PPT1 (palmitoyl thioesterase I protein), CLN6 (neuronal ceroid lipofuscinosis protein 6), or CLN8 expression or activity. The diseases include neurodegenerative diseases such as neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late infantile NCL, juvenile NCL (Batten disease), and adult NCL.

[0039] In certain embodiments, the mammal is human.

[0040] Certain embodiments of the present disclosure provide a method for administering an agent to the eye of an individual, comprising transducing ocular cells with a viral vector described herein, such that the transduced ocular cells express the therapeutic agent and administer the agent in the individual's eye.

[0041] In certain embodiments, the viral vector transduces eye cells.

[0042] In certain embodiments, the transduced ocular cells are within or comprise the retinal outer plexiform layer (OPL).

[0043] Certain embodiments of the present disclosure provide a viral vector or cell as described herein for use in medical treatment or diagnosis.

[0044] Certain embodiments of the present disclosure provide a use of a viral vector or cell, as described herein, to prepare a medicament useful for treating an ocular disease, for example, a genetic ocular disease, in a mammal.

[0045] Certain embodiments of the present disclosure provide a method for identifying peptides that target ocular tissue that comprises using biological selection by phage display in order to identify such peptides.

[0046] The vector may additionally comprise an enzyme, a secreted protein, a nuclear protein or a cytoplasmic protein. As used herein, the term "secreted protein" includes any secreted protein, whether naturally secreted or modified to contain a signal sequence so that it can be secreted. For example, the secreted protein may be betaglucuronidase.

[0047] Nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. Generally speaking, "operably linked" means that the DNA sequences that are linked are contiguous. However, enhancers do not need to be contiguous. Binding is achieved through binding at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. Additionally, multiple copies of the nucleic acids encoding the enzymes can be linked together in the expression vector. Such multiple nucleic acids can be separated by linkers.

[0048] The vector may be an adeno-associated virus (AAV) vector, an adenoviral vector, a retrovirus or a lentivirus vector based on the human immunodeficiency virus or the feline immunodeficiency virus. Examples of such AAVs are found in Davidson et al., PNAS (2000) 97:3428-3432. AAV and lentivirus can provide long-lasting expression while adenovirus can provide transient expression.

[0049] The present disclosure also provides a mammalian cell that contains a vector described herein. The cell could be human and could be from an eye. The cell type can be a population of stem or progenitor cells.

[0050] The present disclosure provides a method of treating a disease, such as a genetic eye disease or cancer in a mammal by administering a polynucleotide, polypeptide, expression vector or cell described herein. Genetic eye disease or cancer may be retinitis pigmentosa, maculopathy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, early onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, oculocutaneous albinism, glaucoma, Stargardt's disease , choroideremia, age-related macular degeneration, SCAT, color blindness, and lysosomal storage diseases that affect the cornea, such as MPS IV and MPS VII.

[0051] The genetic disease may be a lysosomal storage disease or disorder. In certain embodiments, the disease is a deficiency or defect in TPP1 (tripeptidyl peptidase I), CLN3 (Batenin), PPT1 (palmitoyl thioesterase I protein), CLN6 (neuronal ceroid lipofuscinosis protein 6) or CLN8 expression or activity. The genetic disease may be a neurodegenerative disease, for example, a neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late infantile NCL, juvenile NCL (Batten disease) and adult NCL.

[0052] Certain aspects of the disclosure concern polynucleotides, polypeptides, vectors and genetically manipulated cells (modified in vivo) and the use of these. In particular, the disclosure relates to a method for gene or protein therapy that is capable of local administration of a therapeutically effective dose of the therapeutic agent.

[0053] According to one aspect, a cellular expression system for expressing a therapeutic agent in a mammalian recipient is provided. The expression system (also referred to herein as a "genetically modified cell") comprises a cell and an expression vector for expressing the therapeutic agent. Expression vectors include, but are not limited to, viruses, plasmids, and other vehicles for delivering heterologous genetic material to cells. Therefore, the term "expression vector" as used herein refers to a vehicle for delivering heterologous genetic material to a cell. In particular, the expression vector is an adenovirus, adeno-associated virus, or recombinant lentivirus or retrovirus vector.

[0054] The expression vector additionally includes a promoter to control transcription of the heterologous gene. The promoter may be an inducible promoter (described below). The expression system is suitable for administration to the mammalian recipient. The expression system may comprise a plurality of non-immortalized genetically modified cells, each cell containing at least one recombinant gene encoding at least one therapeutic agent.

[0055] The cellular expression system can be formed in vivo. According to yet another aspect, a method for treating a mammalian receptor in vivo is provided. The method includes introducing an expression vector to express a heterologous gene product into a patient's cell in situ, such as through intraocular administration. To form the in vivo expression system, an expression vector for expressing the therapeutic agent is introduced in vivo into the mammalian recipient.

[0056] According to yet another aspect, a method for targeting a mammalian receptor in vivo is provided. The method includes introducing the target protein into the patient in vivo.

[0057] The expression vector for expression of the heterologous gene may include an inducible promoter to control transcription of the heterologous gene product. Accordingly, in situ administration of the therapeutic agent is controlled by exposing the in situ cell to conditions that induce transcription of the heterologous gene.

[0058] The mammalian recipient may have a condition that is amenable to gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase "condition amenable to gene replacement therapy" encompasses conditions such as genetic diseases (e.g., a disease condition that is attributable to one or more genetic defects), acquired conditions (i.e., a pathological condition - gic that is not attributable to an innate defect), cancers and prophylactic processes (i.e. prevention of a disease or an undesirable medical condition). Therefore, as used herein, the term "therapeutic agent" refers to any agent or material, which has a beneficial effect on the mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules with nucleic acid or protein components.

[0059] According to one embodiment, the mammalian recipient has a genetic disease and the exogenous genetic material comprises a heterologous gene that encodes a therapeutic agent for treating the disease. In yet another embodiment, the mammalian recipient has an acquired pathology and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the pathology. According to another embodiment, the patient has a cancer and the exogenous genetic material comprises a heterologous gene encoding an antineoplastic agent. In yet another embodiment, the patient has an unwanted medical condition and the exogenous genetic material comprises a heterologous gene that encodes a therapeutic agent for treating the condition.

[0060] According to yet another embodiment, a pharmaceutical composition is disclosed. The pharmaceutical composition comprises a plurality of the genetically modified cells or polypeptides described above and a pharmaceutically acceptable carrier. The pharmaceutical composition may be for the treatment of a condition amenable to gene replacement therapy and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for the treatment of the condition. The pharmaceutical composition may contain an amount of genetically modified cells or polypeptides sufficient to deliver a therapeutically effective dose of the therapeutic agent to the patient. Exemplary conditions amenable to gene replacement therapy are described below.

