WO2005051413A2

WO2005051413A2 - Disease associated genes

Info

Publication number: WO2005051413A2
Application number: PCT/EP2004/013411
Authority: WO
Inventors: Eric Bertrand; Christine Fritsch; Johannes Voshol
Original assignee: Novartis Ag; Novartis Pharma Gmbh
Priority date: 2003-11-26
Filing date: 2004-11-25
Publication date: 2005-06-09
Also published as: WO2005051413A3

Abstract

The present invention relates to genes whose expression is correlated to diseases related to how the eye focuses light. In particular, the invention relates to methods for the treatment, diagnosing and methods of monitoring diseases disturbing emmetropia, such as myopia or hyperopia using the genes provided by the present invention. In addition, the present invention relates to methods of screening compounds for use in treatment of diseases disturbing emmetropia, such as myopia or hyperopia as well as kits and arrays for use in identifying such diseases.

Description

DISEASE ASSOCIATED GENES

FIELD OF THE INVENTION

The present invention relates generally to the field of ocular diseases related to the refractive status of the eye. The invention further relates to genes which, when varied in their normal expression pattern, are associated with emmetropia disturbing disorders (EDDs), such as myopia or hyperopia. Thus, the present invention relates to the novel use of known genes in diseases disturbing emmetropia. The present invention also relates to methods for diagnosing and observing an EDD. The present invention further relates to methods for identifying agents useful for the treatment of diseases disturbing emmetropia.

BACKGROUND OF THE INVENTION

Ocular axial length is an essential parameter of the eye's refractive status that is tightly controlled throughout development and adulthood in many vertebrates so as to set the focal plane of the eye onto the retina (emmetropia). Disturbance of this control will lead to defocus and vision impairment as the retina drifts behind the focal plane (myopia) or lingers in front of it (hyperopia). The cause of myopia is unknown; both genetic and environmental factors have been implicated. Education background, reading habits and use of computers have been proposed for risk factors involved. Evidence for a genetic component to myopia is provided by population and family studies. At present little is known about the messengers released by the retina to control axial eye growth, especially about those that inhibit axial elongation during development of hyperopia which are of particular interest for potential pharmaceutical treatments. Vision impairment can be easily corrected by glasses, contact lenses or refractive surgery. However, even if vision is corrected the risk for secondary eye diseases, such as, for example, retinal detachment, cataract and glaucoma is markedly increased, especially for people with high myopia. At least in Asia where both incidence and severity of myopia are extraordinary high, pathogenic myopia is one of the major causes for blindness. Thus, development of pharmaceuticals able to prevent myopia would be of high potential value. Although pharmacological studies in animal models have identified some potential targets for intervention of myopia for instance glucagon, dopamine, acetylcholine, retinoic acid, nitric oxide and opiate receptors, it is likely that other targets are also promising and potentially more specific. Muscarinic receptor antagonists have been proven effective at preventing development of myopia in both animal models and humans. The classical antagonist atropine, for example, was shown to prevent the progression of axial myopia in adolescent humans. However, marked ocular side effects and low tolerability limit its use and, thus, search for compounds with less adverse effects and better tolerability is needed. The search for myopia-preventing muscarinic receptor antagonists is hindered because little is known about their precise molecular target and mode of action. The present invention now provides, inter alia, such molecular targets.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method to treat, prevent or ameliorate an emmetropia disturbing disorder (EDD) comprising administering an effective amount of at least one peptide selected from the group consisting of ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK.

In a second aspect, the present invention provides a method to treat, prevent or ameliorate an EDD comprising administering an effective amount an agent modulating the expression, or the activity, of at least one peptide selected from the group consisting of ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK.

In a third aspect, the present invention provides a method for screening a subject for myopia or hyperopia or at risk of developing myopia or hyperopia comprising: a) detecting a level of expression of at least one gene selected from the group consisting of ApoA1, SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject.

In another aspect, the present invention provides a method for monitoring the progression of myopia or hyperopia in a subject comprising: a) detecting a level of expression of at least one gene selected from the group consisting of ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject.

In another aspect, the present invention provides a method for identifying agents for use in the treatment of a myopia or hyperopia comprising: a) contacting cells expressing at least one gene encoding a protein selected from the group consisting of ApoA1 , SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK with a candidate agent; b) detecting a level of expression of at least one of said genes in said cells, or detecting a lower or higher activity of said proteins.

In another aspect, the present invention provides a kit comprising: a) a labeled compound or agent capable of detecting at least one protein selected from the group comprising ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK, or mRNA corresponding to the genes of said proteins, b) means for determining the amount of said proteins or mRNA corresponding to the genes of said proteins; and, optionally, means for comparing the amount of said proteins encoded by or mRNA corresponding to the gene or fragment of the protein, with a standard level of expression of the gene and optionally instructions.

In yet another aspect, the present invention provides agents for use in the treatment of myopia or hyperopia, said agent being capable of regulating the level of expression of at least one gene selected from the group consisting of ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK.

DESCRIPTION OF THE FIGURES

Figure 1 : Apolipoprotein A1 expression after 1 and 7 days of LIH treatment, where axial growth is slower.

Figure 2 A: Dihydropyriminidase related protein-2 expression (Isoform 1) after 1 day and 7 days of FDM and LIM treatments, where axial growth is increased.

Figure 2 B: Dihydropyriminidase related protein-2 expression (Isoform 2) after 1 day and 7 days of FDM and LIM treatment, where axial growth is increased. Figure 3 A: B-creatine kinase expression (Isoform 1 ) after 7 days of LIM treatment, where axial growth is increased.

Figure 3 B: B-creatine kinase expression (Isoform 2) after 1 day of FDM treatment and in

LIM condition after 7 days of treatment, where axial growth is increased.

Figure 3 C: B-creatine kinase expression (Isoform 3) after 7 days of LIM treatment, where axial growth is increased.

Figure 4 A: Tubulin alpha 1 chain expression after 1 day of FDM and LIM treatment and after

7 days of FDM and LIM treatment, where axial growth is increased.

Figure 5 : Gamma enolase expression after 7 days of FDM treatment, where axial growth is increased.

Figure 6 : Visinin expression after 1 day and 7 days is increased in control in the fibrous sclera.

Figure 7: Splicing factor-2 expression after 7 days of LIH treatment, where axial growth is slower.

Figure 8: Interocular difference in refractive error after a 4-day treatment period with

Fenofibrate was calculated. Figure 8 shows % of lens-induced myopia development relative to the binocularly saline-injected control group, which monocularly wore a -7D lens with three different doses 75, 250 and 500 μg.

Figure 9: Interocular difference in refractive error was calculated after a 4-day treatment period with GW7647, and Figure 9 shows % of lens-induced myopia development relative to the binocularly saline-injected control group, which monocularly wore a -7D lens with two different doses 63 and 189 μg.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety.

In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA are used. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively).

The individual proteins/polypeptides referred to herein, i.e. ApoA1 , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK, include any and all forms of these proteins including, but not limited to, partial forms, isoforms, variants, precursor forms, the full length protein, fusion proteins containing the sequence or fragments of any of the above, from human or any other species. Protein homologs or orthologs which would be apparent to one of skill in the art are included in this definition. It is also contemplated that the term refers to proteins isolated from naturally occurring sources of any species such as genomic DNA libraries as well as genetically engineered host cells comprising expression systems, or produced by chemical synthesis using, for instance, automated peptide synthesizers or a combination of such methods. Means for isolating and preparing such polypeptides are well understood in the art. The terms "protein", "peptide" and "polypeptides" are used interchangeably.

The present invention is based on an extensive proteome analysis of chicken retina and sclera (emmetropic, myopic and hyperopic animals) using high-resolution 2D-gel electrophoresis and mass spectrometry (Hoving S, et al., Electrophoresis. 2000;21(13):2617-21). In this analysis, a number proteins have surprisingly been found to be differentially expressed in the myopic or hyperopic conditions:

Apolipoprotein A1 (ApoA1), has been found to be up-regulated in conditions where axial growth of the eye is slower as compared to normal conditions. Thus, ApoA1 acts, in accordance with the present invention, as a stop signal for axial growth of the eye and therefore activation of ApoA1 will prevent the excessive growth associated with myopia, whereas deactivation of ApoAI will enhance growth and prevent too little growth associated with hyperopia. ApoAI is a known protein, however, to the best of the inventors' knowledge, ApoAI has not been linked to ocular diseases related to the refractive status of the eye. ApoAI has been characterized as the main protein component of the high density lipoprotein (HDL) particle (Frank and Marcel, J. Lipid Res. 2000;41: 853-872) which is involved in the transport of cholesterol and other lipids; within the HDL, Apo A1 is a cofactor of Lecithin:cholesterol acyltransferase (LCAT). Apo A1 has also been shown to regulate the activity of endothelial nitric oxide sythase (eNOS) by adjusting the amount of cholesterol within the caveolae (Shaul, Annu. Rev. Physiol. 2002;64:749-74).

Splicing factor-2 (SF-2) has been found to be up-regulated in conditions where axial length growth is slower compared to the control condition. This suggests that Splicing factor-2 might be related to a "stop" signal, which could be activated to stop the excessive growth associated with myopia. Splicing factor-2 is also refered to as SF2, ASF, Alternative splicing factor ASF-1 , pre-mRNA splicing factor SF2 P33 subunit and Splicing factor arginine/serine- rich 1. Splicing factor-2 belongs to the SR family of splicing factors and is extensively phosphorylated on serine residues in the RS domain. Splicing factor-2 plays a role in preventing exon skipping, ensuring the accuracy of splicing and regulating alternative splicing. It interacts with other spliceosomal components, via the RS domains, to form a bridge between the 5' and 3' splice site binding components, u1 snrnp and u2af (for review see Black DL, (2003), Annu Rev Biochem;72:291-336).

Dihydropyrimidinase related protein-2 (DRP-2), B-creatine kinase (B-CK), Tubulin alpha 1 chain, Gamma enolase have been found to be upregulated in conditions where axial growth is faster as compared to a control. Thus, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase act, in accordance with the present invention, as a growth signal for axial growth of the eye and therefore inactivation of these proteins will prevent the excessive growth associated with myopia, whereas activation will enhance growth and prevent too little growth associated with hyperopia.

The nomenclature of the Dihydropyriminidase related protein family has changed several times in the past years. In human and chicken DRP-2 has been refered to as CRMP-2 (collapsin response mediator protein 2) and more recently as TUC-2 (TOAD-64, ULIP, CRMP). Chicken DRP-2 is also known as CRMP-62. The members of the DRP family seem to be mostly involved involved in cellular motility: CRMP-2 is important for neurite outgrowth and CRMP-1 has been associated with cancer cell invasion and metastasis.The function of DRP-2 is regulated by the rho kinases (ROCK1 and 2), the LIM kinase (LIMK1 and 2) and also by a G protein dependent mechanism (Goshima et a!., J. Clin. Invest. 2002;109:993- 998). Several isoforms of DRP-2 were found to be differentially expressed. Creatine kinase plays a central role in energy transduction in tissues with large and fluctuating energy demands. B-CK seems to be a major ATP-regenerating sytem in the photoreceptors (Hemmer et al., J Cell Sci. 1993;106, 671-684). A great number of creatine kinase subspecies with different isoelecric points have been observed (Stolz M et al., Biochem J. 2002;363(3):785-92). Several isoforms of B-CK were found to be differentially expressed. Heterodimers of alpha and beta tubulins represent the major component of microtubules. Microtubules of the eukaryotic cytoskeleton perform a number of important functions and are essential in the control of cell morphology. Tubulin alpha 1 chain expression is predominantly found in differentiated neuronal cells. The enolases (phosphopyruvate hydratase; EC 4.2.1.11) catalyze the interconversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway. The functional enzyme is a dimer made up of subunits referred to as alpha, beta, and gamma. Gamma enolase (Enolase-2) is the major form found in mature neurons and in cells of neuronal origin.

Visinin has been found to be up-regulated in the normal conditions as compared to the conditions where where axial growth of the eye is faster or slower than normal. Thus, Visinin is provided, in accordance with the present invention, as "Image Quality" signal important to maintain emmetropia, i.e. Visinin is related to an "Image Quality" signal which, when activated, prevents the elongation of axial length related to myopia of the eye associated with myopia.

The present invention further provides Rho-kinase (ROCK1 and 2) and LIM-kinase (LIMK1 and 2) as "growth" signal for axial growth of the eye, i.e. interfering with the expression or the function of Rho-kinase and LIM-kinase will prevent the elongation of axial length related to myopia. ROCK1 and ROCK2 are downstream of the small GTPase Rho. The ROCKs are serine/threonine kinases that regulate cytokinesis, smooth muscle contraction, the formation of actin stress fibers and focal adhesions, and the activation of the c-fos serum response element. ROCK1 is also called ROKβ or sometimes p160ROCK wheras ROCK2 is also called ROKα. In some papers ROCK2 has also been called p160 ROCK leading to some confusion in the field, however a recent review has clarified which isoform of ROCK has been studied in the most important papers (Riento and Ridley, Nat Rev Mol Cell Biol. 2003;4(6):446-56). As ROCK2 has been shown to phosphorylate DRP-2 on Threonine 555 (Goshima et al., J. Clin. Invest. 2002;109:993-998). LIMK1 and LIMK2 are part of an intracellular signaling pathway involved in brain development. LIMK1 phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal reorganization. LIMK1 expression is highest in the nervous system especially in the cerebral cortex whereas L1MK2 is expressed predominantly in the lung. The Rac/Pak/LIMK pathway mediates semaphorin-3a induced growth cone collapse (Aizawa et al., Nat Neurosci. 2001 ;(4):367-73). Also, DRP-2 is an essential effector of semaphorin-3a induced growth cone collapse and is phosphorylated downstream of Rac (Arimura et al., J Biol Chem. 2000;275(31):23973-80).

