WO2009083185A2 - Compositions and methods for prophylaxis and treatment of cellular degenerative ophthalmic disorders - Google Patents

Compositions and methods for prophylaxis and treatment of cellular degenerative ophthalmic disorders Download PDF

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WO2009083185A2
WO2009083185A2 PCT/EP2008/010922 EP2008010922W WO2009083185A2 WO 2009083185 A2 WO2009083185 A2 WO 2009083185A2 EP 2008010922 W EP2008010922 W EP 2008010922W WO 2009083185 A2 WO2009083185 A2 WO 2009083185A2
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unc51
kinase
nucleic acid
cell
cells
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PCT/EP2008/010922
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French (fr)
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WO2009083185A3 (en
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Anne Galy
Naomi Berdugo
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Fovea Pharmaceuticals Sa
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the present application relates to novel therapeutic strategies for cellular degenerative ophthalmic diseases, and more specifically diseases associated with photoreceptors degeneration. Said strategies are based on modulating one or more metabolic pathways identified by the inventors, which are correlated with the onset, development and progression of toxicity and apoptosis in eye cells, more specifically in photoreceptors.
  • the invention relates to a novel use of Unc51- Like Kinase modulators, and more specifically of Unc51-Like Kinase inhibitors.
  • the modulators of the invention are useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of cellular degenerative ophthalmic diseases, and more specifically photoreceptors degenerative disorders.
  • the invention relates to the use of modulators of the invention for the manufacture of pharmaceutical compositions giving a beneficial effect.
  • a beneficial effect is disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art.
  • the present Invention relates to compositions and methods for prophylaxis and/or treatment of cellular degenerative ophthalmic diseases, and more specifically degenerative conditions affecting photoreceptors in a mammal using a composition which comprises an effective amount of a Unc51-Like Kinase modulator.
  • the ophthalmic degenerative diseases of the present invention are retinal degenerative diseases, and more specifically are degenerative conditions affecting photoreceptors.
  • retinal degeneration one major cause of blindness is retinal degeneration, and the most common forms of retinal degeneration are retinitis pigmentosa, glaucoma and age- related macular degeneration (AMD) .
  • Retinitis pigmentosa is affecting 1 in 4000 people worldwide and leaving more than 1.5 million people visually handicapped.
  • retinitis pigmentosa is characterized by progressive degeneration of the peripheral retina, leading to night blindness, progressive loss of the peripheral visual field, leading eventually to total blindness, abnormal electroretinogram, ophthalmoscopic changes consisting in dark mosaic like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration (Delyfer et al., 2004, Biol Cell., 96, 261-269) .
  • Retinitis pigmentosa which can be both sporadic and familial, is a disorder which has been linked to a number of different genes. The condition primarily affects the rod cells of the retina, but can eventually lead to loss of peripheral vision and blindness.
  • AMD Age-related macular degeneration
  • AMD affects approximately 15,000,000 people in the United States. It is estimated that total blindness eventually results in 5-10% of these persons. AMD accounts for 17% of new cases of blindness in the United States annually.
  • AMD is primarily a disease of individuals over 65 years old, the incidence of AMD is expected to increase as the population ages. It is characterized by a sudden worsening and distortion of central vision that progresses rapidly, typically with a course of only weeks or months. AMD is characterized by abnormalities in the macular area.
  • the central area (or fovea) of the macula contains the highest density of cone photoreceptors in the retina and mediates high-acuity vision.
  • the disease typically has a preclinical, asymptomatic phase, in which extracellular waste material accumulates in the space between the basement membrane (Bruch's membrane) and the epithelial layer, forming yellow-white spots known as drusen.
  • Advanced forms of AMD includes both dry and wet (or "neovascular” ) AMD.
  • the dry form of AMD is far more common, but the wet form occurs simultaneously with the dry form in about 15 % of cases.
  • Dry AMD is characterized by progressive apoptosis of cells in the epithelial layer, in the overlying photoreceptor cells and in the underlying cells in the choroidal capillary layer.
  • Wet AMD is characterized by choroidal neovascularization with vascular leakage into subretinal spaces. AMD impairs central vision that is required for reading, driving, face recognition, and fine visual tasks.
  • the retina while it is containing neuronal cells [e.g. photoreceptors (rods and cones), ganglion cells, horizontal cells, amacrine cells, bipolar cells] which can be affected by cellular degeneration process, further contains non neuronal cells (e.g. Muller cells or pigmented epithelial cells) which can also degenerate and therefore be the target of the method and compositions of the present invention.
  • neuronal cells e.g. photoreceptors (rods and cones), ganglion cells, horizontal cells, amacrine cells, bipolar cells
  • non neuronal cells e.g. Muller cells or pigmented epithelial cells
  • retinal pigment epithelium is an important cell types in the retina, as it is crucial to the support of the photoreceptor function.
  • the major target of the method and compositions of the present invention are the photoreceptors.
  • a visual input which is capable of activating retinal ganglion cells. If activation by the visual input is successful, then varying degrees of vision should be restored.
  • One suggested method for activating retinal ganglion cells through a visual input is with an electronic stimulator which electrically activates the retinal ganglion cells.
  • electronic stimulator is the focus of numerous research activities, however there remain many problems associated with the use of an electronic stimulator.
  • the lines at present most widely followed by international research are (i) the genetic approach, which seeks to identify the gene or genes responsible for the illness and thus permitting a subsequent intervention by means of modern genetic engineering techniques, (ii) the transplant approach, which aims at perfecting a technique that would make possible the transplant of retinal tissue or, at least, the grafting of healthy cells into diseased retinas, and (iii) the immunological approach, which sets out to verify some theories that assume the illness to be underlain by some alteration of the immunological system.
  • the genetic approach which seeks to identify the gene or genes responsible for the illness and thus permitting a subsequent intervention by means of modern genetic engineering techniques
  • the transplant approach which aims at perfecting a technique that would make possible the transplant of retinal tissue or, at least, the grafting of healthy cells into diseased retinas
  • the immunological approach which sets out to verify some theories that assume the illness to be underlain by some alteration of the immunological system.
  • genes associated with retinal degeneration several groups are specifically expressed in the retina and are critical for
  • RHO rhodopsin
  • cGMP rod cyclic guanosine monophosphate
  • PDE6A and PDE6B rod cyclic guanosine monophosphate phosphodiesterases
  • CNGAl rod cyclic nucleotide gated channel
  • SAG arrestin
  • ATP-binding cassette transporter of rods ABCR
  • CRaIBP cellular retinaldehyde binding protein
  • RPE65 RPE 65 protein
  • RGR RPE- retinal G-protein coupled receptor
  • retinal degeneration code for photoreceptor cell structural proteins, including peripherin (also named retinal degeneration slow, RDS) , rod cell outer membrane protein 1 (ROMl), retinal actin-binding protein fascin (FSCN2) and prominin like-1 (PROM-I) .
  • peripherin also named retinal degeneration slow, RDS
  • ROMl rod cell outer membrane protein 1
  • FSCN2 retinal actin-binding protein fascin
  • PROM-I prominin like-1
  • genes encoding critical metabolic enzymes such as inosine monophosphate dehydrogenase 1 (IMPDHl) and lecithin retinol acyltransferase (LRAT) , are also important for retinal function, and mutations in these genes cause retinal degeneration.
  • IMPDHl inosine monophosphate dehydrogenase 1
  • LRAT lecithin retinol acyltransferase
  • the object of the present invention is to provide a composition that will permit cellular degenerative ophthalmic diseases to be efficiently treated and/or prevented.
  • said composition is comprising pharmacological acceptable carrier.
  • the present invention relates to prevention and/or reduction of eye cells degeneration, more specifically retinal cells degeneration, and even more specifically of photoreceptor cells degeneration.
  • Another purpose of the present invention is to provide a method of preventing and/or treating cellular degenerative ophthalmic diseases, more particularly a method of preventing and/or treating retinal degeneration, and even more specifically a method of preventing and/or treating photoreceptor cells degeneration, that will permit for example a gradual recovery of visual acuity, or at least limit its loss, as well as limiting the loss of visual field, sharpness of images and perception of colours and an electroretinogram (ERG) decrease stabilisation and/or ERG recovery in the long run.
  • said retinal degeneration is photoreceptor cells degeneration.
  • Protein kinases are known to play crucial roles in nearly all aspects of cellular regulation, including cell growth, differentiation, and apoptosis in eukaryotes. Many protein kinases with specific or multiple functions have been characterized, which are different in primary structure, regulation modes and substrate specificities. A large number of protein kinase genes have been identified in the course of genome sequencing projects of various eukaryotes, for example, by sequencing the complete genome of S. cerevisiae and C. elegans (Chervitz et al., 1998, Science, 282, 2022-2028).
  • Phylogenetic analysis of protein kinases allows sub-grouping of structurally related members, which also reflects related function or regulation (Hanks and Hunter, 1995, FASEB J., 9, 576-596) .
  • yeast and nematode also have homologous counterparts in mammals and other species to constitute subfamilies.
  • Unc51-Like kinases are members of an evolutionarily conserved sub-family of ubiquitously expressed serine/threonine-specific protein kinases. Unc-51 was originally discovered in nematode C. elegans as a gene required for axonal elongation and guidance (Ogura et al., 1994, Genes Dev. , 8, 2389-2499; Ogura et al., 1997, Genes Dev., 11, 1801-1811; Tomoda, 1999, Neuron 24, 833-846) and was also linked to endocytosis (Tomoda, et al, 2004, Genes Dev 18, 541-558).
  • Unc-51 gene mutants In Unc-51 gene mutants, most of the neurons exhibit an aberrant axon elongation with premature termination or dysregulated numbers of axons in abnormal. In mammals, Ulkl was shown to be important for axon formation in cerebellar granule neurons (Tomoda et al.,1999, and Tomoda et al., 2004, supra) . ) . More recently, Zhou et al. have shown that Unc51- Like kinases 1 and 2 are involved in filopodia extensions and branching in sensory axons from mouse spinal sensory neurons, this process being mediated by endocytosis (Zhou et al. 2007, PNAS: 104 14. 5842-5847).
  • Unc51-Like kinases 1 and 2 are involved in NGF induced axonal growth, since blocking their expression using RNAi technology, reduces axon lengh and increases axonal branching of neurons stimulated by NGF(ZhOu et al. 2007, PNAS: 104 14. 5842-5847.
  • Unc51-Like kinases 2 has been demonstrated to physically interact with FRS2/3 (Fibroblast Growth Factor Substrate 2 and 3), an adaptor protein to FGF Receptor, therefore antagonizing FGFR/FRS2/3 interaction and consecutive FRS2/3 phosphorylation (Avery et al. 2007 Cell Signal. 19: 177-184).
  • FRS2/3 Fibroblast Growth Factor Substrate 2 and 3
  • Unc51 Mammalian homologue of Unc51 (called UIk-I for Unc-51 kinase 1) have been identified based on sequence homology to the C. elegans Unc-51 gene ( mouse : Yan et al., 1998, Biochem Biophys Res Commun, 246, 222-227, Human : Kuroyanagi et al.,1998, Genomics, 51,76-85 ).
  • ULKl also known as Unc51-Like kinases 1, ATGl, Hs.47061, Unc51.1, EC 2.7.11.1, Serine/threonine-protein kinase ULKl ) contains 1050 amino acids (Kuroyanagi, H. et al. Genomics 51 (1), 76-85 (1998)).
  • ULKl Protein sequence (NP_003556.1) is as follows :
  • ULK2 also known as Unc51-Like kinases 2,Unc51.2 , EC 2.7.11.1 , Serine/threonine-protein kinase ULK2 ) contains 3923 amino acids (Yan,J et al. Oncogene 18 (43), 5850-5859 (1999) ) .
  • ULK2 protein sequence (NP_055498.2) is as follows : MetGluValValGlyAspPheGluTyrSerLysArgAspLeuValGlyHisGlyAlaPhe
  • ULK3 also known as Unc51-Like kinases 3, EC 2.7.11.1 , Serine/threonine-protein kinase ULK3 contains 1763 amino acids predicted ULK3 protein sequence (XP_001134013.1) is as follows :
  • Patent application WO 2002079478 provides means and methods for modulating the biological and biochemical activities of Unc51-Like kinases for treating metabolic disease or condition such as obesity, adipositas, eating disorders, wasting syndrome, pancreatic dysfunctions (e.g. diabetes), energy homeostasis.
  • Patent application WO 2005101013 provides material and methods for screening modulators of neural regeneration such as inhibitors of Unc51-Like kinases, which can be used to stimulate neurogenesis after cerebral ischemia or in Alzheimer disease .
  • Unc51-Like Kinase could be a therapeutic target for treating / preventing disorders characterized by eye cells degeneration, more particularly retinal cells, and even more preferably photoreceptor cells degeneration.
  • the Invention provides a composition comprising at least one Unc51-Like Kinase modulator for treating and/or preventing cellular degenerative ophthalmic disease in a subject in need thereof.
  • the terms “a” and “an” are used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced compounds or steps, unless the context dictates otherwise. More specifically, “at least one” and “one or more” means a number which is one or greater than one, with a special preference for one, two or three.
  • treatment encompasses prophylaxis and/or therapy. Accordingly the compositions and methods of the present invention are not limited to therapeutic applications and can be used in prophylaxis ones. Therefore “treating” or “treatment” of a state, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (ii) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (iii) relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms .
  • patient or “subject in need” refer to a vertebrate, particularly a member of the mammalian species and includes, but is not limited to, domestic animals, sport animals, primates including humans.
  • patient is in no way limited to a special disease status, it encompasses both patients who have already developed a disease of interest and patients who are not sick.
  • Unc51-Like Kinase is intended to designate both Unc51-Like Kinase 1 (ULKl), Unc51-Like Kinase 2 (ULK2) and Unc51-Like Kinase 3 (ULK3) , and can be used herein to designate individually or collectively each of them.
  • Unc51-Like Kinase modulator is intended to designate compound which is able to prevent or limit Unc51-Like Kinase synthesis and/or bioavailability and/or activity.
  • said Unc51-Like Kinase modulator is a Unc51-Like Kinase inhibitor or partial inhibitor.
  • said inhibited or partially inhibited Unc51-Like Kinase is selected in the group consisting of ULKl, ULK2 and ULK3.
  • said inhibited or partially inhibited Unc51-Like Kinase is expressed in photoreceptor cells.
  • ULKl also known as Unc51-Like Kinase 1).
  • NP_003556.1 Its protein sequence (NP_003556.1) is as follows :
  • ULK2 also known as Unc51- Like Kinase 2
  • NP_055498.2 protein sequence
  • ULK3 also known as Unc51- Like Kinase 3
  • XP_001134013.1 protein sequence
  • said Unc51-Like Kinase inhibitor or partial inhibitor can be a direct inhibitor or an indirect inhibitor.
  • said Unc51-Like Kinase inhibitor or partial inhibitor is selected in the group consisting of a small molecule, a nucleic acid molecule (e.g. an antisense to Unc51-Like Kinase -encoding nucleic acid molecules, a siRNA, a miRNA, a ribozyme, a shRNA, a nucleic acid capable of modulating Unc51-Like Kinase nucleic acid expression, a vector more particularly an expression vector expressing at least one nucleic acid capable of modulating Unc51-Like Kinase nucleic acid expression) , an anti-Unc51-Like Kinase antibody.
  • a nucleic acid molecule e.g. an antisense to Unc51-Like Kinase -encoding nucleic acid molecules, a siRNA, a miRNA, a ribozyme, a shRNA, a nucleic acid capable of modulating Unc51-Like Kinase nu
  • Partial inhibitor refers to a compound which acts as the inhibitor but that produces a weak maximum inhibitory response. This term is well known in the art. Such inhibition may be determined using in vitro or in vivo systems, or a combination of both, using methods known to those of ordinary skill in the art (see below) .
  • indirect inhibitor refers according to one example to an agent able to interfere specifically with the Unc51-Like Kinase gene expression and/or Unc51-Like Kinase protein synthesis, and more particularly with the Unc51-Like Kinase mRNA.
  • said Unc51-Like Kinase inhibitor is an indirect inhibitor and is a nucleic acid that is able to modulate, preferably to reduce, the expression level of said Unc51-Like Kinase, and more particularly the Unc51-Like Kinase mRNA level in cells (especially cells of the eye, more especially retinal cells and preferably photoreceptor cells) .
  • the "indirect inhibitor” of the invention is able to modulate, preferably to reduce, the expression level of said Unc51-Like Kinase, and more particularly the Unc ⁇ l-Like Kinase mRNA level in the treated cells e.g. by at least about 10%, about 20%, about 30%, about 50%, about 75%, about 85%, or about 95% (compared to the situation without said indirect inhibitor) .
  • said indirect inhibitor is able to modulate, preferably to reduce (e.g.
  • nucleic acid includes, but is not limited to isolated DNA, RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA, vectors more particularly expression vectors expressing at least one of said isolated nucleic acids (i.e. DNA, RNA, oligonucleotides, antisens, siRNA, miRNA, ribozyme, shRNA) which are capable to modulate, preferably to reduce, the expression levels of said Unc51-Like Kinase, and more precisely are capable of modulating Unc51-Like Kinase nucleic acid expression and/or level (e.g. by modulating the transcription level of mRNA, by controlling mRNA stability/degradation, etc..) .
  • isolated nucleic acids i.e. DNA, RNA, oligonucleotides, antisens, siRNA, miRNA, ribozyme, shRNA
  • Unc51-Like Kinase e.g. by
  • the said nucleic acid can be used naked, i.e. present in the compositions of the invention or administered according to the invention, as such (e.g. DNA, RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) , or vectorized, i.e. (i) incorporated in a vector (e.g. synthetic vectors, for example combined with substances which are known to improve the transfectional efficiency and/or stability of vectors, see below) or (ii) encoded by an expression vector (i.e. is incorporated in the expression vector as heterologous nucleic acid) .
  • a vector e.g. synthetic vectors, for example combined with substances which are known to improve the transfectional efficiency and/or stability of vectors, see below
  • an expression vector i.e. is incorporated in the expression vector as heterologous nucleic acid
  • vector is used to refer to any molecule (e.g. chemical compounds, plasmid, yeast or virus) used to transfer heterologous nucleic acid (e.g. DNA encoding information, RNA molecule, hybrid RNA/DNA and /or nucleic acid analogs) to a host cell.
  • heterologous nucleic acid e.g. DNA encoding information, RNA molecule, hybrid RNA/DNA and /or nucleic acid analogs
  • expression vector refers to a vector which contains heterologous nucleic acid sequences and which, when transformed or transfected into a suitable host cell, directs and/or controls the expression, transcription, and/or translation of said heterologous nucleic acid sequences that are inserted into the said vector.
  • the "expression vector" of the invention when transformed or transfected into a suitable host cell, is able to direct and/or control the expression and transcription of RNA molecules (including RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) .
  • "Expression vector” refers to viral as well as non viral vectors, including extrachromosomal (e.g. episome) , multicopy and integrating vectors (i.e. for being incorporated into the host chromosomes).
  • extrachromosomal e.g. episome
  • multicopy and integrating vectors i.e. for being incorporated into the host chromosomes.
  • vectors generally disclosed and used in gene therapy or vector-based therapy (i.e. which are capable of delivering a nucleic acid to a host organism) as well as expression vectors for use in various expression systems.
  • the vector which can be of plasmid or viral origin can where appropriate be naked or combined with one or more substances which improve the transfectional efficiency and/or stability of the vector.
  • substances are widely documented in the literature available to the skilled man (see, for example, Feigner et al., 1987, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry, 5, 647-654).
  • said substances can be polymers, lipids, in particular cationic lipids, liposomes, nuclear proteins or neutral lipids. These substances can be used alone or in combination.
  • a combination which can be envisaged is that of a recombinant plasmid vector which is combined with cationic lipids (DOGS, DC-CHOL, spermine-chol, spermidine-chol, etc.), lysophospholipides (for example Hexadecylphosphocholine) and neutral lipids (DOPE) .
  • DOGS cationic lipids
  • DC-CHOL spermine-chol
  • spermidine-chol spermidine-chol
  • DOPE neutral lipids
  • plasmids which are derived from pBR322 (Gibco BRL) , pUC (Gibco BRL), pBluescript (Stratagene) , pREP4, pCEP4 ( Invitrogene) , pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pVAX , pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76, 12735-12746) or p Poly (Lathe et al., 1987, Gene 57, 193-201) .
  • a plasmid which is used in the context of the present invention contains an origin of replication which ensures that replication is initiated in a producer cell and/or a host cell (for example, the CoIEl origin will be chosen for a plasmid which is intended to be produced in E. coli and the oriP/EBNAl system will be chosen if it desired that the plasmid should be self-replicating in a mammalian host cell, Lupton and Levine, 1985, MoI. Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815).
