Use of Photomedin products for preventing and/or treating neuronal dysfunctions

Description

The invention provides compositions and methods for treating and/or preventing neuronal dysfunctions, particularly neurodegenerative disorders. The method of the invention involves administering to an individual in need of treatment a composition containing a Photomedin product and/or an effector/modulator thereof.

As the populations ages, an increase in degenerative disorders of the nervous system, including Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), Multiple Sclerosis, and others, is expected. Few treatment options exist for these debilitating diseases and those treatments that are available are of limited efficacy. A need exists for novel approaches to new treatments for these diseases.

Parkinson's disease is a progressive neurodegenerative disorder; symptoms include uncontrolled tremors, stooped posture and gait disturbances. Morphologically, Parkinson's disease is characterized by a loss of the pigmented dopaminergic neurons located in the substantia nigra, resulting in depletion of the neurotransmitter dopamine. Other groups of neurons, such as the noradrenergic neurons of the locus coeruleus, can also be affected (reviewed by Zhang et al. Neurobiol Dis. 2000 7(4):240-250).

The most common current treatments for Parkinson's disease focus on replacement of dopamine, either through direct administration of its immediate precursor, levodopa, or by administration of dopamine receptor agonists or inhibitors of 15 dopamine metabolic enzymes, such as L- deprenyl (Grunblatt et al. J Neurol. 2000;247 Suppl 2:1195-1102). Although

replacement of dopamine affords symptomatic relief, it does not slow the progression of the disease and patients often become refractory to treatment. Levodopa can also cause severe side effects. Therefore, there is a need for new drugs to treat this neurodegenerative disease.

Another example of a neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). ALS is a disorder characterized by progressive weakness and paralysis, eventually leading to death in 1-5 years (reviewed by Borchelt et al. Brain Pathol. 1998;8(4):735-757). The underlying condition that results in these symptoms is the selective degeneration and death of spinal cord motor neurons. Abnormal accumulation of neurofilament proteins is also associated with the disease. Although clinical trials have been conducted to investigate the efficacy of certain neurotrophic factors, an effective treatment for ALS has yet to be found (Mitsumoto Neurology. 1999 22;53(2):248-249). Only one drug (Riluzolek) is currently approved for treatment of ALS, and its effect is only modest (Hurko & Walsh. J Neurol Sci. 2000; 180(1-2):21-28). Genetic studies have provided some information about the cause of ALS.

One of the more common neurologic diseases in human adults is multiple sclerosis. This condition is a chronic, inflammatory CNS disease characterized pathologically by demyelination. There are five main forms of multiple sclerosis: 1 ) benign multiple sclerosis; 2) relapsing-remitting multiple sclerosis (RR-MS); 3) secondary progressive multiple sclerosis (SP- MS); 4) primary progressive multiple sclerosis (PP-MS); and 5) progressive- relapsing multiple sclerosis (PR-MS). It was hypothesized that multiple sclerosis is an autoimmune disease (Compston et al. Lancet. 2002;359 (9313):1221-1231 ; Hafler & Weiner. Immunol Today. 1989; 10(3): 104-107; Olsson T. Curr Opin Neurol Neurosurg. 1992;5(2): 195-202). Another theory regarding the pathogenesis of multiple sclerosis is that a virus, bacteria or other agent, precipitates an inflammatory response in the CNS, which leads to either direct or indirect myelin destruction, potentially with an induced autoimmune component (Lampert. Am J Pathol. 1978;91(1 ): 176-208., Martyn. Br Med Bull. 1997;53(1 ):24-39).

There is a need for novel approaches to treat neurodegenerative disorders. In this invention, we disclose novel and surprising neuroprotective functions for proteins referred to as Photomedins. The Photomedins of the invention are described in Fig. 5. Thus, the invention relates to the use of these genes and proteins in the prevention and/or treatment of neurodegenerative disorders.

Furutani et al., 2005, Biochem J. 2005;389(Pt 3):675-84) describe the identification of two novel olfactomedin-family extracellular proteins named Photomedin-1 and Photomedin-2. These proteins were secreted as disulphide-bonded dimers (Photomedin-1) or oligomers/multimers (Photomedin-2) with O-linked carbohydrate chains, although Photomedin-1 was proteolytically processed in the middle of the molecule after secretion. They found that Photomedin-1 was selectively expressed in the outer segment of photoreceptor cells in the retina and that Photomedin-2 was expressed in all retinal neurons. They also found that Photomedins preferentially bound to chondroitin sulphate-E and heparin. However, a function of Photomedins, in particular a function in vivo, has not been described.

WO 03/099318 describes the use of Photomedin products for preventing and/or treating pancreatic disorders, particularly those related to diabetes as well as neurodegenerative disorders. WO 2005/051408 describes the use of Photomedin for stimulating and/or inducing the differentiation of development of insulin producing cells from progenitor cells.

In this invention we found that Photomedins, in particular Photomedin-1 , act as an survival factor for neuronal cells. Thus, Photomedin products and effectors/molecules thereof are suitable as neuroprotective agents for inhibiting and/or preventing death of neuronal cells and/or for promoting survival and/or regeneration of neuronal cells. Further Photomedin products are suitable for detecting and/or verifying or for the treatment, alleviation and/or prevention of neuronal dysfunctions such as neurodegenerative disorders and/or neuronal

injury or trauma.

According to the present invention, Photomedin products and effectors/modulators thereof have been identified as neuroprotective agents. 'Neuroprotection' refers to protection of the central or peripheral nervous system from neuronal loss, axonal loss and/or myelin loss. Providing neuroprotection is one way to effect the prevention and/or treatment of neuronal dysfunctions such as neurodegenerative conditions and neurotrauma.