The present invention provides, in accordance with a first aspect, a method to treat, prevent or ameliorate an ocular disorder related to ocular refractive status comprising administering an effective amount of at least one peptide selected from the group consisting of ApoAI , SF- 2.DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK. The disease is preferably a EDD, such as myopia or hyperopia. In a preferred embodiment, the present invention provides a method for the treatment of myopia comprising administering an effective amount of ApoAI , SF-2 or Visinin. In another preferred embodiment, the present invention provides a method for the treatment of hyperopia comprising administering an effective amount of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK. An "effective amount" is the amount peptide sufficient to treat, prevent or ameliorate an ocular disease related to ocular refractive status, such as for instance EDDs or myopia or hyperopia. The determination of a therapeutically effective amount is well within the capability of those skilled in the art.

In one embodiment, the present invention provides the use of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK or LIMK as a medicament. In a preferred embodiment, the present invention provides the use of ApoAI, SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK or LIMK for the manufacture of a medicament for the treatment of an EDD, such as for instance hyperopia or myopia. In another preferred embodiment, the present invention provides the use of ApoAI, SF-2 or Visinin for the manufacture of a medicament for the treatment of myopia. In another preferred embodiment, the present invention provides the use of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK for the manufacture of a medicament for the treatment of hyperopia.

In a related aspect, the present invention provides a method to treat, prevent or ameliorate an ocular disorder related to ocular refractive status comprising administering an effective amount of an agent inhibiting or increasing the expression of at least one peptide selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK. The disease is preferably an EDD, such as myopia or hyperopia. In a preferred embodiment, the present invention provides a method for the treatment of myopia comprising administering an effective amount of an agent increasing the expression of ApoAI , SF-2 or Visinin. In a more preferred embodiment, the present invention provides a method to treat, prevent or ameliorate an ocular disorder related to ocular refractive status, such as myopia, comprising administering an agent increasing the expression of ApoAI , said agent being selected among PPARα agonists. In another preferred embodiment, the present invention provides a method for the treatment of myopia comprising administering an effective amount of an agent inhibiting the expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK.

In one embodiment, the present invention provides the use of an agent inhibiting the expression of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK or LIMK for the manufacture of a medicament for the treatment of an EDD, such as hyperopia or myopia. In a preferred embodiment, the present invention provides the use of an agent increasing the expression of ApoAI , SF-2 or Visinin for the manufacture of a medicament for the treatment of myopia. In a more preferred embodiment, the present invention provides the use of an agent increasing the expression of ApoAI , said agent being selected among PPARα agonists. In another preferred embodiment, the present invention provides the use of an agent inhibiting the expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK for the manufacture of a medicament for the treatment of myopia. In another preferred embodiment, the present invention provides the use of an agent inhibiting the expression of ApoAI , SF-2 or Visinin for the manufacture of a medicament for the treatment of hyperopia. In another preferred embodiment, the present invention provides the use of an agent increasing the expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK for the manufacture of a medicament for the treatment of hyperopia.

The "agent", in accordance with the present invention, may be any molecule that is capable of decreasing or increasing the level of mRNA corresponding to, or protein encoded by, at least one of the disclosed genes. The agent may for instance be a small molecule identified by a screening assay as disclosed hereinbelow.

Examples of inhibitory agents can be antibody or an inhibitory nucleic acid as described herein below.

As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments such as Fa, F(ab')2, and Fv, which are capable of binding the epitopic determinant. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. The polypeptides or peptides used to immunize an animal can be derived from the translation of RNA or synthesized chemically, and can be conjugated to a carrier protein. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize an animal (e.g., a mouse, goat, chicken, rat or a rabbit). Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (Nature, Vol. 256, pp. 495-497 (1975); and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, Vol. 4, p. 72 (1983); Cole et al., Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 2026-2030 (1983)), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). The term "humanized antibody" as used herein, refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability. Techniques for production of humanized antibodies are known in the art and are disclosed for instance in U.S. Patent Nos. 5,932,448; 5,693,762; 5,693,761 ; 5,585,089; 5,530,101 ; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661 ,016; and 5,770,429. The term "inhibitory nucleic acids" refers to any nucleic acid molecule that is capable of decreasing the level of mRNA corresponding to, or protein encoded by, at least one of the disclosed genes. Examples of such nucleic acid molecules include, but are not limited to, antisense nucleic acids, ribozymes, double-stranded RNAs.

As used herein, the term "antisense" refers to nucleotide sequences that are complementary to a portion of an RNA expression product of at least one of the disclosed genes. "Complementary" nucleotide sequences refer to nucleotide sequences that are capable of base-pairing according to the standard Watson-Crick complementarity rules. When introduced into a host cell, antisense nucleotide sequences specifically hybridize with the cellular mRNA and/or genomic DNA corresponding to the gene(s) so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation within the cell. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases. The isolated nucleic acid molecule comprising the antisense nucleotide sequence can be delivered, e.g., as an expression vector, which when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the encoded mRNA of the gene(s). Alternatively, the isolated nucleic acid molecule comprising the antisense nucleotide sequence is an oligonucleotide probe which is prepared ex vivo and, which, when introduced into the cell, results in inhibiting expression of the encoded protein by hybridizing with the mRNA and/or genomic sequences of the gene(s). Preferably, the oligonucleotide contains chemical modifications. Examples of modified nucleic acid molecules for use as antisense nucleotide sequences are artificial intemucleotide linkages such as phosphoramidate, phosporothioate and methylphosphonate analogs of DNA as described, e.g., in U.S. Patent No. 5,176,996; 5,264,564; and 5,256,775 or modifications at the 2' position such as for instance 2'-methoxyethoxy (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. General approaches to preparing oligomers useful in antisense therapy are described, e.g., in Van der Krol, BioTechniques, Vol. 6, pp. 958-976 (1988); and Stein et al., Cancer Res., Vol. 48, pp. 2659- 2668 (1988). The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941 , each of which is herein incorporated by reference.

"Ribozymes" are RNA molecules that specifically cleave other single-stranded RNA in a manner similar to DNA restriction endonucleases. By modifying the nucleotide sequences encoding the RNAs, ribozymes can be synthesized to recognize specific nucleotide sequences in a molecule and cleave it as described, e.g., in Cech, J. Amer. Med. Assn., Vol.260, p. 3030 (1988). Accordingly, only mRNAs with specific sequences are cleaved and inactivated. Two basic types of ribozymes include the "hammerhead"-type as described for example in Rossie et al., Pharmac. Ther., Vol. 50, pp. 245-254 (1991); and the hairpin ribozyme as described, e.g., in Hampel et al., Nucl. Acids Res., Vol. 18, pp. 299-304 (1999) and U.S. Patent No. 5,254,678. Intracellular expression of hammerhead and hairpin ribozymes targeted to mRNA corresponding to at least one of the disclosed genes can be utilized to inhibit protein encoded by the gene. Ribozymes can either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety. Ribozymes can either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundance in a cell (Cotten et al., 1989 EMBO J. 8:3861-3866). In particular, a ribozyme coding DNA sequence, designed according to conventional, well known rules and synthesized, for example, by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the anticodon stem and loop of a gene encoding a tRNA, which can then be transformed into and expressed in a cell of interest by methods routine in the art. Preferably, an inducible promoter (e.g., a glucocorticoid or a tetracycline response element) is also introduced into this construct so that ribozyme expression can be selectively controlled. For saturating use, a highly and constituently active promoter can be used. tDNA genes (i.e., genes encoding tRNAs) are useful in this application because of their small size, high rate of transcription, and ubiquitous expression in different kinds of tissues. Therefore, ribozymes can be routinely designed to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly the abundance of virtually any RNA species in a cell can be modified or perturbed. Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety. In another embodiment, the agent is an inhibitoty agent being selected among double- stranded RNA (dsRNA), i.e., sense-antisense RNA, capable of decreasing the expression of a gene in accordance with the present invention through an RNA interference (RNAi) mechanism. Interference with the function and expression of endogenous genes by ds RNA has been shown in various organisms such as C. elegans as described, e.g., in Fire et al., Nature, Vol. 391 , pp. 806-811 (1998); drosophilia as described, e.g., in Kennerdell et al., Cell, Vol. 95, No. 7, pp. 1017-1026 (1998); and mouse embryos as described, e.g., in Wianni et al., Nat. Cell Biol., Vol. 2, No. 2, pp. 70-75 (2000). Suitable ds RNA include for instance small interfering RNAs (si RNAs) as described e.g. in Elbashir, Methods 26 (2002), 199-213. Typically, such si RNAs have a length of about 19 to 25 nucleotides and a short overhang at the 3' end. Other suitable RNAs include short hairpin RNAs (shRNAs) (Brummelkamp.T, Science (2002) 296, 550-553). The design of ds RNA capable of inhibiting any given target gene via RNAi is well within the capability of those skilled in the art. In a first step, for instance, a set of dsRNAs complementary to the target gene may be designed and preselected for RNAi potency using a software for the prediction of the RNAi potency. Such a software is known in the art and available e.g. from oligonucleotide suppliers such as Dharmacon or Qiagen. A number of dsRNAs with a good prediction score can subsequently be selected, synthesized and tested in a suitable assay for RNAi interference activity, using e.g. an RT-PCR assay or a reporter gene assay as described in Hueskens et al., Nucleic Acid Research 31 (2003), e102. The double-stranded RNA can be synthesized by in vitro transcription of single-stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double-stranded RNA can also be synthesized from a cDNA vector construct in which the gene of interest is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal. The effect of the inhibitory agent according to the present invention will typically result in decreasing the level of mRNA corresponding to, or protein encoded by at least 10%, 33%, 50%, 90%, 95% or 99% when as compared to an untreated control. The effect can for instance be assayed in a chicken model as described hereinbelow.

In another embodiment, the agent is a compound capable of increasing the expression of ApoAI.

ApoAI is known to be up-regulated by PPARα. Thus, in a preferred embodiment, a compound capable of increasing the expression of ApoAI may be selected among peroxisome proliferators-activated receptor (PPAR) α agonists.

PPARs belong to the superfamily of nuclear receptors that are ligand-activated transcription factors (Issemann et al., 1990, Nature, 347, 645-650). Among the three different subtypes identified thus far, PPARα, PPARβ (NUC-1 or PPARδ), and PPARγ, PPARα mediates the lipid-lowering activity of the fibrate drugs (staels et al., 1998, Circulation 98: 2088-2093). PPARα is considered a major regulator of intra- and extracellular fatty acid metabolism. Fibrate activation of PPARα increases plasma levels of HDL, decreases VLDL synthesis and secretion, and reduces triglyceride levels. PPARα has been shown to exert these effectes by negatively interfering with NF-κB and activator protein-1 (AP-1) (Pineda Torra atal., 1999, Curr Opin. Lipidol., 10, 141-159).

Any of the known PPARα agonists and their derivatives can be used in the present invention. In preferred embodiments, said PPARα agonists are selected among the group consisting of fenofibrate (2-(4-[4-ChlorobenzoylJphenoxy)-2-methylpropanoic acid), WY14643 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid) (Ren et al., 1996, JBC 19: 17167-17173) and ureido-TiBAs such as GW7647 (3) (Brown et al., 2001, Bioorg. Med. Chem. Lett. 11 : 1225-1227)

In another specific embodiment, an agent capable of increasing ApoAI expression is GW501516 and functional derivatives, said compound GW501516 being a PPAR beta/gamma agonist capable of increasing ApoAI specific efflux or intracellular cholesterol (Dressel et al., Molecular endocrinology, 2003, 17: 2477-2493). ln another specific embodiment, an agent capable of increasing ApoAI expression is selected among the group consisting of statins. As used herein, the term "statins" refers to the competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which increases the levels of ApoAI (Martin et al., 2001 , J din Invest 107:1421-1432, Schaeffer et al., 1999, Atherosclerosis, 144:177-184).

In another specific embodiment, an agent capable of increasing ApoAI expression is selected among the group consisting of thiazolidinedione derivatives like pioglitazone, which increase ApoAI secretion (Sakamoto er a/., 2000, BBRC, 278: 704-711).

The effect of the agent increasing the expression of ApoAI according to the present invention will typically result in increasing the level of mRNA corresponding to, or protein encoded by at least 10%, 33%, 50%, 90%, 95% or 99% when as compared to an untreated control. The effect can for instance be assayed in a chicken model as described hereinbelow.

In a another aspect, the present invention provides a method for screening a subject for myopia or hyperopia or at risk of developing myopia or hyperopia comprising: a) detecting a level of expression of at least one gene selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject. A higher level of expression of ApoAI or SF-2 is indicative of a condition where the growth of the eye is slower compared to the control condition, whereas a lower level of expression of ApoAI or SF-2 is indicative of a condition where the growth of the eye is faster compared to the control condition. A lower level of expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK is indicative of a condition where the growth of the eye is slower compared to the control condition, whereas a higher level of expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK is indicative of a condition where the growth of the eye is faster compared to the control condition. A high level of Visinin is indicative that the eye is .emmetropia "Subject" can be any human or nonhuman organism.