[0061] According to another aspect, a method for forming the pharmaceutical composition described above is provided. The method includes introducing an expression vector to express a heterologous gene product into a cell to form a genetically modified cell and placing the genetically modified cell in a pharmaceutically acceptable carrier.

[0062] These and other aspects, as well as various advantages and uses will be more evident with reference to the detailed description and the attached figures.

[0063] As used herein, the term "enzyme", a "secreted protein", a "nuclear protein" or a "cytoplasmic protein" includes biologically active or inactive variants or fragments of these polypeptides. A "variant" of one of the polypeptides is a polypeptide that is not completely identical to a native protein. Such a variant protein can be obtained by altering the amino acid sequence through the insertion, deletion or substitution of one or more amino acids. The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide with substantially the same, or improved, qualities compared to the native polypeptide. The replacement may be a conserved replacement. A "conserved substitution" is a substitution of one amino acid for another amino acid with a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest possible change in the charge of the amino acid or size of the amino acid's side chain (alternatively, in the size, charge, or type of chemical group within the side chain), such that the peptide generally maintains its spatial conformation, but the biological activity is altered. For example, common conserved changes might be Asp to Glu, Asn, or Gln; His for Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to replace other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine with non-polar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine with uncharged polar side chains; aspartate and glutamate with acidic side chains; and lysine, arginine and histidine with basic side chains.

[0064] Amino acid changes are achieved by altering the codons of the corresponding nucleic acid sequence. It is known that these polypeptides can be obtained based on the replacement of some amino acids with other amino acids in the polypeptide structure, in order to modify or improve biological activity. For example, through the substitution of alternative amino acids, small conformational changes can be imparted to a polypeptide, which results in greater activity. Alternatively, amino acid substitutions in certain polypeptides can be used to provide residues, which can then be linked to other molecules to provide molecule-peptide conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.

[0065] The hydropathic index of amino acids can be used to confer the interactive biological function in a polypeptide, in which it has been found that certain amino acids can be replaced by other amino acids that have similar hydropathic indexes and still retain a similar biological activity . Alternatively, substitution of similar amino acids can be made based on hydrophilicity, particularly where the desired biological function in the polypeptide to be generated is intended for use in immunological modalities. The greater local average hydrophilicity of a "protein", as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Therefore, it should be noted that substitutions can be made based on the hydrophilicity attributed to each amino acid.

[0066] When using the hydrophilicity index or the hydropathic index, which assigns values to each amino acid, it is preferred to perform amino acid substitutions in which these values are ± 2, with ± 1 being particularly preferred and those with ± 0, 5, being the most preferred substitutions.

[0067] The variant protein has at least 50%, at least about 80%, or even at least about 90%, although less than 100%, contiguous amino acid sequence homology or identity with the amino acid sequence of a corresponding native protein.

[0068] The amino acid sequence of the color variant polypeptide responds essentially to the amino acid sequence of the native polypeptide. As used herein "essentially corresponds to" refers to a polypeptide sequence that will elicit a biological response substantially the same as the response generated by the native protein. Such a response may be at least 60% of the level generated by the native protein, and may further be at least 80% of the level generated by the native protein.

[0069] A variant may include amino acid residues that are not present in the corresponding native protein or deletions in relation to the corresponding native protein. A variant may also be a "fragment" that is truncated compared to the corresponding native protein, that is, only a portion of a complete protein. Protein variants also include peptides with at least one D-amino acid.

[0070] The variant protein can be expressed from an isolated DNA sequence that encodes the variant protein. "Recombinant" is defined as a peptide or nucleic acid produced by genetic engineering processes. It should be noted that it is well known in the art that, due to the redundancy of the genetic code, individual nucleotides can be easily exchanged in a codon, and still result in an identical amino acid sequence. The terms "protein", "peptide" and "polypeptide" are used interchangeably herein.

[0071] The present disclosure provides methods of treating a disease in a mammal by administering an expression vector to a cell or patient. For gene therapy methods, one skilled in the art of molecular biology and gene therapy would be able to determine, without undue experimentation, the appropriate dosages and routes of expression vector administration used in the innovative methods of the present disclosure.

[0072] According to one embodiment, the cells are transformed or otherwise genetically modified in vivo. Mammalian recipient cells are transformed (i.e., transduced or transfected) in vivo with a vector containing exogenous genetic material for the expression of a heterologous (e.g., recombinant) gene encoding a therapeutic agent and the therapeutic agent is administered. in situ.

[0073] As used herein, "exogenous genetic material" refers to a nucleic acid or an oligonucleotide, natural or synthetic, that is not naturally found in cells; or if it is naturally found in cells, it is not transcribed or expressed at biologically significant levels by cells. Thus, "exogenous genetic material" includes, for example, a non-naturally occurring nucleic acid that can be transcribed into antisense RNA, as well as a "heterologous gene" (i.e., a gene that encodes a protein that is not expressed or is expressed at biologically insignificant levels in a naturally occurring cell of the same type).

[0074] In certain embodiments, the mammalian recipient has a condition that is amenable to gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase "condition amenable to gene replacement therapy" encompasses conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more genetic defects), acquired conditions (i.e., a pathological condition that is not attributable to an innate defect), cancers, and prophylactic processes (i.e., prevention of a disease or undesirable medical condition). Therefore, as used herein, the term "therapeutic agent" refers to any agent or material, which has a beneficial effect on the mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules with nucleic acid (e.g., antisense RNA) or protein components.

[0075] A number of genetic eye diseases are known. Effective therapeutic agents against these diseases are also known, as it is the protein/enzyme that is known to be deficient in these disorders. In certain embodiments, the disease or condition is retinitis pigmentosa, maculopathies, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, oculocutaneous albinism, glaucoma, Stargardt's disease, choroideremia, age-related macular degeneration, SCAT, color blindness, and lysosomal storage diseases that affect the cornea, such as MPS IV and MPS VII.

[0076] In certain embodiments, the disease or condition may be a lysosomal storage disease (LSD). In certain embodiments, the disease is a deficiency or defect in TPP1 (tripeptidyl peptidase I), CLN3 (Batenin), PPT1 (palmitoyl thioesterase protein I), CLN6 (neuronal ceroid lipofuscinosis protein 6) or expression or activity of CLN8. The disease may be a neurodegenerative disease, such as neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late infantile NCL, juvenile NCL (Batten disease) and adult NCL.

[0077] As used herein, "acquired pathology" refers to a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural or molecular biological state. Exemplary acquired pathologies are provided in Table 2. Therapeutic agents effective against these diseases are also provided. TABLE 2. Potential Gene Therapies for Acquired Pathologies. Corresponding Proteins Encoded by the Genes are set out in Table 1

[0078] Alternatively, the condition amenable to gene replacement therapy is a prophylactic process, that is, a process for the prevention of disease or an undesirable medical condition. Thus, the present disclosure encompasses a cellular expression system for administering a therapeutic agent that has a prophylactic function (i.e., a prophylactic agent) to the mammalian recipient.