In this invention, Zebrafish was used as model system for neurodegenerative disorders. Zebrafish has become a popular model vertebrate for the study of developmental processes as well as for pharmacological and toxicological studies over the last decade (Rubinstein A.L., (2003) Curr Opin Drug Discov Devel. 6: 218-223; Grunwald D.J. and Eisen J.S., (2002) Nat Rev Genet. 3: 717-724). In this organism, large numbers of transparent embryos which rapidly develop outside of their mother are readily available. Transgenic lines expressing fluorescent proteins under the control of tissue-specific promoters allow to rapidly assess the effects of pharmacological treatments or gene loss- and gain-of- function treatments. Suppressing gene function in zebrafish embryos using antisense oligonucleotides, modified Peptide Nucleic Acids (mPNAs) or other antisense compounds with good efficiency and specificity yields phenotypes which are usually indistinguishable from genetic mutants in the same gene (Nasevicius A. and Ekker S.C., (2000) Nat Genet. 26: 216-220; Effimov et al., NAR 26; 566-575; Urtishak K.A: et al., 5th international conference on zebrafish development and genetics, Madison/WI 2002, abstr. #17). Thus, zebrafish embryos represent a relevant model to identify genes or compounds which control neuronal cell formation in humans. Zebrafish is known as established model system for human nervous system neurodegenerative diseases. Compounds that induce neurotoxicity in humans caused similar neurotoxicity in zebrafish. These results support the use of zebrafish as a predictive model for assessing neurotoxicity (Parng C,

et al., J Pharmacol Toxicol Methods. 2006 May 6).

Transgenic zebrafish as models for neurodegenerative disorders have been described in WO 02/082043. In particular, McKinley ET.et al Brain Res MoI Brain Res. 2005 Nov 30;141(2):128-137 show that the mechanism for dopaminergic neuron toxicity that is induced by a neurotoxin in mammals is proven to be conserved in zebrafish larvae. Also, the mechanism of spinal muscular atrophy (SMA) is similar in zebrafish and humans (Winkler et al., Genes Dev. 2005 Oct 1 ;19(19):2320-30). Further, zebrafish is used for studying the pathways underlying Alzheimer disease (Grilli et al., Funct Neurol. 2003 Jul-Sep;18(3):145-148; Tomasiewicz et al., J Neurosci Res. 2002 Dec 15;70(6):734-45)

The present invention relates to the use of a pharmaceutical composition comprising a Photomedin product and/or an effector/modulator of said Photomedin product for the manufacture of a neuroprotective agent. Preferably, the composition contains pharmaceutically acceptable carriers, diluents and/or additives.

According to the present invention, the pharmaceutical composition comprising the Photomedin product and/or the effector/modulator thereof may be used for diagnostic or therapeutic applications in vivo and/or in vitro. More particularly, the composition may be used as a neuroprotective agent for the treatment of neurodegenerative disorders, particularly neurodegenerative disorders of the central nervous system, e.g. involving the brain and/or the spinal cord, or of the peripheral nervous system. The neurodegenerative disorder may be selected from Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (MS), Multiple Sclerosis or other diseases as indicated in more detail below. Further, the composition is suitable for the treatment of neuronal injury or trauma. Furthermore, the composition is suitable for diagnostic purposes, e.g. in order for determining and/or monitoring the status and/or function of neuronal cells.

The closely related secreted Photomedin proteins (Photomedin-1 and Photomedin-2) are evolutionary conserved and present in the vertebrate organisms from zebrafish to human (see Fig. 5). In zebrafish, Photomedin-1 and Photomedin-2 have four homologues (zPhotomedin-1a, zPhotomedin-1 b, zPhotomedin-2a, and zPhotomedin-2b).

As used herein, the term "Photomedin product" includes Photomedin homologous proteins and nucleic acid molecules coding therefor, which are obtainable from vertebrate, e.g. fish, avian or mammalian species. Particularly preferred are nucleic acids encoding the human Photomedin protein and variants thereof. The invention particularly relates to a nucleic acid molecule encoding a polypeptide contributing to neuroprotection, wherein said nucleic acid molecule comprises

(a) the nucleotide sequence of human Photomedin, in particular Photomedin-1 , and/or a sequence complementary thereto,

(b) a nucleotide sequence which hybridizes at 50 ⁰ C in a solution containing 1 x SSC and 0.1 % SDS to a sequence of (a),

(c) a sequence corresponding to the sequences of (a) or (b) within the degeneration of the genetic code, (d) a sequence which encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the amino acid sequences of the human Photomedin proteins,

(e) a sequence which differs from the nucleic acid molecule of (a) to (d) by mutation and wherein said mutation causes an alteration, deletion, duplication and/or premature stop in the encoded polypeptide or

(f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 15-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.

As used herein, Photomedin proteins include purified natural, synthetic, or recombinant Photomedin-1 or Photomedin-2 and variants thereof. Variants include insertion, substitution and deletion variants and chemically modified

derivatives. Variants also include recombinant proteins, for example but not limited to hybrids or fusions of Photomedin-1 or Photomedin-2 and other proteins. Also included are proteins or peptides substantially homologous to the human Photomedin-1 or Photomedin-2 protein having the amino acid sequence published as GenBank Accession Number XP_034000 (Photomedin-1 ) or GenBank Accession Number NP_872293 (Photomedin- 2). The term "Photomedin product" also includes nucleic acids, e.g. RNA or DNA coding for the above described Photomedin-1 or Photomedin-2 protein product. The term "Photomedin product" also includes Photomedin-1 or Photomedin-2 homodimers or heterodimers of a Photomedin-1 or Photomedin-2 protein product and another protein.