The term "sample" as used herein, is used in its broadest sense. A biological sample from a subject may comprise blood, urine, brain tissue, primary cell lines, immortilized cell lines, or other biological material with which protein activity or gene expression may be assayed. A biological sample may include, for example, blood, tumors or other specimens from which total RNA may be purified for gene expression profiling using, for example, conventional glass chip microarray technologies such as Affymetrix chips, RT-PCR or other conventional methods. The samples may be ocular cells, such as keratocytes and/or lens epithelial cells, which are preferably derived from the eye of a subject having myopia or hyperopia or at risk of developing myopia or hyperopia or from body fluids such as for instance sera, plasma or vitreous humor or aqueous humor. Conventional screening assays (both in vitro and in vivo) may be used to identify modulators that inhibit protein activity and/or inhibit gene expression. Protein activity levels, e.g., enzymatic activity levels, can be assayed in a subject using a biological sample from the subject using conventional enzyme activity assays. Gene expression (e.g. mRNA levels) may also be determined using methods familiar to one of skill in the art, including, for example, conventional Northern analysis or commercially available microarrays. Additionally, the effect of test compound inhibition of protein levels can be detected with an ELISA antibody- based assay or fluorescent labelling reaction assay. These techniques are readily available and are familiar to one skilled in the art. In the methods of the invention, the level of expression of one of the disclosed genes in a subject with myopia or hyperopia or at risk of developing myopia or hyperopia preferably differs from the level of expression of the gene in normal subject by a statistically significant amount. In presently preferred embodiments, at least about a 1.5-fold difference in expression levels is observed. In some embodiments, the expression levels of a gene differ by at least 2, 3-, 4-, 5-, 10- or 100-fold or more in the diseased tissue compared to the non-diseased tissue. A threshold level of expression may be determined in the methods of the invention. It is sometimes desirable to determine the level of expression of 2 or more of the disclosed genes. RNA can be isolated from the samples by methods well known to those skilled in the art as described, e.g., in Ausubel et al., Current Protocols in Molecular Biology, Vol. 1 , pp.4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc. (1996). Methods for detecting the level of expression of mRNA are well known in the art and include, but are not limited to, northern blotting, reverse transcription PCR, real time quantitative PCR and other hybridization methods. A particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of the disclosed genes involves hybridization of labeled mRNA to an ordered array of oligonucleotides. Such a method allows the level of transcription of a plurality of these genes to be determined simultaneously to generate gene expression profiles or patterns. The gene expression profile derived from the sample obtained from the subject can be compared with the gene expression profile derived from the sample obtained from the disease-free subject to determine whether the genes are over-expressed in the sample from the subject relative to the genes in the sample obtained from the disease-free subject, and thereby determine whether the subject has or is at risk of developing an EDD, such as myopia or hyperopia.

In another aspect, the treatment of a subject with a therapeutic agent, such as those described in the present invention, can be monitored by detecting the level of expression of mRNA or protein encoded by at least one of the genes encoding ApoAI , SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK, or the activity of at least one of said proteins. These measurements will indicate whether the treatment is effective or whether it should be adjusted or optimized. Accordingly, one or more of the genes described herein can be used as a marker for the efficacy of a drug during clinical trials. Thus, the present invention provides a method for monitoring the progression of myopia or hyperopia in a subject comprising: a) detecting a level of expression of at least one gene selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject. The level of expression of said gene is indicative of conditions as described above and can be measured as above. Preferred differences are as described above.

In a another aspect, the present invention provides methods for identifying agents for use in the treatment of a myopia or hyperopia comprising: a) contacting cells expressing at least one gene encoding a protein selected from the group consisting of ApoAI , SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, and Gamma enolase, Visinin, ROCK and LIMK with a candidate agent; b) detecting a level of expression of at least one of said genes in said cells, or detecting a lower or higher activity of said proteins.

In a preferred embodiment, the level of expression of at least one of said genes in the presence of the candidate agent is compared with a level of expression of at least one of said genes in cells that are not contacted with the candidate agent, wherein a lower expression level of ApoAI , SF-2 or Visinin, or a greater expression level of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of hyperopia. Similarly, a greater expression level of ApoAI , SF-2 or Visinin, or a lower expression level of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of myopia. In another preferred embodiment, the activity of at least one of said proteins in the presence of the candidate agent is compared with the activity of at least one of said proteins in cells that are not contacted with the candidate agent, wherein a lower activity of ApoAI , SF-2 or Visinin, or a higher activity of of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of hyperopia. Similarly, a higher activity of ApoAI , SF-2 or Visinin, or a lower activity of of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of myopia.

Cell-based and/or cell-free assays can for instance be used to identify compounds which are capable of interacting with one of the proteins ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK. Cell-based and/or cell-free assays can also be used to identify compounds which modulate the interaction between the encoded protein and its binding partner such as a target peptide. In one embodiment, cell-based and/or cell-free assays for identifying such compounds comprise a cell expressing a protein encoded by any one of the genes of the invention or a reaction mixture containing a protein encoded by one of the disclosed genes and a test compound or a library of test compounds in the presence or absence of the binding partner, e.g., a biologically inactive target peptide, or a small molecule. Accordingly, a cell-based and/or cell-free method for identifying agents useful in the treatment of an EDD, such as myopia or hyperopia is provided which comprises contacting a protein or functional fragment thereof or the protein binding partner with a test compound or library of test compounds and detecting the formation of complexes. For detection purposes, the protein can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker. Interaction of a test compound with the protein or fragment thereof or the protein-binding partner can then be detected by measuring the level of the two labels after incubation and washing steps. The presence of the two labels is indicative of an interaction. Interaction between molecules can also be assessed by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB), which detects surface plasmon resonance, an optical phenomenon. Detection depends on changes in the mass concentration of mass macromolecules at the biospecific interface and does not require labeling of the molecules. In one useful embodiment, a library of test compounds can be immobilized on a sensor surface, e.g., a wall of a micro-flow cell. A solution containing the protein, functional fragment thereof, or the protein-binding partner is then continuously circulated over the sensor surface. An alteration in the resonance angle, as indicated on a signal recording, indicates the occurrence of an interaction. This technique is described in more detail in BIAtechnology Handbook by Pharmacia.

A considerable change (potentiation or inhibition) in the interaction of the protein and binding partner in the presence of the test compound compared to the interaction in the absence of the test compound indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of the proteins activity for the test compound. The components of the assay can be combined simultaneously or the protein can be contacted with the test compound for a period of time, followed by the addition of the binding partner to the reaction mixture. The efficacy of the compound can be assessed by using various concentrations of the compound to generate dose response curves. A control assay can also be performed by quantitating the formation of the complex between the protein and its binding partner in the absence of the test compound. Formation of a complex between the protein and its binding partner can be detected by using detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled protein or its binding partner, by immunoassay or by chromatographic detection. In preferred embodiments, the protein or its binding partner can be immobilized to facilitate separation of complexes from uncomplexed forms of the protein and its binding partner and automation of the assay. Complexation of the protein to its binding partner can be achieved in any type of vessel, e.g., microtitre plates, micro- centrifuge tubes and test tubes. In particularly preferred embodiments, the protein can be fused to another protein, e.g., glutathione-S-transferase to form a fusion protein which can be adsorbed onto a matrix, e.g., glutathione sepharose beads (Sigma Chemical. St. Louis, Mo.), which are then combined with the labeled protein partner, e.g., labeled with ³⁵S, and test compound and incubated under conditions sufficient to formation of complexes. Subsequently, the beads are washed to remove unbound label, and the matrix is immobilized and the radiolabel is determined. The aforementioned cell-free assays are particularly useful with proteins encoded by the Genes of the invention. Another method for immobilizing proteins on matrices involves utilizing biotin and streptavidin. For example, the protein can be biotinylated using biotin NHS (N-hydroxy-succinimide), using well known techniques and immobilized in the well of streptavidin-coated plates. Cell-free assays can also be used to identify agents which are capable of interacting with a protein encoded by the at least one gene and modulate the activity of the protein encoded by the gene. In one embodiment, the protein is incubated with a test compound and the catalytic activity of the protein is determined. In another embodiment, the binding affinity of the protein to a target molecule can be determined by methods known in the art.

Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of ApoAI or SF-2 or DRP-2 or B-CK or Tubulin alpha 1 chain or Gamma enolase or Visinin or ROCK and LIMK or of a combination of thereof. In a related aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of an agent capable of regulating expression of the target of the invention as described above or of an agent identified in a screening assay as described above. More specifically, the present invention relates to a pharmaceutical composition comprising an effective amount of a PPARα agonist as described above. More preferably, said PPARα agonist is selected among fenofibrate, WY-14643 or GW7647.

One embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for the treatment of a disease related to ocular refractive status , such as for instance EDDs or myopia or hyperopia. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. The pharmaceutical compositions encompassed by the invention may be administered by any number of routes: e.g. Transscleral delivery of bioreactive protein to the choroid and retina, Ambati et al. (2000) Investigative Ophthalmology and Visual Science, 41 , 1186. The formulation of topical ophthalmic preparations, including ophthalmic solutions, suspensions and ointments is well known to those skilled in the art (see Remington's Pharmaceutical Sciences, 18th Edition, Chapter 86, pages 1581-1592 Mack Publishing Company, 1990). Other modes of administration are available, including intracameral injections (which may be made directly into the anterior chamber or directly into the vitreous chamber), subconjunctival injections and retrobulbar injections, and methods and means for producing ophthalmic preparations suitable for such modes of administration are also well known. As used in this application, "extraocular" refers to the ocular surface and the (external) space between the eyeball and the eyelid. Examples of extraocular regions include the eyelid fornix or cul-de-sac, the conjunctiva! surface and the corneal surface. This location is external to all ocular tissue and an invasive procedure is not required to access this region. Examples of extraocular systems include inserts and "topically" applied drops, gels or ointments which may be used to deliver therapeutic material to these regions. Extraocular devices are generally easily removable, even by the patient. The following patents disclose extraocular systems which are used to administer drugs to the extraocular regions. Higuchi et al. discloses in U.S. Pat. Nos. 3,981 ,303, 3,986,510 and 3,995,635, a biodegradable ocular insert which contains a drug. The insert can be made in different shapes for retention in the cul-de-sac of the eyeball, the extraocular space between the eyeball and the eyelid. Several common biocompatible polymers are disclosed as suitable for use in fabricating this device. These polymers include zinc alginate, poly (lactic acid), poly (vinyl alcohol), poly (anhydrides) and poly (glycolic acid). The patents also describe membrane coated devices with reduced permeation to the drug and hollow chambers holding the drug formulation. Theeuwes, U.S. Pat. No. 4,217,898, discloses microporous reservoirs which are used for controlled drug delivery. These devices are placed extraocularly in the ocular cul-de-sac. Among the polymer systems of interest include poly (vinylchloride)-co-poly (vinyl acetate) copolymers. Kaufman discloses in U.S. Pat. Nos. 4,865,846 and 4,882,150 an ophthalmic drug delivery system which contains at least one bio-erodible material or ointment carrier for the conjunctival sac. The patent discloses polymer systems, such as, poly (lactide), poly (glycolide), poly (vinyl alcohol) and cross linked collagen, as suitable delivery systems. In the presently described use of ApoAI, SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or of an agent in accordance with the present invention, protein product for the treatment of retinal disease or injury it is also advantageous that a topically applied ophthalmic formulation include an agent to promote the penetration or transport of the therapeutic agent into the eye. Such agents are known in the art. For example, Ke et al., U.S. Pat. No. 5,221 ,696 disclose the use of materials to enhance the penetration of ophthalmic preparations through the cornea. Intraocular systems are those systems which are suitable for use in any tissue compartment within, between or around the tissue layers of the eye itself. These locations include subconjunctival (under the ocular mucous membrane adjacent to the eyeball), orbital (behind the eyeball), and intracameral (within the chambers of the eyeball itself). In contrast to extraocular systems, an invasive procedure consisting of injection or implantation is required to access these regions. The following patents disclose intraocular devices. Wong, U.S. Pat. No. 4,853,224, discloses microencapsulated drugs for introduction into the chamber of the eye. Polymers which are used in this system include polyesters and polyethers. Lee, U.S. Pat. No. 4,863,457, discloses a biodegradable device which is surgically implanted intraocularly for the sustained release of therapeutic agents. The device is designed for surgical implantation under the conjunctiva (mucous membrane of the eyeball). Krezancaki, U.S. Pat. No. 4,188,373, discloses a pharmaceutical vehicle which gels at human body temperature. This vehicle is an aqueous suspension of the drug and gums or cellulose derived synthetic derivatives. Haslam et al. discloses in U.S. Pat. Nos. 4,474,751 and 4,474,752 a polymer-drug system which is liquid at room temperature and gels at body temperature. Suitable polymers used in this system include polyoxyethylene and polyoxy propylene. Davis et al. disclose in U.S. Pat. No. 5,384,333 a biodegradable injectable drug delivery polymer which provides long term drug release. The drug composition is made up of a pharmaceutically active agent in a biodegradable polymer matrix, where the polymer matrix is a solid at temperatures in the range 20 degree, to 37 degree. C. and is flowable at temperatures in the range 38degree. to 52degree. C. The drug delivery polymer is not limited to the delivery of soluble or liquid drug formulations. For example, the polymer can be used as a matrix for stabilizing and retaining at the site of injection drug-containing microspheres, liposomes or other particulate-bound drugs. A particularly suitable vehicle for intraocular injection is sterile distilled water in which the ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or an agent in accordance with the present invention is formulated as a sterile, isotonic solution, properly preserved. Yet another ophthalmic preparation may involve the formulation of the ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or an agent in accordance with the present invention with an agent, such as injectable microspheres or liposomes, that provides for the slow or sustained release of the peptide or agent which may then be delivered as a depot injection. Other suitable means for the intraocular introduction of ApoAI, SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or an agent in accordance with the present invention includes, implantable drug delivery devices or which contain the ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or an agent in accordance with the present invention. The ophthalmic preparations of the present invention, particularly topical preparations, may include other components, for example ophthalmically acceptable preservatives, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, antioxidants and surfactants, as are well known in the art. For example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Sufficient tonicity enhancing agent is advantageously added so that the formulation to be instilled into the eye is hypotonic or substantially isotonic. Suitable preservatives include, but are not limited to, benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide may also be used as preservative. Suitable cosolvents include, but are not limited to, glycerin, propylene glycol and polyethylene glycol. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta -cyclodextrin or hydroxypropyl- beta -cyclodextrin. Suitable surfactants or wetting agents include, but are not limited to, sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol and the like. The buffers can be conventional buffers such as borate, citrate, phosphate, bicarbonate, or Tris-HCI. The formulation components are present in concentrations that are acceptable to the extraocular or intraocular site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8. Additional formulation components may include materials which provide for the prolonged ocular residence of the extraocularly administered therapeutic agent so as to maximize the topical contact and promote absorbtion. Suitable materials include polymers or gel forming materials which provide for increased viscosity of the ophthalmic preparation. Chitosan is a particularly suitable material as an ocular release-rate controlling agent in sustained release liquid ophthalmic drug formulations (see U.S. Pat. No. 5,422,116, Yen, et. al.). The suitability of the formulations of the instant invention for controlled release (e.g., sustained and prolonged delivery) of an ophthalmic treating agent in the eye can be determined by various procedures known in the art, e.g., as described in Journal of Controlled Release, 6:367-373, 1987, as well as variations thereof. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, aiginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration may be formulated m aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuhc, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0. 1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of ApoAI, SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or an agent in accordance with the present invention, such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as, for instance, the chicken model described hereinbelow. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient, for example ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or fragments thereof, or an agent in accordance with the present invention, or antibodies to said peptides, agonists, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patents 5,008,114; 5,505,962; 5,641 ,515; 5,681 ,811 ; 5,700,486; 5,766,633; 5,792,451 ; 5,853,748; 5,972,387; 5,976,569; and 6,051 ,561.