[0079] In summary, the term "therapeutic agent" includes, but is not limited to, the agents listed in the Tables above, as well as their functional equivalents. As used herein, the term "functional equivalent" refers to a molecule (e.g., a peptide or protein) that has the same or enhanced beneficial effect on the mammalian receptor as the therapeutic agent of which it is considered. a functional equivalent. As will be appreciated by one skilled in the art, functionally equivalent proteins can be produced by recombinant techniques, for example, by expressing a "functionally equivalent DNA". As used herein, the term "functionally equivalent DNA" refers to a non-naturally occurring DNA which encodes a therapeutic agent. For example, many, if not all, of the agents disclosed in Tables 1-2 have known amino acid sequences, which are encoded by naturally occurring nucleic acids. However, due to the degeneration of the genetic code, more than one nucleic acid can encode the same therapeutic agent. Accordingly, the present disclosure encompasses therapeutic agents encoded by naturally occurring DNAs, as well as non-naturally occurring DNAs, which encode the same protein as encoded by the naturally occurring DNA.

[0080] The therapeutic agents disclosed above and the conditions amenable to gene replacement therapy are merely illustrative and are not intended to limit the scope of the present disclosure. The selection of a suitable therapeutic agent for the treatment of a condition is considered to be within the scope of one skilled in the art without excessive experimentation.

SCREENING METHODS

[0081] The present disclosure provides methods for screening and identifying amino acid sequences that aim, for example, to specifically target, a specific area, such as the vasculature of the central nervous system. This method can be used to identify targeting sequences that are specific to specific disease states. In other words, targeting sequences can be identified and used to treat specific diseases.

AAV VECTORS

[0082] Adeno-associated virus (AAV) is a small (20 nm) non-pathogenic virus that is useful in treating human diseases, such as Parkinson's disease and recessive genetic diseases. A construct is generated that surrounds a promoter linked to a target gene with AAV inverted terminal repeat (ITR) sequences.

[0083] In one embodiment, a viral vector of dissemination is an AAV vector. An "AAV" vector refers to an adeno-associated virus, and can be used to refer to the naturally occurring wild-type virus itself or its derivatives. The term encompasses all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, unless otherwise required. As used herein, the term "serotype" refers to an AAV that is identified and distinguished from other AAVs based on the reactivity of the capsid protein with defined antisera; for example, there are many known serotypes of primate AAVs ( for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV-Rh74 and AAVRh10, and modified capsids of these serotypes). For example, the AAV-2 serotype is used to refer to an AAV that contains capsid proteins encoded from the AAV-2 cap gene and a genome containing 5' and 3' ITR sequences from the same AAV serotype. -two. The AAV pseudotype refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs from a second serotype. The rAAV pseudotype would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. The rAAV pseudotype is produced using standard techniques described in the art. As used herein, for example, rAAV1 may be used to refer to an AAV with both capsid proteins and 5'-3' ITRs of the same serotype or may refer to an AAV with serotype 1 capsid proteins. and 5'-3' ITRs from a different AAV serotype, e.g., AAV serotype 2. For each example illustrated in this document, the description of vector design and production describes the capsid serotype and the 5'-3' ITR sequences. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").

[0084] An "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably all capsid proteins of a wild-type AAV) and a polynucleotide encapsulated. If the particle comprises the heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be administered to a mammalian cell), it is typically referred to as "rAAV".

[0085] In one embodiment, AAV expression vectors are constructed using known techniques to provide, at least as operably linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a region of transcriptional termination. The control elements are selected to be functional in a mammalian cell. The resulting construct, which contains the operably linked components, is flanked (5' and 3') with functional AAV ITR sequences.

[0086] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" are meant the art-recognized regions found at each end of the AAV genome that function together in cis as origins of DNA replication and as signals packaging for the virus. AAV ITRs, together with the AAV rep coding region, provide efficient excision, rescue, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.

[0087] The nucleotide sequences of the AAV ITR regions are known. See, for example, Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K.I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.). As used herein, an "AAV ITR" need not have the wild-type nucleotide sequence represented, but may be altered, for example, by insertion, deletion, or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRh10 and capsids. modified from these serotypes. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV vector do not necessarily need to be identical or derived from the same AAV serotype or isolated in order to function as intended, i.e. to allow excision and recovery of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the genome of the recipient cell when AAV Rep gene products are present in the cell.

[0088] In one embodiment, the AAV ITRs may be derived from any of several AAV serotypes, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV- Rh74 (Rhesus monkey-derived AAV) AND AAVRh10. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV expression vector do not necessarily need to be identical or derived from the same AAV serotype or isolated in order to function as intended, i.e. to allow excision and recovery of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the genome of the recipient cell when AAV Rep gene products are present in the cell.

[0089] In one embodiment, AAV capsids can be derived from any of several AAV serotypes, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV- Rh74 (Rhesus monkey-derived AAV) and AA-VRh10, and the AAV ITRs are derived from AAV serotype 2. In certain embodiments, the AAV capsid is the AAV2 capsid. Suitable DNA molecules for use in AAV vectors will be less than about 5 kilobases (kb), less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about of 3 kb, less than about 2.5 kb in size and are known in the art.

[0090] In some embodiments of the disclosure, DNA molecules for use in AAV vectors will contain one or more copies of a unique siRNA sequence. As used herein, the term multiple copies of siRNA sequences means at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least at least 9 copies and at least 10 copies. In some embodiments, the DNA molecules for use in the AAV vectors will contain multiple siRNA sequences. As used herein, the term multiple siRNA sequences means at least 2 siRNA sequences, at least 3 siRNA sequences, at least 4 siRNA sequences, at least 5 siRNA sequences, at least 6 siRNA sequences, at least 7 siRNA sequences, at least 8 siRNA sequences, at least 9 siRNA sequences and at least 10 siRNA sequences. In some embodiments, suitable DNA vectors of the disclosure will contain a sequence encoding the siRNA molecule of the disclosure and a filler fragment. Suitable filler fragments of the disclosure include sequences known in the art, including, without limitation, sequences that do not encode an expressed protein molecule; sequences encoding a normal cellular protein that would have no detrimental effect on the cell types in which it was expressed; and sequences that would not encode a functional siRNA duplex molecule.