The present invention also refers to proteins which have a high degree of identity to the biologically active Photomedin protein product having the amino acid sequence published as GenBank Accession Number XP_034000 (Photomedin-1) or GenBank Accession Number NP_872293 (Photomedin-2), that is preferably in excess of 70%, most preferably in excess of 80%, and even more preferably in excess of 90% or 95% over the entire protein. The degree of identity between the mouse and the human protein is about 91%, and it is contemplated that preferred mammalian Photomedin proteins will have a similarly high degree of identity. Also included are proteins which are hybrids between Photomedin-1 and another protein which retain the protective effect on neuronal cells found in Photomedin-1. The percentage of homology or percent identity between a Photomedin product and a human Photomedin protein or nucleic acid may be determined according to standard procedures, e.g. by using the BLAST algorithm. Preferably, it is calculated as the percentage of nucleotide or amino acid residues found in the smaller of the two sequences that align with identical nucleotide or amino acid residues in the sequence being compared, when four gaps in a length of 100 nucleotides or amino acids may be introduced to assist in that alignment. Also included as substantially homologous is any Photomedin protein product which may be isolated by virtue of cross-reactivity with antibodies to the Photomedin protein product

or whose genes may be isolated through hybridization with the gene or with segments of the gene encoding the Photomedin protein product.

As used herein, the term "effector/modulator" of a Photomedin product refers to any substance that modulates, induces or e.g. stimulates or inhibits the expression and/or function of Photomedin. Eξxamples of effectors/modulators of Photomedin products particularly refer to biologically active nucleic acids such as antisense molecules, ribozymes, siRNA molecules or precursors thereof. Further, the term encompasses antibodies or antibody fragments, aptamers, or scaffold proteins specifically directed against a Photomedin protein product. For diagnostic applications, the term "effector/modulator" also refers to hybridization probes and/or primers.

In one embodiment of the invention, functional assays in zebrafish were performed with Photomedin. A. Antisense oligonucleotides against Photomedin were designed to study the function of the Photomedin genes in zebrafish. Whole-mount in situ hybridizations were performed according to standard protocols as known to those skilled in the art and as described previously (for example, Pelton, R.W. et al., (1990) Development 110,609- 620; BeIo, J. A. et al., (1997) Mech. Dev. 68, 45-57). It was found in this invention that the zebrafish Photomedin-1 polypeptide and genes expressing those are expressed in the developing nervous system (see Fig. 2).The nucleic acid sequence encoding the Photomedin-2 protein is expressed in the boundary between midbrain and hindbrain (MHB) (see Fig. 1 ). Photomedin- 1 is expressed in the deep layers in the dorsal midline in early developmental stages (see Fig. 2). At 17 hpf, Photomedin-1 is expressed in the nervous system ganglia, and at 24 hpf expression can be viewed exclusively in the spinal cord, brain and eyes (see Fig. 2 and Examples for more detail).

In another embodiment, we injected wild type zebrafish one cell stage embryos with morpholino oligonucleotides directed to Photomedin-1 a or Photomedin-1 b. Upon injection with these oligonucleotides, the function of Photomedin-1 was

completed inhibited (see Rg. 3 and .Examples for more detail). In these fish embryos with inhibited function of Photomedin-1 (1a or 1b) or Photomedin-2 (2a or 2b), massive cell death was detected specifically in the brain and spinal cord (see Fig. 3 and Fig. 4, respectively, and Examples for more detail). Thus, the secreted protein Photomedin is required for neural cell survival during embryogenesis. Thus, Photomedins have a neuroprotective function, providing that Photomedins protect neuronal cells from cell death and degeneration. Thus, Photomedin products may be used for treatment of neurological disorders caused by excessive cell death, such as Alzheimer disease, Parkinson, ALS, Multiple sclerosis, and others. Neuroprotection thus comprises treating a neurodegenerative disease and/or delaying the death of neuronal cells associated with a neuronal dysfunction, e.g. a neurodegenerative disease.

According to this invention, the Photomedin product and/or modulator/effector thereof may be administered

(i) as a pharmaceutical composition, e.g. locally or systemically

(ii) via implantation of Photomedin protein product expressing cells and/or

(iii) via gene therapy as described in more detail below.

The compositions of the present invention may be used for the treatment and/or prevention of neuronal dysfunctions. For example, they may be suitable for delaying the onset of clinical symptoms and/or delaying the progress of neurodegenerative disorders. Further, they are suitable for providing neuroprotection to the central and/or peripheral nervous system.