In another aspect, kits are provided for detecting the level of expression of at least one gene selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK in a sample. For example, the kit can comprise a labeled compound or agent capable of detecting a protein encoded by, or mRNA corresponding to, at least one gene selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK, or a fragment of the protein, means for determining the amount of protein encoded by or mRNA corresponding to the gene or fragment of the protein; and means for comparing the amount of protein encoded by or mRNA corresponding to the gene or fragment of the protein, obtained from the subject sample with a standard level of expression of the gene, e.g., from a disease-free subject. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect protein encoded by or mRNA corresponding to the gene. ln a another aspect, the present invention provides gene therapy methods for the treatment of a disease related to ocular refractive status, such as for instance EDDs or myopia or hyperopia.

For instance, nucleic acids comprising a sequence encoding at least one peptide selected from the group consisting of ApoAI, SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK are administered by way of gene therapy to promote the restoration of the affected function. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. In a preferred aspect, the gene therapy vector comprises at least one peptide selected from the group consisting of ApoAI , SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK and expresses at least one peptide selected from the group consisting of ApoAI, SF-2, DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK or fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the coding region of at least one peptide selected from the group consisting of ApoAI, SF-2, DRP-2, B- CK, Tubulin alpha 1 chain, Gamma enolase, Visinin, ROCK and LIMK, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy. In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, e.g., U.S. Pat. No. 4,980,286 and others mentioned infra), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell- surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., U.S. Patents 5,166,320; 5,728,399; 5,874,297; and 6,030,954, all of which are incorporated by reference herein in their entirety) (which can be used to target cell types specifically expressing the receptors), etc. The nucleic acid can also be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (see, e.g., U.S. Patents 5,413,923; 5,416,260; and 5,574,205; and Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, a viral vector comprising a promoter and/or enhancer and at least one gene encoding a protein selected from the group of proteins consisting of ApoAI , SF-2 and Visinin or of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK, or a fragment thereof, is used for the treatment of an EDD, such as for instance hyperopia or myopia. In another embodiment, the present invention provides use of a viral vector comprising a promoter and/or enhancer and at least one gene encoding a protein selected from the group of proteins consisting of ApoAI , SF-2 and Visinin or of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK and LIMK, or a fragment thereof, for the manufacture of a medicament for the treatment of an EDD, such as for instance myopia or hyperopia. For example, a retroviral or lentiviral vector can be used (see, e.g., U.S. Patents 5,219,740; 5,604,090; and 5,834,182). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Methods for conducting adenovirus-based gene therapy are described in, e.g., U.S. Patents 5,824,544; 5,868,040; 5,871 ,722; 5,880,102; 5,882,877; 5,885,808; 5,932,210; 5,981 ,225; 5,994,106; 5,994,132; 5,994,134; 6,001 ,557; and 6,033,8843, all of which are incorporated by reference herein in their entirety. Adeno- associated virus (AAV) has also been proposed for use in gene therapy. Methods for producing and utilizing AAV are described, e.g., in U.S. Patents 5,173,414; 5,252,479; 5,552,311 ; 5,658,785; 5,763,416; 5,773,289; 5,843,742; 5,869,040; 5,942,496; and 5,948,675, all of which are incorporated by reference herein in their entirety. The following Examples illustrate the present invention, without in any way limiting the scope thereof.

EXAMPLES

Chicken are a very useful model in this field, as their ocular axial length can be manipulated by altering their visual environment (Schaeffel et al., Vision Res. 1988;28(5):639-57; Irving et al., Ophthalmic Physiol Opt. 1992;2(4):448-459; Schmid et al., Vision Res. 1996;36(7):1023- 36). Negative lenses, which place the image behind the retina, accelerate axial eye growth, whereas positive lenses slow it down. In this model, as in primates (Hung et al., Nat Med. 1995;1 (8):761-5; Smith et al., Vision Res. 1999 39(8):1415-35,), the retina and the sclera are known to be key players in the control of axial length and tissue samples can be obtained which are suited for proteomic studies. In an effort to analyze the molecular mechanisms of emmetropization, we undertook an extensive proteome analysis of chicken retina and sclera (emmetropic, myopic and hyperopic animals) using high-resolution 2D-gel electrophoresis and mass spectrometry (Hoving S et al., Electrophoresis. 2000;21 (13):2617-21 ).

Example 1

10-day old chicks were subjected to three types of experimental manipulations: Form deprivation myopia (FDM) with translucent goggles, lens-induced myopia (LIM) with -15D lenses and the opposite condition, lens-induced hyperopia (LIH) with + 15D lenses. FDM and LIM induced an elongation whereas LIH induced a shortening of the ocular globe. These treatments were applied only to one eye of each chick, the contralateral eye as well as eyes from animals without any visual manipulations being used as a control (CTL). The treatment was applied either for 1 day or 7 days, and the retina and sclera of both eyes were dissected. For the analysis of treatment-induced differential protein expression in retina and sclera solubilized tissue samples were separated by high-resolution two-dimensional electrophoresis (Hoving S et al., Electrophoresis. 2000;21(13):2617-21). The 2D gels thus obtained were stained in Sypro Ruby, digitized and analyzed to localize differentially expressed spots. Image analysis was performed using the Melanie 3 (Genebio, Geneva Switzerland) and Progenesis (Non-Linear Dynamics, Newcastle UK) software packages. Spots were selected if the normalized expression level was significantly different between control and any of the treatments at the p< 0.05 level (ANOVA). Since the digitized images sometimes contain artefacts, due to incomplete resolution of spots or background staining, differences were always checked and corrected, if necessary, by visual analysis of the original gels. Finally these spots were picked, subjected to trypsin digestion and identified by peptide mass fingerprinting using MALDI mass spectrometry or by partial sequencing using nanoelectrospray MSMS whenever necessary.

Example 2

One of the chick retinal protein that is differentially expressed has been identified as Apolipolipoprotein A1 (Apo A1 see Table I) which is upregulated after 1 day and 7 days of treatment with a +15 Diopter lens (Fig. 1), where axial length growth is slower compared to the control condition. This suggests that Apolipoprotein A1 might be related to a "stop" signal, which could be activated to stop the excessive growth associated with myopia. Chicken Apo A1 is thought to be the functional equivalent of both mammalian Apo A1 and Apo E (Dawson et al., J Biol Chem. 1986;261(13):5681-4).

4 chick retinal proteins that are differentially expressed were identified as Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase (see table I) which are all up-regulated after 1 day and/or 7 days of treatment with a -15 Diopter lens (Figs. 2, 3, 4, 5) where axial length growth is increased compared to the control condition. This suggest that Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase might be related to a "growth" signal which could be antagonized to stop the excessive growth associated with myopia.

Example 3

Methods

Animals

Male White Leghorn chickens (Gallus domesticus) were obtained from a local hatchery (company Weiss in Kirchberg, Germany) on day one after hatching. They were raised in groups of minimally ten animals in large cages at a 12-hour light / 12-hour dark cycle in the animal facility. The room temperature was kept at 30°C during the first week post-hatching and at 28°C thereafter. To accustom the chickens to human voices a radio played during the light period. Water and food was supplied ad libitum. Intravitreal injections

For a period of 4 successive days injections were performed daily under ether anesthesia. Two peroxisome proliferator-activated receptor alpha agonists (PPAR) (Fenofibrate and GW7647, Sigma). The 2 compounds were dissolved in vehicle solution: 70% saline + 30% DMSO . 12.5 μl of each freshly produced solution were injected monocularly with a 0.3 mm (30G) x 8 mm syringe cannula into the vitreous through skin, sclera, choroid and retina close to the margin of the upper orbit. The contralateral control eyes received 12.5 μl of vehicle solution (70% saline + 30% DMSO). 70% ethanol was used to disinfect feathers and skin surrounding the injection site and to sterilize needles between injections. Chickens were foraging normally approximately 10 minutes subsequent to the injections.

The following doses were injected (doses per eye):

Fenofibrate: 75 μg, 250 μg, 500 μg

Gw7647: 63 μg, 189 μg

Biometry

(A) Infrared photoretinoscopy

Refractive errors (RE) were measured by means of semiautomatic infrared (IR) photoretinoscopy (e.g. Schaeffel, Farkas and Howland, 1987; Schaeffel, Wilhelm and Zrenner, 1993; Schaeffel et al., 1994). The refraction procedure requires a real-time video board (Matrox Meteor II/4, The Imaging Source) to grab and store single video frames. An IR-sensitive video camera (Canon Infrared G-20PR) equipped with a Zeiss lens (Carl Zeiss 1.4 / 85) and a computer monitor were used to display the video frames. The IR photoretinoscope used consisted of 13 light emitting diodes (LEDs) mounted on a semicircle shaped metal disc. The retinoscope was fastened to the camera's lens by an adapter and covered the lower part of the camera's aperture. The LEDs were positioned at 4 eccentricities (distances to the edge of the LED disc and the optical axis of the camera, respectively) (eccentricities: 3 mm (2 LEDs), 9 mm (2 LEDs), 15 mm (3 LEDs), 21 mm (6 LEDs)). They were run at 12 volt. (B) A-scan ultrasonography

To measure axial ocular dimension a modified low-frequency A-scan ultrasound biometer (Echorule, Phakosystems Inc., 3M) with a transducer probe was used. The probe was designed for the human eye. Therefore its best spatial resolution was at about 2 cm from the transducer tip. To be able to measure chicken eyes the probe was elongated using a rubber tube (length 12 mm, inner diameter 4 mm, outer diameter 6 mm). The tube was filled with tap water and its tip covered with parafilm. Care was taken to avoid air bubbles being trapped in the tube. Measurements were performed in alert hand-held chickens. The cornea was locally anesthetized by one drop of a 2% Xylocain solution (AstraZeneca GmbH, Wedel). For measurements the tip of the rubber tube was moistened with the Xylocain solution and the cornea was touched for about one second. To determine the eye's anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD) and axial length (AL) ultrasound traces were temporarily frozen. Axial length was defined as the distance from the corneal surface to the retino-vitreal interface (equivalent to the sum of ACD, LT and VCD). Each eye was measured three to four times.

Devices to induce myopia: Minus lenses

The ophthalmic minus lenses used were modified human contact lenses (diameter: 10 mm, material: PMMA, Hecht Contactlinsen GmbH Germany). A power of -7 diopters (D) was chosen because this lens power is located within the linear compensation range for imposed defocus in chickens (Irving, Sivak and Callender, 1992).

The minus lenses were fixed in front of the eyes of the chickens via velcro ring supports. The devices were mounted on velcro rings using crazy glue and two-component glue, respectively. The counterpart rings were glued on the feathers surrounding the eye under light ether anesthesia (using crazy glue without sharp vapours).