  • an origin of replication which ensures that replication is initiated in a producer cell and/or a host cell
  • the CoIEl origin will be chosen for a plasmid which is intended to be produced in E. coli and the oriP/EBNAl system will be chosen if it desired that the plasmid should be self-replicating in a mammalian host cell, Lupton and Levine, 1985, MoI. Cell. Biol
  • the plasmid can additionally comprise a selection gene which enables the transfected cells to be selected or identified (complementation of an auxotrophic mutation, gene encoding resistance to an antibiotic, etc.).
  • the plasmid can contain additional elements which improve its maintenance and/or its stability in a given cell (e.g. cer sequence, which promotes maintenance of a plasmid in monomeric form (Summers and Sherrat, 1984, Cell 36, 1097-1103), sequences for integration into the cell genome.
  • Suitable "viral vectors” which can be used within the context of the present invention may be derived from a variety of different viruses (e.g. retrovirus, adenovirus, AAV, poxvirus, herpes virus, measle virus, foamy virus and the like) .
  • the term "viral vector” encompasses vector DNA/RNA as well as viral particles generated thereof.
  • Viral vectors can be replication-competent, or can be genetically disabled so as to be replication-defective or replication-impaired.
  • replication-competent as used herein encompasses replication-selective and conditionally- replicative viral vectors which are engineered to replicate better or selectively in specific host cells.
  • the recombinant vector in use in the invention is a recombinant adenoviral vector (for a review, see “Adenoviral vectors for gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press) . It can be derived from a variety of human or animal (canine, avian, bovine, murine, ovine, porcine, simian, etc.) sources and any serotype can be employed from the adenovirus serotypes 1 through 51.
  • CAV-I or CAV-2 adenoviruses of canine origin of the DAV adenovirus of avian origin or of the Bad type 3 adenovirus of bovine origin (Zakharchuk et al., 1993, Arch. Virol., 128: 171-176; Mittal et al., 1995, J. Gen. Virol., 76: 93-102).
  • an adenoviral vector of human origin which is preferably derived from a serotype C-adenovirus, in particular human adenoviruses 2 (Ad2), 5 (Ad5) , 6 (Ad6) , 11 (AdIl), 24 (Ad24) and 35 (Ad35) .
  • human adenoviruses 2 (Ad2), 5 (Ad5) , 6 (Ad6) , 11 (AdIl), 24 (Ad24) and 35 (Ad35) Nevertheless, three serotypes D- adenoviruses (Ad8, Adl9, and Ad37) have been shown to cause a severe ocular disease, epidemic keratoconjunctivitis (EKC) (Ford et al., 1987, Epidemiol.
  • EKC epidemic keratoconjunctivitis
  • the adenoviral vector in use in the present invention can be replication-competent.
  • Numerous examples of replication-competent adenoviral vectors are readily available to those skill in the art (see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727).
  • the adenoviral vector in use in the invention is replication-defective (see for example WO94/28152; Lusky et al., 1998, J. Virol 72, 2022-2032).
  • Preferred replication- defective adenoviral vectors are El-defective (see for example US 6,136,594 and US 6,013,638), with an El deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of the human adenovirus type 5 disclosed in the GeneBank under the accession number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280-285) .
  • the cloning capacity can further be improved by deleting additional portion (s) of the adenoviral genome (all or part of the non essential E3 region or of other essential E2, E4 regions). Insertion of a nucleic acid in any location of the adenoviral vector can be performed through homologous recombination as described in Chartier et al. (1996, J. Virol. 70, 4805-4810).
  • the expression vector is an Adeno-associated virus (AAV), i.e. a viral vector system based on a non pathogenic and replication-defective virus.
  • AAV Adeno-associated virus
  • the AAV genome is a linear, single-stranded DNA molecule containing about 4681 nucleotides.
  • the AAV genome generally comprises an internal non repeating genome flanked on each end by inverted terminal repeats (ITRs) .
  • ITRs are approximately 145 base pairs (bp) in length.
  • the ITRs have multiple functions, including as origins of DNA replication, and as packaging signals for the viral genome.
  • the internal non repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes.
  • the rep and cap genes code for viral proteins that allow the virus to replicate and package into a virion.
  • one family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight.
  • the AAV cap region encodes at least three proteins, VPl, VP2 and VP3.
  • AAV has been engineered to deliver genes of interest by deleting the internal non repeating portion of the AAV genome (i.e. the rep and cap genes) and inserting a heterologous gene between the ITRs.
  • the heterologous nucleic acid sequence is typically functionally linked to a heterologous promoter (constitutive promoter, tissue- or cell-specific promoter or inducible promoter) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.
  • AAV is a helper-dependent virus; that is, it requires coinfection with a helper virus, or triple transfection with plasmids expressing viral proteins (e.g. adenovirus, herpesvirus or vaccinia), in order to form AAV virions.
  • AAV In the absence of coinfection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to replicate and package its genome into an infectious AAV virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells coinfected with a canine adenovirus.
  • rAAV chimeric serotypes wherein the vector is flanked by AAV-2 ITRs but encapsidated in an AAV-I, 2, 3, 4 or -5 capsid have been studied (Auricchio et al., 2001, Hum MoI Genet 10, 3075-81,; Rabinowitz et al., 2002, J Virol 76, 791-801; Yang et al. , 2002, J Virol 76, 7651-60). It was shown that their subretinal delivery results in a quantitative transgene expression hierarchy with rAAV-4 and -5 capsids being the most efficient.
  • AAV2.8 or AAV2.9 are six to eightfold more efficient to transduce photoreceptors at least in rodent models, than AAV2.5 (Auricchio et al., 2007, J. virol. 81, 20, 11372-11380) .
  • the inventors of US 20040208847 have further demonstrated that a recombinant AAV of serotype 4 delivered in the subretinal space of a non human primate leads to exclusive transduction of retinal pigment epithelial (RPE) cells.
  • RPE retinal pigment epithelial
  • nucleic acid i.e. DNA, RNA, RNAi, ribozyme, oligonucleotides, antisense, siRNA, miRNA, shRNA, etc..
  • AAV capsid protein or an expression vector, including AAV recombinant vector, engineered to express such an AAV capsid on its surface
  • said AAV capsid can have a structure similar to that of a natural AAV or with a few changes, such as, for example, VPl, VP2 and VP3 coming from more than one single AAV serotype, or such as a capsid made of an AAV capsid protein in which one or several amino acid have been deleted, added or modified.
  • an AAV capside by replacing part of it with a sequence from the AAV-5, 8 or 9 capsid protein, in such a way that the expressed proteins are able to form a capsid which retains the natural tropism of AAV-5, 8 or 9 towards retinal cells, and thus allows to target the delivery of the nucleic acid to this special type of cells such as photoreceptors. Therefore, the present invention concerns nucleic acids (including both viral and non viral expression vectors) which are characterized by the fact that they exhibit an AAV-5, 8 or 9 capsid protein.
  • AAV-5, 8 or 9 are part of the vector, in such a way that they are exposed at its surface, thereby enabling the targeting of retinal cells, and more specifically of photoreceptor cells.
  • the AAV-5, 8 or 9 capsid protein can be integrated into the viral particle (for example, in the case of a native or chimeric AAV capsid) , or simply bound to the particle, by any physical means (for example, in the case of a non-viral vector) .
  • the vector in use in the invention is a poxviral vector (see for example Cox et al. in "Viruses in Human Gene Therapy” Ed J. M. Hos, Carolina Academic Press) .
  • poxviral vector see for example Cox et al. in "Viruses in Human Gene Therapy” Ed J. M. Hos, Carolina Academic Press
  • suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol.
  • the Wyeth strain and the highly attenuated attenuated virus derived thereof including MVA (for review see Mayr, A., et al., 1975, Infection 3, 6-14) and derivates thereof (such as MVA vaccinia strain 575 (ECACC V00120707 - US 6,913,752), NYVAC (see WO 92/15672 - Tartaglia et al., 1992, Virology, 188, 217-232).
  • the vector may also be obtained from any other member of the poxviridae, in particular fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); canarypox (e.g.
  • the nucleic acid is preferably inserted in the thymidine kinase gene (tk) .
  • tk thymidine kinase gene
  • other insertion sites are also appropriate, e.g. in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189- 4198), in the KlL locus, in the u gene (Zhou et al., 1990, J. Gen. Virol.
  • the heterologous nucleic acid can be inserted in anyone of the identified deletions I to VII as well as in the D4R locus, but insertion in deletion II or III is preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038 ; Sutter et al., 1994, Vaccine 12, 1032-1040) .
  • the heterologous nucleic acid is preferably introduced in the intergenic region situated between ORFs 7 and 9 (see for example EP 314 569 and US 5,180,675).
  • the viral vector according to the invention derives from retroviruses which have the property of infecting, and in most cases integrating into, dividing cells and in this regard are particularly appropriate for use in relation to cancer.
  • a recombinant retrovirus according to the invention generally contains the LTR sequences, an encapsidation region and the nucleotide sequence according to the invention, which is placed under the control of the retroviral LTR or of an internal promoter such as those described below.
  • the recombinant retrovirus can be derived from a retrovirus of any origin (murine, primate, feline, human, etc.) and in particular from the MOMuLV (Moloney murine leukemia virus) , MVS (Murine sarcoma virus) or Friend murine retrovirus (Fb29) . It is propagated in an encapsidation cell line which is able to supply in trans the viral polypeptides gag, pol and/or env which are required for constituting a viral particle. Such cell lines are described in the literature (PA317, Psi CRIP GP + Am-12 etc.).
  • the retroviral vector according to the invention can contain modifications, in particular in the LTRs (replacement of the promoter region with a eukaryotic promoter) or the encapsidation region (replacement with a heterologous encapsidation region, for example the VL30 type) (see FR9408300 and FR9705203).
  • the nucleic acid in use in the invention is in a form suitable for its expression in a host cell or organism, which means that the nucleic acid (encoding more specifically RNA) are placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism.
  • expression in a host cell or organism means at least RNA (including RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) synthesis into the said host cell or organism.
  • regulatory sequence refers to any sequence that allows, contributes or modulates the expression of a nucleic acid in a given host cell, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA ...) into the host cell.
  • RNA Ribonucleic acid
  • oligonucleotides oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA
  • shRNA oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA
  • the regulatory sequences may, for example, be a mammalian or viral promoter, such as a constitutive, regulable or cell/tissue specific promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT) , adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters.
  • HPRT hypoxanthine phosphoribosyl transferase
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV) , simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • CMV cytomegalovirus
  • simian virus e.g., SV40
  • papilloma virus e.g., SV40
  • HSV40 human immunodeficiency virus
  • HSV40 human immunodeficiency virus
  • Rous sarcoma virus Rous sarcoma virus
  • cytomegalovirus cytomegalovirus
  • LTR long terminal repeats
  • Regulable promoters are expressed in the presence of an inducing agent.
  • the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill in the art.
  • the promoter will be selected in order to be cell or tissue specific (e.g. retinal cell-specific promoter, photoreceptor- specific promoter, retinal ganglion cell or retinal pigmented epithelium specific promoter etc... ) .
  • RNA polymerase III promoters will be used for expressing shRNA.
  • RNA polymerase III is one of three types of nuclear RNA polymerases present in eucaryotic cells. It synthesizes RNA molecules, some of which are involved in protein synthesis, pre-mRNA splicing, tRNA processing, and the control of RNA polymerase II elongation, whereas some others have still unknown functions. Like other RNA polymerases, RNA polymerase III cannot recognize its target promoters directly. Instead it is recruited to specific promoter sequences through the help of transcription factors. There are three basic types of RNA polymerase III promoters, called types 1, 2, and 3.
  • RNA polymerase III may be recruited to these promoters as part of a large complex (holo RNA polymerase III) containing the polymerase and its initiation factors, in vitro the reaction can be divided into several steps.
  • the promoter elements are- recognized by DNA binding factors, which then recruit a factor known as TFIIIB.
  • TFIIIB itself then directly contacts RNA polymerase III.
  • TFIIIB contains TBP, Bdpl, and Brfl (Brfl-TFIIIB) , and the other TBP, Bdpl, and Brf2 (Brf2- TFIIIB) .
  • Polymerase III promoter used for shRNA expression are U6 promotor, H6 promotor, Hl promotor normally regulating Hl RNA, the RNA component of human RNase P.
  • MRP regulating sequences 5' flanking sequences of human MRP/7, sequences controlling the expression of B2 snRNA and 7SK snRNA, Yl and Y3 RoRNA promotors, telomerase TERl proximal regulating sequences, (reviewed in Geiduschek EP et al. J MoI Biol. 2001 Jun 29;310(l) :l-26.
  • Photoreceptor-specific promoters include, for example, beta-subunit of cGMP phosphodiesterase (beta-PDE) promoter (Lerner et al., 2002, J. Biol. Chem. , 277, 25877-25883), interphotoreceptor retinoid-binding protein (IRBP) promoter (Glushakova et al. MoI. Vis. 2006 :12, 298-309), rhodopsin Kinase (Young et al. 2005, MoI. Vis. 11:1041-1051, Young et al. 2007 Genomics 90, (2 ): 236-248, Sharhrockh C.
  • beta-PDE beta-subunit of cGMP phosphodiesterase
  • IRBP interphotoreceptor retinoid-binding protein
  • the gene expression sequence shall include, as necessary, 5 1 non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined nucleic acid.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • the regulatory elements controlling the expression of the nucleic acid molecule of the invention may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g.
  • nuclear localization signal sequences e.g. nuclear localization signal sequences
  • processing e.g. splicing signals
  • stability e.g. introns and non-coding 5' and 3' sequences
  • translation e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine-Dalgarno sequences, etc.
  • antisense nucleic acid molecules that can hybridize to, or are complementary to, the nucleic acid molecule, nucleotide sequence, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein (e.g. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence) .
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an Unc51-Like Kinase (or fragments or fragment combination thereof) .
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to an "untranslated region" of the coding strand of a nucleotide sequence encoding a Unc51-Like Kinase.
  • the term “untranslated region” refers to 5 1 and 3' sequences that flank the coding region and that are not translated into amino acids.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of a Unc51-Like Kinase mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of Unc51-Like Kinase mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Unc51- Like Kinase mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g. an antisense oligonucleotide
  • an antisense nucleic acid e.g. an antisense oligonucleotide
  • can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids e.g.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 carboxy ⁇ nethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethylurac
  • the antisense nucleic acid can be produced biologically in situ using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e. RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following section) .
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ after administration of an expression vector as indicated above, such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding Unc51-Like Kinase to thereby inhibit expression of the protein (e.g. by inhibiting transcription and/or translation) .
  • the hybridization can be by conventional nucleotide complementarily to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically .
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g. by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense compound is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% complementary to a nucleic acid molecule encoding Unc51-Like Kinase.
  • the invention relates to short interfering nucleic acid ( siNA) such as short interfering RNA (siRNA) , double- stranded RNA (dsRNA) , micro-RNA (miRNA) , and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Unc51-Like Kinase genes expression and/or activity.
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • siNA short interfering nucleic acid
  • RNAi can occur via selective intracellular degradation of RNA and/or by translational repression.
  • “Molecules capable of mediating RNA interference” refers to an at least partly double-stranded RNA molecule, optionally including one or more nucleotide analogs or modifications, having a structure characteristic of molecules that can mediate inhibition of gene expression through RNAi. These molecules include a portion that is substantially complementary to a target gene.
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the said siNA molecule of the invention, in a manner which allows expression of the siNA molecule.
  • the vector can contain sequence (s) encoding both strands of a siNA molecule comprising a duplex.
  • the vector can also contain sequence (s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule.
  • Non- limiting examples of such expression vectors have been provided above.
  • the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase siRNA.
  • siRNA refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3 1 end of the sense strand and/or the antisense strand.
  • siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
  • siRNA may be divided into five groups (non-functional, semifunctional, functional, highly functional, and hyper- functional) based on the level or degree of silencing that they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions where the siRNA is transfected into said cell line at a concentration from 1OnM to 100 nM and the level of silencing is tested at a time of roughly 24 hours after transfection, and not exceeding 72 hours after transfection. In this context, “non-functional siRNA” are defined as those siRNA that induce less than 50% ( ⁇ 50%) target silencing. "Semi-functional siRNA” induce 50-79% target silencing. “Functional siRNA” are molecules that induce 80-95% gene silencing.
  • Highly-functional siRNA are molecules that induce greater than 95% gene silencing.
  • “Hyperfunctional siRNA” are a special class of molecules which are defined as those molecules that (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.
  • the methods of the invention comprise administering the Unc51-Like Kinase- specific siRNA to a mammal in an amount and for a period of time sufficient to indirectly inhibit or reduce Unc51-Like Kinase expression, and preferably inhibit or reduce Unc51-Like Kinase mRNA level.
  • siRNAs lead to post-transcriptional silencing of the mRNA to which they are homologous.
  • RNA interference or siRNA is a method based on small-interfering RNAs that can lead to the silencing of specific genes. RNA interference technology is well known in the art and is described for example in US20020162126, or Hannon et al., 2002, Nature, 11, 418, 244-251.
  • siRNA is mediated by RNA-induced silencing complex or RISC, which is a sequence specific, multi-component nuclease that degrades mRNAs and contains short RNAs.
  • RISC RNA-induced silencing complex
  • Complementary portions of siRNA, that hybridize to form the double-stranded structure typically have substantial or complete identity.
  • the double- stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands.
  • siRNAs of the invention may comprise one or more strands of polymerized ribonucleotide and may include modifications to either the phosphate-sugar backbone or the nucleoside.
  • bases may be modified to block the activity of enzyme adenosine deaminase, an enzyme that plays a role in RNA-editing.
  • RNA duplex formation can be initiated either before or after administration into a host organism or cell for effective inhibition of or reduction in the expression of the target gene.
  • siRNAs are complementary to certain portions of a particular mRNA, e.g., a target gene.
  • the sequence of a siRNA of the invention can correspond to the entire length of or only a portion of a target gene. In one embodiment the length of the siRNA, i.e.
  • the length of each individual strand of the double-stranded structure, as well as the length of the duplex comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In another embodiment, the length of the siRNA is about 25-50 nucleotides. In yet another embodiment, the length of the siRNA is greater than 50 nucleotides.
  • siRNA Finder computer program "BLOCK-iTTM RNAi Designer” Invitrogen, "HP Flexible siRNA Design” Qiagen
  • the mRNA sequence encoding Unc51-Like Kinase of the invention can be determined for example according to the rules of degenerative code or based on the nucleic acid sequences provided herein.
  • the siRNA can be administered to a subject or generated in situ using an expression vector into which a nucleic acid has been subcloned which encodes the said siRNA.
  • siRNA of the present invention is selected in the group consisting in : -for ULKl :
  • siRNA of the present invention is selected in the group consisting in :
  • CACGGTGTACAAGGCCTACGC CCTCACGGAGATTGAGATCCT CAGAACCTCATGGCCCGAGCT TAGCAACCGGGTCATCGAGCT CCCGCGCCTGGACGGCTTCAT TTCGTAGCTCTTGCACCCTTC CGGCACGTACGCCACGGTGTA TCCCGAGACTGCCGGGACCTA CCGCGCCTGGACGGCTTCATC GACTGTCGGAATCTGTTCGTA CAGCGGAAGGAGGCAATTAAG TTAGGTTAATCCCTCAAGCAT CGGCAGTATGACGCCCGCGTG GGCCCGAGCTGAATACTTGAA AGGCGGGAGCTGCTTCACACT CAGCGGCAGTATGACGCCCGC AGCAACCGCCCTGGTGGTGCA CCCGTGGGATGAGAAGCACGT CAGCGGCACGTACGCCACGGT CCTTCCTTCCAATCA CAGCAATTAGCTAGCGCCCTG ATCCTCAAGGGCATTCGACAT TTCCTGCATGAACGGAATAATA
  • siRNA of the present invention are selected in the group consisting in the siRNA (sense and antisense) sequences disclosed in Figure 1 (ULKl), Figure 2 (ULK2) and Figure 3 (ULK3).
  • the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase miRNA (microRNA) .
  • miRNA is usually used to refer to endogenous RNAs that are naturally expressed, similar molecules or precursors thereof that either mimic the sequence of naturally occurring miRNAs or are specifically designed to hybridize to a target transcript so as to result in a duplex structure containing one or more bulges can be introduced into, and expressed within, cells and can cause translational repression.