In various embodiments, the neurodegenerative disease may be selected from Alzheimer disease, Parkinson disease, Creutzfeldt-Jakob disease, Huntington disease, Multiple sclerosis, Alper's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Alexander disease, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease, BSE, Canavan disease, Cockayne syndrome, Corticobasal degeneration, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy,

Olivopontocerebellar Atrophy, Pelizaeus-Merzbacher Disease, Progressive Supranuclear Palsy, Pick's disease, Postpolyomielitus syndrome, Primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, Shy-Drager Syndrome, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson- Olszewski disease, Tabes dorsalis, CIDP(Chronic Inflammatory Demyelinating Polyneuropathy, Guillian-Barre Neuropathy, Diabetic Neuropathy, Tumor related neuropathy, HIV Neuropathy, Hepatitis Neuropathy, Lyme Neuropathy, Alcohol Neuropathy, Agent Orange Neuropathy, Charcot-Marie-Tooth Disease, Carpal Tunnel Syndrome, Chemotherapy-Induced Neuropathy, radiation-induced Neuropathy, acute disseminated encephalomyelitis, transverse myelitis, demyelinating genetic diseases, virus-induced demyelination, Progressive Multifocal Leucoencephalopathy, Human Lymphotrophic T-cell Virus I (HTLVI)- associated myelopathy, a nutritional metabolic disorder/ acute glaucoma, chronic glaucoma, close angle glaucoma, open-angle glaucoma, optic neuritis, or systemic lupus erythematosus.

In a preferred embodiment, the medicament comprises a Photomedin protein product as an active ingredient. The Photomedin protein product may be a native or recombinant protein including fusion hybrid proteins. The protein may be a glycosylated or non-glycosylated protein.

Preferably, the protein is a recombinant protein which may be obtained by cloning the gene encoding the protein in a suitable vector introducing the vector into a host cell and expressing the gene in the host cell. The host cell may be a eukaryotic host cell, e.g. an insect cell or a mammalian cell including a human cell, or a prokaryotic cell, e.g. an E.coli cell. Exemplary methods for producing recombinant Photomedin products, vectors, host cells and culture growth conditions for the expression of Photomedin protein products are known to those skilled in the art.

Preferably, the Photomedin protein is a human Photomedin protein, e.g. the human Photomedin protein-1 or -2 or a variant thereof.

Photomedin protein product variants may be prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or by in vitro chemical synthesis of the desired polypeptide. It will be appreciated by those skilled in the art that many combinations of deletions, insertions, and substitutions can be made resulting in a protein product variant presenting Photomedin-1 or Photomedin-2 biological activity. Mutagenesis techniques for the replacement, insertion or deletion of one or more selected amino acid residues are well known to one skilled in the art Photomedin-1 or Photomedin-2 substitution variants have at least one amino acid residue of the human or mouse Photomedin-1 or Photomedin-2 amino acid sequence removed and a different residue inserted in its place. Such substitution variants include allelic variants, which are characterized by naturally occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change.

Chemically modified derivatives of Photomedin-1 or Photomedin-2 protein products also may be prepared by one of skill in the art given the disclosures herein. The chemical moieties most suitable for derivatization include water soluble polymers. A water soluble polymer is desirable because the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Preferably, the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. A particularly preferred water-soluble polymer for use herein is polyethylene glycol. One may specifically desire an N-terminal chemically modified protein.

The present invention contemplates use of derivatives which e.g. are prokaryote-expressed Photomedin-1 or Photomedin-2, or variants thereof, linked to at least one polyethylene glycol molecule, as well as use of Photomedin-1 or Photomedin-2, or variants thereof, attached to one or more polyethylene glycol molecules via an acyl or alkyl linkage.

The present invention also discloses use of derivatives which are e.g. prokaryote-expressed Photomedin-1 or Photomedin-2, or variants thereof, linked to at least one hydrophobic residue, for example fatty acid molecule, as well as use of Photomedin-1 or Photomedin-2, or variants thereof, attached to one or more hydrophobic residues. For example, patent application published as WO 03/010185, which is hereby incorporated by reference, describes a method for producing acylated polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are then to be acylated in a subsequent in vitro step.

In a further preferred embodiment, the agent comprises a polynucleotide encoding a Photomedin protein product. A polynucleotide encoding a Photomedin protein product can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such nucleic acid sequences are set forth, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), by Ausubel et al., eds (Current Protocols in Molecular Biology, Current Protocols Press, 1994), and by Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc., San Diego, CA, 1987). Chemical synthesis of a nucleic acid sequence which encodes a Photomedin protein product can also be accomplished using methods well known in the art, such as those set forth by Engels et al. (Angew. Chem. Intl. Ed., 28:716-734, 1989).

Included within the scope of this invention are Photomedin-1 or

Photomedin-2 product polynucleotides with the native signal sequence and other pre-pro sequences as well as polynucleotides wherein the native signal sequence is deleted and replaced with a heterologous signal sequence. The heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell. For prokaryotic host cells that do not recognize and process the native Photomedin-1 or Photomedin-2 signal sequence, the signal sequence may be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin 11 leaders. For yeast secretion, the native Photomedin-1 or Photomedin-2 signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders. In mammalian cell expression the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable. Expression and cloning vectors generally include a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.

Also encompassed by the invention is the use of polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those of the polynucleotide encoding the proteins of the invention, under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as described in Wahl G.M. et al., (1987; Methods Enzymol. 152: 399-407) and Kimmel A.R. (1987; Methods Enzymol. 152: 507-511), and may be used at a defined stringency. Preferably, hybridization under stringent conditions means that after washing for 1 h with 1 x SSC and 0.1% SDS at 50 ⁰ C, preferably at 55°C, more preferably at 62°C and most preferably at 65°C, particularly for 1 h in 0.2 x SSC and 0.1% SDS at 50 ⁰ C, preferably at 55 ⁰ C, more preferably at 62 ⁰ C and most preferably at 65°C, a positive hybridization signal is observed. Altered nucleic acid sequences encoding the proteins which are encompassed by the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent protein.

The encoded proteins may also contain deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of the protein is retained.