Experimental procedure

All experiments were started on day 8 post-hatching. For a period of 4 successive days monocular intravitreal injections were performed daily. The contralateral control eyes received the vehicle. The control groups received vehicle into both eyes. Directly after the first injection ophthalmic -7D lenses were attached monocularly, in front of the compound- injected eyes. Lenses were cleaned daily and were worn for 4 days. Measurements (refractive error, axial eye dimensions and weight) were performed before the first injection (baseline measurements) and after the 4-day treatment period. Each group contained 7-9 animals.

Data analysis

After the 4-day treatment period interocular differences (differences between the treated eyes and their contralateral control eyes) were calculated for the two ocular parameters (refractive error and axial length). Subsequently the percentage of lens-induced myopia development relative to the binocularly vehicle-injected control group, which monocularly wore a -7D lens, was calculated. Mean % of lens-induced myopia + SEMs are presented.

Statistics:

The statistical significance of the data was analyzed using t test. Significance levels: p<0.001 , ** : p<0.01 , * : p<0.05.

Results

The effect of Fenofibrate, a PPARalpha agonist, on lens-induced myopia

Effect on refractive development

After the 4-day treatment period with Fenofibrate, the interocular difference in refractive error was calculated. Subsequently the % of lens-induced myopia development relative to the binocularly saline-injected control group, which monocularly wore a -7D lens, was calculated. % of lens-induced myopia development + SDs is shown in figure 8.

The data show that suppression of lens-induced myopia was dose-dependent (t test: significant dose-response effect (p<0.05)). The lowest dose tested (75 μg) already reduced lens-induced myopia by 11.87% (Lens-induced myopia: 88.13+ 29.63% vs control: 100+37.21%). 250 μg suppressed lens-induced myopia by 12.15% (Lens-induced myopia: 87.85± 28.57% vs control: 100+37.21%); 500 μg induced 28.83% inhibition (Lens-induced- myopia: 71.17+ 20.81% vs control: 100+37.21%).

The axial length measures are correlated with the refractive errors measures (data not shown).

The effect of GW7647, a potent human PPARalpha agonist, on lens-induced myopia

Effect on refractive development

After the 4-day treatment period with GW7647, the interocular difference in refractive error was calculated. Subsequently the % of lens-induced myopia development relative to the binocularly saline-injected control group, which monocularly wore a -7D lens, was calculated. % of lens-induced myopia development + SDs is shown in figure 9.

The data show that 189 μg treatment of GW7647 suppressed lens-induced myopia by 28.58% (Lens-induced myopia: 71.42+ 33.93% vs control: 100+28.29%); There was no significative difference between the treated group compared to the control group, but the % of inhibition is similar to Fenofibrate.

Table I Swissprot linked Swissprot linked chicken Genbank human Genbank reference chicken homolog human Apolipoprotein A-l precursor P08250 M25559 P02647 M1 1791 M17961 (homolog) M 18746 P02649 M 12529 M96012 (ortholog) Splicing factor-2 AJ448756 Q07955 BC010264 Dihydropyrimidinase related Q90635 U 17277 Q16555 U 17279 protein-2 B-creatine kinase P05122 X03509 P12277 L47647 Tubulin alpha 1 chain P02552 V00388 P05209 AF081484 Gamma enolase 057391 AB004291 P09104 M22349 ROCK-1 Q98SN6 AF347075 Q 13464 D87931 ROCK-2 Q98SN5 AF347076 Q9UQN5 U43195 LIMK-1 Q8QFP AW355608 P53667 D26309 LIMK-2 P53666 D26310 P53671 D45906 Visinin P22728 M84729 P35243 BT009838

Table II: Sequences

Apolipoprotein A1 :

Chicken amino acid sequence:

>sw| P08250|APA1_CHICK Apolipoprotein A-I precursor (Apo-AI)

MRGVLVTLAVLFLTGTQARSFWQHDEPQTPLDRIRDMVDVYLETVKASGKDAIAQFESSA

VGKQLDLKLADNLDTLSAAAAKLREDMAPYYKEVREM LKDTEALRAELTKDLEEVKEKI

RPFLDQFSAK TEELEQYRQRLTPVAQELKELTKQKVELMQAKLTPVAEEARDRLRGHVE

ELRKNLAPYSDE RQKLSQKLEEIREKGIPQASEYQAKVMEQLSNLREKMTPLVQEFRER

LTPYAENLKNRLISFLDELQKSVA

Chicken nucleotide sequence: 1 atgagaggcg tgctggtgac cctcgctgtg ctcttcctga cgggcatcca ggcccgctcc 61 ttctggcagc acgatgagcc ccagacgccc ctggaccgca ttcgggatat ggtggacgtc

121 tacctggaga cggtgaaggc cagcggcaag gatgccatcg cccagttcga gtcctctgct 181 gtgggcaaac agcttgacct gaagctggct gacaacctgg acacgctgag tgccgcggct

241 gccaagctgc gtgaggacat ggctccctac tacaaggagg tgcgcgagat gtggctgaag

301 gacaccgagg ctctgcgtgc tgagctcacc aaggacctgg aggaggtgaa ggagaagatc

361 cggcccttcc tggaccagtt ctccgccaag tggacggagg agctggagca gtaccgccag

421 cgcctgacgc ccgtggctca gaagctgaag gagctcacca agcagaaggt ggagctgatg

481 caggccaagc tgaccccggt ggctgaggag gcgcgggatc gtctgcgtgg gcacgtggag

541 gagctgcgca aaaacctggc gccatacagc gatgagctgc ggcagaagct gagccagaag

601 ctggaggaga tccgtgagaa gggcatcccc caggcttccg agtaccaggc caaggtgatg

661 gagcagctca gcaacctgcg tgagaagatg acgcctctgg tgcaggaatt cagggagcgc

721 ctcaccccct atgctgagaa cctcaagaac cgcttgatct ccttcctgga cgaactccag

781 aagtccgtgg cctga

Corresponding human amino acid sequence:

>sw| P02647 |APA1_HUAN Apolipoprotein A-I precursor (Apo-AI) - Homo sapiens

(Huma )

>sw| P02649 |APE_HU AN Apolipoprotein E precursor (Apo-E) - Homo sapiens

(Human)

- Splicing factor-2:

The sequences provided therein are predicted from the two contiguous chicken ESTs CB016853 and BU110078 (Genbank references) .

Predicted chicken amino acid sequence

>SF2_ESTcontig

MSGGGVIRGPAGNNDCRIYVGN PPDIRTKDIEDVFYKYGAIRDID KNRRGGPPFAFV

EFEDPRDAEDAVYGRDGYBYDGYRLRVEFPRSGRGTGRGGGGGGGGGAPRGRYGPPSRR

SEYRVIVSGLPPSGS QD KDHMREAGDVCYADVFRDGTGWEFVRKEDMTYAVRKLDN

TKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRSRSRSNSRSRSYSPRRSRGSPR

YSPRHSRSRSRT

Predicted Chicken nucleotide sequence 1 atgtccggag ggggcgtgat ccgcggccca gccggcaata acgactgccg catctacgtg 61 ggcaacctgc cccccgacat ccgcaccaag gacatcgagg acgtcttcta caaatacggc

121 gccatccgcg acatcgacct gaagaaccgc cgcggagggc cgcccttcgc cttcgtggag

181 tttgaggacc ccagggacgc ggaggacgcc gtctacgggc gggacggtta cgattacgat

241 gggtatcgcc tccgcgtgga gttccctcgc agcggccggg gcaccggcag aggaggcggc

301 ggcggcggag ggggcggagc cccgcggggc aggtacggcc ccccgtcccg gcgatcggag

361 tacagagtga tcgtctcggg gctgcctcca agtggaagtt ggcaggattt aaaggatcac

421 atgcgtgaag caggtgatgt atgttatgct gatgttttcc gagatggcac tggtgtcgtg

481 gagtttgtac ggaaggaaga tatgacctac gctgtgcgaa aactggataa cactaagttt

541 agatctcatg agggagaaac tgcctacatc cgtgttaaag ttgatggtcc aagaagtcca

601 agctatggaa gatctcggtc acgcagccgt agtcgtagca gaagccgtag tcgaagcaac

661 agcagaagcc gcagttattc cccaagaaga agcagaggat ctccacgcta ctctccccgc 721 cacagcagat cccgatctcg tacataa

Corresponding human amino acid sequence

>swall IQ07955 I SFR1_HUMAN Splicing factor, arginine/serine-rich 1 (pre-mRNA splicing factor SF2, P33 subunit) (Alternative splicing factor ASF-1) . Dihydropyriminidase related protein-2:

Chicken amino acid sequence:

>s |Q90635|DPY2_CHICK Dihydropyrimidinase related protein-2 (DRP-2) (Collapsin response mediator protein CRMP-62)

MSYQGKKNIPRITSDRLLIKGGKIVNDDQSFYADIYMEDGLIKQIGENLIVPGGVKTIEA HGRMVIPGGIDVHTRFQMPEQGMTSADDFFQGTKAALAGGTTMIIDHVVPEPGTSLLTAF DQWRE ADSKSCCDYSLHVDITE HKGVQEE EALVKDHGVNSFLVYMAFKDRFQLSDSQ IYEVLSVIRDIGATAQVHAENGDIIAEEQQRILELGITGPEGHVLSRPEEVEAEAVNRAI TIANQTNCPLYITKVMSKSAAEVIAQARKKGTVVYGEPITASLGTDGSHYWSK AKAAA FVTSPPLSPDPTTPDFLNSLLSCGDLQVTGSAHCTFNTAQKAVGKDNFTLIPEGTNGTEE RMSIIWDKAVVTGKMDENQFVAVTSTNAAKIFNLYPRKGRIAVGSDADLVIWDPDSVKTI SAKTHNISLEYNIFEGMECRGSPLVVISQGKIVLEDGNLHVTEGSGRYIPRKPFPDFVYK RIKARSRLAELRGVPRGLYDGPVCEVSVTPKTVTPASSAKTSPAKQQAPPVRN HQSGFS LSGAQIDDNIPRRTTQRIVAPPGGRANITSLG

Chicken nucleotide sequence: 1 atgtcttacc aggggaagaa gaacatcccg cggatcacga gcgatcgtct tctgatcaaa 61 ggtgggaaga tcgtcaatga cgaccagtct ttctatgcag acatctacat ggaagatggg 121 ctcatcaagc aaatagggga gaatctcatc gtgcccggag gagtgaaaac catcgaagcc 181 cacggcagga tggtgattcc tggagggatc gatgttcaca cccgcttcca gatgccagag 241 caggggatga cttctgctga tgacttcttc caagggacca aggctgcgct ggctgggggc 301 accaccatga tcattgatca tgtggttcct gagcccggga ccagtttgtt gactgcattt 361 gaccagtgga gagaatgggc agacagcaaa tcgtgctgtg attactccct gcacgtggac 421 atcaccgagt ggcacaaagg cgtccaggag gagatggaag ctctggttaa agatcacggt 481 gtgaattcat tcctggttta tatggctttc aaagaccgct tccagctgtc tgactcgcag 541 atctatgagg tcctgagtgt tatccgggat attggtgcca cagctcaagt gcatgctgag 601 aatggtgaca tcattgccga ggagcagcaa aggatcctgg agctgggaat cacaggccct 661 gaagggcacg tgttgagcag acctgaagag gtggaggcag aagctgtgaa ccgggcaatc 721 accatcgcca accaaaccaa ctgccctctc tatatcacca aggtgatgag caaaagcgct 781 gctgaagtca ttgctcaagc caggaaaaag ggcactgtgg tgtatgggga gcccatcaca 841 gccagcttgg gtactgatgg atctcattac tggagcaaga attgggcgaa ggcggccgct 901 tttgtcactt ccccaccact gagtcctgac ccaaccactc ctgatttcct caactcactg 961 ctgtcctgcg gggacctcca agttactggc agtgcccact gcaccttcaa cactgctcag

1021 aaagctgttg ggaaggacaa cttcacccta atccccgagg ggaccaacgg gactgaggag

1081 aggatgtcca tcatctggga caaggctgtg gtgactggca agatggatga gaaccagttt

1141 gtggctgtga ccagcacaaa tgcagctaaa atctttaacc tgtacccacg gaagggccgt

1201 attgcagtgg gctctgatgc tgatttggtt atctgggatc ctgacagcgt caagacaatc

1261 tctgccaaga cccacaacat atctctggag tacaacatct ttgaaggcat ggagtgtcgt

1321 gggtctcccc tggtggttat cagccaaggg aagatcgtgc tggaggatgg gaatctccac

1381 gtaacggagg ggtctgggag gtatatcccc aggaaaccat tccctgactt cgtttacaag

1441 cgcatcaaag caaggagcag gctggccgag ctgcggggtg tgcctcgagg tctctatgat

1501 gggccggtct gcgaagtgtc ggtgacgccg aagacggtca ctcctgcatc ttcagctaaa

1561 acatctcctg ccaaacagca agccccacct gtgaggaacc tgcaccagtc tggctttagc

1621 ttgtctggcg cacagatcga cgacaacatc ccacgccgca ccacccagcg catcgtggca

1681 ccgccgggcg gacgtgccaa catcaccagc ttgggttaa

Corresponding human amino acid sequence:

>s |Q16555|DPY2_HUMAN Dihydropyrimidinase related protein-2 (DRP-2) (Collapsin response mediator protein 2) (CRMP-2) (N2A3) - homo sapiens (Human)

B-Creatine kinase: Chicken amino acid sequence:

>s | P05122|KCRB_CHICK Creatine kinase, B chain (EC 2.7.3.2) (B-CK)

MPFSNSHNLLKMKYSVDDEYPDLSVHNNHMAKVLTLDLYKKLRDRQTSSGFTLDDVIQTG

VDNPGHPFIMTVGCVAGDEESYEVFKELFDPVIEDRHGGYKPTDEHKTDLNADNLQGGDD

LDPNYVLSSRVRTGRSIRGFCLPPHCSRGERRAIEKLSVEALGS GGDLKGKYYALRNMT

DAEQQQLIDDHFLFDKPVSPLLLASGMARDWPDARGI HNDNKTFLVWINEEDHLRVISM

QKGGNMKEVFTRFCTGLTQIETLFKSKNYEFMWNPHLGYILTCPSNLGTGLRAGVHIKLP

NLGKHEKFGEVLKRLRLQKRGTGGVDTAAVGGVFDVSNADRLGFSEVELVQMVVDGVKLL

IEMEKRLEKGQSIDDLMPAQK

Chicken nucleotide sequence: 1 atgcccttct caaacagcca caacctcctg aagatgaagt actccgttga cgatgagtac 61 cccgacctga gcgtgcacaa caatcacatg gccaaggtgc tgaccctgga cctgtacaag 121 aagttgaggg acaggcagac ttccagtgga tttacgctgg atgatgtcat tcagaccggg 181 gttgacaacc caggccaccc cttcataatg acagtaggat gcgtagctgg cgacgaagaa 241 tcctatgaag tgtttaagga actctttgat ccggttattg aggacaggca cggtggctac 301 aaaccaactg atgagcacaa gaccgacctg aatgctgata acctgcaggg tggtgacgac 361 ttggatccta attacgtgct gagctctcgt gtgagaactg gcaggagcat ccgtggattc 421 tgccttcccc ctcattgcag caggggagag agacgggcta ttgaaaagct ctctgttgaa 481 gctctgggca gtctgggagg tgatctcaag gggaagtact atgccctaag gaacatgact 541 gatgcagagc agcagcagct gattgatgat cacttcttgt ttgacaaacc tgtttctcct 601 cttctgttgg catctgggat ggcacgagat tggcctgatg ccaggggtat ctggcacaat 661 gacaacaaga ccttccttgt ttggatcaat gaggaggatc acctcagggt tatttccatg 721 cagaaaggtg gcaacatgaa ggaagtgttt actcgcttct gtacagggct aacacagata 781 gaaactctct tcaagtccaa aaactacgag ttcatgtgga acccacactt gggctacatc 841 ctgacctgcc catccaacct tggaacgggg ctccgtgctg gtgtgcacat caagctacca 901 aaccttggga agcatgagaa gtttggagaa gtcctcaaga ggcttcggct gcagaaacga 961 ggcacaggtg gtgtggacac agctgcagtc ggaggggtgt ttgatgtatc caatgctgat 1021 cgtcttggct tctctgaggt agagctggtg cagatggtgg tagatggtgt gaagctgctc 1081 attgaaatgg aaaaacgcct tgagaaaggc cagtccattg atgacctcat gccagctcag 1141 aaataa

Corresponding human amino acid sequence:

>sw|P12277]KCRB_HUMAN Creatine kinase, B chain (EC 2.7.3.2) (B-CK) Homo sapiens (Human)

Tubulin alpha 1 chain:

Chicken amino acid sequence:

>sw| P02552]TBA1_CHICK Tubulin alpha-1 chain (Fragment)

ETIGGGDDSFNTFFSETGAGKHVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDA

ANNYARGHYTIGKEIID VLDRIRKLADQCTGLQGFSVFHSFGGGTGSGFTS LMERLSV

DYGKKSKLEFSIYPARQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDI

ERPTYTNLNRLIGQIVSSITASLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAE

KAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKR

TIQFVDWCPTGFKVGINYQPPTVVPGGDLAKVQRAVCMLSNTTAIAEA ARLDHKFDLMY

AKRAFVHWYVGEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY

Chicken nucleotide sequence 1 gaaaccatcg gcggggggga cgactccttc aacaccttct tcagcgagac gggggccggc 61 aagcacgtgc cccgtgccgt ctttgtggac ctggagccca ccgtgatcga tgaggtgcgc 121 acggggacgt accggcagct cttccacccc gagcagctga tcacgggcaa ggaggatgcg 181 gccaacaact acgcccgggg gcactacacc atcggcaagg agatcatcga cctggtgctg 241 gaccgcatcc gcaagctggc tgaccagtgc acggggctgc agggcttctc ggtgttccac 301 agcttcgggg gcggcaccgg ctccggcttc acctcgctgc tgatggagcg cctgtcggtg 361 gactacggca agaagtccaa gctggagttc tccatctacc cggcccgtca ggtgtccacg 421 gccgtggtgg agccctacaa ctccatcctc accacccaca ccacgctgga gcactccgac 481 tgcgccttca tggtggacaa cgaggccatc tacgacatct gtcgcaggaa cctggacatc 541 gagcgcccca cctacaccaa cctcaacagg ctgatagggc agatcgtgtc ctccatcaca 601 gcctcactgc gcttcgatgg ggctctgaac gtcgacctga cggagttcca gaccaacctg 661 gtgccctacc ctcgcatcca cttcccattg gccacgtatg cgccggtgat ctctgctgag 721 aaggcctacc acgagcagct gtcggtggct gagatcacca acgcgtgctt cgagccggcc 781 aaccagatgg tgaagtgcga cccgcggcac ggcaagtaca tggcgtgctg cctgctgtac 841 cgcggggacg tggtgcccaa agatgtcaac gccgccatcg ccaccatcaa gaccaagcgc 901 accattcagt tcgtagactg gtgcccaact ggtttcaagg tgggcatcaa ctaccagcct 961 cccaccgtgg tgcccggggg ggacctggcc aaggtgcagc gcgccgtgtg catgctgagc 1021 aacaccacgg ccatcgccga ggcgtgggcg cgcttggacc acaagtttga cctgatgtac 1081 gccaagcgcg ccttcgtgca ctggtatgtg ggggagggca tggaggaggg ggagttctca 1141 gaggctcggg aggatatggc tgccctggag aaggattacg aggaggtggg ggtggattcg 1201 gtggagggtg agggtgagga ggaaggagag gagtactga

Corresponding human amino acid sequence:

>s |P05209|TBAl_HUMAN Tubulin alpha-1 chain (Alpha-tubulin 1) - Homo sapiens (Human)

Gamma enolase:

Chicken amino acid sequence:

>sp|057391|ENOG_CHICK Gamma enolase (EC 4.2.1.11) (2-phospho-D-glycerate hydro-lyase) (Neural enolase) (NSE) - Gallus gallus (Chicken)

MAVERIHAREILDSRGNPTVEVDLYTHKG FRAAVPSGASTGIYEALELRDNDKSRFLGK

GVLQAVDHINSTVAPAIVGSGLSVVDQEKIDNLMLEMDGTENKSKFGANAILGVSLAVCK

AGAAEKDVPLYRHIADLAGNSDLILPVPAFNVINGGSHAGNKLAMQEFMILPVGAESFRD

AMRIGAEVYHNLKSVIKEKYGKDATNVGDEGGFAPNILENSEALELLKEAIDKAGYTDKI

VIGMDVAASEFYRDGKYDLDFKSPDDPSRYISADELGDLYQSFVRAYPVLSIEDPFDQDD

WEAWSKFTANVGIQIVGDDLTVTNPKRIERAVEEKACNCLLLKVNQIGSVTEAIQACKLA

QENGWGVMVSHRSGETEDTFIADLVVALCTGQIKTGAPCRSERLAKYNQLMRIEEELGDE

ARFAGHNFRNPSVL

Chicken nucleotide sequence: 1 atggcagttg agaggatcca tgcccgagag atcctggatt ctcgtgggaa ccccactgta 61 gaggtggacc tgtacacaca caaaggcatg tttcgagcag cggtccccag cggagcatcc 121 actggcatct atgaagcatt ggagctacga gataatgaca agtcacgttt cctcggaaaa 181 ggggtcctgc aggccgtgga tcatatcaac agcactgtcg ccccagctat cgtgggctct 241 ggcctctctg ttgtagacca agagaagata gacaatctga tgcttgagat ggacggcaca 301 gagaacaaat ccaagtttgg tgccaatgcc attctgggag tttcactggc tgtgtgcaag 361 gcaggagctg cagagaagga tgtccccctg taccggcaca ttgctgacct ggcaggcaac 421 tccgatctca tccttcctgt gccagctttc aatgtgatca atggaggttc ccatgcaggc 481 aacaaattgg caatgcagga gttcatgatc ctacctgtgg gagctgaaag cttccgcgat 541 gccatgcgca ttggagctga agtctatcac aatctcaaga gtgttatcaa ggagaagtat 601 ggcaaggatg ctactaatgt gggtgacgag ggaggatttg ctcctaacat cctggaaaac 661 agtgaagctc tggagctcct caaggaagct attgacaagg ctggctacac agacaagatc 721 gttatcggta tggatgtggc agcctctgag ttctatcgag atggcaaata tgaccttgac 781 ttcaagtccc cagatgaccc aagccgctac atttctgcag atgagttggg tgacctctat 841 caaagctttg tacgtgctta tccagtgctt tccattgagg atccctttga ccaagatgac 901 tgggaggcct ggtccaagtt cacagccaac gttgggattc agatcgtggg agacgacctg 961 acagtgacaa accccaagcg cattgagcga gctgttgaag agaaggcatg caactgcctc

1021 ctgctcaaag tcaaccagat tggatctgtc acggaggcca tccaagcctg taagttggcc

1081 caggagaatg gctggggtgt gatggtgagt caccgatctg gggagaccga agacactttc

1141 attgctgatc tggtcgtagc actgtgcact gggcagataa agactggtgc cccctgcagg

1201 tctgaacgcc tggctaaata caaccagctc atgaggattg aggaggagct tggcgatgaa

1261 gcacgctttg ctggacacaa ctttcgcaac ccaagtgttc tttga

Corresponding human amino acid sequence: >sp|P09104 |ENOG_HUMAN Gamma enolase (EC 4.2.1.11) (2-phospho-D-glycerate hydro-lyase) (Neural enolase) (NSE) (Enolase 2) - Homo sapiens (Human)

ROCK1 :

Chicken amino acid sequence:

>trembl | Q98SN6 | Rho-associated coiled-coil forming kinase 1 (Fragment)

MKAEDYEVVKVIGRGAFGEVQLVRHKSSRRVYAMKLLSKFEMIKRSDSAFF EERDIMAF

ANSPWVVQLFYAFQDDRYLYMVMEYMPGGDLVNLMSNYDVPEK ARFYTAEVVLALDAIH

SMGFIHRDVKPDNMLLDKAGHLKLADFGTC KMNKEGMVRCDTAVGTPDYISPEVLKSQG

GDGYYGRECD SVGVFLYEMLVGDTPFYADSLVGTYSKIMNHKNSLTFPDDNEISKEAK

NLICAFLTDREVR GRNGVEEIKRHLFFKDDQ AWETLRDTVAPVVPDLSSDIDTSNFDD

IDEDKGEEETFPIPKAFVGNQLPFVGFTYYSNRRYLSVSAENSNDNRMGSSMDKSVMENM

QKMIYDLEEQLHNEMQLKDEMEQKCRSSNITLDKIMKELDEEGNQRKNLELPVSQIEKEK

MALQHKINDYQRKIDQESEKRRNVENEVSTLKDQMEDLKKISQHSQITNEKITQLQKQLE

EANDLLRTESETAVRLRKGNTEMSKSLSQVESLNRELQERCRVLESAKLQVEKDYYQLQA

ALESERRDRSHGSE IGELQVRITTLQEELKNLKNNLERVEAERKQAQDRLNHSEKEKNN

LEIDLNYKLKSLQDRLEQEVNEHKVTKARLTCKHQSIEEAKSVAMCEMEKKVKEERAARE

KAENRIVQAEKQCSMLEFDLKQSQQKVEHLLQQKERLEDEVKNLSLQLEQETNKRIMAQN

ELKAQAFEADNLKGSEKQLKQEINTLLEAKRLLEVELAQLAKQYRGNEGQMRELQDQLEA

EQYFSTLYKTQVKELKEEIDEKNKETQRKMQELQNEKETLTTQLDLAETKAESEQLARAL

LEEQYFELSQESKKAASRHRQEMTD

Chicken nucleotide sequence (EST): 1 atgaaagctg aagattatga agtggtgaag gtgattggca gaggggcatt tggggaagtt 61 cagttggtaa ggcataaatc atcaagaaga gtgtatgcta tgaaacttct gagcaaattt 121 gagatgataa aaagatcaga ttctgcgttc ttctgggaag aaagggatat tatggctttt 181 gctaatagtc cctgggttgt tcagttgttt tacgcattcc aagatgatcg ttacctttac 241 atggtcatgg aatacatgcc tggtggagac cttgtgaatt taatgagtaa ctatgatgtt 301 cctgagaaat gggcaagatt ttatactgct gaagttgttc ttgcattaga tgcaattcac 361 tcaatgggct ttatacacag agatgtgaag cctgacaata tgctgctaga taaagctggt 421 catttaaaac tagcagattt tggtacttgt atgaaaatga acaaggaagg tatggtacga 481 tgtgatacag ctgtagggac accagactat atctctcctg aagtattgaa atcccagggt 541 ggtgatggtt actatggacg agaatgtgac tggtggtcag ttggagtttt tttgtatgag 601 atgcttgtag gtgacacacc tttttatgca gattctttgg ttggaacata cagtaagatt 661 atgaaccaca agaactccct taccttccct gatgacaacg agatctctaa agaggcaaaa 721 aaccttattt gtgccttttt aactgatagg gaagtgagac taggacgaaa tggcgtagaa 781 gaaattaaac gacatctttt cttcaaagat gatcagtggg cttgggagac tctcagagac 841 actgtagcac cagttgtgcc cgacttaagt agtgacatag acactagtaa ttttgatgat 901 atcgatgaag acaaaggaga ggaagaaaca tttcccattc caaaagcatt tgtgggcaac 961 cagcttcctt ttgttggatt tacatattac agcaaccgtc ggtatttatc tgtctctgca