[0091] In one embodiment, DNA molecules suitable for use in AAV vectors will be less than about 5 kilobases (kb) in size and will include, for example, a filler sequence and a sequence encoding an siRNA molecule of the disclosure . For example, in order to prevent any packaging of AAV genomic sequences containing the rep and cap genes, a plasmid containing the rep and cap DNA fragment can be modified by the inclusion of a filler fragment as is known in the art. in the AAV genome that causes the DNA to exceed the length for optimal packaging. Thus, the helper fragment is not packaged into AAV virions. This is a safety feature that ensures that only a recombinant AAV vector genome that does not exceed the optimal packaging size is packaged into virions. An AAV helper fragment incorporating a filler sequence may exceed the wild-type genome length of 4.6 kb, and lengths above 105% of the wild type will generally not be packaged. The filler fragment can be derived, for example, from such non-viral sources as the Lac-Z or beta-galactosidase gene.

[0092] In one embodiment, the selected nucleotide sequence is operably linked to control elements that direct their transcription or expression in the individual in vivo. Such control elements may comprise control sequences normally associated with the selected gene. Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early (CMVIE) promoter region, a Rous sarcoma virus (RSV) promoter, pol II promoters, of pol III, synthetic promoters, hybrid promoters and the like. Furthermore, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use in this document. Such promoter sequences are commercially available from, for example, Stratagene (San Diego, Calif.).

[0093] In one embodiment, both heterologous promoters and other control elements, such as retina-specific and inducible promoters, enhancers and the like, will be of specific use. Examples of heterologous promoters include the CMV promoter. Examples of retinal-specific promoters include IRBP (interphotoreceptor retinoid-binding protein) promoter, hGRK1 (human rhodopsin kinase) promoter, IRBPe/GNAT2 (an interphotoreceptor retinoid-binding protein promoter enhancer element), and a minimal human transducin alpha subunit promoter sequence are examples of a retina-specific promoter.

[0094] In one embodiment, the AAV expression vector harboring the DNA molecule of interest, bound by the AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome that has had the AAV major open reading frames excised from it. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remains to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art.

[0095] Alternatively, AAV ITRs can be excised from the viral genome or an AAV vector containing the same and fused 5' and 3' to a selected nucleic acid construct that is present in another vector using standard ligation techniques. For example, binding can be achieved in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 gg/mL BSA, 10 mM-50 mM NaCl, and 40 μM ATP. , 0.01-0.02 (Weiss) unit of T4 DNA ligase at 0 °C (for "sticky end" binding), or 1 mM ATP, 0.30.6 (Weiss) unit of T4 DNA ligase at 14 °C (for "blunt end" connection). "Stick-end" intermolecular ligations are typically performed at DNA concentrations of 30100 μg/mL (total final concentration 5-100 nM). AAV vectors that contain ITRs. In particular, several AAV vectors are described herein, which are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225 and 53226.

[0096] Furthermore, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared using conventional methods.

[0097] In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as through transfection. Various transfection techniques are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid administration using high-speed microprojectiles.

[0098] In one embodiment, host cells suitable for the production of rAAV virions include microorganisms, yeast cells, insect cells and mammalian cells, which can be, or have been, used as receptors for a DNA molecule. heterologous. The term includes the progeny of the original cell that was transfected. Thus, a "host cell", as used herein, generally refers to a cell that has been transfected with an exogenous DNA sequence. Cells of the stable human cell line, 293 (readily available, for example, through the American Type Culture Collection under ATCC accession number CRL1573), can be used in the practice of the present disclosure. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with type 5 adenovirus DNA fragments, and expresses the Ela and Elb adenoviral genes. The 293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV virions.

[0099] In one embodiment, host cells containing the AAV expression vectors described above are depicted capable of providing AAV auxiliary functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce virions of rAAV. AAV helper functions are generally AAV-derived coding sequences that can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to supplement necessary AAV functions that are missing from AAV expression vectors. Thus, AAV auxiliary functions include one or both of the main AAV ORFs (open reading frames), i.e., the rep and cap coding regions, or functional homologs thereof.

[00100] These Rep expression products have been shown to have many functions, including, among others: recognition, binding and cutting of the AAV origin of DNA replication; DNA helicase activity and transcription modulation of AAV (or other heterologous) promoters. Cap expression products provide the necessary packaging functions. AAV auxiliary functions are used in this document to complement in-trans AAV functions that are absent from AAV vectors.

[00101] In one embodiment, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct, prior to or simultaneously with transfection of the AAV expression vector. The term "AAV helper construct" generally refers to a nucleic acid molecule that includes nucleotide sequences that provide AAV functions deleted from an AAV vector that must be used to produce an transduction vector for delivery of a nucleotide sequence of interest. AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are required for lytic AAV replication; however, the helper constructs lack AAV ITRs and cannot replicate or package. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 that encode both Rep and Cap expression products. Several other vectors have been described, which encode expression products. Rep and/or Cap.

[00102] By "AAV rep coding region" is meant the technically recognized region of the AAV genome that encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. It has been shown that these expression products Rep have many functions, including recognition, binding and cutting of the AAV origin of DNA replication, DNA helicase activity and the modulation of transcription from AAV (or other heterologous) promoters. Rep expression products are collectively required for AAV genome replication. Suitable homologues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication.

[00103] By "AAV cap coding region" is meant the technically recognized region of the AAV genome that encodes the capsid proteins VP1, VP2 and VP3, or functional homologues thereof. These Cap expression products provide the packaging functions that are collectively required for packaging the viral genome. In certain embodiments, modifications, such as insertions, are made in AAV2 capsid proteins at P34-A35, T138-A139, A139-P140, G453-T454, N587-R588 and/or R588-Q589. In certain embodiments, insertions are made in D384, G385, I560, T561, N562, E563, E564, E565, N704 and/or Y705 of AAV9.

[00104] In one embodiment, both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes that confer antibiotic resistance or sensitivity, confer color or alter the antigenic characteristics of those cells that have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium. Several selectable marker genes that are useful in the practice of disclosure include the hygromycin B resistance gene (encoding Aminoglycoside phosphotransferase (APH)) which allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Mo.). Other suitable markers are known to those skilled in the art.

[00105] In one embodiment, the host cell (or packaging cell) is made capable of providing non-AAV-derived functions, or "accessory functions", to produce rAAV virions. Accessory functions are non-AAV-derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, accessory functions include at least those non-AAV proteins and RNAs that are required in AAV replication, including those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Virus-based accessory functions can be derived from any of the known helper viruses.

[00106] In one embodiment, accessory functions can be introduced and then expressed in host cells using methods known to those skilled in the art. Commonly, accessory functions are provided by infection of host cells with an unrelated helper virus. Several suitable helper viruses are known, including adenoviruses; herpesviruses, such as herpes simplex viruses types 1 and 2; and vaccinia virus. Non-viral accessory functions will also find use herein, such as those provided by cellular synchronization using any of several known agents.

[00107] In one embodiment, accessory functions are provided using an accessory function vector. Accessory function vectors include nucleotide sequences that provide one or more accessory functions. An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon, or cosmid. Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments that, when associated with appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions.