Furthermore, the invention relates to Photomedin fragments, e.g. peptide fragments of the proteins or derivatives thereof such as cyclic peptides, retro-inverso peptides or peptide mimetics having a length of at least 4, preferably at least 6 and up to 300 amino acids.

For example, the fragments may contain at least one Photomedin CXCXCX(9) C motif, where C is cysteine and X is any amino acid. The CXCXCX(9)C motive is specific for N-termini of mature Photomedins 1 and 2 and conserved across species. It is involved in cysteine bond formation and dimerisation of Photomedins. Preferably, these fragments comprise amino acids 21-260 of human Photomedin-2 or Photomedin-1 or at least 100 contiguous amino acids thereof or a sequence having an identity of at least

85%, preferably at least 90% and more preferably at least 95% thereto.

In another embodiment of this invention, peptides could be fragments encompassing the olfactomedin domain of Photomedin-2 or Photomedin-1 , or parts of it. For example, these fragments comprise amino acids 494-744 of mouse Photomedin-2 (olfactomedin-like 2B, Accession Number NP_796042); amino acids 498-748 of human Photomedin-2 (olfactomedin- like 2B, GenBank Accession Number NP_056256); amino acids 445-679 of mouse Photomedin-1 (olfactomedin-like 2A, GenBank Accession Number NP_766442), or amino acids 202-436 of human Photomedin-1 (Olfactomedin-like 2A GenBank Accession Number AAH54001 ) or at least 100 contiguous amino acids thereof or a sequence having an identity of at least 85%, preferably at least 90% and more preferably at least 95% thereto.

Also included within the scope of the present invention are allelic variants of the genes encoding the proteins of the invention. As used herein, an 'allele ¹ or 'allelic sequence' is an alternative form of the gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structures or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, are generally ascribed to natural deletions, additions or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others, one or more times in a given sequence.

In order to express a biologically active protein, the nucleotide sequences encoding the proteins may be inserted into appropriate expression vectors, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods, which are well known to those skilled in the art, may be used to construct expression vectors containing sequences encoding the proteins and the appropriate transcriptional and translational control elements. Regulatory elements include for example a promoter, an initiation codon, a stop codon, a mRNA stability regulatory element, and a polyadenylation signal. Expression of a polynucleotide can be assured by (i) constitutive promoters such as the Cytomegalovirus (CMV) promoter/enhancer region, (ii) tissue specific promoters such as the insulin promoter (see, Soria B. et al., (2000), Diabetes 49: 157-162), SOX2 gene promoter (see Li M. et al., (1998) Curr. Biol. 8: 971-974), Msi-1 promoter (see Sakakibara S. and Okano H., (1997) J. Neuroscience 17: 8300-8312), alpha-cardia myosin heavy chain promoter or human atrial natriuretic factor promoter (Klug M.G. et al., (1996) J. Clin. Invest 98: 216-224; Wu J. et al., (1989) J. Biol. Chem. 264: 6472-6479) or (iii) inducible promoters such as the tetracycline inducible system. Expression vectors can also contain a selection agent or marker gene that confers antibiotic resistance such as the neomycin, hygromycin or puromycin resistance genes. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic

recombination. Such techniques are described in Sambrook J. et al., (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel F.M. et al., (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

In a further embodiment of the invention, natural, modified or recombinant nucleic acid sequences encoding the proteins of the invention and homologous proteins may be ligated to a heterologous sequence to encode a fusion protein. Heterologous sequences are preferably located at the N- and/or C-terminus of the fusion protein.

A variety of expression vector/host systems, as known in the art, may be utilized to contain and express sequences encoding the proteins or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, adenovirus, adeno-associated virus, lentiverus, retrovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or PBR322 plasmids); or animal cell systems.

The presence of polynucleotide sequences of the invention in a sample can be detected by DNA-DNA or DNA-RNA hybridization and/or amplification using probes or portions or fragments of said polynucleotides. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences specific for the gene to detect transformants containing DNA or RNA encoding the corresponding protein. As used herein 'oligonucleotides' or Oligomers' refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting polynucleotide sequences include oligo-labeling, nick translation, end-labeling of RNA probes, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

The presence of Photomedin proteins in a sample can be determined by immunological methods or activity measurement. A variety of protocols for detecting and measuring the expression of proteins, using either polyclonal or monoclonal antibodies specific for the protein or reagents for determining protein activity are known in the art. {Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox D.E. et al. (1983; J. Exp. Med. 158: 1211-1226).

Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like.

Specific constructs of interest include anti-sense molecules, which will block the expression of the proteins of the invention, or expression of dominant negative mutations. A detectable marker, such as for example lac-Z, may be introduced in the locus of the genes of the invention, where up-regulation of expression of the genes of the invention will result in an easily detected change in phenotype.

One may also provide for expression of the genes of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development. In addition, by providing expression of the proteins of the invention in cells in which they are not normally produced, one can induce changes in cell behavior.

The data disclosed in this invention show that the Photomedin nucleic acids and proteins and effector/modulator molecules thereof are useful in diagnostic and therapeutic applications implicated, in neurodegenerative disorders. Hence, diagnostic and therapeutic uses for the proteins of the invention nucleic acids and proteins of the invention are, for example but not limited to, the following: (i) protection of nervous system (neuroprotection and protection of all cell types comprising nervous system and cell types interacting with the nervous system ) in vitro and in vivo, (ii) nervous tissue regeneration (regeneration of nervous system and regeneration of all cell types composing nervous system and cell types interacting with the nervous system) in vitro and in vivo, (iii) small molecule drug target, (iv) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (v) diagnostic and/or prognostic marker, (vi) protein therapy, (vii) gene therapy (gene delivery/gene ablation), and / or (viii) research tools.