1021 gaaaattcca atgataacag aatgggctcc agtatggata aaagtgtgat ggaaaatatg

1081 cagaagatga tctatgattt ggaagaacag ctacataatg aaatgcagct gaaagatgaa

1141 atggagcaaa agtgcaggtc ttcaaacata acgctagaca agataatgaa agaactggat

1201 gaagagggaa atcaaagaaa gaacctagaa ttaccagtgt cccagattga gaaggaaaag

1261 atggcactgc agcataagat aaacgactac caaaggaaaa ttgaccaaga gagtgaaaag

1321 agacggaatg tggaaaatga agtgtctacg ctaaaagatc aaatggaaga cttgaaaaaa

1381 atcagccaac attcacaaat tacaaatgag aagataacac agttacaaaa acaactagaa

1441 gaagccaatg atttgctacg gacagaatca gaaacagcag taagactgag gaagggtaac

1501 acagaaatga gcaagtcatt aagtcaagta gaatcactga atagagaact gcaggaaagg

1561 tgtagggttc tagaaagtgc aaaactgcag gtggaaaagg attattatca gctgcaagca

1621 gctttggaat cagaacgaag ggacagaagt catggatctg aaatgattgg agaactacaa

1681 gttcggatta caactttaca agaagaatta aaaaacctta aaaataatct tgaaagagtg

1741 gaagcagaaa ggaaacaagc acaggatagg cttaatcatt cagaaaagga aaagaacaat

1801 ttggagatag atttgaacta caaactcaag tctttacaag accgtttaga acaggaagta

1861 aatgaacaca aagtgactaa agctcgatta acttgcaaac atcagtcaat agaggaggca

1921 aaatcagttg caatgtgcga aatggaaaaa aaagtaaagg aagaaagggc agcaagagaa

1981 aaggcagaga atcgaatagt tcaggctgaa aaacagtgtt ccatgttaga gtttgacctg 2041 aagcagtctc agcagaaagt ggaacacctt cttcagcaaa aggaaagact ggaggatgaa

2101 gtaaagaatc tgtcacttca gctagagσaa gagacaaata agcgaataat ggcacagaat

2161 gaattaaagg cacaagcttt tgaagcagat aacttgaagg gttctgagaa gcaactgaaa

2221 caagaaatca atacgttatt ggaagcaaaa agattactgg aagttgagtt ggctcaactt

2281 gctaagcagt acagaggaaa tgaaggtcaa atgcgtgagc ttcaggatca gcttgaagcg

2341 gagcagtatt tttcgacact ttacaaaact caggttaagg aactgaagga agaaattgat

2401 gaaaagaata aagaaactca aagaaaaatg caggaattgc aaaatgagaa agaaacactc

2461 actacgcagc tggatttagc tgaaaccaaa gctgagtcgg agcagttggc acgggcactt

2521 cttgaagagc agtattttga gttgagtcag gagagcaaaa aagcagcatc acgacatagg

2581 caagaaatga cagataa

Corresponding human amino acid sequence:

>trembl I Q13464 I Rho-associated, coiled-coil containing protein kinase plδOROCK

ROCK2:

Chicken amino acid sequence:

>trembl I Q98SN5 I Rho-associated coiled coil forming kinase 2 (Fragment)

MLLDKHGHLKLADFGTC KMDETGMVRCDTAVGTPDYISPEVLKSQGGDGYYGRECD S

VGVFLFEMLVGDTPFYADSLVGTYSKIMDHKNSLHFPDDVEISKHAKNLICAF TDRDVR

LGRNGVEEIKHHPFFKSDQ N DNIRETAAPVVPELSSDIDSSNFDDIEDDKGDVETFPI

PKAFVGNQLPFIGFTYYRDNLLLSDSSQSCRENESVHSSKNEFQKKLSKLEEQLSNELQA

KDELEQKYRSANTRLEKIVKELDEEITSRKNVESAVRQLEREKALLQHKNTEYQRKAEHE

ADKKRNLENEVNSLKDQLEDLKKRNQNSQISNEKINQLQRQLDEANSLLRSESETAARLR

KNQTESTKQIQQLEANNRELQDKNC LENAKLKEKDFLNLQSALESERRDRSHGSEIIS

DLQGRISS EEEVKNGKSALAKLEMEKRQLQEKLTDLEKEKSNMEIDMTYKFKVMQQNLE

QEEAEHKATKARLADKNKIYESIEEAKSEAMKEMEKKLLEERALKQKVENRLLEAEKQRS

MLDCDLKQSQQKINELLRQKDKLNEDVKNLTLKIEQETQKRCLTQNDLKMQTQQVNTLKM

SEKQLKQENNHLQEIKLSLEKQNNELRKERQDADGQMKELQDQLEAEQYFSTLYKTQVRE

LKEECEEKTKLCKEMQQKIQELQDERDSLAAQLEITLTKADSEQLARSIAEE

Chicken nucleotide sequence (EST): 1 atgctattag ataaacatgg gcacctgaag ctggcagatt ttggcacttg catgaaaatg 61 gatgaaacag gtatggtgcg ctgtgacacg gctgttggaa cccccgacta catatccccc 121 gaagttctga aatcacaagg aggtgatggt tattatggac gggagtgtga ctggtggtcc 181 gtaggagttt tcctttttga gatgctagtt ggtgatactc ctttttatgc agactcttta 241 gtagggacat acagtaaaat tatggatcac aagaactcat tacatttccc agatgatgta 301 gaaatctcta agcatgcaaa gaacctcatc tgtgccttct taactgacag ggacgtgcga 361 cttgggagaa atggggtaga agaaataaag catcaccctt tctttaagag tgatcagtgg 421 aattgggaca acatccgaga gactgctgct cctgttgttc ctgagcttag cagtgatata 481 gacagcagta attttgatga cattgaggat gacaagggag acgtggaaac ctttccaatc 541 cccaaagcct ttgtgggaaa ccagctgcct tttataggat ttacctacta tagagataat 601 ttgttgctaa gtgactcctc tcagtcttgc agagaaaatg aatcggtgca ttctagtaaa 661 aatgagtttc agaaaaaact aagcaagtta gaagaacaac tcagcaatga attgcaagcc 721 aaagatgaac tagaacagaa atacaggtct gctaataccc gtttagagaa gatagtgaaa 781 gagctagatg aagagataac ttcaaggaaa aacgtagagt ctgcagtcag acagctagaa 841 agggagaagg ctcttcttca gcataagaat acagaatacc agagaaaagc agaacatgaa 901 gcagataaga aacgcaattt ggaaaatgag gttaacagtt tgaaagatca gcttgaagat 961 ttaaaaaaga ggaatcagaa ctctcaaata tccaatgaga aaatcaatca actacaaaga

1021 cagttggatg aggccaactc tttgctgcga tcggagtctg aaactgcagc caggttaagg

1081 aaaaaccaga cggaaagtac aaagcaaatc cagcagttgg aagctaataa tcgagaacta

1141 caggataaga actgcctgct agagaacgct aaactcaaac tggagaagga ctttctcaat

1201 cttcagtcag ctctagaatc agaaaggaga gatcgaagtc atggatcaga gattatcagt

1261 gatttacaag gtcgaatatc gagcctggaa gaagaagtga aaaatggaaa gagtgcatta

1321 gccaaactag aaatggagaa aaggcaattg caggaaaaac ttacagattt agaaaaggaa

1381 aagagcaaca tggaaataga tatgacatac aaattcaaag ttatgcagca gaaccttgag

1441 caagaggaag ctgaacataa agctacaaaa gcacgattgg cagacaaaaa taagatttat 1501 gaatctatag aggaggcaaa atctgaagcc atgaaagaaa tggagaagaa acttt ggaa 1561 gagagagctt tgaagcaaaa agtagaaaat cggttgttag aagctgaaaa gcagcgctcc 1621 atgttggatt gtgatctcaa acaatcacag cagaaaataa atgaactcct taggcaaaag 1681 gataaactaa atgaagatgt taaaaacttg actttaaaaa tagaacaaga aacacaaaag 1741 cgttgtctca ctcaaaacga cctcaagatg caaacacaac aggtcaacac cttaaaaatg 1801 tcagagaagc agctaaaaca ggagaataat catcttcagg aaatcaaatt aagcctggag 1861 aaacaaaaca atgaactccg caaagaacgt caagatgcag atggacaaat gaaagagctt 1921 caagaccaac ttgaagctga acagtatttc tcgactctgt ataagacgca agttcgagaa 1981 cttaaagaag aatgtgagga aaagaccaaa ctttgtaaag agatgcagca aaagatacaa 2041 gagttacagg acgagagaga ttctttggca gcacagctag aaatcacttt gacaaaagca 2101 gattcagaac aacttgcacg ttccattgct gaagaaca

Corresponding human amino acid sequence:

>trembl I Q9UQN5 I Rho kinase

LIMK1:

Chicken amino acid sequence:

>trembl | Q8QFP8 | LIM kinase 1

MRLMLLCCT RDEPMGEEEGTDLPVCASCGQGIFDGQYLQA NADWHADCFRCGECGASL SHQYYEKDGRLYCKKDY ARFGELCHGCAEQITKGLVMVAGEQKYHPECFSCLNCRAFIG DGDTYALVERSKLYCGHCYYQMWTPVIEQILPDSPGSRIPHTVTLVSIPACSDGKRGFS VSIDPHCGAQGCGAEHSRTVRVREVDPDCISPDVKNSIHVGDRILEINGTPIGHVPLDEI DLLIQETSRLLQLTIEHDPHEPLPRDLALPCSPLPDPHSPLRSPVPAPHGDLGTMRQRAV MRSCSTDKSPGSSSVGSPASQRKDIGRSESLRVVSRAHRIFRPSDLIHGEVLGKGCFGQA IKVTHRETGEVMVMKELIRFDEETQRTFLKEVKVMRCLEHPNVLKFIGVLYKEKRLNFIT EYIKGGTLRGLIKSMDSHYP SQRVSFAKDIAAGMAYLHSMNIIHRDLNSHNCLVRENKS VWADFGLARLMVDEKNQPEHLQNLKKPDRKKRYTVVGNPY MAPEMINGRSYDEKVDIF SFGIVLCEIIGRVSADPDYLPRTTDFG NVRGFLERYCPPACPPSFFPIAACCCDLDPEK RPSFSKLEQWLETLRMHLDIRLPLSSQLEQLTCAF ETHRRGEGGLPPHPELPDTAPHLH PL

Chicken nucleotide sequence: 1 gctccgagtc gctgcgcgtc gtctctcgcg ctcaccgcat cttccgcccc tctgacctga 61 tccatggcga ggtgctgggc aagggctgct tcggccaggc catcaaggtg acgcacaggg 121 aaacgggcga ggtgatggtt atgaaggagc tgatccgctt tgatgaggag acgcagagga 181 ccttcctcaa agaggtgaag gtgatgcgct gcctggagca ccccaacgtg ctgaagttca 241 tcggagtgct ttacaaggag aagcggctca acttcatcac tgagtacata aaggggggca 301 ccttgcgggg cctcatcaag agcatggaca gtcactaccc ctggagccag cgggtcaagc 361 ttcgccaaag acattgctgc tggcatggcc ctacctgcac tccatgaaca tcatccancg 421 cgacctcaac tcccacaact gcctgg

Corresponding human amino acid sequence:

>sw|P53667|LIKl HUMAN LIM domain kinase 1 (EC 2.7.1.37) (LIMK-1)

- LIMK2:

Chicken amino acid sequence:

>sw|P53666|LIK2_CHICK LIM domain kinase 2 (EC 2.7.1.-) (LIMK-2)