  • double-stranded duplex molecule structurally similar or identical to siRNAs, or hairpin precursors that can be processed intracellularly in a similar manner to naturally occurring miRNA precursors, can be introduced into cells and can mediate RNAi via translational repression (see, Doench, J., et al., 2003, Genes and Dev., 17, 438-442) .
  • RNAi-inducing entity that mediates RNAi by repressing translation of a target transcript, and that consists of or comprises a strand that binds to a target transcript to form a duplex containing one or more bulges, is said herein to act via an miRNA translational repression pathway, and the strand that binds to the target may be referred to as an miRNA-like molecule.
  • Endogenous miRNAs can also mediate cleavage of RNA targets (i.e., they can act in an siRNA-like manner) if they have sufficient complementarity to the target. Further description of miRNAs and the mechanism by which they ' are believed to mediate silencing is found in Bartel, 2004, Cell, 116, 281-297.
  • miRNA of the present invention are defined by using the sequences provided above for siRNA.
  • the said Unc51-Like Kinase inhibitor or partial inhibitor may have a short hairpin structure having a sticky portion at the 3' terminus and is an Unc51-Like Kinase shRNA (short hairpin RNA).
  • shRNA refers to short hairpin RNA which functions as RNAi and/or siRNA species but differs in that shRNA species are double stranded hairpin-like structure for increased stability.
  • shRNA are molecule of about 20 or more base pairs in which a single-stranded RNA partially contains a palindromic base sequence and forms a double-strand structure therein (i.e., a hairpin structure).
  • Such palindromic sequence is composed of a antisense sequence (that anneals to the target mRNA) in 5' to 3' (sequence a) orientation and an sense sequence in reverse orientation (3' to 5') (sequence b) separated by a linker sequence or loop sequence (that is unrelated to the target mRNA).
  • shRNA may preferably have a 3 1 protruding end.
  • the length of the double-stranded portion is not particularly limited, but is preferably about 10 or more nucleotides, and more preferably about 20 or more nucleotides.
  • the 3 ? protruding end may be preferably DNA, more preferably DNA of at least 2 nucleotides in length, and even more preferably DNA of 2-4 nucleotides in length.
  • the "sequence a" and "sequence b" are selected in the group consisting in the siRNA sequences listed above.
  • the said hairpin loop sequence of the present invention is selected in the group consisting in :
  • shRNA of the present invention is selected in the group consisting in all combinations of antisense sequences "a” with each hairpin loop sequence described above and sense sequence "b” corresponding to the antisenses, therefore forming a group of palindromic sequences .
  • the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase-specific ribozyme having specificity for a Unc51-Like Kinase-encoding nucleic acid. It can include one or more sequences complementary to the nucleotide sequence of a Unc51-Like Kinase cDNA and a sequence having known catalytic sequence responsible for mRNA cleavage (see US 5,093,246 or Haselhoff and Gerlach, 1988, Nature, 334, 585-591).
  • Unc51-Like Kinase gene expression can further be inhibited by targeting nucleotide sequences complementary to the regulatory region of the Unc51-Like Kinase (e.g. the Unc51-Like Kinase promoter and/or enhancers) to form triple helical structures that prevent transcription of the Unc51-Like Kinase gene in target cells (Helene, 1991, Anticancer Drug Des., 6, 569-84; Maher, 1992, Bioassays, 14, 807-15).
  • nucleotide sequences complementary to the regulatory region of the Unc51-Like Kinase e.g. the Unc51-Like Kinase promoter and/or enhancers
  • Unc51-Like Kinase The nucleic acid sequences encoding Unc51-Like Kinase are known in the art and thus are available to the skilled man : see for example references (Kuroyanagi, H. et al. Genomics 51 (1), 76-85 (1998), Yan,J et al. Oncogene 18 (43), 5850-5859 (1999) ) .
  • cDNA nucleic acid sequence encoding ULKl (NM_003565.1) is :
  • AACGTAAGGAAATAAACCTTTGGAATTGTTGAAAAAAAAAAAAAAAAAAAAA cDNA nucleic acid sequence encoding ULK2 (NM_014683.2 ) is :
  • CATTGTTTCTCTATTAAATTATTTTCTCTATT Figure 4 provides a table with correspondences between sequences from nucleic acid and corresponding polypeptides.
  • the term "indirect inhibitor” as used herein refers to an anti-Unc51-Like Kinase antibody or an inhibitory peptide.
  • antibody refers to an immunoglobulin molecule or immunologically active portion thereof, i. e., an antigen-binding portion.
  • immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F (ab 1 ) 2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively.
  • the antibody can be a polyclonal, monoclonal, recombinant, e.g.
  • the antibody can be coupled to a toxin or imaging agent.
  • a full-length Unc51-Like Kinase protein or, antigenic peptide fragment of Unc51-Like Kinase can be used as an immunogen or can be used to identify anti- Unc51-Like Kinase antibodies made with other immunogens, e. g. , cells, membrane preparations, and the like.
  • the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of Unc51-Like Kinase located on the surface of the protein, e. g., hydrophilic regions, as well as regions with high antigenicity.
  • chimeric, humanized, and completely human antibodies are also within the scope of the invention. Chimeric, humanized, but most preferably, completely human antibodies are desirable for applications which include repeated administration, e. g., therapeutic treatment of human patients.
  • the invention provides methods (also referred to herein as "screening assays") for identifying modulators of the present invention (especially Unc51-Like Kinase inhibitors, including Unc51-Like Kinase partial inhibitors), i.e. candidate or test compounds or agents (e.g. proteins, peptides, peptidomimetics, peptoids, small molecules, nucleic acids, oligonucleotides or any other drugs) which have a modulating effect on Unc51-Like Kinase activity, and more specifically on Unc51-Like Kinase expression (indirect inhibitors).
  • “candidate or compounds or agents” are synonyms.
  • a test substance that modulates (preferably inhibits) Unc51-Like Kinases activity can be evaluated as follows: quantification of Unc51-Like Kinases activity by liquid-phase electrophoretic differencial mobility of Unc51-Like Kinases fluorescent substrate with respect to its phosphorylated or dephosphorylated status (see US 7,105,304).
  • Unc51-Like Kinases substrate used can be for example a peptide that upon phosphorylation by ULK, shows an electrophoretic migration advance as compared to dephosphorylated substrate, which shows a retardation. The substrate/product chromotagraphic separation is performed using a microfluidic device. Quantification of phospho- versus non-phospho substrate, using the fluorescence, gives a precise evaluation of Unc51-Like Kinases activity (NOVASCREEN-CALIPER LabChip 3000 (see US 7,105,304)
  • the following method is for example usefull and well known in the art for measuring these properties : total Unc51- Like Kinase quantification using a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) (Invitrogen) .
  • ELISA Enzyme Linked-Immuno-Sorbent Assay
  • a mouse monoclonal antibody specific for Unc51-Like Kinase is coated onto the wells of the microtiter strips provided. Samples, including a standard containing Unc51-Like Kinase, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the Unc51-Like Kinase antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for Unc51-Like Kinase is added to the wells.
  • an in vivo assay in which the capacity of test substance to reduce photoreceptor induced degeneration in vivo is quantified after intravitreal injection of the test substance.
  • Mouse animal models can display a photoreceptor degeneration induced by different mutations, e . g. rdl mouse mutated in Phospho-di- esterase (Bowes et al., 1993, PNAS, 90, 2955-2959); Zaprinast induced photoreceptor degeneration (Vallaza-Deschamps et al., 2005, Eur. J.
  • Rhodopsin P23H mutated mouse or rat Rhodopsin P23H mutated mouse or rat (Ollson, 1992, Neuron, 9, 815-830 and Lewin, 1998, Nat. Med., 4, 967-971).
  • Photoreceptor survival quantification is assessed by histological section, co- labelling with photoreceptor specific marker (rhodopsin) , outer nuclear layer thickness quantification and cell counting. Electroretinogram is used to assess functionality of surviving photoreceptors (Frasson et al., 1999, Nat Med., 5, 1183-7) .
  • modulators of Unc51-Like Kinase expression are identified.
  • a cell or cell free mixture is contacted with a test substance and the expression of Unc51-Like Kinase mRNA or protein evaluated relative to the level of expression of Unc51-Like Kinase mRNA or protein in the absence of the test substance.
  • expression of Unc51-Like Kinase mRNA or protein is greater in the presence of the test substance than in its absence, the test substance is identified as a stimulator of Unc51-Like Kinase mRNA or protein expression.
  • Unc51-Like Kinase mRNA or protein when expression of Unc51-Like Kinase mRNA or protein is less (statistically significantly less) in the presence of the test substance than in its absence, the candidate compound is identified as an inhibitor of Unc51-Like Kinase mRNA or protein expression.
  • the level of Unc51-Like Kinase mRNA or protein expression can be determined by quantitative RTPCR or Western-blotting respectively using an antibody specific to Unc51-Like Kinase (Abnova, Evertest Biotech) .
  • the sources for test substances to be screened include natural sources, such as a cell extract (e.g., invertebrate cells including, but not limited to, bacterial, fungal, algal, and plant cells) and synthetic sources, such as chemical compound libraries or biological libraries such as antibody substance or peptide libraries. Agents are screened for the ability to either stimulate or inhibit the activity.
  • natural sources such as a cell extract (e.g., invertebrate cells including, but not limited to, bacterial, fungal, algal, and plant cells) and synthetic sources, such as chemical compound libraries or biological libraries such as antibody substance or peptide libraries.
  • synthetic sources such as chemical compound libraries or biological libraries such as antibody substance or peptide libraries.
  • Agents are screened for the ability to either stimulate or inhibit the activity.
  • libraries used for the identification of small molecule modulators, including: (i) chemical libraries, (ii) natural product libraries, and (iii) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules
  • Chemical libraries consist of random chemical structures, or analogs of known compounds, or analogs of compounds that have been identified as "hits” or "leads” in prior drug discovery screens, some of which may be derived from natural products or from non-directed synthetic organic chemistry.
  • Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.
  • Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof.
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or “lead”) to optimize the capacity of the "hit” to modulate activity.
  • the methods of the invention comprise steps of culturing a neuronal cell in the presence and absence of the modulators as set out above; measuring and comparing cell growth or survival or differentiation in the presence and absence of the modulator; and selecting a modulator that promotes increased survival or growth or differentiation of said neuronal cell.
  • the cell is selected from the group consisting of a hippocampal neuron or neural stem cell, a subventricular neuron or neuron stem cell, a cortical neuron or neuron stem cell, and a neuroblastoma cell.
  • the neuronal cell is a retinal cell selected from the group consisting of photoreceptors (rods and cones), ganglion cells, horizontal cells, amacrine cells, bipolar cells.
  • the methods of the invention comprise steps of culturing a non neuronal retinal cell in the presence and absence of the modulators as set out above; measuring and comparing cell growth or survival or differentiation in the presence and absence of the modulator; and selecting a modulator that promotes increased survival or growth or differentiation of said cell.
  • the cell is selected from the group consisting Muller cells and pigmented epithelial cells.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating (activator or inhibitor) agent of the invention can be identified using a cell-based or a cell free assay, and the ability of the said agent to (i) inhibit the expression of a Unc51-Like Kinase protein and/or (ii) reduce the Unc51-Like Kinase mRNA levels can be confirmed in vivo, e. g. , in an animal such as a mouse model (e.g., animal models of retinal degeneration disclosed above such as P23H rat, P23H mouse or rdl mouse) .
  • a mouse model e.g., animal models of retinal degeneration disclosed above such as P23H rat, P23H mouse or rdl mouse
  • This invention further pertains to novel compounds identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a Unc51-Like Kinase direct inhibitor, a Unc51-Like Kinase indirect inhibitor) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel compounds identified by the above-described screening assays can be used for treatments as described herein.
  • an agent identified as described herein e.g., a Unc51-Like Kinase direct inhibitor, a Unc51-Like Kinase indirect inhibitor
  • said Unc51-Like Kinase inhibitor is an indirect inhibitor and is a nucleic acid.
  • the (Unc51-Like Kinase) modulator themselves also are aspects of the invention, as are mixtures of these modulators; mixtures of modulators with any other neurotropic factors; and pharmaceutical compositions comprising the modulators in pharmaceutically acceptable carriers.
  • Non limiting examples of neurotropic factors according to the invention are NGF, BDNF, NT-3, 4, 5, or 6, CNTF, IGFI, IGFII, GDNF, GPA, bFGF, TGFB, and apolipoprotein E.
  • compositions are within the scope of the present invention.
  • Such pharmaceutical compositions are compositions as disclosed above admixed with pharmaceutically or physiologically acceptable formulations.
  • the compositions of the present invention are comprising the Unc51-Like Kinase modulators of the Invention combined with further ingredients that are physiologically tolerable and do not typically produce adverse reactions when administered to a subject in need thereof (e.g. human).
  • These ingredients are preferably "pharmaceutically or physiologically acceptable" as approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, and more particularly in humans.
  • the present invention relates to a composition containing an effective non-toxic dose of the compounds of the present invention (i.e. the Unc51-Like Kinase modulator) as well as pharmaceutically or physiologically acceptable carriers or solvents.
  • the pharmaceutical compositions are obtained by blending a therapeutically active amount of at least one compound of the present invention with a pharmaceutically or physiologically acceptable carrier, which may have different forms depending on the desired administration route.
  • carrier applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which an active compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition. According to one embodiment of the present invention carriers are suitable for immediate-release, i.e., release of most or all of the active ingredients over a short period of time, such as 60 minutes or less, and make rapid absorption of the drug possible.
  • the composition of the invention is delivered to areas within the eye, most preferably in the vicinity of the retina. It can be accomplished by injection, canula or other invasive device designed to introduce precisely metered amounts of a desired ophthalmic composition to a particular compartment or tissue within the eye (e.g. posterior chamber or retina) .
  • An intraocular injection may be into the vitreous (intravitreal) , or under the conjunctiva (subconjunctival), or behind the eye (retrobulbar) , into the sclera, or under the Capsule of Tenon (sub- Tenon) , and may be in a depot form.
  • Other intraocular routes of administration and injection sites and forms are also contemplated and are within the scope of the invention.
  • the combination product of the invention will be delivered by sub-retinal or intra-vitreal injection.
  • the ophthalmic composition is intraocularly injected (e.g. into the vitreous or sub retinal) to treat or prevent an ophthalmic condition related to cellular degenerative conditions, and more specifically retinal degenerative diseases.
  • the active agents should be concentrated to minimise the volume for injection.
  • the volume for injection is less than about 5 ml. Volumes such as this may require compensatory drainage of the vitreous fluid to prevent increases in intraocular pressure and leakage of the injected fluid through the opening formed by the delivery needle. More preferably, the volume injected is between about 1.0 ml and 0.05 ml. Most preferably, the volume for injection is approximately 0.1 ml.
  • a concentration of the compound of the present invention less than about 20 mg/ml may be injected.
  • a dose of about 10 mg/ml is administered.
  • Sample concentrations include, but are not limited to, about 5 ⁇ g/ml to about 50 ⁇ g/ml; about 25 ⁇ g/ml to about 100 ⁇ g/ml; about 100 ⁇ g/ml to about 200 ⁇ g/ml; about 200 ⁇ g/ml to about 500 ⁇ g/ml; about 500 ⁇ g/ml to about 750 ⁇ g/ml; about 500 ⁇ g/ml up to 1 mg/ml etc. preferred 50mg/ml.
  • sample concentrations include, but are not limited to, 10 3 , l ⁇ 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 10 11 to 10 12 genome copy/ml.
  • Intraocular injection may be achieved by a variety of methods well known in the art.
  • the eye may be washed with a sterilising agent such as Betadine® and the- pharmaceutical composition of the Invention is injected in an appropriate carrier with a fine gauge needle (e.g. 27 gauge) at a position in the eye such that the compound will settle to the posterior pole towards the ventral surface.
  • a fine gauge needle e.g. 27 gauge
  • preliminary vitrectomy may be necessary.
  • Local anaesthetic or general anaesthetic may be necessary.
  • the syringe used in practicing the method of this invention is suitably one which can accommodate a 21 to 40 gauge needle and is preferably of a small volume, for example 1.5 ml, or more preferably 0.1 ml.
  • the needle and syringe may be of the type where the needle is removable from the syringe, it is preferred that the arrangement is of a unitary syringe/needle construction. This would clearly limit the possibility of disengagement of the needle from the syringe. It is also preferred that the arrangement be tamper evident.
  • the pharmaceutical composition of the present invention may therefore be provided in the form of a single unit dose, or separated unit doses each containing part of the combination product, in a pre-prepared syringe ready for administration.
  • a suitable style of syringe is, for example, sold under the name of Uniject® manufactured by Becton Dickinson and Company.
  • the material is expelled through the needle into the eye by pressure applied to the sides of a pliable reservoir supplying the needle, rather than by a plunger.
  • the construction of the reservoir and needle forms a single unit.
  • Topical application of composition of the invention for the treatment or prevention of disorders according to the present invention may be as ointment, gel or eye drops.
  • the topical ophthalmic composition may further be an in situ gellable aqueous formulation.
  • Such a formulation comprises a gelling agent in a concentration effective to promote gelling upon contact with the eye or with lacrimal fluid in the exterior of the eye.
  • Suitable gelling agents include, but are not limited to, thermosetting polymers such as tetra- substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamine) ; polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa- carrageenan and iota-carrageenan) , chitosan and alginate gums.
  • thermosetting polymers such as tetra- substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamine) ; polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa- carrageenan and iota-carrageenan) , chitosan and alginate gums.
  • in situ gellable as used herein embraces not only liquids of low viscosity that form gels upon contact with the eye or with lacrimal fluid in the exterior of the eye, but also more viscous liquids such as semi-fluid and thixotropic gels that exhibit substantially increased viscosity or gel stiffness upon administration to the eye.
  • a topical ophthalmic composition for the treatment of disorders as disclosed herein, a therapeutically effective amount of the composition of the invention is placed in an ophthalmological vehicle as is known in the art.
  • topical ophthalmic formulations containing steroids are disclosed in US 5,041,434, whilst sustained release ophthalmic formulations of an ophthalmic drug and a high molecular weight polymer to form a highly viscous gel have been described in US 4,271,143 and US 4,407,792.
  • GB 2007091 describes an ophthalmic composition in the form of a gel comprising an aqueous solution of a carboxyvinyl polymer, a water-soluble basic substance and an ophthalmic drug.
  • US 4,615,697 discloses a controlled release composition and method of use based on a bioadhesive and a treating agent, such as an anti- inflammatory agent.
  • composition of the invention may be also administered as a slow release formulation, with a carrier formulation such as microspheres, microcapsules, liposomes, etc., as a topical ointment or solution, an intravenous solution or suspension, or in an intraocular injection, as known to one skilled in the art to treat or prevent ophthalmic disorders.
  • a carrier formulation such as microspheres, microcapsules, liposomes, etc.
  • topical ointment or solution such as a topical ointment or solution, an intravenous solution or suspension, or in an intraocular injection, as known to one skilled in the art to treat or prevent ophthalmic disorders.
  • sustained release or controlled release
  • controlled release is meant that the therapeutically active component is released from the formulation at a controlled rate such that therapeutically beneficial levels (but below toxic levels) of the component are maintained over an extended period of time ranging from e.g., about 12 to about 24 hours, thus, providing, for example, a 12 hour or a 24 hour dosage
  • a time-release drug delivery system may be administered intraocularly to result in sustained release of the combination product over a period of time.
  • the composition may be in the form of a vehicle, such as a micro- or macro-capsule or matrix of biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, ' polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and other polymers such as those disclosed in US Patents Nos.
  • biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, ' polyacrylonitriles,
  • a microscopic or macroscopic ophthalmic composition may be administered through a needle, or may be implanted by suturing within the eye, eg intravitreal cavity or sub-retinal space.
  • Delayed or extended release properties may be provided through various formulations of the vehicle (coated or uncoated microsphere, coated or uncoated capsule, lipid or polymer components, unilamellar or multilamellar structure, and combinations of the above, etc.).
  • the formulation and loading of microspheres, microcapsules, liposomes, etc and their ocular implantation are standard techniques known by one skilled in the art.
  • the invention also provides a method for the treatment or prophylaxis of ophthalmic disorders, said method comprising the step of administering a composition of the Invention in a biocompatible, biodegradable matrix, for example in the form of a gel or polymer which is preferably suited for insertion into the retina or into a cavity of the eye, anterior or posterior, as an implant.
  • a composition of the Invention in a biocompatible, biodegradable matrix, for example in the form of a gel or polymer which is preferably suited for insertion into the retina or into a cavity of the eye, anterior or posterior, as an implant.
  • the pharmaceutical composition may be incorporated in any known biocompatible biodegradable matrix as a liquid, or in the form, for example, of a micelle using known chemistry or as microparticles .