[00108] In one embodiment, the nucleic acid sequences that provide the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. . In this regard, adenovirus-derived accessory functions have been extensively studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. Specifically, the early adenoviral gene regions E1a, E2a, E4, VAI RNA, and possibly E1b are thought to participate in the accessory process. Herpesvirus-derived accessory functions have been described. Accessory functions derived from vaccinia viruses have also been described.

[00109] In one embodiment, as a consequence of infection of the host cell with a helper virus or transfection of the host cell with an accessory function vector, accessory functions are expressed, which transactivate the AAV helper construct to produce Rep and/or Cap proteins of AAV. Rep expression products excise recombinant DNA (including the DNA of interest) from the AAV expression vector. Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication occurs, and the DNA is packaged into rAAV virions.

[00110] In one embodiment, following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as CsC1 gradients. Furthermore, if the infection is employed to express accessory functions, the residual helper virus can be inactivated using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C, for example, for 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable, while helper adenovirus is heat labile. The resulting rAAV virions are then ready for use for DNA delivery into the individual's eye.

[00111] Viral vector delivery methods include, but are not limited to, intraocular, subretinal and intravitreal routes. In general, rAAV virions can be introduced into eye cells using in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cell will be removed from the individual, transduced with rAAV virions, and reintroduced into the individual. Alternatively, syngeneic or xenogeneic cells can be used where the cells will not generate an inappropriate immune response in the individual.

[00112] Suitable methods for administering and introducing transduced cells into an individual have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with eye cells, for example, in suitable media, and screening for those cells harboring the DNA of interest can be screened using conventional techniques such as Southern blots and/or PCR, or through the use of selectable markers. The transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and the composition introduced into the individual by various techniques, such as by means of grafting, intramuscular, intravenous, subcutaneous and intraperitoneal injection.

[00113] In one embodiment, for in vivo administration, rAAV virions are formulated into pharmaceutical compositions and, in general, will be administered intraocularly, for example, by subretinal injection.

[00114] In one embodiment, the pharmaceutical compositions will comprise genetic material sufficient to produce a therapeutically effective amount of the nucleic acid of interest, that is, in an amount sufficient to reduce or attenuate the symptoms of the disease state in question, or a sufficient quantity to confer the desired benefit. The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80 and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may include, for example, acid salts of minerals, such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in such vehicles. A complete discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[00115] As is evident to those skilled in the art, in view of the teachings of this specification, an effective amount of the viral vector that should be added can be determined empirically. Administration can be carried out in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages of administration are well known to those skilled in the art and will vary with the viral vector, the composition of the therapy, the target cells and the individual being treated. Single and multiple administrations can be performed at the dose level and pattern selected by the treating physician.

[00116] It should be understood that more than one transgene can be expressed by the administered viral vector. Alternatively, separate vectors, each expressing one or more different transgenes, can also be administered to the eye as described herein. Furthermore, it is also intended that the viral vectors administered by the methods of the present disclosure will be combined with other suitable compositions and therapies.

METHODS FOR INTRODUCING GENETIC MATERIAL INTO CELLS

[00117] Exogenous genetic material (e.g., a cDNA encoding one or more therapeutic proteins) is introduced into the cell ex vivo or in vivo through genetic transfer methods, such as transfection or transduction, to provide a cell with genetically modified. Various expression vectors (i.e., vehicles to facilitate the administration of exogenous genetic material into a target cell) are known to one of skill in the art.

[00118] As used herein, "cell transfection" refers to the acquisition by a cell of new genetic material through the incorporation of added DNA. Thus, transfection refers to the insertion of nucleic acid into a cell using physical or chemical methods. Various transfection techniques are known to those skilled in the art, including: co-precipitation of DNA with calcium phosphate; DEAE-dextran; electroporation; transfection mediated by cationic liposomes; and microparticle bombardment facilitated by tungsten particles. Co-precipitation of DNA with strontium phosphate is another possible transfection method.

[00119] On the other hand, "cell transduction" refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. An RNA virus (i.e., a retrovirus) for transferring a nucleic acid into a cell is referred to herein as a transducing chimeric retrovirus. The exogenous genetic material contained within the retrovirus is incorporated into the genome of the transduced cell. A cell that has been transduced with a chimeric DNA virus (e.g., an adenovirus carrying a cDNA encoding a therapeutic agent) will not have exogenous genetic material incorporated into its genome, but will be able to express exogenous genetic material that is maintained extrachromosomally within the cell.

[00120] Typically, the exogenous genetic material includes the heterologous gene (generally in the form of a cDNA comprising the exons encoding the therapeutic protein) together with a promoter to control transcription of the new gene. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (e.g., enhancers) necessary to obtain the desired gene transcriptional activity. For the purposes of this discussion an "enhancer" is simply any untranslated DNA sequence that functions contiguous with the coding sequence (in cis) to alter the basal transcription level dictated by the promoter. Exogenous genetic material can be introduced into the cellular genome immediately downstream of the promoter so that the promoter and the coding sequence are operably linked to allow transcription of the coding sequence. A retroviral expression vector may include an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.

[00121] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, a gene under the control of a constitutive promoter is expressed under all cell growth conditions. Exemplary constitutive promoters include the promoters for the following genes that encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol - rol mutase, the actin promoter and other constitutive promoters known to those skilled in the art. Furthermore, many viral promoters function constitutively in eukaryotic cells. These include: the SV40 early and late promoters; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the herpes simplex virus thymidine kinase promoter, among many others. Therefore, any of the aforementioned constitutive promoters can be used to control transcription of a heterologous gene insertion.

[00122] Genes that are under the control of inducible promoters are expressed only or to a higher degree in the presence of an inducing agent (for example, transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metallic ions). Inducible promoters include responsive elements (REs) that stimulate transcription when their inducing factors are turned on. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters that contain a specific ER can be chosen in order to obtain an inducible response and, in some cases, the ER itself can be linked to a different promoter, thereby conferring inducibility to the recombinant gene. In this way, through the selection of the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and the level of expression of a therapeutic agent in the genetically modified cell. If the gene encoding the therapeutic agent is under the control of an inducible promoter, in situ administration of the therapeutic agent is triggered by exposing the in situ genetically modified cell to conditions to permit transcription of the therapeutic agent, e.g. intraperitoneal injection of specific inducers of inducible promoters that control the transcription of the agent. For example, in situ expression by genetically modified cells of a therapeutic agent encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate metal ions (i.e., induction) in situ.

[00123] Therefore, the amount of therapeutic agent that is administered in situ is regulated by controlling factors such as: (1) the nature of the promoter used to direct transcription of the inserted gene, (i.e., whether the promoter is constitutive or inducible, strong or weak); (2) the number of copies of the exogenous gene that is inserted into the cell; (3) the number of transduced/transfected cells that is administered (e.g., implanted) to the patient; (4) the size of the implant (e.g., grafted or encapsulated expression system); (5) the number of implants; (6) the period of time that the transduced/transfected cells or implants are left in place; and (7) the rate of production of the therapeutic agent by the genetically modified cell. The selection and optimization of these factors for the administration of a therapeutically effective dose of a specific therapeutic agent are considered to be within the scope of one of skill in the art, without unnecessary experimentation, taking into account the factors disclosed above and the clinical profile of the patient. .