For example, but not limited to, the polynucleotides and the proteins of the invention and particularly the human polynucleotides and polypeptides may be useful in preventing or regulating the degeneration of tissues or preventing the cell death of tissues when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from neurodegenerative disorders.

The Photomedin nucleic acids and proteins and effectors/modulators thereof are useful in diagnostic and therapeutic applications implicated in various embodiments as described below. For example, but not limited to Photomedin polynucleotides may be useful in gene therapy, and the Photomedin proteins

may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from neuronal dysfunctions, e.g. neurodegenerative disorders as described above.

The Photomedin nucleic acids and proteins and effectors/modulators thereof may be administered either as a monotherapy or as a combination therapy with other pharmaceutical agents. For example, they may be administered together with other pharmaceutical agents suitable for the treatment and/or prevention of neuronal dysfunction, e.g. neurodegenerative disorders. Further, they may be administered together with pharmaceutical agents which have an immunosuppressive activity, e.g. antibodies, polypeptides and/or peptidic or non-peptidic low molecular weight substances. Preferred examples of immunosuppressive agents are listed in the following Table 1.

Table 1 : Exemplary agents for immune suppression

The combination therapy may comprise coadministration of the medicaments during the treatment period and/or separate administration of single medicaments during different time intervals in the treatment period.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may comprise Photomedin nucleic acids and/or the proteins and/or effectors/modulators thereof such as antibodies, mimetics, agonists, antagonists or inhibitors of the proteins or nucleic acids as an active ingredient. 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 utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means. Preferably, the composition is administered by injection, e.g. by subcutaneous, intravenous or intramuscular injection.

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 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 compounds, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of preadipocyte cell lines or in animal models, usually mice, rabbits, dogs or pigs. 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 the Photomedin nucleic acids or proteins or fragments thereof or antibodies, which is sufficient for treating a specific 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 therapeutic and toxic 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 from 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 0.01 to 100,000 μg, up to a total dose of about 1 g, depending upon the route of administration. An especially preferred dosage is from 70 ng to 1 μg per kg body weight per injection. 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 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.

The Figures show:

Fig. 1 shows expression of Photomedin-2 homologues in developing zebrafish embryo (in-situ hybridisation) (shown in purple). A first expression can be noticed at 24 hours post fertilisation (hpf). Top panel: Photomedin-2a is expressed as a stripe aligning otic vesicle (ear primordia). Bottom panel: Photomedin-2b is expressed in the boundary between Midbrain and Hindbrain (MHB).

Fig. 2 shows expression of Photomedin-1 homologues in zebrafish (in-situ hybridisation) (shown in purple).

Fig. 2a shows expression of Photomedin-1 a at indicated developmental stages. At 10 hpf side view shows Photomedin-1 b is expressed in the deep layers in the dorsal midline (top left). Top (top right) and side (bottom left) of 17 hpf embryo views show expression in the nervous system ganglia. Bottom left: side view and 24 hpf shows expression in the spinal cord, brain and eyes.

Fig. 2b shows expression of Photomedin-1 b at indicated developmental

stages. At 10 hpf , side view shows Photomedin-1 b is expressed in the deep layers in the dorsal midline (top left), similar to Photomedin-1a. View from top at 12 hpf shows Photomedin-1b expression in the nervous system (top right); side view at 19 hpf shows expression in the posterior part of the body (bottom left); side view and 24 hpf shows expression in the spinal cord, brain and eyes.

Fig. 3a shows the phenotype of morpholino (MO) knockout of Photomedin- 1a and b in the head: Side view of the head region of 36 hpf zebrafish embryos injected with control MO (Photomedin2a, left), Photomedin-1 b MO (middle) and Photomedin-1a MO (right ). Top row: bottom light. Bottom row: acridine orange stain to visualize apoptotic cells (green). Note the abundant dead cells in the heads of Photomedin-1 b MO (middle) and Photomedin-1a MO (right ) injected embryos.

Fig. 3b Shows the phenotype of morpholino (MO) knockout of Photomedin- 1a and b in the whole body and the tail. Top row: side view of the whole 36 hpf zebrafish embryos injected with control MO (Photomedin2a, left), Photomedin-1 b MO (middle) and Photomedin-1a MO (right). Bottom row: side view of the tail region of the embryos, injected as above.acridine orange stain to visualize apoptotic cells (green). Note the abundant dead cells in the spinal cord of Photomedin-1 b MO (middle) and Photomedin-1a MO (right ) injected embryos.

Fig. 4 shows that Photomedin-2b is necessary for survival of the neural cells in the zebrafish embryo.

Fig. 4a. shows the expression of Photomedin-2b in the nervous system (in- situ hybridisation) (shown in purple).. Top view, 24 hpf embryo.

Fig. 4b. shows the top view of the trunk region of 36 hpf embryo injected with Photomedin-1b MO (light).

Fig. 4c. shows the same view as in Fig. 4b, but here acridine orange staining to vizualise dead cells was used (shown in green). Dead cells are localized to the neural tissue (compare to Fig. 4a).

Fig. 4d. shows the top view of the trunk region of 36 hpf embryo injected with control (Photomedin-2a) MO.

Fig. 4e. shows the same view as in fig. 4d, with acridine orange staining to vizualise dead cells (shown in green). Virtually no dead cells can be seen, which confirms the specificity of phenotype shown on fig.4c.