  • Slow or extended-release delivery systems include any of a number of biopolymers (biological-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, can be utilized with the compositions described herein to provide a continuous or long term source of therapeutic compound (i.e. the 4EBPs modulator) .
  • implants used in the method of the present invention are formulated with composition of the invention entrapped within the bio-erodible polymer matrix. Release of the therapeutic compounds is achieved by erosion of the polymer followed by exposure of previously entrapped compound to the vitreous, and subsequent dissolution and release of compound.
  • the release kinetics achieved by this form of drug release are different than that achieved through formulations which release drug through polymer swelling, such as with hydrogels such as methylcellulose .
  • the therapeutic compound is not released through polymer erosion, but through polymer swelling, which releases active compound as liquid diffuses through the pathways exposed.
  • the parameters which determine the release kinetics include the size of the therapeutic compound particles, the water solubility of the active compound, the ratio of active compound to polymer, the method of manufacture, the surface area exposed, and the erosion rate of the polymer.
  • biocompatible, non-biodegradable polymers of particular interest include polycarbamates or polyureas, particularly polyurethanes, polymers which may be cross-linked to produce non- biodegradable polymers such as cross-linked poly (vinyl acetate) and the like.
  • ethylene-vinyl ester copolymers having an ester content of 4% to 80% such as ethylene-vinyl acetate (EVA) copolymer, ethylene-vinyl hexanoate copolymer, ethylene-vinyl propionate copolymer, ethylene-vinyl butyrate copolymer, ethylene-vinyl pentantoate copolymer, ethylene-vinyl trimethyl acetate copolymer, ethylene-vinyl diethyl acetate copolymer, ethylene- vinyl 3-methyl butanoate copolymer, ethylene-vinyl 3-3- dimethyl butanoate copolymer, and ethylene-vinyl benzoate copolymer.
  • EVA ethylene-vinyl acetate
  • EVA ethylene-vinyl acetate
  • ethylene-vinyl hexanoate copolymer ethylene-vinyl propionate copolymer
  • Additional exemplary naturally occurring or synthetic non- biodegradable polymeric materials include poly (methylmethacrylate) , poly (butylmethacrylate) , plasticized poly (vinylchloride) , plasticized poly (amides) , plasticized nylon, plasticized soft nylon, plasticized poly (ethylene terephthalate) , natural rubber, silicone, poly (isoprene) , poly (isobutylene) , poly (butadiene) , poly (ethylene) , poly (tetrafluoroethylene) , poly (vinylidene chloride), poly (acrylonitrile, cross-linked poly (vinylpyrrolidone) , poly (trifluorochloroethylene) , chlorinated poly (ethylene) , poly (4, 4'- isopropylidene 'diphenylene carbonate), vinylidene chloride-acrylonitrile copolymer, vinyl chloridediethyl fumarate copolymer, silicone, silicone rubbers (especially the medical grade), poly
  • Diffusion of the composition from the implant may also be controlled by the structure of the implant.
  • diffusion of the pharmaceutical composition from the implant may be controlled by means of a membrane affixed to the polymer layer comprising the drug.
  • the membrane layer will be positioned intermediate to the polymer layer comprising the pharmaceutical composition and the desired site of therapy.
  • any of the composition used in the method of the invention will dwell in the ocular environment will depend, inter alia, on such factors as the physicochemical and/or pharmacological properties of the compounds employed in the formulation, the concentration of the compound employed, the bioavailability of the compound, the disease to be treated, the mode of administration and the preferred longevity of the treatment. Where that balance is struck will often depend on the longevity of the effect required in the eye and the ailment being treated.
  • the frequency of treatment according to the method of the invention is determined according to the disease being treated, the deliverable concentration of the composition and the method of delivery. If delivering the combination product by intravitreal injection, the dosage frequency may be monthly. Preferably, the dosage frequency is every three months. The frequency of dosage may also be determined by observation, with the dosage being delivered when the previously delivered combination product is visibly cleared. In general, an effective amount of the compound is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • compositions prepared for used in the method of the present invention to prevent or treat ophthalmic disorders will preferably have dwell times from hours to many months and possibly years, although the latter time period requires special delivery systems to attain such duration and/or alternatively requires repetitive administrations.
  • the combination product for use in the method of the invention will have a dwell time (ie duration in the eye) of hours (i.e. 1 to 24 hours), days (i.e. 1, 2, 3, 4, 5, 6 or 7 days) or weeks (i.e. 1, 2, 3, 4 weeks).
  • the combination product will have a dwell time of at least a few months such as, 1 month, 2 months, 3 months, with dwell times of greater than 4, 5, 6, 7 to 12 months being achievable.
  • the method or use of the invention can be carried out alone, or. in conjunction with one or more conventional therapeutic modalities (such as photodynamic therapy, laser surgery, laser photocoagulation or one or more biological or pharmaceutical treatments. These methods are well known from the skilled man in the art and widely disclosed in the literature) .
  • the use of multiple therapeutic approaches provides the patient with a broader based intervention.
  • the method of the invention can be preceded or followed by a surgical intervention.
  • it can be preceded or followed by photodynamic therapy, laser surgery, laser photocoagulation.
  • Those skilled in the art can readily formulate appropriate therapy protocols and parameters which can be used.
  • the present Invention further concerns a method for improving the treatment of a patient which is undergoing one or more conventional treatment as listed above, which comprises co-treatment of said patient along with at least one Unc51-Like Kinase modulator of the present invention.
  • the present invention further concerns a method for treating cell degenerative ophthalmic conditions, and related diseases, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one Unc51-Like Kinase modulator of the present ' invention.
  • the present Invention further concerns a method for improving the treatment of a patient which is undergoing one or more conventional treatment as listed above, which comprises co-treatment of said patient along with at least one Unc51-Like Kinase inhibitor of the present invention, and preferably said inhibitor is an indirect inhibitor.
  • the present invention further concerns a method for treating cell degenerative ophthalmic conditions, and related diseases, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one Unc51-Like Kinase modulator of the present invention (preferably an indirect Unc51-Like Kinase inhibitor) , effective to limit cellular degeneration in the eye, and more preferably retinal cell degeneration, and most preferably photoreceptor cell degeneration.
  • Unc51-Like Kinase modulator of the present invention preferably an indirect Unc51-Like Kinase inhibitor
  • terapéuticaally effective amount in the context of degenerative diseases or conditions described herein refers to an amount effective to achieve measurable improvement (compared to an untreated control) as assessed by any relevant medical parameter used to evaluate subjects receiving treatment for the disease or condition.
  • the term "cell degenerative ophthalmic conditions, and related diseases” relates to a disorder characterized by retinal cell degeneration, i.e. degeneration affecting one or more of retinal cells, and more preferably photoreceptors (rods and/or cones) .
  • the term "cell degenerative ophthalmic conditions, and related diseases” relates more specially to a disorder characterized by photoreceptor cell degeneration. It is defined herein as any condition marked by a decrease in photoreceptor cell number and/or function.
  • the disorder is age-related macular degeneration.
  • age-related macular degeneration is defined as an age- related disorder which causes a decrease in visual acuity and possible loss of central vision.
  • the disorder is retinitis pigmentosa.
  • Other disorders characterized by photoreceptor cell degeneration according to the invention include edema (e.g. macular and retinal edema), ischemic conditions and uveitis.
  • a mammalian subject is treated with a therapeutically effective amount of a modulator of the invention, which is an amount that is sufficient to induce a desired response in the treated subject.
  • a biologically or therapeutically effective amount of a modulator may be the amount that interferes with physiological activity of the treated mammal in a non-lethal manner.
  • examination of the fundus of the eye aims at assessing the condition of the retina and to look for the presence of the characteristic pigment spots on the retinal surface, which in the illness assume a characteristic "osteoblast-like" appearance; examination of the visual field makes it possible to evaluate the sensitivity of the various parts of the retina to light stimuli; visual fields ' can be measured by means of microperimetry, Goldmann dynamic perimetry, and photopic and scotopic (dark-adapted) automated static perimetry.
  • electroretinogram EMG consists of recording the electrical activity of the retina in response to particular light stimuli, thus making possible distinct valuations of the functionality of the two different types of photoreceptors (-i.e. cone cells and rod cells).
  • the electroretinogram is a very important examination for diagnosing retinitis pigmentosa, because, even when the illness is in its initial stages, the resulting trace is almost always either very flat or altogether absent; Even more importantly, multi-focal ERG make it possible to detect defects and benefits on few photoreceptors.
  • Retinal thickness is determined by optical coherence tomography. Fluorangiography is performed by means of the intravenous injection of a fluorescent substance and subsequent photography of the retina at different times. Due to blood circulation, in fact, the fluorescent substance arrives at the retina, where it colours the arteries, the capillaries and the veins and thus renders them visible, as also the functional state of their walls; visus examination permits a valuation of visual acuity and consists of the patient reading letters of different sizes at a distance of three metres.
  • the compound or composition of the present invention allows to improve at least one disease related parameter selected in the group of parameters measured by examination of the fundus of the eye, examination of the visual field, electroretinograms, fluorangiography, and visus examination.
  • subject in need thereof refers to a mammal preferably a human.
  • the invention described herein may include one or more range of values (e.g. size, concentration, etc).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

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Abstract

The present application relates to novel therapeutic strategies for cellular degenerative diseases, and more specifically neurodegenerative diseases. Said strategies are based on modulating one or more metabolic pathways identified by the inventors, which are correlated with the onset, development and progression of toxicity and apoptosis in cells, especially nerve cells, and are particularly pertinent in neurodegenerative diseases. The invention relates to a novel use of Unc51-Like Kinase modulator, and more specifically of Unc51-Like Kinase inhibitors. The compounds of the invention are useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of cellular degenerative diseases, and more specifically neurodegenerative disorders.

Description

COMPOSITIONS AND METHODS FOR PROPHYLAXIS AND TREATMENT OF CELLULAR DEGENERATIVE OPHTHALMIC DISORDERS
The present application relates to novel therapeutic strategies for cellular degenerative ophthalmic diseases, and more specifically diseases associated with photoreceptors degeneration. Said strategies are based on modulating one or more metabolic pathways identified by the inventors, which are correlated with the onset, development and progression of toxicity and apoptosis in eye cells, more specifically in photoreceptors. The invention relates to a novel use of Unc51- Like Kinase modulators, and more specifically of Unc51-Like Kinase inhibitors. The modulators of the invention are useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of cellular degenerative ophthalmic diseases, and more specifically photoreceptors degenerative disorders. The invention relates to the use of modulators of the invention for the manufacture of pharmaceutical compositions giving a beneficial effect. A beneficial effect is disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art. More specifically, the present Invention relates to compositions and methods for prophylaxis and/or treatment of cellular degenerative ophthalmic diseases, and more specifically degenerative conditions affecting photoreceptors in a mammal using a composition which comprises an effective amount of a Unc51-Like Kinase modulator. And in special embodiment, the ophthalmic degenerative diseases of the present invention are retinal degenerative diseases, and more specifically are degenerative conditions affecting photoreceptors.
The following description is provided to aid in understanding the invention but is not admitted to be prior art to the invention
Diseases of the nervous system, including the central nervous system (CNS) and peripheral nervous system, are conditions frequently marked by a decrease in neuronal cell number and/or function. Additionally, because of the absence of endogenous repopulation, effective recovery of function following neuronal-related disease is either extremely limited or absent .
For example, one major cause of blindness is retinal degeneration, and the most common forms of retinal degeneration are retinitis pigmentosa, glaucoma and age- related macular degeneration (AMD) . Retinitis pigmentosa is affecting 1 in 4000 people worldwide and leaving more than 1.5 million people visually handicapped. First described over a hundred years ago, retinitis pigmentosa is characterized by progressive degeneration of the peripheral retina, leading to night blindness, progressive loss of the peripheral visual field, leading eventually to total blindness, abnormal electroretinogram, ophthalmoscopic changes consisting in dark mosaic like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration (Delyfer et al., 2004, Biol Cell., 96, 261-269) . The most prominent pathological finding of retinitis pigmentosa is a continuing decrease in the number of photoreceptor cells, often followed by alterations in the retinal pigmented epithelium and retinal glia with the appearance of intraretinal pigment deposits which appear around the mid-peripheral retina of individuals (Berson, 1996, PNAS, 93, 4526-4528) . Retinitis pigmentosa, which can be both sporadic and familial, is a disorder which has been linked to a number of different genes. The condition primarily affects the rod cells of the retina, but can eventually lead to loss of peripheral vision and blindness.
Age-related macular degeneration (AMD) affects approximately 15,000,000 people in the United States. It is estimated that total blindness eventually results in 5-10% of these persons. AMD accounts for 17% of new cases of blindness in the United States annually. Because AMD is primarily a disease of individuals over 65 years old, the incidence of AMD is expected to increase as the population ages. It is characterized by a sudden worsening and distortion of central vision that progresses rapidly, typically with a course of only weeks or months. AMD is characterized by abnormalities in the macular area. The central area (or fovea) of the macula contains the highest density of cone photoreceptors in the retina and mediates high-acuity vision. The disease typically has a preclinical, asymptomatic phase, in which extracellular waste material accumulates in the space between the basement membrane (Bruch's membrane) and the epithelial layer, forming yellow-white spots known as drusen. Advanced forms of AMD includes both dry and wet (or "neovascular" ) AMD. The dry form of AMD is far more common, but the wet form occurs simultaneously with the dry form in about 15 % of cases. Dry AMD is characterized by progressive apoptosis of cells in the epithelial layer, in the overlying photoreceptor cells and in the underlying cells in the choroidal capillary layer. Wet AMD is characterized by choroidal neovascularization with vascular leakage into subretinal spaces. AMD impairs central vision that is required for reading, driving, face recognition, and fine visual tasks.
Additionally, the retina while it is containing neuronal cells [e.g. photoreceptors (rods and cones), ganglion cells, horizontal cells, amacrine cells, bipolar cells] which can be affected by cellular degeneration process, further contains non neuronal cells (e.g. Muller cells or pigmented epithelial cells) which can also degenerate and therefore be the target of the method and compositions of the present invention. For example, retinal pigment epithelium is an important cell types in the retina, as it is crucial to the support of the photoreceptor function. It performs several complex tasks, including phagocytosis of shed outer segments of rods and cones, vitamin A metabolism, synthesis of mucopolysaccharides involved in the metabolite exchange in the subretinal space, transport of metabolites, regulation of angiogenesis, absorption of light, enhancement of resolution of images, and the regulation of many other functions in the retina through secreted proteins such as proteases and protease inhibitors. However, the major target of the method and compositions of the present invention are the photoreceptors.
Today, one possible method for restoring vision, or at least for limiting its loss, in patients afflicted with a cellular (e.g. photoreceptors) degeneration disorder is through the use of a visual input which is capable of activating retinal ganglion cells. If activation by the visual input is successful, then varying degrees of vision should be restored. One suggested method for activating retinal ganglion cells through a visual input is with an electronic stimulator which electrically activates the retinal ganglion cells. Known as a "retina chip" or "silicon retina, " electronic stimulator is the focus of numerous research activities, however there remain many problems associated with the use of an electronic stimulator. For example, spatial resolution problems associated with the stimulating array may occur, as well as toxicity which often accompanies the use of a foreign object in the body. Due to these complications, use of electronic stimulators is still years away from clinical application. Additionally, although retinitis pigmentosa was identified and classified about midway through last century, very little concrete progress has so far been achieved regarding either possible cures or understanding of the causes that determine and regulate its courses. The lines at present most widely followed by international research are (i) the genetic approach, which seeks to identify the gene or genes responsible for the illness and thus permitting a subsequent intervention by means of modern genetic engineering techniques, (ii) the transplant approach, which aims at perfecting a technique that would make possible the transplant of retinal tissue or, at least, the grafting of healthy cells into diseased retinas, and (iii) the immunological approach, which sets out to verify some theories that assume the illness to be underlain by some alteration of the immunological system. Among known genes associated with retinal degeneration, several groups are specifically expressed in the retina and are critical for retinal cell survival and function. The most commonly seen retinitis pigmentosa mutations occur in genes important for phototransduction. These genes include rhodopsin (RHO) , the α- and β-subunits of rod cyclic guanosine monophosphate (cGMP) phosphodiesterases (PDE6A and PDE6B) , the α-subunit of rod cyclic nucleotide gated channel (CNGAl) and arrestin (SAG) . Mutations in genes important for visual cycles are also associated with photoreceptor cell degeneration. These include ATP-binding cassette transporter of rods (ABCR) , cellular retinaldehyde binding protein (CRaIBP), RPE 65 protein (RPE65) and RPE- retinal G-protein coupled receptor (RGR) , all encoding proteins that are expressed mainly in retinal pigment epithelium (RPE) cells. These proteins are involved in recycling the rhodopsin chromophore 11-cis-retinaldehyde, which absorbs light to initiate the phototransduction cascade. Another group of genes defective in retinal degeneration code for photoreceptor cell structural proteins, including peripherin (also named retinal degeneration slow, RDS) , rod cell outer membrane protein 1 (ROMl), retinal actin-binding protein fascin (FSCN2) and prominin like-1 (PROM-I) . In addition, genes encoding critical metabolic enzymes, such as inosine monophosphate dehydrogenase 1 (IMPDHl) and lecithin retinol acyltransferase (LRAT) , are also important for retinal function, and mutations in these genes cause retinal degeneration.
There thus remains a strong need for a treatment for patients suffering from degeneration of eye cells, e.g. retinal cells and more specifically degeneration of photoreceptor cells.
The object of the present invention is to provide a composition that will permit cellular degenerative ophthalmic diseases to be efficiently treated and/or prevented. In preferred embodiment said composition is comprising pharmacological acceptable carrier. According to one special embodiment, the present invention relates to prevention and/or reduction of eye cells degeneration, more specifically retinal cells degeneration, and even more specifically of photoreceptor cells degeneration.
Another purpose of the present invention is to provide a method of preventing and/or treating cellular degenerative ophthalmic diseases, more particularly a method of preventing and/or treating retinal degeneration, and even more specifically a method of preventing and/or treating photoreceptor cells degeneration, that will permit for example a gradual recovery of visual acuity, or at least limit its loss, as well as limiting the loss of visual field, sharpness of images and perception of colours and an electroretinogram (ERG) decrease stabilisation and/or ERG recovery in the long run. According to one preferred embodiment, said retinal degeneration is photoreceptor cells degeneration.
Protein kinases are known to play crucial roles in nearly all aspects of cellular regulation, including cell growth, differentiation, and apoptosis in eukaryotes. Many protein kinases with specific or multiple functions have been characterized, which are different in primary structure, regulation modes and substrate specificities. A large number of protein kinase genes have been identified in the course of genome sequencing projects of various eukaryotes, for example, by sequencing the complete genome of S. cerevisiae and C. elegans (Chervitz et al., 1998, Science, 282, 2022-2028). Phylogenetic analysis of protein kinases allows sub-grouping of structurally related members, which also reflects related function or regulation (Hanks and Hunter, 1995, FASEB J., 9, 576-596) . Of interest, most of the kinases found in yeast and nematode also have homologous counterparts in mammals and other species to constitute subfamilies.
Unc51-Like kinases (ULK) are members of an evolutionarily conserved sub-family of ubiquitously expressed serine/threonine-specific protein kinases. Unc-51 was originally discovered in nematode C. elegans as a gene required for axonal elongation and guidance (Ogura et al., 1994, Genes Dev. , 8, 2389-2499; Ogura et al., 1997, Genes Dev., 11, 1801-1811; Tomoda, 1999, Neuron 24, 833-846) and was also linked to endocytosis (Tomoda, et al, 2004, Genes Dev 18, 541-558). In Unc-51 gene mutants, most of the neurons exhibit an aberrant axon elongation with premature termination or dysregulated numbers of axons in abnormal. In mammals, Ulkl was shown to be important for axon formation in cerebellar granule neurons (Tomoda et al.,1999, and Tomoda et al., 2004, supra) . ) . More recently, Zhou et al. have shown that Unc51- Like kinases 1 and 2 are involved in filopodia extensions and branching in sensory axons from mouse spinal sensory neurons, this process being mediated by endocytosis (Zhou et al. 2007, PNAS: 104 14. 5842-5847). Furthermore, Unc51-Like kinases 1 and 2 are involved in NGF induced axonal growth, since blocking their expression using RNAi technology, reduces axon lengh and increases axonal branching of neurons stimulated by NGF(ZhOu et al. 2007, PNAS: 104 14. 5842-5847. Unc51-Like kinases 2 has been demonstrated to physically interact with FRS2/3 (Fibroblast Growth Factor Substrate 2 and 3), an adaptor protein to FGF Receptor, therefore antagonizing FGFR/FRS2/3 interaction and consecutive FRS2/3 phosphorylation (Avery et al. 2007 Cell Signal. 19: 177-184). Mammalian homologue of Unc51 (called UIk-I for Unc-51 kinase 1) have been identified based on sequence homology to the C. elegans Unc-51 gene ( mouse : Yan et al., 1998, Biochem Biophys Res Commun, 246, 222-227, Human : Kuroyanagi et al.,1998, Genomics, 51,76-85 ).