[00124] In addition to at least one promoter and at least one heterologous nucleic acid encoding the therapeutic agent, the expression vector may include a selection gene, for example, a neomycin resistance gene, to facilitate selection of cells that were transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), the other vector containing a selection gene. The selection of a suitable promoter, enhancer, selection gene and/or signal sequence (described below) is considered to be within the scope of one skilled in the art, without undue experimentation.

[00125] The therapeutic agent can be targeted for administration to an extracellular, intracellular or membrane location. If it is desirable for the gene product to be secreted by the cells, the expression vector is designed to include a secretion "signal" sequence suitable for secretion of 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. In a similar way, the expression vector can be constructed to include "retention" signal sequences for anchoring the therapeutic agent within the plasma membrane of the cell. For example, all membrane proteins have hydrophobic transmembrane regions, which prevent translocation of the membrane protein and do not allow the protein to be secreted. The construction of an expression vector including signal sequences for targeting a gene product to a specific location is considered to be within the scope of one skilled in the art without the need for undue experimentation.

[00126] The following discussion is directed to various uses of the present disclosure. For example, the present disclosure has utility as an expression system suitable for detoxifying intra- and/or extracellular toxins in situ. By binding or omitting the appropriate signal sequence to a gene encoding a therapeutic agent capable of detoxifying a toxin, the therapeutic agent can be targeted for administration to the extracellular environment, the cellular plasma membrane, or an intracellular location. In one embodiment, the exogenous genetic material containing a gene encoding an intracellular detoxifying therapeutic agent further includes sequences encoding surface receptors to facilitate transport of extracellular toxins into the cell where they can be detoxified intracellularly by the therapeutic agent. . Alternatively, cells can be genetically modified to express the detoxifying therapeutic agent anchored within the cellular plasma membrane, such that the active portion extends into the extracellular environment. The active portion of the therapeutic agent bound to the membrane detoxifies toxins, which are present in the extracellular environment.

[00127] In addition to the therapeutic agents described above, some of which are directed toward intracellular retention, the present disclosure also encompasses agents intended for administration to the extracellular environment and/or agents intended to be anchored in the cellular plasma membrane.

[00128] The selection and optimization of a particular expression vector to express a specific gene product in an isolated cell is achieved by obtaining the gene, potentially with one or more appropriate control regions (e.g., promoter, insertion sequence) ; preparing a construction vector comprising the vector into which the gene is inserted; transfecting or transducing cells cultured in vitro with the vector construct; and determine whether the gene product is present in the cultured cells. In certain embodiments, a virus from the adeno-associated virus family is used. In certain embodiments, an expression vector for gene therapy based on AAV2, AAV4 and/or AAV5 is used.

[00129] Thus, as will be evident to one skilled in the art, a variety of suitable viral expression vectors are available for transferring exogenous genetic material into cells. The selection of an appropriate expression vector for expressing a therapeutic agent for a particular condition amenable to gene replacement therapy and the optimization of conditions for insertion of the selected expression vector into the cell are within the scope of one skilled in the art without the need of undue experimentation.

[00130] In an alternative embodiment, the expression vector is in the form of a plasmid, which is transferred to target cells by one of a variety of methods: physical (e.g., microinjection, electroporation, scrape loading, microparticle bombardment) or by cellular uptake as a chemical complex (e.g. calcium or strontium co-precipitation, lipid complexation, ligand complexation). Several commercial products are available for complexing cationic liposomes including LipofectinTM (Gibco-BRL, Gaithersburg, Md.) and TransfectamTM (ProMega, Madison, Wis.). However, the efficiency of transfection by these methods is highly dependent on the nature of the target cell and, therefore, conditions for optimal transfection of nucleic acids into cells using the procedures mentioned above must be optimized. Such optimization is within the scope of one skilled in the art without the need for undue experimentation.

[00131] The present disclosure also provides various methods for making and using the genetically modified cells described above. As used herein, the term "isolate" means a cell or a plurality of cells that have been removed from their naturally occurring in vivo location. Methods for removing cells from a patient, as well as methods for maintaining isolated cells in culture, are known to those skilled in the art.

[00132] The present disclosure also provides methods for genetically modifying cells of a mammalian recipient in vivo. According to one embodiment, the method comprises introducing an expression vector for expressing a heterologous gene product into the cells of the mammalian recipient in situ by, for example, injecting the vector into the recipient.

[00133] In one embodiment, the genetically modified cell preparation contains a quantity of cells sufficient to deliver a therapeutically effective dose of the therapeutic agent to the recipient in situ. Determination of a therapeutically effective dose of a specific therapeutic agent for a known condition is within the scope of one skilled in the art without the need for undue experimentation. Thus, in determining the effective dose, one skilled in the art would consider the condition of the patient, the severity of the condition, as well as the results of clinical studies of the specific therapeutic agent being administered.

[00134] If the genetically modified cells are not already present on a pharmaceutically acceptable carrier, they are placed on such a carrier prior to administration to the recipient. Such pharmaceutically acceptable carriers include, for example, isotonic saline and other buffers as appropriate for the patient and therapy.

[00135] More than one recombinant gene can be introduced into each genetically modified cell in the same or different vectors, thus allowing the expression of multiple therapeutic agents by a single cell.

EXAMPLE 1 Preparation of Modified AAVs Targeting Eye Cells

[00136] The present inventors have designed AAVs that are modified to target ocular cells after local administration. To generate an adeno-associated virus (AAV) that targets ocular cells, the inventors first used in vivo phage display selection to identify peptide motifs that preferentially bind to ocular cells. A phage display library was injected into the eyes of mice, and eye cells were subsequently isolated along with binding phage. The isolated phage was then amplified and reinjected, and after five rounds of such in vivo selection, DNA sequencing of the recovered phage revealed an enrichment of peptide motifs distinct from the initial phage library.

[00137] In mice, the peptide motifs GSTPPPM (SEQ ID NO: 1) and GETRAPL (SEQ ID NO: 4) were identified. To confirm the affinity of these phages for ocular cells, each phage was individually reinjected intravenously into mice and visualized. According to the selection results, each selected phage accumulated in the eye beyond the background levels observed for the controls.