MAGPPGEEV RCLGCGDLIAAGQRLYRMVNEAWHISCFRCSECQDPLTNWYYEKDGKLYC

HKDY GKFGESCHGCSLLMTGPVMVAGEYKYHPECFACMSCKVIIEDGDTYALVQHSTLY

CGKCHNQIVLTPMIEKHSTESLREQLPYTLTLISMPAATDGKRGFSVSVEGGCSSYATGV

QVKEVNRMHISPDVRNAIHPADRILEINGAPIRTLQVEEVEDLIRKTSQTLQLLIEHDPV

SQRLDRLRLDSRLPTHIKSPISPHSISPLDIKENLEGTLRRRSLRRSNSISKSPGPSSPK

EPLLLSRDISRSESLRSSSSCSQQIFRPCDLIHGEVLGKGFFGQAIKVTHKATGIVMVMK ELIRCDEETQKTFLTEVKVMRSLDHPNVLKFIGVLYKDKKLNLLTEYIEGGTLKDFLRNA DPFPWQQKVSFAKGIASGMAYLHSMCIIHRDLNSHNCLIKLDKTWVADFGLSRLIVEER KKPTLEKPSAKKRTLRKSDRKKRYTVVGNPY MAPEMLNGQSYDETVDIFSFGIVLCEII GQVYADPDCLPRTLDFGLNVKLF EKFVPADCPPAFFPLAAICCRLEPESRPPFSKLEDS FEALSLYLGELAIPLPSELEELDHNVSVQYGLNRDKLPENTT

Chicken nucleotide sequence: 1 atggcggggc cgccaggtga agaagtctgg agatgcctgg gatgtgggga cctcattgct 61 gctggtcagc gcctctacag gatggttaat gaagcttggc atatttcctg tttccggtgt 121 tctgaatgcc aggatcccct cacgaactgg tactatgaga aagatgggaa gctgtattgc 181 cacaaagact actgggggaa gtttggggaa tcttgccatg gctgctctct gctgatgact 241 ggacctgtga tggtggctgg agaatacaag tatcaccctg agtgctttgc ttgtatgagc 301 tgcaaagtga tcattgaaga tggggacact tacgcactgg tgcagcattc cactctctac 361 tgtggaaaat gccataacca gattgtgctg acaccgatga tagaaaaaca ttccactgag 421 tctctgcgtg agcagctgcc ttatacactg acactcatct ccatgccagc agctactgat 481 ggcaagaggg gcttctctgt gtctgttgag ggcggctgct ccagctatgc cactggcgtc 541 caagtgaagg aagttaacag gatgcacatc agcccagatg tccgaaatgc catccaccct 601 gcagatcgta tcctggaaat taatggagct cccattcgta cattacaggt ggaggaggtg 661 gaggacttga ttcgcaagac aagccagaca cttcagctgc tgatagagca tgaccctgtc 721 tcacagcgct tagacaggct tcggttggac tcccggcttc ccacccacat aaagtcaccc 781 atctctcccc actccatctc tcccctggac atcaaggaga atctggaagg aacgctccgc 841 cgacgatccc tcaggcgaag taacagcatt tctaagtctc ctggccccag ttctccaaag 901 gaacccctcc ttctgagccg tgacatcagt cgctctgaat ctctacgttc ctcttctagc 961 tgctcccagc aaatcttccg gccgtgtgat ctcattcatg gagaagtact aggaaaagga

1021 ttttttggac aagcaatcaa ggtgactcac aaagcaacag gaaaggtgat ggtgatgaaa

1081 gagctgattc gctgtgatga ggaaacacag aagacttttt tgacagaggt gaaagtgatg

1141 cgcagcctgg atcaccccaa tgtgctgaag ttcattggtg tactgtacaa ggacaagaag

1201 ctcaatctcc tcactgagta catcgagggt ggcaccctga aggacttcct ccgcaatgcg

1261 gacccgtttc cctggcagca gaaggtcagc tttgccaaag gaattgcctc tggaatggct

1321 tacttgcact ccatgtgcat cattcacaga gacctgaatt cacataactg cctaatcaag

1381 ttggataaga cagtggtggt ggctgacttc ggtctgtctc ggctgattgt ggaggaaaga

1441 aaaaagccca ctctggagaa gccatctgcc aagaaacgaa ccctgcggaa gagtgacagg

1501 aagaagcgct acacggtggt tggcaacccg tactggatgg ccccagagat gctaaatgga

1561 cagagctatg atgagacagt ggacatcttt tcatttggga tagttctttg tgagatcata

1621 ggccaggttt atgctgaccc agactgtctc ccacgcacac tggattttgg ccttaatgtt

1681 aagctgttct gggaaaagtt tgttcctgct gactgtcctc cagccttttt ccctctggct

1741 gccatttgct gtagactgga accagagagc aggccacctt tttccaagct tgaagattcc

1801 tttgaagctc tctctctcta cctgggagaa cttgccattc ccctcccatc tgagctggag

1861 gagctggatc acaacgtgag tgtgcagtac ggactgaacc gtgacaagtt acctgagaac

1921 acaacctag

Corresponding human amino acid sequence:

>s | P53671|LIK2 HUMAN LIM domain kinase 2 (EC 2.7.1.-) (LIMK-2)

Visinin:

Chicken amino acid sequence:

>sw| P22728 |VISI_CHICK Visinin

GNSRSSALSREVLQELRASTRYTEEELSR YEGFQRQCPDGRIRCDEFERIYGNFFPNSE

PQGYARHVFRSFDTNDDGTLDFREYIIALHLTSSGKTHLKLE AFSLFDVDRNGEVSKSE

VLEIITAIFKMIPEEERLQLPEDENSPQKRADKL AYFNKGENDKIAEGEFIDGVMKNDA

IMRLIQYEPKK

Chicken nucleotide sequence: 1 atggggaaca gccgcagcag cgctctgtcc cgcgaggtcc tccaggagct gcgcgcctcc 61 acgcgttaca cggaggagga gctgagccgt tggtacgaag gcttccagcg gcagtgcccc 121 gacggccgca tccgctgcga tgaattcgag cgcatttacg gcaacttctt ccccaattcg 181 gagcctcagg gctacgcccg ccacgtcttc aggagcttcg acaccaacga cgacggcacg 241 ttggacttca gggagtacat catagcgctg cacctcacct cgtccgggaa gacccacctg 301 aagctcgagt gggccttctc cttgttcgac gtggaccgca acggggaggt cagcaagagc 361 gaggtgctgg agatcatcac ggccatcttc aagatgatcc cggaggagga gcggctgcag 421 ctgcccgagg atgagaacag cccccaaaaa cgggccgaca aactgtgggc ctatttcaac 481 aaaggggaga acgataagat cgccgagggg gaattcattg acggggtgat gaagaacgac 541 gccattatgc gcctcatcca atacgaaccc aagaaataa

Corresponding human amino acid sequence:

>s | P35243 |RECO_HUMAN Recoverin (Cancer associated retinopathy protein) (CAR protein)

Claims

We claim:

1. A method to treat, prevent or ameliorate an EDD comprising administering an effective amount of a peptide selected from the group consisting of Apolipolipoprotein A1 , Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Visinin, Rho-Kinase and LIM-Kinase and mixtures thereof, to a subject in need of such treatment, prevention or amelioration.

2. A method according to claim 1 wherein said peptide is Apolipolipoprotein A1, Splicing factor-2 or Visinin and wherein said disease is myopia.

3. The method according to Claim 1 , wherein said peptide is Apolipoprotein A1.

4. A method according to claim 1 wherein said peptide is selected from the group consisting of Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase and LIM-Kinase and wherein said disease is hyperopia.

5. Use of ApoAI , SF-2 or Visinin for the manufacture of a medicament for the treatment of myopia.

6. Use of ApoAI for the manufacture of a medicament for the treatment of myopia.

7. Use of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK for the manufacture of a medicament for the treatment of hyperopia.

8. A method to treat, prevent or ameliorate an EDD comprising administering an effective amount an agent inhibiting or increasing the expression of at least one peptide selected from the group consisting of Apolipolipoprotein A1 , Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Visinin, Rho- Kinase and LIM-Kinase and mixtures thereof, to a subject in need of such treatment, prevention or amelioration.

9. A method according to claim 8 wherein said agent is an inhibitory nucleic acid.

10. A method according to claim 8 wherein said agent is an agent capable of increasing the expression of Apolipolipoprotein A1 , Splicing factor-2 or Visinin and wherein said disease is myopia.

11. The method according to Claim 10, wherein said peptide is Apolipoprotein A1 and wherein said agent is selected among PPARα agonists.

12. The method according to Claim 11 , wherein said agent is selected among the group consisting of fenofibrate, WY-14643 and GW7647(3).

13. A method according to claim 8 or 9 wherein said agent is an agent capable of inhibiting a peptide selected from the group consisting of Dihydropyrimidinase related protein-2, B- creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase and LIM-Kinase and wherein said disease is myopia.

14. Use of an agent increasing the expression of ApoAI , SF-2 or Visinin for the manufacture of a medicament for the treatment of myopia.

15. Use according to Claim 14, wherein said agent is an agent increasing ApoAI expression and is selected among PPARα agonists.

16. Use according to Claim 15, wherein said agent is selected among the group consisting of fenofibrate, WY-14643 and GW7647(3).

17. Use of an agent inhibiting the expression of DRP-2, B-CK, Tubulin alpha 1 chain, Gamma enolase, ROCK or LIMK for the manufacture of a medicament for the treatment of myopia.

18. A method for screening a subject for myopia or hyperopia or at risk of developing myopia or hyperopia comprising: a) detecting a level of expression of at least one gene selected from the group consisting of Apolipolipoprotein A1 , Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase and Visinin, Rho-Kinase and LIM-Kinase in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject.

19. A method for monitoring the progression of myopia or hyperopia in a subject comprising: a) detecting a level of expression of at least one gene selected from the group consisting of Apolipolipoprotein A1, Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase in a sample obtained from said subject, b) comparing said level of expression with the level of expression of said gene(s) in a sample obtained from a disease-free subject.

20. A method according to claim 18 or 19 wherein a lower expression level of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a greater expression level of Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase or LIM-Kinase in the subject sample compared to the sample from the disease-free subject is indicative of the subject having myopia or at risk of developing myopia.

21. A method according to Claim 18 or 19 wherein a greater expression level of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a lower expression level of Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase or LIM-Kinase in the subject sample compared to the sample from the disease-free subject is indicative of the subject having hyperopia or at risk of developing hyperopia.

22. A method according to any of claims 18, 20 or 21 wherein the level of expression of the gene is determined by detecting the level of expression of a mRNA corresponding to the gene.

23. A method according to any of claims 18, 20 or 21 wherein the level of expression of the gene is determined by detecting the amount of a protein encoded by the gene.

24. A method for identifying agents for use in the treatment of a myopia or hyperopia comprising: a) contacting cells expressing at least one gene encoding a protein selected from the group consisting of Apolipolipoprotein A1, Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase, Visinin, Rho-Kinase and LIM-Kinase with a candidate agent; b) detecting a level of expression of at least one of said genes in said cells, or detecting a lower or higher activity of said proteins.

25. A method according to claim 24 further comprising comparing the level of expression of at least one of said genes in the presence of the candidate agent with a level of expression of at least one of said genes in cells that are not contacted with the candidate agent, wherein a lower expression level of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a greater expression level of Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase and LIM-Kinase in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of hyperopia.

26. A method according to claim 24 further comprising comparing the level of expression of at least one of said genes in the presence of the candidate agent with a level of expression of at least one of said genes in cells that are not contacted with the candidate agent, wherein a greater expression level of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a lower expression level of Dihydropyrimidinase related protein- 2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase and LIM- Kinase in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of myopia.

27. A method according to claim 24 further comprising comparing the activity of said proteins in the presence of the candidate agent with the activity of said proteins in cells that are not contacted with the candidate agent, wherein a lower activity of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a higher activity of of Dihydropyrimidinase related protein-2, B-creatine kinase Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase or LIM-Kinase in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of hyperopia.

28. A method according to claim 24 further comprising comparing the activity of said proteins in the presence of the candidate agent with the activity of said proteins in cells that are not contacted with the candidate agent, wherein a higher activity of Apolipolipoprotein A1 , Splicing factor-2 or Visinin, or a lower activity of Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, Gamma enolase, Rho-Kinase or LIM-Kinase in the cells contacted with the candidate agent compared to the cells that are not contacted is indicative of an agent useful in the treatment of myopia.

29. A pharmaceutical composition comprising an effective amount of a peptide selected from the group consisting of Apolipolipoprotein A1 , Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase, Visinin, Rho-Kinase and LIM-Kinase or of a mixtures thereof.

30. A pharmaceutical composition comprising an effective amount of an agent according to claims 8, 9 or 24 to 28.

31. Use of a viral vector comprising a promoter and/or enhancer and at least one gene encoding a protein selected from the group of proteins consisting of Apolipolipoprotein A1, Splicing factor-2, Dihydropyrimidinase related protein-2, B- creatine kinase, Tubulin alpha 1 chain, and Gamma enolase, Visinin, Rho-Kinase and LIM-Kinase, or a fragment thereof, for the treatment of hyperopia or myopia.

32. The use of claim 31 wherein said vector is an adenoviral vector or an Adeno- associated viral vector.

33. A kit comprising a) a labeled compound or agent capable of detecting at least one protein selected from the group comprising Apolipolipoprotein A1 , Splicing factor-2, Dihydropyrimidinase related protein-2, B-creatine kinase, Tubulin alpha 1 chain, and Gamma enolase, or mRNA corresponding to the genes of said proteins, b) means for determining the amount of said proteins or mRNA corresponding to the genes of said proteins; and, optionally c) means for comparing the amount of said proteins encoded by or mRNA corresponding to the gene or fragment of the protein, with a standard level of expression of the gene and optionally instructions.