In fact, three different Unc51-Like kinases (ULK) proteins have been identified in mammals (ULKl to 3) :
ULKl (also known as Unc51-Like kinases 1, ATGl, Hs.47061, Unc51.1, EC 2.7.11.1, Serine/threonine-protein kinase ULKl ) contains 1050 amino acids (Kuroyanagi, H. et al. Genomics 51 (1), 76-85 (1998)).
ULKl Protein sequence (NP_003556.1) is as follows :
MetGluProGlyArgGlyGlyThrGluThrValGlyLysPheGluPheSerArgLysAsp LeuIleGlyHisGlyAlaPheAlaValValPheLysGlyArgHisArgGluLysHisAsp LeuGluValAlaValLysCysIleAsnLysLysAsnLeuAlaLysSerGlnThrLeuLeu GlyLysGluIleLysIleLeuLysGluLeuLysHisGluAsnlleValAlaLeuTyrAsp PheGlnGluMetAlaAsnSerValTyrLeuValMetGluTyrCysAsnGlyGlyAspLeu AlaAspTyrLeuHisAlaMetArgThrLeuSerGluAspThrlleArgLeuPheLeuGln GInIIeAIaGIyAIaMetArgLeuLeuHisSerLysGlyIIeIIeHisArgAspLeuLys ProGlnAsnlleLeuLeuSerAsnProAlaGlyArgArgAlaAsnProAsnSerlleArg ValLysIleAlaAspPheGlyPheAlaArgTyrLeuGlnSerAsnMetMetAlaAlaThr LeuCysGlySerProMetTyrMetAlaProGluVallleMetSerGlnHisTyrAspGly LysAlaAspLeuTrpSerlleGlyThrlleValTyrGlnCysLeuThrGlyLysAlaPro
PheGlnAlaSerSerProGlnAspLeuArgLeuPheTyrGluLysAsnLysThrLeuVal
ProThrlleProArgGluThrSerAlaProLeuArgGlnLeuLeuLeuAlaLeuLeuGln
ArgAsnHisLysAspArgMetAspPheAspGluPhePheHisHisProPheLeuAspAla
SerProSerValArgLysSerProProValProValProSerTyrProSerSerGlySer
GlySerSerSerSerSerSerSerThrSerHisLeuAlaSerProProSerLeuGlyGlu
MetGlnGlnLeuGlnLysThrLeuAlaSerProAlaAspThrAlaGlyPheLeuHisSer
SerArgAspSerGlyGlySerLysAspSerSerCysAspThrAspAspPheValMetVal
ProAlaGlnPheProGlyAspLeuValAlaGluAlaProSerAlaLysProProProAsp
SerLeuMetCysSerGlySerSerLeuValAlaSerAlaGlyLeuGluSerHisGlyArg
ThrProSerProSerProProCysSerSerSerProSerProSerGlyArgAlaGlyPro
PheSerSerSerArgCysGlyAlaSerValProIleProValProThrGlnValGlnAsn
TyrGlnArglleGluArgAsnLeuGlnSerProThrGlnPheGlnThrProArgSerSer
AlalleArgArgSerGlySerThrSerProLeuGlyPheAlaArgAlaSerProSerPro
ProAlaHisAlaGluHisGlyGlyValLeuAlaArgLysMetSerLeuGlyGlyGlyArg
ProTyrThrProSerProGlnValGlyThrlleProGluArgProGlyTrpSerGlyThr
ProSerProGlnGlyAlaGluMetArgGlyGlyArgSerProArgProGlySerSerAla
ProGluHisSerProArgThrSerGlyLeuGlyCysArgLeuHisSerAlaProAsnLeu
SerAspLeuHisValValArgProLysLeuProLysProProThrAspProLeuGlyAla
ValPheSerProProGlnAlaSerProProGlnProSerHisGlyLeuGlnSerCysArg
AsnLeuArgGlySerProLysLeuProAspPheLeuGlnArgAsnProLeuProProIle
LeuGlySerProThrLysAlaValProSerPheAspPheProLysThrProSerSerGln
AsnLeuLeuAlaLeuLeuAlaArgGlnGlyValValMetThrProProArgAsnArgThr
LeuProAspLeuSerGluValGlyProPheHisGlyGlnProLeuGlyProGlyLeuArg
ProGlyGluAspProLysGlyProPheGlyArgSerPheSerThrSerArgLeuThrAsp
LeuLeuLeuLysAlaAlaPheGlyThrGlnAlaProAspProGlySerThrGluSerLeu
GlnGluLysProMetGluIleAlaProSerAlaGlyPheGlyGlySerLeuHisProGly
AlaArgAlaGlyGlyThrSerSerProSerProValValPheThrValGlySerProPro
SerGlySerThrProProGlnGlyProArgThrArgMetPheSerAlaGlyProThrGly
SerAlaSerSerSerAlaArgHisLeuValProGlyProCysSerGluAlaProAlaPro
GluLeuProAlaProGlyHisGlyCysSerPheAlaAspProIleAlaAlaAsnLeuGlu
GlyAlaValThrPheGluAlaProAspLeuProGluGluThrLeuMetGluGlnGluHis
ThrGluIleLeuArgGlyLeuArgPheThrLeuLeuPheValGlnHisValLeuGluIle
AlaAlaLeuLysGlySerAlaSerGluAlaAlaGlyGlyProGluTyrGlnLeuGlnGlu SerValValAlaAspGlnlleSerLeuLeuSerArgGluTrpGlyPheAlaGluGlnLeu ValLeuTyrLeuLysValAlaGluLeuLeuSerSerGlyLeuGlnSerAlalleAspGln IleArgAlaGlyLysLeuCysLeuSerSerThrValLysGlnValValArgArgLeuAsn GluLeuTyrLysAlaSerValValSerCysGlnGlyLeuSerLeuArgLeuGlnArgPhe PheLeuAspLysGlnArgLeuLeuAspArglleHisSerlleThrAlaGluArgLeuIle PheSerHisAlaValGlnMetValGlnSerAlaAlaLeuAspGluMetPheGlnHisArg GluGlyCysValProArgTyrHisLysAlaLeuLeuLeuLeuGluGlyLeuGlnHisMet LeuSerAspGlnAlaAspIleGluAsnValThrLysCysLysLeuCysIleGluArgArg LeuSerAlaLeuLeuThrGlylleCysAla
(ii) ULK2 (also known as Unc51-Like kinases 2,Unc51.2 , EC 2.7.11.1 , Serine/threonine-protein kinase ULK2 ) contains 3923 amino acids (Yan,J et al. Oncogene 18 (43), 5850-5859 (1999) ) .
ULK2 protein sequence (NP_055498.2) is as follows : MetGluValValGlyAspPheGluTyrSerLysArgAspLeuValGlyHisGlyAlaPhe
AlaValValPheArgGlyArgHisArgGlnLysThrAspTrpGluValAlalleLysSer IleAsnLysLysAsnLeuSerLysSerGlnlleLeuLeuGlyLysGluIleLysIleLeu LysGluLeuGlnHisGluAsnlleValAlaLeuTyrAspValGlnGluLeuProAsnSer ValPheLeuValMetGluTyrCysAsnGlyGlyAspLeuAlaAspTyrLeuGlnAlaLys GlyThrLeuSerGluAspThrlleArgValPheLeuHisGlnlleAlaAlaAlaMetArg IleLeuHisSerLysGlyllelleHisArgAspLeuLysProGlnAsnlleLeuLeuSer TyrAlaAsnArgArgLysSerSerValSerGlylleArglleLysIleAlaAspPheGly PheAlaArgTyrLeuHisSerAsnMetMetAlaAlaThrLeuCysGlySerProMetTyr MetAlaProGluVallleMetSerGlnHisTyrAspAlaLysAlaAspLeuTrpSerlle GlyThrVallleTyrGlnCysLeuValGlyLysProProPheGlnAlaAsnSerProGln AspLeuArgMetPheTyrGluLysAsnArgSerLeuMetProSerlleProArgGluThr SerProTyrLeuAlaAsnLeuLeuLeuGlyLeuLeuGlnArgAsnGlnLysAspArgMet AspPheGluAlaPhePheSerHisProPheLeuGluGlnGlyProValLysLysSerCys ProValProValProMetTyrSerGlySerValSerGlySerSerCysGlySerSerPro SerCysArgPheAlaSerProProSerLeuProAspMetGlnHisIleGlnGluGluAsn LeuSerSerProProLeuGlyProProAsnTyrLeuGlnValSerLysAspSerAlaSer ThrSerSerLysAsnSerSerCysAspThrAspAspPheValLeuValProHisAsnlle SerSerAspHisSerCysAspMetProMetGlyThrAlaGlyArgArgAlaSerAsnGlu
PheLeuValCysGlyGlyGlnCysGlnProThrValSerProHisSerGluThrAlaPro
IleProValProThrGlnlleArgAsnTyrGlnArglleGluGlnAsnLeuThrSerThr
AlaSerSerGlyThrAsnValHisGlySerProArgSerAlaValValArgArgSerAsn
ThrSerProMetGlyPheLeuArgProGlySerCysSerProValProAlaAspThrAla
GlnThrValGlyArgArgLeuSerThrGlySerSerArgProTyrSerProSerProLeu
ValGlyThrlleProGluGlnPheSerGlnCysCysCysGlyHisProGlnGlyHisAsp
SerArgSerArgAsnSerSerGlySerProValProGlnAlaGlnSerProGlnSerLeu
LeuSerGlyAlaArgLeuGlnSerAlaProThrLeuThrAspIleTyrGlnAsnLysGln
LysLeuArgLysGlnHisSerAspProValCysProSerHisThrGlyAlaGlyTyrSer
TyrSerProGlnProSerArgProGlySerLeuGlyThrSerProThrLysHisLeuGly
SerSerProArgSerSerAspTrpPhePheLysThrProLeuProThrllelleGlySer
ProThrLysThrThrAlaProPheLysIleProLysThrGlnAlaSerSerAsnLeuLeu
AlaLeuValThrArgHisGlyProAlaGluGluGlnSerLysAspGlyAsnGluProArg
GIuCysAlaHisCysLeuLeuValGlnGlySerGluArgGlnArgAiaGluGlnGlnSer
LysAlaValPheGlyArgSerValSerThrGlyLysLeuSerAspGlnGlnGlyLysThr
ProIleCysArgHisGlnGlySerThrAspSerLeuAsnThrGluArgProMetAspIle
AlaProAlaGlyAlaCysGlyGlyValLeuAlaProProAlaGlyThrAlaAlaSerSer
LysAlaValLeuPheThrValGlySerProProHisSerAlaAlaAlaProThrCysThr
HisMetPheLeuArgThrArgThrThrSerValGlyProSerAsnSerGlyGlySerLeu
CysAlaMetSerGlyArgValCysValGlySerProProGlyProGlyPheGlySerSer
ProProGlyAlaGluAlaAlaProSerLeuArgTyrValProTyrGlyAlaSerProPro
SerLeuGluGlyLeuIleThrPheGluAlaProGluLeuProGluGluThrLeuMetGlu
ArgGluHisThrAspThrLeuArgHisLeuAsnValMetLeuMetPheThrGluCysVal
LeuAspLeuThrAlaMetArgGlyGlyAsnProGluLeuCysThrSerAlaValSerLeu
TyrGlnlleGlnGluSerValValValAspGlnlleSerGlnLeuSerLysAspTrpGly
TrpValGluGlnLeuValLeuTyrMetLysAlaAlaGlnLeuLeuAlaAlaSerLeuHis
LeuAlaLysAlaGlnlleLysSerGlyLysLeuSerProSerThrAlaValLysGlnVal
ValLysAsnLeuAsnGluArgTyrLysPheCysIleThrMetCysLysLysLeuThrGlu
LysLeuAsnArgPhePheSerAspLysGlnArgPhelleAspGluIleAsnSerValThr
AlaGluLysLeuIleTyrAsnCysAlaValGluMetValGlnSerAlaAlaLeuAspGlu
MetPheGlnGlnThrGluAspIleValTyrArgTyrHisLysAlaAlaLeuLeuLeuGlu
GlyLeuSerArglleLeuGlnAspProAlaAspIleGluAsnValHisLysTyrLysCys
SerlleGluArgArgLeuSerAlaLeuCysHisSerThrAlaThrVal (iii) ULK3 (also known as Unc51-Like kinases 3, EC 2.7.11.1 , Serine/threonine-protein kinase ULK3 ) contains 1763 amino acids predicted ULK3 protein sequence (XP_001134013.1) is as follows :
MetAlaGlyProGlyTrpGlyProProArgLeuAspGlyPhelleLeuThrGluArgLeu GlySerGlyThrTyrAlaThrValTyrLysAlaTyrAlaLysLysAspThrArgGluVal ValAlalleLysCysValAlaLysLysSerLeuAsnLysAlaSerValGluAsnLeuLeu ThrGluIIeGIuIleLeuLysGlyIleArgHisProHisIleValGlnLeuLysAspPhe GlnTrpAspSerAspAsnlleTyrLeuIleMetGluPheCysAlaGlyGlyAspLeuSer ArgPhelleHisThrArgArglleLeuProGluLysValAlaArgValPheMetGlnGln LeuAlaSerAlaLeuGlnPheLeuHisGluArgAsnlleSerHisLeuAspLeuLysPro GlnAsnlleLeuLeuSerSerLeuGluLysProHisLeuLysLeuAlaAspPheGlyPhe AlaGlnHisMetSerProTrpAspGluLysHisValLeuArgGlySerProLeuTyrMet AlaProGluMetValCysGlnArgGlnTyrAspAlaArgValAspLeuTrpSerMetGly VallleLeuTyrGluAlaLeuPheGlyGlnProProPheAlaSerArgSerPheSerGlu LeuGluGluLysIleArgSerAsnArgVallleGluLeuProLeuArgProLeuLeuSer ArgAspCysArgAspLeuLeuGlnArgLeuLeuGluArgAspProSerArgArglleSer PheGlnAspPhePheAlaHisProTrpValAspLeuGluHisMetProSerGlyGluSer LeuGlyArgAlaThrAlaLeuValValGlnAlaValLysLysAspGlnGluGlyAspSer AlaAlaAlaLeuSerLeuTyrCysLysAlaLeuAspPhePheValProAlaLeuHisTyr GluValAspAlaGlnArgLysGluAlalleLysAlaLysValGlyGlnTyrValSerArg AlaGluGluLeuLysAlalleValSerSerSerAsnGlnAlaLeuLeuArgGlnGlyThr SerAlaArgAspLeuLeuArgGluMetAlaArgAspLysProArgLeuLeuAlaAlaLeu GluValAlaSerAlaAlaMetAlaLysGluGluAlaAlaGlyGlyGluGlnAspAlaLeu AspLeuTyrGlnHisSerLeuGlyGluLeuLeuLeuLeuLeuAlaAlaGluProProGly ArgArgArgGluLeuLeuHisThrGluValGlnAsnLeuMetAlaArgAlaGluTyrLeu LysGluGlnValLysMetArgGluSerArgTrpGluAlaAspThrLeuAspLysGluGly LeuSerGluSerValArgSerSerCysThrLeuGln.
Patent application WO 2002079478 provides means and methods for modulating the biological and biochemical activities of Unc51-Like kinases for treating metabolic disease or condition such as obesity, adipositas, eating disorders, wasting syndrome, pancreatic dysfunctions (e.g. diabetes), energy homeostasis.
Patent application WO 2005101013 provides material and methods for screening modulators of neural regeneration such as inhibitors of Unc51-Like kinases, which can be used to stimulate neurogenesis after cerebral ischemia or in Alzheimer disease .
SUMMARY OF THE INVENTION
The Inventors have now surprisingly shown that Unc51-Like Kinase could be a therapeutic target for treating / preventing disorders characterized by eye cells degeneration, more particularly retinal cells, and even more preferably photoreceptor cells degeneration.
According to a first embodiment, the Invention provides a composition comprising at least one Unc51-Like Kinase modulator for treating and/or preventing cellular degenerative ophthalmic disease in a subject in need thereof.
As used herein throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced compounds or steps, unless the context dictates otherwise. More specifically, "at least one" and "one or more" means a number which is one or greater than one, with a special preference for one, two or three.
The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term" .
The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
As used herein, the term "comprising", "containing" when used to define products, compositions and methods, is intended to mean that the products, compositions and methods include the referenced compounds or steps, but not excluding others.
As used herein, the term "treatment" or "treating" encompasses prophylaxis and/or therapy. Accordingly the compositions and methods of the present invention are not limited to therapeutic applications and can be used in prophylaxis ones. Therefore "treating" or "treatment" of a state, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (ii) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (iii) relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms .
The terms "patient" or "subject in need" refer to a vertebrate, particularly a member of the mammalian species and includes, but is not limited to, domestic animals, sport animals, primates including humans. The term "patient" is in no way limited to a special disease status, it encompasses both patients who have already developed a disease of interest and patients who are not sick.
As used herein, the term "Unc51-Like Kinase" is intended to designate both Unc51-Like Kinase 1 (ULKl), Unc51-Like Kinase 2 (ULK2) and Unc51-Like Kinase 3 (ULK3) , and can be used herein to designate individually or collectively each of them.
As used herein, the term "Unc51-Like Kinase modulator" is intended to designate compound which is able to prevent or limit Unc51-Like Kinase synthesis and/or bioavailability and/or activity.
According to one preferred embodiment, said Unc51-Like Kinase modulator is a Unc51-Like Kinase inhibitor or partial inhibitor.
According to another preferred embodiment, said inhibited or partially inhibited Unc51-Like Kinase is selected in the group consisting of ULKl, ULK2 and ULK3.
According to an advantageous embodiment, said inhibited or partially inhibited Unc51-Like Kinase is expressed in photoreceptor cells.