[00138] Peptide-modified AAVs were generated by inserting peptides identified from phage display selection into the AAV2 capsid. Peptides were inserted between positions 587 and 588 of the VP3 capsid protein to produce clones. AAV-WT (wild type, no insertion) served as control virus. The 587/588 site is located in a domain of the VP3 capsid protein involved in the binding of AAV2 with its main receptor, heparin sulfate proteoglycan (HSPG) and the insertion of peptides in this site can alter AAV tropism without compromising the viability of viruses. The modified capsid proteins packaged the AAV vector genomes with genomic titers comparable to those of wild-type viruses. AAV2 wild-type capsid sequence

[00139] The wild-type capsid sequence of AAV2 is described below in SEQ ID NO: 8. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS- RGLVLPGYKYL GPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHA- DAEFQERLKE - KRPVEHSPVEPDSSSGTG KAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNT- MATGSGAPMAD NNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWAL- PTYNNHLYKQISSQSGA SNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR- LNFKLFNIQV KEVTQNDGTTTIANNLTSTVQVFT DSEYQLPYVLGSAHQGCL- PPFPADVFMVPQ YGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDV- PFHSSYAHSQS LDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQS- RNWLPGPCYR QQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHK - DDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTN- LQRGNRQAAT ADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGG- FGLKHPPPQ ILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIE- WELQKENSKRWNPEIQYTSNYNKSVNVDF TVDTNGVYSEPRPIG- TRYLTRNL* (SEQ IDNO:8)

[00140] In the example below, the AAV2 wild-type capsid sequence with inserted motif (SEQ ID NO: 1) that targets eye cells is in italics and the underlined amino acids are spacers. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS- RGLVLPGYKYL GPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHA- DAEFQERLKE DTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGK- KRPVEHSPVEPDSSSGTG KAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAA PSGLGTNT- MATGSGAPMAD NNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWAL- PTYNNHLYKQISSQSga SFYCLEYFPSQMLRTGNNFTFSYTFEDV- PFHSSYAHSQS LDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQS- RNWLPGPCYR QQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHK- DDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTN- LQRGNAAGS TPPPMAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWA- KIPHTDGHFHPSPL MGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFIT- QYSTGQVSVEIEWELQKE NSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL* (SEQ ID NO:9)

[00141] The AAV2 wild-type capsid sequence with the inserted motif of SEQ ID NO: 4 is represented below in SEQ ID NO: 10. The amino acid motif that targets eye cells is in italics, and the underlined amino acids are spacers. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDS- RGLVLPGYKYL GPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHA- DAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTA- PGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP- DPQPLGQPPAA PSGLGTNTMATGSGAPMADNNEGAD- GVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQIS- SQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWG- FRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPY- VLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSS- FY CLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMN- PLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG- PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPA- MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTN- PVATEQYGSVSTNLQRGNAAA GET RAPL AARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTD- GHFHPSPL MGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFIT- QYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV- DTNGVYSEPRPIGTRYLTRNL* (SEQ ID NO:10)

[00142] The wild-type AAV9 capsid sequence is described below in SEQ ID NO: 11. MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNAR- GLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDN- PYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVE- EAAKTAPGKKRPVE QSPQEPDSSAGIGKSGAQPAKKRLNF- GQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGA- DGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNS- TSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWG- FRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDS dyqlpy- KQGTGRDNVDADKVMITNEEEIKTTN- PVATESYGQVATNHQSAQAQAQTGWVQNQGILPG- MVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQI- LIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKR- WNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL* (SEQ ID NO:11)

Materials and methods:

[00143] In vivo bioselection: 1 μL of Ph.D.TM library of 7 phages (titer 2.0x 1013 pfu/mL from NEB) was injected into the subretinal space of mouse eyes. After 30 minutes, the retina and RPE of injected eyes were separated, washed with 5 ml of cold PBS 3-5 times. The retina and RPE were homogenized in 500 μL of PBS. The recovered phages were titrated, amplified, purified and reinjected into additional eyes for the next round of in vivo selection. The entire procedure was repeated for four rounds. Afterwards, 50 randomly selected clones were sequenced to determine enriched motifs.

[00144] Construction of peptide-modified AAV2 capsids: The plasmid for cloning modified capsids was developed from pXX2, containing the wild-type AAV2 Rep and Cap. A plasmid with a DNA fragment encoding the amino acids AAAstopA and the NotI and AscI restriction sites inserted between amino acid positions 587 and 588 of AAV2 Cap was constructed as the backbone plasmid. dsDNA insertions encoding selected peptides were cloned into the NotI and AscI site as peptide-modified pXX2.

[00145] AAV2 production and titration: Plates of 293T cells were co-transfected with three plasmids: pXX2 or peptide-modified pXX2, which provided the AAV2 Rep and Cap proteins; pHelper, which contained the helper functions of the adenovirus; and a vector plasmid, which contained the AAV2 ITRs and the transgene of interest. Twenty plates of 150 mm in diameter were cotransfected with 90 μg of pXX2, pHelper, and vector plasmid DNA in a molar ratio of 1:1:1. After incubation for 60 hours, the virus was purified with iodixanol gradients and further purification through a Mustang Q membrane. Recombinant AAV titers were determined by real-time PCR.

EXAMPLE 2 Treatment of Genetic Eye Disease Through Gene Therapy

[00146] Two methods of administering viral vectors to the eye were tested in order to test the administration of gene therapy agents in vivo. The modes of administration were by injection of viral vectors subretinal (Fig. A-D) and intravitreal (Fig. E-I).

[00147] The virus used in this study was AAV2/2. As control, wild-type AAV2/2.CMV. eGFP was used and the test agent was PMAAV-GST. CMV. EGFP. The virus was injected into five-week-old female C57BL/6 mice. Mice were subretinal injected with virus (2.26 x 109 vg/eye). Three weeks later, the mice were anesthetized and their pupils were dilated. They were placed on a stereotaxic platform and dynamic fluorescent background photographs were taken. Four weeks later, the mice were sacrificed and the eyes were enucleated and fixed in 4% paraformaldehyde. Tissue sections were cut and mounted. eGFP expression was observed under a fluorescent microscope.

[00148] Three weeks later, subretinal injection of wild-type AAV2/2.CMV.eGFP and PMAAV-GST.CMV.eGFP, fluorescent imaging in live animals showed relative infection efficiencies. The targeting peptides used in this experiment were GSTPPPM (SEQ ID NO: 1). Spotty regions of GFP positivity were observed in AAV2/2 injection eyes, whereas there were broad and uniform GFP signals in PMAAV-GST eyes (Fig. 2). In other experiments, the target peptide was GETRAPL (SEQ ID NO: 4).

[00149] Four weeks after subretinal injection, PMAAV-GST.CMV.eGFP targeted the retinal pigment epithelium (RPE) and Outer Nuclear Layer (ONL) layers and some cells in the ganglion cell layer (GCL). AAV2/2 targeted the outer plexiform layer (OPL) primarily ( Fig. 3 ).