Fig. 5. shows the phylogenetic sequence alignment of Photomedin family members Hs - Homo Sapiens, Mm - Mus Musculus, and Dr -Danio Rerio.

Fig. 6 shows that Photomedin-2 is N-glycosylated and forms disulfide- bonded dimers.

Fig. 6a (Top) shows a drawing of the Photomedin structure. Gray - signal peptide, rectangle -N-terminal domain, containing tandem CXCXCX(9)C motives (X denotes any aminoacid), OLF- olfactomedin domain. Arrows - two predicted sites of N-glycosylation. Middle, bottom - scheme of N-Flag and N-His constructs (drawn not to scale).

Fig. 6b shows that N-deglycosylation reduces the molecular weight of Photomedin-2 N-Flag. 293 HEK cells were transiently transfected with N-

Flag construct, media was changed to serum-free one day after transfection.

Serum-free media was collected 2 days after transfection and concentrated.

Concentrated media were treated with N-glycosidaseF or left untreated. For

Western blot, 4 μg total protein of treated (*) and untreated sample were loaded per lane and anti-Flag M2 AP (alkaline phosphatase) coupled antibody was used.

Fig. 6c shows that Photomedin-2 forms dimers. N-His and N-Flag were

transfected separately or co-transfected (N-Flag + N-His) into 293 HEK cells. Serum-free media was collected and concentrated. 4 μg of total protein from double N-His and N-Flag transfection, N-Flag or N-His transfection were immunoprecipitated with Flag M2 agarose (lanes 1 ,3,4, respectively) half of each sample was loaded to the gel and hybridised with either anti-Flag or anti-His antibody. Lane 2: 2μg of concentrated supernatant of N-Flag and N- His transfection were mixed and immunoprecipitated with Flag M2 agarose. Lanes 5,6,7: 2 μg of concentrated crude supernatants were loaded per lane, as indicated. Note, that Flag immunoprecipitation pulls down N-His only when N-His and N-Flag are coexpressed in the same cells (lane 1), but not when they are mixed in solution (lane 2).

The examples illustrate the invention:

Example 1: In situ hybridisations

24 hr old zebrafish were fixed in parapholmaldehyde and processed for whole mount in situ hybridization with antisense DIG-labeled RNA probes for

Photomedin (PH)Ia, PH 1b, PH2a and PH2b, as described in (Hauptman G, Gerster.T. 1994. Trends Genet. 10:266.). For probe synthesis, PCR was performed using zebrafish 24 hours post fertilization (hpf) cDNA, prepared using Superscript Reverse Transcriptase (Invitrogen), according to manufacturer's instructions.

The following primers were used: Photomedin-1a (PH1a):

SEQ ID NO:1 Forward: δ'-CGCCGAGATGTGGAGGATTGTG-S';

SEQ ID NO:2 Reverse: δ'-CAGGTTGTTCATTTGAGACACC-S';

Photomedin-1 b (PH1 b):

SEQ ID NO:3 Forward: δ'-CATGGTGGATTTGCTGGAAGGA-S' SEQ ID NO:4 Reverse: δ'-CTCGCTCTTAGACGCTTTGTAG-S'

Photomedin-2a (PH2a):

SEQ ID NO:5 Forward: δ'-GAGCAGGTGGAAGAAGAAAAAG-S'

SEQ ID NO:6 Reverse: 5'-TGTTGCTAAAAGGAAGATGAGG-S'

Photomedin-2b (PH2b):

SEQ ID NO:7 Forward: 5'-TCTTGAGGAATCTTCATAATGGG-S'

SEQ ID NO:8 Reverse: δ'-GGGGAGTTTGTAGGAGTTGCTG-S'

PCR products were cloned and used for synthesis of DIG-labeled RNA probes for PH 1a, PH 1b, PH2a and PH2b were used as templates.

We found in this invention that RNAs encoding the zebrafish Photomedin-1 (PH1a and PH1b) proteins are expressed in the deep layers of the developing embryo and localize to the nervous system, including spinal cord, brain and eyes at 24 hpf (see Fig.2 a,b). We found in this invention that RNA encoding the zebrafish Photomedin-2a (PH2a) is specifically expressed in the otic vesicle (see Fig.1 , top) at 24 hpf and that the RNA encoding the zebrafish Photomedin-2b (PH2b) is expressed in the mid-hindbrain boundary (see Fig.1 , bottom).

Example 2: Identification of the Photomedin nucleic acid and protein sequences

The term "polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number" relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number. The term "GenBank Accession number" relates to NCBI GenBank database entries (Ref.: Benson D.A. et al., (2000) Nucleic Acids Res. 28: 15-18).

Sequences homologous to the mouse sequences were identified using the publicly available program BLASTP 2.2.3 of the non-redundant protein data base of the National Center for Biotechnology Information (NCBI) (see, Altschul S.F. et al., (1997) Nucleic Acids Res. 25: 3389-3402).

Photomedin proteins and nucleic acid molecules coding therefore are obtainable from vertebrate species, e.g. mammals or fish. Particularly preferred are nucleic acid molecules and proteins encoced thereby comprising

human and mouse.