In one embodiment, it is ULKl (also known as Unc51-Like Kinase 1). Its protein sequence (NP_003556.1) is as follows :
MetGluProGlyArgGlyGlyThrGluThrValGlyLysPheGluPheSerArgLysAsp
LeuIleGlyHisGlyAlaPheAlaValValPheLysGlyArgHisArgGluLysHisAsp LeuGluValAlaValLysCysIleAsnLysLysAsnLeuAlaLysSerGlnThrLeuLeu GlyLysGluIleLysIleLeuLysGluLeuLysHisGluAsnlleValAlaLeuTyrAsp PheGlnGluMetAlaAsnSerValTyrLeuValMetGluTyrCysAsnGlyGlyAspLeu AlaAspTyrLeuHisAlaMetArgThrLeuSerGluAspThrlleArgLeuPheLeuGln GlnlleAlaGlyAlaMetArgLeuLeuHisSerLysGlyllelleHisArgAspLeuLys ProGlnAsnlleLeuLeuSerAsnProAlaGlyArgArgAlaAsnProAsnSerlleArg ValLysIleAlaAspPheGlyPheAlaArgTyrLeuGlnSerAsnMetMetAlaAlaThr LeuCysGlySerProMetTyrMetAlaProGluVallleMetSerGlnHisTyrAspGly LysAlaAspLeuTrpSerlleGlyThrlleValTyrGlnCysLeuThrGlyLysAlaPro PheGlnAlaSerSerProGlnAspLeuArgLeuPheTyrGluLysAsnLysThrLeuVal ProThrlleProArgGluThrSerAlaProLeuArgGlnLeuLeuLeuAlaLeuLeuGln ArgAsnHisLysAspArgMetAspPheAspGluPhePheHisHisProPheLeuAspAla SerProSerValArgLysSerProProValProValProSerTyrProSerSerGlySer GlySerSerSerSerSerSerSerThrSerHisLeuAlaSerProProSerLeuGlyGlu MetGlnGlnLeuGlnLysThrLeuAlaSerProAlaAspThrAlaGlyPheLeuHisSer SerArgAspSerGlyGlySerLysAspSerSerCysAspThrAspAspPheValMetVal ProAlaGlnPheProGlyAspLeuValAlaGluAlaProSerAlaLysProProProAsp SerLeuMetCysSerGlySerSerLeuValAlaSerAlaGlyLeuGluSerHisGlyArg
ThrProSerProSerProProCysSerSerSerProSerProSerGlyArgAlaGlyPro
PheSerSerSerArgCysGlyAlaSerValProIleProValProThrGlnValGlnAsn
TyrGlnArglleGluArgAsnLeuGlnSerProThrGlnPheGlnThrProArgSerSer
AlalleArgArgSerGlySerThrSerProLeuGlyPheAlaArgAlaSerProSerPro
ProAlaHisAlaGluHisGlyGlyValLeuAlaArgLysMetSerLeuGlyGlyGlyArg
ProTyrThrProSerProGlnValGlyThrlleProGluArgProGlyTrpSerGlyThr
ProSerProGlnGlyAlaGluMetArgGlyGlyArgSerProArgProGlySerSerAla
ProGluHisSerProArgThrSerGlyLeuGlyCysArgLeuHisSerAlaProAsnLeu
SerAspLeuHisValValArgProLysLeuProLysProProThrAspProLeuGlyAla
ValPheSerProProGlnAlaSerProProGlnProSerHisGlyLeuGlnSerCysArg
AsnLeuArgGlySerProLysLeuProAspPheLeuGlnArgAsnProLeuProProIle
LeuGlySerProThrLysAlaValProSerPheAspPheProLysThrProSerSerGln
AsnLeuLeuAlaLeuLeuAlaArgGlnGlyValValMetThrProProArgAsnArgThr
LeuProAspLeuSerGluValGlyProPheHisGlyGlnProLeuGlyProGlyLeuArg
ProGlyGluAspProLysGlyProPheGlyArgSerPheSerThrSerArgLeuThrAsp
LeuLeuLeuLysAlaAlaPheGlyThrGlnAlaProAspProGlySerThrGluSerLeu
GlnGluLysProMetGluIleAlaProSerAlaGlyPheGlyGlySerLeuHisProGly
AlaArgAlaGlyGlyThrSerSerProSerProValValPheThrValGlySerProPro
SerGlySerThrProProGlnGlyProArgThrArgMetPheSerAlaGlyProThrGly
SerAlaSerSerSerAlaArgHisLeuValProGlyProCysSerGluAlaProAlaPro
GluLeuProAlaProGlyHisGlyCysSerPheAlaAspProIleAlaAlaAsnLeuGlu
GlyAlaValThrPheGluAlaProAspLeuProGluGluThrLeuMetGluGlnGluHis
ThrGluIleLeuArgGlyLeuArgPheThrLeuLeuPheValGlnHisValLeuGluIle
AlaAlaLeuLysGlySerAlaSerGluAlaAlaGlyGlyProGluTyrGlnLeuGlnGlu
SerValValAlaAspGlnlleSerLeuLeuSerArgGluTrpGlyPheAlaGluGlnLeu
ValLeuTyrLeuLysValAlaGluLeuLeuSerSerGlyLeuGlnSerAlalleAspGln
IleArgAlaGlyLysLeuCysLeuSerSerThrValLysGlnValValArgArgLeuAsn
GluLeuTyrLysAlaSerValValSerCysGlnGlyLeuSerLeuArgLeuGlnArgPhe
PheLeuAspLysGlnArgLeuLeuAspArglleHisSerlleThrAlaGluArgLeuIle
PheSerHisAlaValGlnMetValGlnSerAlaAlaLeuAspGluMetPheGlnHisArg
GluGlyCysValProArgTyrHisLysAlaLeuLeuLeuLeuGluGlyLeuGlnHisMet
LeuSerAspGlnAlaAspIleGluAsnValThrLysCysLysLeuCysIleGluArgArg
LeuSerAlaLeuLeuThrGlylleCysAla . In another embodiment, it is ULK2 (also known as Unc51- Like Kinase 2). Its protein sequence (NP_055498.2) is as follows :
MetGluValValGlyAspPheGluTyrSerLysArgAspLeuValGlyHisGlyAlaPhe
AlaValValPheArgGlyArgHisArgGlnLysThrAspTrpGluValAlalleLysSer
IleAsnLysLysAsnLeuSerLysSerGlnlleLeuLeuGlyLysGluIleLysIleLeu
LysGluLeuGlnHisGluAsnlleValAlaLeuTyrAspValGlnGluLeuProAsnSer
ValPheLeuValMetGluTyrCysAsnGlyGlyAspLeuAlaAspTyrLeuGlnAlaLys
GlyThrLeuSerGluAspThrlleArgValPheLeuHisGlnlleAlaAlaAlaMetArg
IleLeuHisSerLysGlyllelleHisArgAspLeuLysProGlnAsnlleLeuLeuSer
TyrAlaAsnArgArgLysSerSerValSerGlylleArglleLysIleAlaAspPheGly
PheAlaArgTyrLeuHisSerAsnMetMetAlaAlaThrLeuCysGlySerProMetTyr
MetAlaProGluVallleMetSerGlnHisTyrAspAlaLysAlaAspLeuTrpSerlle
GlyThrVallleTyrGlnCysLeuValGlyLysProProPheGlnAlaAsnSerProGln
AspLeuArgMetPheTyrGluLysAsnArgSerLeuMetProSerlleProArgGluThr
SerProTyrLeuAlaAsnLeuLeuLeuGlyLeuLeuGlnArgAsnGlnLysAspArgMet
AspPheGluAlaPhePheSerHisProPheLeuGluGlnGlyProValLysLysSerCys
ProValProValProMetTyrSerGlySerValSerGlySerSerCysGlySerSerPro
SerCysArgPheAlaSerProProSerLeuProAspMetGlnHisIleGlnGluGluAsn
LeuSerSerProProLeuGlyProProAsnTyrLeuGlnValSerLysAspSerAlaSer
ThrSerSerLysAsnSerSerCysAspThrAspAspPheValLeuValProHisAsnlle
SerSerAspHisSerCysAspMetProMetGlyThrAlaGlyArgArgAlaSerAsnGlu
PheLeuValCysGlyGlyGlnCysGlnProThrValSerProHisSerGluThrAlaPro
IleProValProThrGlnlleArgAsnTyrGlnArglleGluGlnAsnLeuThrSerThr
AlaSerSerGlyThrAsnValHisGlySerProArgSerAlaValValArgArgSerAsn
ThrSerProMetGlyPheLeuArgProGlySerCysSerProValProAlaAspThrAla
GlnThrValGlyArgArgLeuSerThrGlySerSerArgProTyrSerProSerProLeu
ValGlyThrlleProGluGlnPheSerGlnCysCysCysGlyHisProGlnGlyHisAsp
SerArgSerArgAsnSerSerGlySerProValProGlnAlaGlnSerProGlnSerLeu
LeuSerGlyAlaArgLeuGlnSerAlaProThrLeuThrAspIleTyrGlnAsnLysGln
LysLeuArgLysGlnHisSerAspProValCysProSerHisThrGlyAlaGlyTyrSer
TyrSerProGlnProSerArgProGlySerLeuGlyThrSerProThrLysHisLeuGly
SerSerProArgSerSerAspTrpPhePheLysThrProLeuProThrllelleGlySer
ProThrLysThrThrAlaProPheLysIleProLysThrGlnAlaSerSerAsnLeuLeu AlaLeuValThrArgHisGlyProAlaGluGluGlnSerLysAspGlyAsnGluProArg GluCysAlaHisCysLeuLeuValGlnGlySerGluArgGlnArgAlaGluGlnGlnSer LysAlaValPheGlyArgSerValSerThrGlyLysLeuSerAspGlnGlnGlyLysThr ProIleCysArgHisGlnGlySerThrAspSerLeuAsnThrGluArgProMetAspIle AlaProAlaGlyAlaCysGlyGlyValLeuAlaProProAlaGlyThrAlaAlaSerSer LysAlaValLeuPheThrValGlySerProProHisSerAlaAlaAlaProThrCysThr HisMetPheLeuArgThrArgThrThrSerValGlyProSerAsnSerGlyGlySerLeu CysAlaMetSerGlyArgValCysValGlySerProProGlyProGlyPheGlySerSer ProProGlyAlaGluAlaAlaProSerLeuArgTyrValProTyrGlyAlaSerProPro SerLeuGluGlyLeuIleThrPheGluAlaProGluLeuProGluGluThrLeuMetGlu ArgGluHisThrAspThrLeuArgHisLeuAsnValMetLeuMetPheThrGluCysVal LeuAspLeuThrAlaMetArgGlyGlyAsnProGluLeuCysThrSerAlaValSerLeu TyrGlnlleGlnGluSerValValValAspGlnlleSerGlnLeuSerLysAspTrpGly TrpValGluGlnLeuValLeuTyrMetLysAlaAlaGlnLeuLeuAlaAlaSerLeuHis LeuAlaLysAlaGlnlleLysSerGlyLysLeuSerProSerThrAlaValLysGlnVal ValLysAsnLeuAsnGluArgTyrLysPheCysIleThrMetCysLysLysLeuThrGlu LysLeuAsnArgPhePheSerAspLysGlnArgPhelleAspGluIleAsnSerValThr AIaGIuLysLeuIIeTyrAsnCysAlaValGluMetValGlnSerAlaAlaLeuAspGlu MetPheGlnGlnThrGluAspIleValTyrArgTyrHisLysAlaAlaLeuLeuLeuGlu GlyLeuSerArglleLeuGlnAspProAlaAspIleGluAsnValHisLysTyrLysCys SerlleGluArgArgLeuSerAlaLeuCysHisSerThrAlaThrVal.
In another embodiment, it is ULK3 (also known as Unc51- Like Kinase 3). Its protein sequence (XP_001134013.1) is as follows :
MetAlaGlyProGlyTrpGlyProProArgLeuAspGlyPhelleLeuThrGluArgLeu GlySerGlyThrTyrAlaThrValTyrLysAlaTyrAlaLysLysAspThrArgGluVal ValAlalleLysCysValAlaLysLysSerLeuAsnLysAlaSerValGluAsnLeuLeu ThrGluIIeGIuIleLeuLysGlylleArgHisProHisIleValGlnLeuLysAspPhe GlnTrpAspSerAspAsnlleTyrLeuIleMetGluPheCysAlaGlyGlyAspLeuSer ArgPhelleHisThrArgArglleLeuProGluLysValAlaArgValPheMetGlnGln LeuAlaSerAlaLeuGlnPheLeuHisGluArgAsnlleSerHisLeuAspLeuLysPro GlnAsnlleLeuLeuSerSerLeuGluLysProHisLeuLysLeuAlaAspPheGlyPhe AIaGInHisMetSerProTrpAspGluLysHisValLeuArgGlySerProLeuTyrMet AlaProGluMetValCysGlnArgGlnTyrAspAlaArgValAspLeuTrpSerMetGly VallleLeuTyrGluAlaLeuPheGlyGlnProProPheAlaSerArgSerPheSerGlu LeuGluGluLysIleArgSerAsnArgVallleGluLeuProLeuArgProLeuLeuSer ArgAspCysArgAspLeuLeuGlnArgLeuLeuGluArgAspProSerArgArglleSer PheGlnAspPhePheAlaHisProTrpValAspLeuGluHisMetProSerGlyGluSer LeuGlyArgAlaThrAlaLeuValValGlnAlaValLysLysAspGlnGluGlyAspSer AlaAlaAlaLeuSerLeuTyrCysLysAlaLeuAspPhePheValProAlaLeuHisTyr GluValAspAlaGlnArgLysGluAlalleLysAlaLysValGlyGlnTyrValSerArg AlaGluGluLeuLysAlalleValSerSerSerAsnGlnAlaLeuLeuArgGlnGlyThr SerAlaArgAspLeuLeuArgGluMetAlaArgAspLysProArgLeuLeuAlaAlaLeu GluValAlaSerAlaAlaMetAIaLysGluGluAlaAlaGlyGlyGluGlnAspAlaLeu AspLeuTyrGlnHisSerLeuGlyGluLeuLeuLeuLeuLeuAlaAlaGluProProGly ArgArgArgGluLeuLeuHisThrGluValGlnAsnLeuMetAlaArgAlaGluTyrLeu LysGluGlnValLysMetArgGluSerArgTrpGluAlaAspThrLeuAspLysGluGly LeuSerGluSerValArgSerSerCysThrLeuGln.
According to the present invention, said Unc51-Like Kinase inhibitor or partial inhibitor can be a direct inhibitor or an indirect inhibitor.
According to one special embodiment, said Unc51-Like Kinase inhibitor or partial inhibitor (including direct and indirect inhibitors) is selected in the group consisting of a small molecule, a nucleic acid molecule (e.g. an antisense to Unc51-Like Kinase -encoding nucleic acid molecules, a siRNA, a miRNA, a ribozyme, a shRNA, a nucleic acid capable of modulating Unc51-Like Kinase nucleic acid expression, a vector more particularly an expression vector expressing at least one nucleic acid capable of modulating Unc51-Like Kinase nucleic acid expression) , an anti-Unc51-Like Kinase antibody.
"Partial inhibitor" refers to a compound which acts as the inhibitor but that produces a weak maximum inhibitory response. This term is well known in the art. Such inhibition may be determined using in vitro or in vivo systems, or a combination of both, using methods known to those of ordinary skill in the art (see below) . The term "indirect inhibitor" as used herein, refers according to one example to an agent able to interfere specifically with the Unc51-Like Kinase gene expression and/or Unc51-Like Kinase protein synthesis, and more particularly with the Unc51-Like Kinase mRNA. According to another preferred embodiment, said Unc51-Like Kinase inhibitor is an indirect inhibitor and is a nucleic acid that is able to modulate, preferably to reduce, the expression level of said Unc51-Like Kinase, and more particularly the Unc51-Like Kinase mRNA level in cells (especially cells of the eye, more especially retinal cells and preferably photoreceptor cells) .
According to special embodiments, the "indirect inhibitor" of the invention is able to modulate, preferably to reduce, the expression level of said Unc51-Like Kinase, and more particularly the Uncδl-Like Kinase mRNA level in the treated cells e.g. by at least about 10%, about 20%, about 30%, about 50%, about 75%, about 85%, or about 95% (compared to the situation without said indirect inhibitor) . In another embodiment, said indirect inhibitor is able to modulate, preferably to reduce (e.g. by at least about 10%, about 20%, about 30%, about 50%, about 75%, about 85%, or about 95%), the expression level, and more particularly the mRNA level, of at least one Unc51-Like Kinase selected in the group consisting in ULKl, ULK2 and ULK3. These inhibitory characterisations of the indirect inhibitor can be tested using well known molecular biology methods, such as for example Western blots, QRTPCR, and the like.
The term "nucleic acid" includes, but is not limited to isolated DNA, RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA, vectors more particularly expression vectors expressing at least one of said isolated nucleic acids (i.e. DNA, RNA, oligonucleotides, antisens, siRNA, miRNA, ribozyme, shRNA) which are capable to modulate, preferably to reduce, the expression levels of said Unc51-Like Kinase, and more precisely are capable of modulating Unc51-Like Kinase nucleic acid expression and/or level (e.g. by modulating the transcription level of mRNA, by controlling mRNA stability/degradation, etc..) . Those terms are widely used in the art and are well known from the skilled person.
It should be understood that according to the invention, the said nucleic acid can be used naked, i.e. present in the compositions of the invention or administered according to the invention, as such (e.g. DNA, RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) , or vectorized, i.e. (i) incorporated in a vector (e.g. synthetic vectors, for example combined with substances which are known to improve the transfectional efficiency and/or stability of vectors, see below) or (ii) encoded by an expression vector (i.e. is incorporated in the expression vector as heterologous nucleic acid) .
The term "vector" is used to refer to any molecule (e.g. chemical compounds, plasmid, yeast or virus) used to transfer heterologous nucleic acid (e.g. DNA encoding information, RNA molecule, hybrid RNA/DNA and /or nucleic acid analogs) to a host cell. As used herein, the term "expression vector" (or alternatively "recombinant vector") refers to a vector which contains heterologous nucleic acid sequences and which, when transformed or transfected into a suitable host cell, directs and/or controls the expression, transcription, and/or translation of said heterologous nucleic acid sequences that are inserted into the said vector. In preferred embodiments, the "expression vector" of the invention when transformed or transfected into a suitable host cell, is able to direct and/or control the expression and transcription of RNA molecules (including RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) . "Expression vector" refers to viral as well as non viral vectors, including extrachromosomal (e.g. episome) , multicopy and integrating vectors (i.e. for being incorporated into the host chromosomes). Particularly important in the context of the invention are vectors generally disclosed and used in gene therapy or vector-based therapy (i.e. which are capable of delivering a nucleic acid to a host organism) as well as expression vectors for use in various expression systems.
The vector which can be of plasmid or viral origin, can where appropriate be naked or combined with one or more substances which improve the transfectional efficiency and/or stability of the vector. These substances are widely documented in the literature available to the skilled man (see, for example, Feigner et al., 1987, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry, 5, 647-654). By way of non-limiting illustration, said substances can be polymers, lipids, in particular cationic lipids, liposomes, nuclear proteins or neutral lipids. These substances can be used alone or in combination. A combination which can be envisaged is that of a recombinant plasmid vector which is combined with cationic lipids (DOGS, DC-CHOL, spermine-chol, spermidine-chol, etc.), lysophospholipides (for example Hexadecylphosphocholine) and neutral lipids (DOPE) .
The choice of the "plasmids" (non viral vectors) which can be used within the context of the present invention is huge. They are known to the skilled man and, while a number of them are available commercially, it is also possible to construct them or to modify them using the techniques of genetic manipulation. Examples which may be mentioned are the plasmids which are derived from pBR322 (Gibco BRL) , pUC (Gibco BRL), pBluescript (Stratagene) , pREP4, pCEP4 ( Invitrogene) , pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pVAX , pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76, 12735-12746) or p Poly (Lathe et al., 1987, Gene 57, 193-201) . Preferably, a plasmid which is used in the context of the present invention contains an origin of replication which ensures that replication is initiated in a producer cell and/or a host cell (for example, the CoIEl origin will be chosen for a plasmid which is intended to be produced in E. coli and the oriP/EBNAl system will be chosen if it desired that the plasmid should be self-replicating in a mammalian host cell, Lupton and Levine, 1985, MoI. Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815). The plasmid can additionally comprise a selection gene which enables the transfected cells to be selected or identified (complementation of an auxotrophic mutation, gene encoding resistance to an antibiotic, etc.). Naturally, the plasmid can contain additional elements which improve its maintenance and/or its stability in a given cell (e.g. cer sequence, which promotes maintenance of a plasmid in monomeric form (Summers and Sherrat, 1984, Cell 36, 1097-1103), sequences for integration into the cell genome.
Suitable "viral vectors" which can be used within the context of the present invention may be derived from a variety of different viruses (e.g. retrovirus, adenovirus, AAV, poxvirus, herpes virus, measle virus, foamy virus and the like) . As used herein, the term "viral vector" encompasses vector DNA/RNA as well as viral particles generated thereof. Viral vectors can be replication-competent, or can be genetically disabled so as to be replication-defective or replication-impaired. The term "replication-competent" as used herein encompasses replication-selective and conditionally- replicative viral vectors which are engineered to replicate better or selectively in specific host cells.
In one aspect, the recombinant vector in use in the invention is a recombinant adenoviral vector (for a review, see "Adenoviral vectors for gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press) . It can be derived from a variety of human or animal (canine, avian, bovine, murine, ovine, porcine, simian, etc.) sources and any serotype can be employed from the adenovirus serotypes 1 through 51. More particular mention may be made of the CAV-I or CAV-2 adenoviruses of canine origin, of the DAV adenovirus of avian origin or of the Bad type 3 adenovirus of bovine origin (Zakharchuk et al., 1993, Arch. Virol., 128: 171-176; Mittal et al., 1995, J. Gen. Virol., 76: 93-102). However, preference will be given to an adenoviral vector of human origin which is preferably derived from a serotype C-adenovirus, in particular human adenoviruses 2 (Ad2), 5 (Ad5) , 6 (Ad6) , 11 (AdIl), 24 (Ad24) and 35 (Ad35) . Nevertheless, three serotypes D- adenoviruses (Ad8, Adl9, and Ad37) have been shown to cause a severe ocular disease, epidemic keratoconjunctivitis (EKC) (Ford et al., 1987, Epidemiol. Rev., 9,244-261), and natural tropism of Ad37 has already been explored as adenovirus vectors for therapy of ocular diseases (US 20020193327); these adenoviruses strains with natural tropism for eye are preferred. Such adenoviruses are available from the American Type Culture Collection (ATCC, Rockville, Md.), and have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them (see for example US 6,133,028; US 6,110,735; WO 02/40665; WO 00/50573; EP 1016711; Vogels et al., 2003, J. Virol. 77, 8263-8271). The adenoviral vector in use in the present invention can be replication-competent. Numerous examples of replication-competent adenoviral vectors are readily available to those skill in the art (see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727). For example, they can be engineered from a wild-type adenovirus genome by deletion in the ElA CR2 domain (see for example WO00/24408) and/or by replacement of the native El and/or E4 promoters with heterologous promoter (constitutive promoter, tissue- or cell-specific promoter or inducible promoter) Alternatively, the adenoviral vector in use in the invention is replication-defective (see for example WO94/28152; Lusky et al., 1998, J. Virol 72, 2022-2032). Preferred replication- defective adenoviral vectors are El-defective (see for example US 6,136,594 and US 6,013,638), with an El deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of the human adenovirus type 5 disclosed in the GeneBank under the accession number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280-285) . The cloning capacity can further be improved by deleting additional portion (s) of the adenoviral genome (all or part of the non essential E3 region or of other essential E2, E4 regions). Insertion of a nucleic acid in any location of the adenoviral vector can be performed through homologous recombination as described in Chartier et al. (1996, J. Virol. 70, 4805-4810).