[00150] All publications, patents and patent applications are incorporated into this document by reference. While in the foregoing specification the present invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that some of the details described in this document may vary considerably without departing from the basic principles of the invention.

[00151] The use of the terms "a", "an" and "the" and similar referents in the context of describing the invention should be interpreted to encompass both the singular and the plural, unless otherwise indicated in this document or in case of clear contradiction due to the context. The terms "comprising", "having", "including" and "containing" shall be construed as open-ended terms (i.e. meaning "including, but not limited to") unless otherwise indicated. Citation of ranges of values in this document is intended only to serve as a simplified method for referring individually to each separate value included in the range, unless otherwise indicated in this document, and each separate value is incorporated into the specification as if it had been individually cited. in this document. All methods described in this document may be performed in any appropriate order unless otherwise indicated in this document or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") set forth herein, is intended only to further illuminate the invention and does not represent a limitation on the scope of the invention unless otherwise claimed. No language used in the specification should be construed as indicating any element not claimed to be essential to the practice of the invention.

[00152] Embodiments of the present invention are described in this document, including the best way known to the inventors to carry out the invention. Variations of these modalities may become apparent to those skilled in the art after reading the aforementioned description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced other than as specifically described herein. Therefore, this invention includes all modifications and equivalents of the subject matter cited in the claims attached hereto, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations thereof is encompassed by the invention, unless otherwise indicated herein or clearly contradicted by the context.

Claims (23)

1. Modified adeno-associated virus (AAV) capsid protein, characterized in that it comprises an eye cell targeting peptide, wherein the targeting peptide comprises a sequence of up to 10 amino acids in length that comprises GSTPPPM (SEQ ID NO: 1), in an amino to carboxy orientation or in a carboxy to amino orientation, and wherein the targeting peptide directs an AAV to an eye cell. 2. Capsid protein according to claim 1, characterized in that the targeting peptide is 8 or 9 amino acids in length. 3. Capsid protein according to claim 1, characterized by the fact that the targeting peptide is inserted between the AAV2 capsid residues at positions P34-A35, T138-A139, A139-P140, G453-T454, N587 -R588 and/or R588-Q589 of SEQ ID NO: 8. 4. Capsid protein according to claim 1, characterized by the fact that the targeting peptide is inserted after the AAV9 capsid residues at positions D384, G385, I560, T561, N562, E563, E564, E565 , N704 and/or Y705 of SEQ ID NO: 11. 5. Capsid protein according to claim 1, characterized by the fact that the capsid protein comprises or consists of SEQ ID NO: 9. 6. Capsid protein according to claim 1 or 2, characterized in that the targeting peptide targets a diseased ocular cell. 7. Capsid protein according to claim 6, characterized by the fact that the target peptide targets an ocular cell in an individual who has retinitis pigmentosa, maculopathies, Leber congenital amaurosis, early-onset severe retinal dystrophy, achromate - psyia, retinoschisis, ocular albinism, oculocutaneous albinism, glaucoma, Stargardt's disease, choroideremia, age-related macular degeneration, spinocerebellar ataxia type 7 (SCAT), color blindness, and lysosomal storage diseases affecting the cornea. 8. Capsid protein according to any one of claims 1 to 7, characterized in that the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74 (Rhesus monkey-derived AAV), AAVRh10, or modified capsids of these serotypes. 9. Nucleic acid sequence, characterized by the fact that it encodes the modified capsid, as defined in any one of claims 1 to 8. 10. Nucleic acid sequence according to claim 9, characterized in that it comprises the nucleic acid sequence GGGTCGACGCCGCCTCCTATG (SEQ ID NO: 2). 11. Viral vector particle, characterized by the fact that it comprises the capsid protein, as defined in any one of claims 1 to 8. 12. Viral vector particle according to claim 11, characterized in that the viral vector further comprises a nucleic acid sequence that encodes a nucleic acid of interest. 13. Viral vector particle according to claim 12, characterized by the fact that the nucleic acid of interest is a therapeutic agent. 14. Viral vector particle according to claim 13, characterized in that the therapeutic agent is an enzyme or an RNAi molecule. 15. Viral vector particle according to claim 13, characterized by the fact that the therapeutic agent is ataxin 7 mirRNA, a 65 kDa protein specific to the retinal pigment epithelium (RPE 65), an inhibitor of vascular endothelial growth factor (VEGF) or soluble VEGF receptor 1 (sFifl), (Rab-escort protein 1) REP1, L-opsin, rhodopsin (Rho), phosphodiesterase 6(3 (PDE6I3), ATP-binding cassette, subfamily A, member 4 (ABCA4), lecithin retinol acyltransferase (LRAT), retinal degeneration, Peripherin/slow (RDS/Peripherin), Mer protein tyrosine kinase (MERTK), Inosine-5 prime-monophosphate dehydrogenase, type I (IMPDHI), guanylate cyclase 2D (GUCY2D ), aryl hydrocarbon-interacting protein type 1 (AIPL 1), retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIPI), Inosine-5-prime-monophosphate dehydrogenase, type I (guanine nucleotide-binding protein IMPDH1, alpha transduction activity polypeptide 2 (GNAT2), voltage-dependent cyclic nucleotide beta 3 (CNGB3), retinoschisin 1 (Rsl), ocular albinism type 1 (OA1), oculocutaneous albinism type 1 tyrosinase ( OCA1), P21 WAF-1/Cipl, platelet-derived growth factor (PDGF), endostatin, angiostatin, arylsulfatase B or 13-glucuronidase. 16. Pharmaceutical composition, characterized by the fact that it comprises the viral vector particle, as defined in any one of claims 11 to 15, and a pharmaceutically acceptable carrier. 17. Use of the viral vector, as defined in any one of claims 11 to 15, characterized by the fact that it is for the preparation of a medicine for the treatment of an eye disease in a mammal. 18. Use according to claim 17, characterized by the fact that the mammal is a human being. 19. Use, according to claim 17 or 18, characterized by the fact that the disease is retinitis pigmentosa, maculopathies, Leber's congenital amaurosis, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, ocular albinism, oculocutaneous albinism. neo, glaucoma, Stargardt disease, choroideremia, age-related macular degeneration, SCAT, color blindness, or lysosomal storage diseases that affect the cornea, such as MPS IV and MPS VII. 20. Use, according to claim 17, characterized by the fact that the medicine when made available to the individual's eye transduces the ocular cells so that the ocular cells express the therapeutic agent. 21. Use according to claim 17, characterized by the fact that the transduced ocular cells are within or comprise the outer plexiform layer of the retina (OPL). 22. Viral vector according to any one of claims 11 to 15, characterized by the fact that it is for use in medical treatment or diagnosis. 23. Viral vector according to any one of claims 11 to 15, characterized by the fact that it is for use in the prophylactic or therapeutic treatment of an eye disease in a mammal.