To identify all possible zebrafish, human and mouse ortologues of Photomedin, sequence databases (NCBI non redundant protein database [ftp://ftp.ncbi.nih.gov/blast/db], EST section of NCBI Genbank (see Boguski et al., 1993, Nat Genet. 4: 332-333). dbEST-database for "expressed sequence tags", and zebrafish genome draft assembly 2 [http://www.ensembl.org/Danio rerio/1) were searched using the blastall programm (version 2.2.6, Altschul et al. 1997, Nucleic Acids Res. 25: 3389- 3402). Starting from the blast hits candidate genes were assembled and translated as necessary using the programms genewise (version 2.2.0, see http://www.ebi.ac.uk/Wise2/). getorf, est2genome, and showseq (from the EMBOSS package version 2.7.1 , see http://www.hqmp.mrc.ac.uk/Software/EMBOSS/). The resulting candidate protein sequences were compared to similar mouse and human proteins in multiple alignments made with the clustalw programm (version 1.83, see Thompson et al., 1994, Nucleic Acids Research, 22: 4673-4680) to verify the homology to Photomedin family; see Fig. 5.

Example 3. Loss-of-function experiments in zebrafish

Zebrafish were raised, maintained, and crossed as described (see, Westerfield M., (1995) The Zebrafish Book. Eugene, Oregon: Univ.of Oregon Press). Staging was performed according to Kimmel CB. et al., (1995) Dev Dyn 203: 253-310. Development of zebrafish embryos was carried out at 28°C. The age of embryos is indicated as hours post fertilization (hpf).

For loss-of function experiments, antisense morpholino oligonucleotides (Genetools) were used. If available from the assembly data (see Example 1 and Fig. 5), translation start sites were selected for antisense oligonucleotide targeting. Otherwise splice donor sites identified by alignment of zebrafish EST data or mouse protein data to zebrafish genomic sequence were used for antisense oligonucleotides targeting. The following morpholino

oligonucleotides were used: PH2a: 2 splice donor blockers

SEQ ID NO:9 S'-AATGTATCTTACCTGACTTAATATA-a' SEQ ID NO:10 δ'-CTGGTCACCTTGATATTATCTTTTC-S' PH2b 1 translation blocker

SEQ ID NO: 11 5'-ATAACAGTAATCCCATTATGAAGAT-3 ^I PH 1a 1 translation blocker

SEQ ID NO: 12 5'-GCTCCACAATCCTCCACATCTCGGC-3' PH1b 2 splice donor blockers SEQ ID NO:13 5'-TTACCTCTTCTAATGTGTTCATCTG-3' SEQ ID NO: 14 5 ^I -TGTGTTACCTGTGCTTTACTGTCTG-3 ^I

Photomedin or control antisense oligonucleotides were injected into fertilized one-cell stage embryos as described (see, for example, Nasevicius & Ekker, 2000, Nat Genet 26: 216-220; Urtishak et al., 2003, Dev Dyn. 228: 405-413). Injected embryos were analysed at different stages of development.(Fig.3 and Fig 4). We found in this invention that 'translation blocker' morpholino blocks the expression of maternal as well as zygotic RNA whereas the 'splice blocker' morpholino blocks only zygotic one. Therefore, surprisingly, the loss-of function phenotype of Photomedin-1a (PH 1a) is significantly more severe than the correspondant loss-of-function phenotype of Photomedin-1b (PH1 b), although the pattern of expression of these genes is quite similar (see Fig.2).

Example 4. Acridine orange stain of living zebrafish for identification of apoptotic cells.

Acridine orange staining is a standard simple technique which allows to visualise dead cells in zebrafish embryos: see, for example, ( Parng C, Seng WL, Semino C, McGrath P. Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1 ):41- 8). Zebrafish were incubated in 5 ng/ml acridine orange solution in water for 30 min, washed twice, put into tricaine working solution until the movement ceases, illuminated with blue light (argon lamp) and photographed using

Leica Stereomicroscope equipped with epifluorescence. Acridine orange stains apoptotic cells in the head and neural tube region of morpholino- injected PhMa and PhMb embryos (see Fig.3 and Fig.4, respectively).

Example 5: Expression of N-terminal Photomedin fragments

Photomedin-2 and Photomedin-1 contain a signal peptide followed by N- terminal cystein-rich domain, separated by a spacer region from C-terminal olfactomedin domain. Photomedin-2 contains two potential N-glycosylation sites at amino acid residues 187 and 213 (Fig.6 a), top). The N-terminal domain contains two cystein stretches CXCXCX(9)C, conserved between homologues in vertebrates (including zebrafish, mouse, rat and human), suggesting N-terminal disulfide-bonded dimer formation (see Figure 5). To test this directly, we expressed the N-terminal 240 amino acids of Photomedin-2 with C-terminally located 6XHis or Flag-Tag ( N-His and N- Flag in Fig 6 A) in the 293 HEK cells. Products of both constructs migrated as double bands (Fig.6). Nonreducing western blots of secreted Photomedin-2 expression constructs in 293 HEK cells detect the double bands of twice the molecular weight than under reducing conditions. We cotransfected N-His and N-Flag into 293 HEK cells, collected the supernatants and performed immunoprecipitation with Flag M2 agarose (Sigma), Fig. 6c, lane 1 ). Western blot with anti-his or anti-Flag antibodies shows that Flag immunoprecipitation pulls down N-His only when N-His and N-Flag are coexpressed in the same cells (lane 1 ), but not when they are mixed in solution (lane 2), confirming intracellular dimer formation by N- terminal domain of Photomedin-2. Treatment with N-glycanase (N- glycosidaseF) removes the higher molecular weight band of N-Flag and slightly increases mobility of the lower (Fig. 6b), suggesting that Photomedin-2 is N-glycosylated.

For the purpose of the present invention, it will be understood by the person having average skill in the art, that any combination of any feature mentioned throughout the specification is explicitly disclosed herewith.