According to another embodiment, the expression vector is an Adeno-associated virus (AAV), i.e. a viral vector system based on a non pathogenic and replication-defective virus. The AAV genome is a linear, single-stranded DNA molecule containing about 4681 nucleotides. The AAV genome generally comprises an internal non repeating genome flanked on each end by inverted terminal repeats (ITRs) . The ITRs are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including as origins of DNA replication, and as packaging signals for the viral genome. The internal non repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package into a virion. In particular, one family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VPl, VP2 and VP3. AAV has been engineered to deliver genes of interest by deleting the internal non repeating portion of the AAV genome (i.e. the rep and cap genes) and inserting a heterologous gene between the ITRs. The heterologous nucleic acid sequence is typically functionally linked to a heterologous promoter (constitutive promoter, tissue- or cell-specific promoter or inducible promoter) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included. AAV is a helper-dependent virus; that is, it requires coinfection with a helper virus, or triple transfection with plasmids expressing viral proteins (e.g. adenovirus, herpesvirus or vaccinia), in order to form AAV virions. In the absence of coinfection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to replicate and package its genome into an infectious AAV virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells coinfected with a canine adenovirus. Interestingly, several publications (Reichel et al., 1996, Hum MoI Genet 5, 591-594; Reichel et al., 1998, Hum Gene Ther, 9, 81-86 ; Bennett and Maguire, 1999, Proc Natl Acad Sci, 96, 9920-9925) have shown that in the retina, following subretinal delivery, AAV-2 vectors transduce retinal pigmented epithelium and photoreceptor cells, and were successful in delivering ribozymes, photoreceptor genes, and neurotrophic factors in mice and rat models of retinal degeneration (AIi et al., 2000, Nat Genet 25, 306-310 ; Lau et al. 2000, Invest Ophthalmol Vis Sci 41, 3622-3633; LaVail et al. , 2000, Proc Natl Acad Sci USA, 97, 11488-93 ; Green et al., 2001, MoI Ther 3, 507-515 ; Liang et al, . 2001, MoI Ther 3, 241-248) . Visual function was restored in a canine model of childhood blindness using a rAAV-2 carrying a wtRPE65 gene (Acland et al. 2001, Nat Genet 28, 92-95; WO 02/082904). Furthermore, it was recently demonstrated that a subretinal injection in two retinal degenerative rat models, with AAV5 expressing the anti-apoptotic factor XIAP provides an efficient protection against photoreceptor loss and consecutive blindness (Leonard K. C. et al. 2007, PLOS One, 3,1-8). Taken together these publications provide critical preclinical data supporting these vectors for human applications. Recent data confirmed that sub-retinal injection of AAV2 and 5 target RPE and photoreceptor cells in mice (Pang et al. 2007 Oct 18 Vision Res. Comparative analysis of in vivo and in vitro AAV vector transduction in the neonatal mouse retina: Effects of serotype and site of administration, Epub ahead of print) as well as in non-human primates using opsin promoters (see below) (Mancuso K et al., 2007, J Opt Soc Am A Opt Image Sci Vis., 24, 1411-141 6). rAAV chimeric serotypes wherein the vector is flanked by AAV-2 ITRs but encapsidated in an AAV-I, 2, 3, 4 or -5 capsid have been studied (Auricchio et al., 2001, Hum MoI Genet 10, 3075-81,; Rabinowitz et al., 2002, J Virol 76, 791-801; Yang et al. , 2002, J Virol 76, 7651-60). It was shown that their subretinal delivery results in a quantitative transgene expression hierarchy with rAAV-4 and -5 capsids being the most efficient. More recently, it was shown that AAV2.8 or AAV2.9 (first number corresponds to genome serotype and second number to the capsid) are six to eightfold more efficient to transduce photoreceptors at least in rodent models, than AAV2.5 (Auricchio et al., 2007, J. virol. 81, 20, 11372-11380) . The inventors of US 20040208847 have further demonstrated that a recombinant AAV of serotype 4 delivered in the subretinal space of a non human primate leads to exclusive transduction of retinal pigment epithelial (RPE) cells. Since the primate eye is anatomically very similar to the human eye, rAAV-mediated nucleic acid transfer in the eye of non human primates is highly relevant with respect to future clinical development in humans. Recently, phase I human clinical trials have been successfully performed by three groups of laboratories, in Leber Congenital Amaurosis patients harbouring RPE65 mutation. Retina transduction with AAV2-4 expressing RPE65 normal gene gave significative functional vision rescue (Bainbridge JW et al., N Engl J Med. 2008 May 22;358 (21) :2231-9, Maguire AM et al. N Engl J Med. 2008 May 22;358 (21) :2240-8, Hauswirth W et al. Hum Gene Ther. 2008 Sep 7.) Therefore, according to the present invention, its is further possible to deliver the nucleic acid (i.e. DNA, RNA, RNAi, ribozyme, oligonucleotides, antisense, siRNA, miRNA, shRNA, etc..) specifically to retinal cells by using an AAV capsid protein (or an expression vector, including AAV recombinant vector, engineered to express such an AAV capsid on its surface) as targeting molecule. In special embodiment, said AAV capsid can have a structure similar to that of a natural AAV or with a few changes, such as, for example, VPl, VP2 and VP3 coming from more than one single AAV serotype, or such as a capsid made of an AAV capsid protein in which one or several amino acid have been deleted, added or modified. For example, it is possible to derive an AAV capside by replacing part of it with a sequence from the AAV-5, 8 or 9 capsid protein, in such a way that the expressed proteins are able to form a capsid which retains the natural tropism of AAV-5, 8 or 9 towards retinal cells, and thus allows to target the delivery of the nucleic acid to this special type of cells such as photoreceptors. Therefore, the present invention concerns nucleic acids (including both viral and non viral expression vectors) which are characterized by the fact that they exhibit an AAV-5, 8 or 9 capsid protein. This means that at least one of VPl, VP2 and VP3 of AAV-5, 8 or 9 are part of the vector, in such a way that they are exposed at its surface, thereby enabling the targeting of retinal cells, and more specifically of photoreceptor cells. The AAV-5, 8 or 9 capsid protein can be integrated into the viral particle (for example, in the case of a native or chimeric AAV capsid) , or simply bound to the particle, by any physical means (for example, in the case of a non-viral vector) .
In another aspect, the vector in use in the invention is a poxviral vector (see for example Cox et al. in "Viruses in Human Gene Therapy" Ed J. M. Hos, Carolina Academic Press) . According to special embodiment it is selected in the group consisting of vaccinia virus, suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), the Wyeth strain and the highly attenuated attenuated virus derived thereof including MVA (for review see Mayr, A., et al., 1975, Infection 3, 6-14) and derivates thereof (such as MVA vaccinia strain 575 (ECACC V00120707 - US 6,913,752), NYVAC (see WO 92/15672 - Tartaglia et al., 1992, Virology, 188, 217-232). Determination of the complete sequence of the MVA genome and comparison with the Copenhagen VV genome has allowed the precise identification of the seven deletions (I to VII) which occurred in the MVA genome (Antoine et al., 1998, Virology 244, 365-396), any of which can be used to insert the nucleic acid of interest. The vector may also be obtained from any other member of the poxviridae, in particular fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); canarypox (e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); pigeonpox; swinepox and the like. By way of example, persons skilled in the art may refer to WO 92 15672 which describes the production of expression vectors based on poxviruses capable of expressing such heterologous nucleotide sequence. The basic technique for inserting the nucleic acid in a poxviral genome is described in numerous documents accessible to the man skilled in the art (Paul et al., 2002, Cancer gene Ther. 9, 470-477; Piccini et al . , 1987, Methods of Enzymology 153, 545-563) . Usually, one proceed through homologous recombination between overlapping sequences (i.e. desired insertion site) present both in the viral genome and a plasmid carrying the nucleic acid to insert. When using the Copenhagen vaccinia virus, the nucleic acid is preferably inserted in the thymidine kinase gene (tk) . However, other insertion sites are also appropriate, e.g. in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189- 4198), in the KlL locus, in the u gene (Zhou et al., 1990, J. Gen. Virol. 71, 2185-2190) or at the left end of the vaccinia virus genome where a variety of spontaneous or engineered deletions have been reported in the literature. When using MVA, the heterologous nucleic acid can be inserted in anyone of the identified deletions I to VII as well as in the D4R locus, but insertion in deletion II or III is preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038 ; Sutter et al., 1994, Vaccine 12, 1032-1040) . When using fowlpox virus, although insertion within the thymidine kinase gene may be considered, the heterologous nucleic acid is preferably introduced in the intergenic region situated between ORFs 7 and 9 (see for example EP 314 569 and US 5,180,675).
According to another embodiment, the viral vector according to the invention derives from retroviruses which have the property of infecting, and in most cases integrating into, dividing cells and in this regard are particularly appropriate for use in relation to cancer. A recombinant retrovirus according to the invention generally contains the LTR sequences, an encapsidation region and the nucleotide sequence according to the invention, which is placed under the control of the retroviral LTR or of an internal promoter such as those described below. The recombinant retrovirus can be derived from a retrovirus of any origin (murine, primate, feline, human, etc.) and in particular from the MOMuLV (Moloney murine leukemia virus) , MVS (Murine sarcoma virus) or Friend murine retrovirus (Fb29) . It is propagated in an encapsidation cell line which is able to supply in trans the viral polypeptides gag, pol and/or env which are required for constituting a viral particle. Such cell lines are described in the literature (PA317, Psi CRIP GP + Am-12 etc.). The retroviral vector according to the invention can contain modifications, in particular in the LTRs (replacement of the promoter region with a eukaryotic promoter) or the encapsidation region (replacement with a heterologous encapsidation region, for example the VL30 type) (see FR9408300 and FR9705203).
Preferably, the nucleic acid in use in the invention is in a form suitable for its expression in a host cell or organism, which means that the nucleic acid (encoding more specifically RNA) are placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism. According to preferred embodiment, "expression in a host cell or organism" means at least RNA (including RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA) synthesis into the said host cell or organism. As used herein, the term "regulatory sequence" refers to any sequence that allows, contributes or modulates the expression of a nucleic acid in a given host cell, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. RNA, oligonucleotides, antisense, siRNA, miRNA, ribozyme, shRNA ...) into the host cell. It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on factors such as the host cell, the vector and the level of gene expression desired. The regulatory sequences may, for example, be a mammalian or viral promoter, such as a constitutive, regulable or cell/tissue specific promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT) , adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV) , simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include regulable promoters. Regulable promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art. According to preferred embodiment, the promoter will be selected in order to be cell or tissue specific (e.g. retinal cell-specific promoter, photoreceptor- specific promoter, retinal ganglion cell or retinal pigmented epithelium specific promoter etc... ) .
According to another embodiment, RNA polymerase III promoters will be used for expressing shRNA. RNA polymerase III is one of three types of nuclear RNA polymerases present in eucaryotic cells. It synthesizes RNA molecules, some of which are involved in protein synthesis, pre-mRNA splicing, tRNA processing, and the control of RNA polymerase II elongation, whereas some others have still unknown functions. Like other RNA polymerases, RNA polymerase III cannot recognize its target promoters directly. Instead it is recruited to specific promoter sequences through the help of transcription factors. There are three basic types of RNA polymerase III promoters, called types 1, 2, and 3. Although in vivo, RNA polymerase III may be recruited to these promoters as part of a large complex (holo RNA polymerase III) containing the polymerase and its initiation factors, in vitro the reaction can be divided into several steps. First, the promoter elements are- recognized by DNA binding factors, which then recruit a factor known as TFIIIB. TFIIIB itself then directly contacts RNA polymerase III. In human cells there are at least two versions of TFIIIB. One contains TBP, Bdpl, and Brfl (Brfl-TFIIIB) , and the other TBP, Bdpl, and Brf2 (Brf2- TFIIIB) . Polymerase III promoter used for shRNA expression are U6 promotor, H6 promotor, Hl promotor normally regulating Hl RNA, the RNA component of human RNase P. MRP regulating sequences: 5' flanking sequences of human MRP/7, sequences controlling the expression of B2 snRNA and 7SK snRNA, Yl and Y3 RoRNA promotors, telomerase TERl proximal regulating sequences, (reviewed in Geiduschek EP et al. J MoI Biol. 2001 Jun 29;310(l) :l-26.
Photoreceptor-specific promoters include, for example, beta-subunit of cGMP phosphodiesterase (beta-PDE) promoter (Lerner et al., 2002, J. Biol. Chem. , 277, 25877-25883), interphotoreceptor retinoid-binding protein (IRBP) promoter (Glushakova et al. MoI. Vis. 2006 :12, 298-309), rhodopsin Kinase (Young et al. 2005, MoI. Vis. 11:1041-1051, Young et al. 2007 Genomics 90, (2 ): 236-248, Sharhrockh C. et al., 2007, IOVS, 48,9,3954-3961), rhodopsin promoter (Lewin et al. 1998, Nat. Med., 4, 967-971, Gouras et al. 1994, Vis. Neurosci, 11, 1227-1231, Woodford et al. 1994, Exp Eye Res., 58, 631-635, Glushakova et al . MoI. Vis. 2006, 12, 298-309), peripherin/rds (Moritz et al., 2002, Gene, 298,173-182), guanylate cyclase-E (Dude et al., 1998, MoI Cell Biochem. , 189,63-70; Johnston et al., 1997, Gene 193, 219-227), alpha subunit of rod transducin (Ahmad et al., 1994, J Neurochem. , 62, 396-399), arrestin (Mani et al., 1999, J Biol Chem. 274, 15590-15597 ; Kikuchi et al. 1993, MoI Cell Biol. 13, 4400-4408, cone-specific promoters such as promoter sequences of red and green visual pigment (Wang et al. 1992, Neuron, 9; 429-440; Shaaban and Deeb,1998, Invest . Ophthalmol . Vis. Sci . 39, 885-896, Alexander et al. 2007, Nat. Med, 13, (6) 685-687, Mancuso et al. 2007 J Opt Soc Am A Opt Image Sci Vis., 24, 1411-6, Li et al. 2007, Cone-specific expression using a human red opsin promoter in recombinant AAV, Sep 28; Vision Res. [Epub ahead of print] ), blue opsin (Chen et al. 1994, PNAS, 91, 2611-2615, Glushakova et al., 2006, Invest Ophthalmol Vis Sci., 47, 3505-13), or cone I arrestin (Zhu et al., 2002, FEBS Lett., 524, 116-122).
In general, the gene expression sequence shall include, as necessary, 51 non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired. Those skilled in the art will appreciate that the regulatory elements controlling the expression of the nucleic acid molecule of the invention may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine-Dalgarno sequences, etc.) into the host cell or organism.
One special class of indirect inhibitor that may be used in conjunction with the present invention is antisense nucleic acid molecules that can hybridize to, or are complementary to, the nucleic acid molecule, nucleotide sequence, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence) . In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an Unc51-Like Kinase (or fragments or fragment combination thereof) . The term "coding region" refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to an "untranslated region" of the coding strand of a nucleotide sequence encoding a Unc51-Like Kinase. The term "untranslated region" refers to 51 and 3' sequences that flank the coding region and that are not translated into amino acids. Given the coding strand sequences encoding Unc51-Like Kinase disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of a Unc51-Like Kinase mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of Unc51-Like Kinase mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Unc51- Like Kinase mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g. an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used) . Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 carboxyτnethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5 methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' methoxycarboxymethyluracil, 5- methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil 5- oxyacetic acid (v) , wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5-methyl-2- thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2, 6-diaminopurine . Alternatively, the antisense nucleic acid can be produced biologically in situ using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e. RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following section) . The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ after administration of an expression vector as indicated above, such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding Unc51-Like Kinase to thereby inhibit expression of the protein (e.g. by inhibiting transcription and/or translation) . The hybridization can be by conventional nucleotide complementarily to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically . For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g. by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). In some embodiments, the antisense compound is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% complementary to a nucleic acid molecule encoding Unc51-Like Kinase.
According to another special class of indirect inhibitor, the invention relates to short interfering nucleic acid ( siNA) such as short interfering RNA (siRNA) , double- stranded RNA (dsRNA) , micro-RNA (miRNA) , and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Unc51-Like Kinase genes expression and/or activity. "RNA interference or RNAi" refers to sequence specific inhibition of gene expression mediated by an at least partly double-stranded RNA molecule that contains a portion that is substantially complementary to a target gene (e.g. to an mRNA transcribed from . the target gene) . RNAi can occur via selective intracellular degradation of RNA and/or by translational repression. "Molecules capable of mediating RNA interference" refers to an at least partly double-stranded RNA molecule, optionally including one or more nucleotide analogs or modifications, having a structure characteristic of molecules that can mediate inhibition of gene expression through RNAi. These molecules include a portion that is substantially complementary to a target gene. In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the said siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence (s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence (s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non- limiting examples of such expression vectors have been provided above.
According to one embodiment of the present invention, the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase siRNA. The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 31 end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. siRNA may be divided into five groups (non-functional, semifunctional, functional, highly functional, and hyper- functional) based on the level or degree of silencing that they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions where the siRNA is transfected into said cell line at a concentration from 1OnM to 100 nM and the level of silencing is tested at a time of roughly 24 hours after transfection, and not exceeding 72 hours after transfection. In this context, "non-functional siRNA" are defined as those siRNA that induce less than 50% (<50%) target silencing. "Semi-functional siRNA" induce 50-79% target silencing. "Functional siRNA" are molecules that induce 80-95% gene silencing. "Highly-functional siRNA" are molecules that induce greater than 95% gene silencing. "Hyperfunctional siRNA" are a special class of molecules which are defined as those molecules that (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics. The methods of the invention comprise administering the Unc51-Like Kinase- specific siRNA to a mammal in an amount and for a period of time sufficient to indirectly inhibit or reduce Unc51-Like Kinase expression, and preferably inhibit or reduce Unc51-Like Kinase mRNA level. siRNAs lead to post-transcriptional silencing of the mRNA to which they are homologous. RNA interference or siRNA is a method based on small-interfering RNAs that can lead to the silencing of specific genes. RNA interference technology is well known in the art and is described for example in US20020162126, or Hannon et al., 2002, Nature, 11, 418, 244-251. It has been shown that siRNA is mediated by RNA-induced silencing complex or RISC, which is a sequence specific, multi-component nuclease that degrades mRNAs and contains short RNAs. Complementary portions of siRNA, that hybridize to form the double-stranded structure, typically have substantial or complete identity. The double- stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands. siRNAs of the invention may comprise one or more strands of polymerized ribonucleotide and may include modifications to either the phosphate-sugar backbone or the nucleoside. Likewise, bases may be modified to block the activity of enzyme adenosine deaminase, an enzyme that plays a role in RNA-editing. RNA duplex formation can be initiated either before or after administration into a host organism or cell for effective inhibition of or reduction in the expression of the target gene. In the methods of the invention, siRNAs are complementary to certain portions of a particular mRNA, e.g., a target gene. The sequence of a siRNA of the invention can correspond to the entire length of or only a portion of a target gene. In one embodiment the length of the siRNA, i.e. the length of each individual strand of the double-stranded structure, as well as the length of the duplex, comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In another embodiment, the length of the siRNA is about 25-50 nucleotides. In yet another embodiment, the length of the siRNA is greater than 50 nucleotides. Based on the mRNA sequence encoding Unc51-Like Kinase of the present invention (see below) , it is possible to use specific algorithm (siRNA Finder computer program, "BLOCK-iT™ RNAi Designer" Invitrogen, "HP Flexible siRNA Design" Qiagen) to design siRNA sequences. The mRNA sequence encoding Unc51-Like Kinase of the invention can be determined for example according to the rules of degenerative code or based on the nucleic acid sequences provided herein. As described for antisense nucleic acids, the siRNA can be administered to a subject or generated in situ using an expression vector into which a nucleic acid has been subcloned which encodes the said siRNA.
According to special embodiment, siRNA of the present invention is selected in the group consisting in : -for ULKl :
CTGAAGGAACTGAAACATGAA CAGGCTGAATGAGCTGTACAA CAGAGGCAGTTCTTTGTTCAA
CCCAAGCACTTTATGCATATA CCCGTCCAGCTTTGACAGTCA TTTGCGTTATATTGTATAATA
CTGAATCAGTAGATACTTGAA ATGCGGCTTCTGCACAGCAAA CTGGATACGTCTTGTAATCTT
CACGGAGAGCCTGCAGGAGAA CGCGCGGTACCTCCAGAGCAA CCCACCCAGCTTTGTCAATCA
CAAGTGCATTAACAAGAAGAA TGCCCTTTGCGTTATATTGTA CAAGCACTTTATGCATATAGA
CACACGCCACATAACΔGACAA AAGGCGGCGTTTGGGACACAA AGCCCTGGATACGTCTTGTAA
CGCCTTCGCGGTGGTCTTCAA CTGGGCCAGGTTAGAGGCAGA CGCGGAACAGCTGGTGCTGTA
CTGGTTGAATGTTCTCTTGAT ACAGCTGGTGCTGTACCTGAA CCTTTGCGTTATATTGTATAA
CTGGTGTTTGTACATACACAT CAAGTTCGAGTTCTCCCGCAA TAGGTGTTTAAGAATTGGTTT
CACTGCCGACTCAAAGCCAAA CAGCCCTGGATACGTCTTGTA CTGCCTGTCGTCCACTGTGAA
AAACTTGTAGGTGTTTAAGAA CCCAGCTTTGTCAATCACCCA
ATGTTTGAATTTATTGCTTCA CTTGTAATCTTTCACACTTTA
GGACUCUUCCUGUGACACA GAAGAACAAGACGUUGGUC GUGCAAGCUGUGCAUUGAG
GUGCAUUAACAAGAAGAAC GCAAGAGCACACGGAGAUC GAUCGCUGACUUCGGCUUC
GGACACCAUCAGGCUCUUC GCUGUGCAUUGAGCGGAGA
GAACAUCCUGCUGUCCAAC GAACCUCGCCAAGUCUCAG
and complementary strands. - for ULK2 :
AAGATTCTGCCAGTACTAGTA GGAAUUAUCAGCGCAUAGA
CTGTGTCACCTCACAGCGAAA GGCUGACUUGUGGAGCAUA
TTGGCCTAAAGTCAGGAACTA GAUUCUGCCAGUACUAGUA
TTGCCTTGAGTATAGCAATAA GACCACUCAUGUGAUAUGC
ATCAACAAGGAAAGACTCCTA GUUAUCAGAUCAACAAGGA
TACGTTGTGTATTATTTGGTT GUAUUGCAAUGGUGGAGAC
GGCCAAUAGUCCUCAAGAC GGAAUCAUCCACAGAGAUC
GCUUAAUGCCUAGUAUUCC GUAACAUGAUGGCUGCAAC
and complementary strands. - for ULK3 :
GGACACUCGUGAAGUGGUA CAAAGTGAATTTAACCTGGAA ATGGGCACTTATGAGGACAAA
GGAGGCAAUUAAGGCAAAG GCCCGCCATCTTGGTACTTTA CATCTTGGTACTTTAGGTTAA
GAAGGACACUCGUGAAGUG CACGTACGCCACGGTGTACAA AAGGCTCTGGACTTCTTTGTA
GACUUUGGUUUCGCACAAC CAGCTCCTCTTTGCACTGGAA CTGGGTGGACCTGGAGCACAT
GGCUCUGGACUUCUUUGUA CCGCAGGATTCTGCCTGAGAA AGCAGCTGCCTTATCACTCTA
GCCACAGAACAUUCUACUG CCAGCGGAAGGAGGCAATTAA GAGGCTGAGTGTAAAGAGTAT
GAACAUUCUACUGAGCUCC AGCCATAAAGTGTGTAGCCAA CTCGTGAAGTGGTAGCCATAA
GGAGCAGGUCAAGAUGAGG TCGTGAAGTGGTAGCCATAAA CCCAGCGGAAGGAGGCAATTA
GUGGUAGCCAUAAAGUGUG ATGAGGACAAAGTGAATTTAA AAAGTGAATTTAACCTGGAAA
GAGAAGAUCCGUAGCAACC CTGAGTGTAAAGAGTATCATT CACTTATGAGGACAAAGTGAA
CACGGAGATTGAGATCCTCAA CAGGTCGTTCTCGGAGCTGGA
CAGGCTGACGCCAAGAATGAA AAGGACACTCGTGAAGTGGTA and complementary strands.
According to preferred embodiment, siRNA of the present invention is selected in the group consisting in :
-for ULKl :
TTCTTTGCAATACTTGAAATA AACCACAAGGACCGCATGGAC GGCAGUUCUUUGUUCAAGC CGGCGCCTTCGCGGTGGTCTT AATACTTGAAATATTGCCACT GUUCUUUGUUCAAGCGUUC GAGGAGACCCTCATGGAGCAA TTGAAATATTGCCACTGTGCT GAGACAGAACCUGGACCUC CTTTGCAATACTTGAAATATT CATGGAGCAAGAGCACACGGA GACAGAACCUGGACCUCAC TACTTGAAATATTGCCACTGT ACCGTGGGCAAGTTCGAGTTC GAGUGCCGUGGCACAGGCG GACCCTCATGGAGCAAGAGCA CCGTGGGCAAGTTCGAGTTCT CTGGTCTTTGGAGGAAAGCAA CCACAAGGACCGCATGGACTT GAGCAACATGATGGCGGCCAC GGAAUUAUCAGCGCAUAGA GACCGTGGGCAAGTTCGAGTT TTGCAATACTTGAAATATTGC GGCUGACUUGUGGAGCAUA TGAAATATTGCCACTGTGCTT ACCCTCATGGAGCAAGAGCAC GAUUCUGCCAGUACUAGUA CCCTCATGGAGCAAGAGCACA CGTGGGCAAGTTCGAGTTCTC GACCACUCAUGUGAUAUGC TACCTCCAGAGCAACATGATG CGGTACCTCCAGAGCAACATG GUUAUCAGAUCAACAAGGA TTTCTTTGCAATACTTGAAAT GCCUCUCCCUCCGAGAUAC GUAUUGCAAUGGUGGAGAC TTTGCAATACTTGAAATATTG GCACUUUAUGCAUAUAGAG GGAAUCAUCCACAGAGAUC GGAGACCCTCATGGAGCAAGA GGCCUGCCUUCCGGAUUCC GUAACAUGAUGGCUGCAAC TCTTTGCAATACTTGAAATAT GUAGAUACUUGAACGAGUC GGCCAAUAGUCCUCAAGAC GCAATACTTGAAATATTGCCA GUGCUGGACCCUUUGUCUA GCUUAAUGCCUAGUAUUCC CAGACTAAATGAGCTGTACAA and complementary strands, -for ULK2
ATGCGAATCCTGCACAGCAAA TTCATATTGCACTAATTTATT AAATCTTTCATATTGCACTAA CATGCGAATCCTGCACAGCAA ACAGGAGCTTAATGCCTAGTA AACCCTGAGCTGTGCACATCT ATCTTTCATATTGCACTAATT AATAAATGTACCTTAGAACTA GATATGCAGCATATTCAGGAA CATATTGCACTAATTTATTAA CTGAGTGTGTGCTGGACCTGA AAACAGGAGCTTAATGCCTAG TCATATTGCACTAATTTATTA ATTCTGCATCACCATGTGCAA AACTCTTCTTGTGACACGGAT CAGAGAAACTCATCTATAATT CCTAGTATTCCCAGAGAAACA TGTCTCAACATTATGATGCTA ACCATGTGCAAGAAACTTACA ACACAACATCTCGTCAGACCA ATCACCATGTGCAAGAAACTT CCCTTCCAGATATGCAGCATA GCAGAGAAACTCATCTATAAT GAACAAGCAGAAGCTCAGAAA ATGTGCAAGAAACTTACAGAA CAGTGTGACTGCAGAGAAACT ACTGTGTCACCTCACAGCGAA TCACCATGTGCAAGAAACTTA CATGTGCAAGAAACTTACAGA TACTGTGTCACCTCACAGCGA TAGCAAGAACTCTTCTTGTGA AATCATTGGCTCTCCTACTAA TAAGGAATTATCAGCGCATAG ATATTGCACTAATTTATTAAA GAGAAACTCATCTATAATTGT CTAGTATTCCCAGAGAAACAT AAGGAATTATCAGCGCATAGA TGTGCAAGAAACTTACAGAAA ATGTCTCAACATTATGATGCT TCCAGATATGCAGCATATTCA ATCAGAACAAGCAGAAGCTCA GGCACAATGGACTTGTTTAAA AGCAGCTGGTGTTGTACATGA CAATCATTGGCTCTCCTACTA AACATCTCGTCAGACCACTCA CAGAACAAGCAGAAGCTCAGA TCCACCATCCCTTCCAGATAT ACTGCAGAGAAACTCATCTAT CTTTCATATTGCACTAATTTA TTAGCCTTGGTTACTCGTCAT ATCATTGGCTCTCCTACTAAG CACCATGTGCAAGAAACTTAC AATACTTGAATAAACCATTCA TCCTCAGGTTCTCCAGTGCCA CAACAATCATTGGCTCTCCTA TTTCATATTGCACTAATTTAT GTGTGACTGCAGAGAAACTCA TTGATGAAATCAACAGTGTGA ACCATCCCTTCCAGATATGCA AATGCCTAGTATTCCCAGAGA AGGAGCTTAATGCCTAGTATT CCAGATATGCAGCATATTCAG CTCTTCTTGTGACACGGATGA GACTGCAGAGAAACTCATCTA TTCCCAGAGAAACATCACCTT TTCCAGATATGCAGCATATTC CTCAGGTTCTCCAGTGCCACA TCCCTTCCAGATATGCAGCAT CCATGCGAATCCTGCACAGCA TGCATCACCATGTGCAAGAAA AATCTTTCATATTGCACTAAT CCTTCCAGATATGCAGCATAT TTAATGCCTAGTATTCCCAGA ATAAATGTACCTTAGAACTAG CTGCAGCCCTGGATGAGATGT GTGATGGAGTATTGCAATGGT CAACATCTCGTCAGACCACTC AGTATTCCCAGAGAAACATCA TTCTTGTGACACGGATGACTT TGCAGCCCTGGATGAGATGTT GAACAAGCAGUGAUUUGAA TCTCAACATTATGATGCTAAG TGGGTCCTCCCAACTATCTAC GAAAUAGCUUAAGAAGAAG AAACTCATCTATAATTGTGCT AACAATCATTGGCTCTCCTAC GCAGGAGCAAGAGUGUUUG TATGCAGCATATTCAGGAAGA CCAACAATCATTGGCTCTCCT GuuuGAUAuuuGGAGUAAG ■ AATCAACAGTGTGACTGCAGA TCTTGTGACACGGATGACTTT GGCACAAUGGACUUGUUUA CATCACCATGTGCAAGAAACT CTGTTAGCCTTGGTTACTCGT GUAUUGAGAGAAGACUGUC ACATCTCGTCAGACCACTCAT ATCAACAGTGTGACTGCAGAG GAACUAGGAUAAUCGGUAC GTCTCAACATTATGATGCTAA TTTATTGATGAAATCAACAGT GCAUGCAUCUUUAAACAGC ACCCTGAGCTGTGCACATCTG TAGCCTTGGTTACTCGTCATG GCUUAAGAAGAAGUGAAAC AACCTGTTAGCCTTGGTTACT TTGTGACACGGATGACTTTGT GGCCUAAGUAGGAUUCUAC CTTCCAGATATGCAGCATATT CTGAGCTGTGCACATCTGCTG GGAAUUAUCAGCGCAUAGA AACAGGAGCTTAATGCCTAGT GAACTCTTCTTGTGACACGGA GGCUGACUUGUGGAGCAUA TCATTGGCTCTCCTACTAAGA ATCTATCAGAACAAGCAGAAG GAUUCUGCCAGUACUAGUA AGAACAAGCAGAAGCTCAGAA TTGCTTCTCCACCATCCCTTC GACCACUCAUGUGAUAUGC TGTGACTGCAGAGAAACTCAT GAAATCAACAGTGTGACTGCA GUUAUCAGAUCAACAAGGA ATTGATGAAATCAACAGTGTG AGAGAAACTCATCTATAATTG GUAUUGCAAUGGUGGAGAC ATCCCTTCCAGATATGCAGCA CACAACATCTCGTCAGACCAC GGAAUCAUCCACAGAGAUC TCAGAACAAGCAGAAGCTCAG TCTTTCATATTGCACTAATTT GUAACAUGAUGGCUGCAAC ATACTTGAATAAACCATTCAC ACAATCATTGGCTCTCCTACT GGCCAAUAGUCCUCAAGAC GCAAGAACTCTTCTTGTGACA GTGTCACCTCACAGCGAAACA GCUUAAUGCCUAGUAUUCC GTGACACGGATGACTTTGTTT TAGTAGCAAGAACTCTTCTTG
and complementary strands -for ULK3
CACGGTGTACAAGGCCTACGC CCTCACGGAGATTGAGATCCT CAGAACCTCATGGCCCGAGCT TAGCAACCGGGTCATCGAGCT CCCGCGCCTGGACGGCTTCAT TTCGTAGCTCTTGCACCCTTC CGGCACGTACGCCACGGTGTA TCCCGAGACTGCCGGGACCTA CCGCGCCTGGACGGCTTCATC GACTGTCGGAATCTGTTCGTA CAGCGGAAGGAGGCAATTAAG TTAGGTTAATCCCTCAAGCAT CGGCAGTATGACGCCCGCGTG GGCCCGAGCTGAATACTTGAA AGGCGGGAGCTGCTTCACACT CAGCGGCAGTATGACGCCCGC AGCAACCGCCCTGGTGGTGCA CCCGTGGGATGAGAAGCACGT CAGCGGCACGTACGCCACGGT CCTGCCCGCCATCTTGGTACT CATCGTCTCCTCTTCCAATCA CAGCAATTAGCTAGCGCCCTG ATCCTCAAGGGCATTCGACAT TTCCTGCATGAACGGAATATC TGGGCAGCGGCACGTACGCCA AGGCCTACGCCAAGAAGGACA AAGTCTGAACAAGGCATCGGT ATGGCCCGAGCTGAATACTTG CTCACGGAGATTGAGATCCTC TGCAATTCCTGCATGAACGGA CGGAACCGCCGCCCGCAGCGA CATCGGTGGAGAACCTCCTCA CCCGAGACTGCCGGGACCTAC CGGTGTACAAGGCCTACGCCA CTGGCAGACTTTGGTTTCGCA CAAGGCATCGGTGGAGAACCT TACGCCAAGAAGGACACTCGT CGCCACGGTGTACAAGGCCTA GAGAACCTCCTCACGGAGATT CATGGCCCGAGCTGAATACTT ATGAGACCTTTGTTACTCGGT CTCAAGGGCATTCGACATCCC ACTCGTGAAGTGGTAGCCATA CCGCGTGGACCTCTGGTCCAT CCCGCCATCTTGGTACTTTAG GAGATCCTCAAGGGCATTCGA ACGCCACGGTGTACAAGGCCT TGAAGTGGATGCCCAGCGGAA ACACTCGTGAAGTGGTAGCCA CGAGATGGTGTGCCAGCGGCA TCCTGCATGAACGGAATATCT :ACTCGTGAAGTGGTAGCCAT GTGGCGCGTGTCTTCATGCAG ATGGTGTGCCAGCGGCAGTAT :TCGCTTCATCCATACCCGCA TAGGTTAATCCCTCAAGCATG CAATTAGCTAGCGCCCTGCAA ;CGGGCGCAGGCGGCCGGAAT CCGAGACTGCCGGGACCTACT CCGGGACCTACTGCAGCGGCT 'GGCCCGAGCTGAATACTTGA CTCCAGGTCGTTCTCGGAGCT GCCATCGTCTCCTCTTCCAAT :CGTAGCAACCGGGTCATCGA CTGCCCGCCATCTTGGTACTT GTGTTGGTGATTTGTAGCCCT
LGCTAGCGCCCTGCAATTCCT TTGGTACTTTAGGTTAATCCC AGGTTAATCCCTCAAGCATGA 'CACGGAGATTGAGATCCTCA TGCCCGCCATCTTGGTACTTT GGCGCGTGTCTTCATGCAGCA ACCGCCGCCCGCAGCGAGGAA CCCGCGTGGACCTCTGGTCCA CTGATACCTTCACAGCCCACA TGGGATGAGAAGCACGTGCTC CCTCTAGGCCACCCACTTCTA TTGGCTCCGAGAAGAGGGCAA TGGCGAGCAGGCTTCTTGGAT AGCTGGAAGAGAAGATCCGTA GCCCTGCACTATGAAGTGGAT ATCAGCGGGTGTTGGTGATTT ACCTCATGGCCCGAGCTGAAT CGCGGGCCTTGTGCTGGAGTT ACTAACCCAGGCTGACGCCAA TGGGTGGACCTGGAGCACATG GTTCTCGGAGCTGGAAGAGAA TTTAGGTTAATCCCTCAAGCA GAAGAGAAGATCCGTAGCAAC ATACTTGAAGGAGCAGGTCAA TCGGAATCTGTTCGTAGCTCT CAATATCTACCTCATCATGGA CACCTGCCCGCCATCTTGGTA CTGGCGAGCAGGCTTCTTGGA ACTGCTGTTGCTGGCAGCGGA AGACTTTGGTTTCGCACAACA CCTCCTCACGGAGATTGAGAT GGGCATGCAGCCCTCTATCAT CGCCATCTTGGTACTTTAGGT TTGTAGCCCTTCTGCCCTTAA TATCTACCTCATCATGGAGTT GACCCTAGAAGAATGATTGGA TTCATCCATACCCGCAGGATT AGCGGGTGTTGGTGATTTGTA ACAAAGAGGGACTGTCGGAAT GAACCTCCTCACGGAGATTGA GTGGGACAGTGACAATATCTA CAGCACTTCTTTCATGAGACC AACGGAATATCTCTCACCTGG GAGGGACTGTCGGAATCTGTT CTCATCTCTCCCACCTCCGTT AAGGCCTACGCCAAGAAGGAC CTCGGTGGTTACTGGGTCCTG ACCTTTGTTACTCGGTGGTTA CTGCCTTATCACTCTACTGCA GGCATGCAGCCCTCTATCATT TAGCGCCCTGCAATTCCTGCA ATCGGTGGAGAACCTCCTCAC CTGCCTGAGAAGGTGGCGCGT CACTATGAAGTGGATGCCCAG GCAGACTTTGGTTTCGCACAA ATCGTCTCCTCTTCCAATCAG GACAATATCTACCTCATCATG GGCAGACTTTGGTTTCGCACA AACCTCCTCACGGAGATTGAG ATTGAGATCCTCAAGGGCATT CAAGATGAGGGAATCTCGCTG GCTGAGTGTAAAGAGTATCAT AAGAATGAAGTGGCCCACTGC TACTTTAGGTTAATCCCTCAA TCGTAGCTCTTGCACCCTTCA CAGCGCGGGCCTTGTGCTGGA ATGAAGTGGATGCCCAGCGGA CAGTGACCCTAGAAGAATGAT CCATCTTGGTACTTTAGGTTA TCGCTTCATCCATACCCGCAG ACCTCCTCACGGAGATTGAGA TTGGTGATTTGTAGCCCTTCT AAGGTGGCGCGTGTCTTCATG AAGTGGTAGCCATAAAGTGTG CCCTCAAGCATGAAAGCTGGA GCCAAGAAGGACACTCGTGAA CAGGTCAAGATGAGGGAATCT TCGGTGGTTACTGGGTCCTGT CTGCATGAACGGAATATCTCT GCACCCTTCAGTGACCCTAGA CGACCTGTCTCGCTTCATCCA CAGCATGGGCACTTATGAGGA ACGGAATATCTCTCACCTGGA TGGCCTAGGGAGCACTCAGGA AGGTCGTTCTCGGAGCTGGAA CCACAGCATGGGCACTTATGA GACAAAGAGGGACTGTCGGAA CACAGCATGGGCACTTATGAG GGCTGAGTGTAAAGAGTATCA GAGATGGCCCGGGACAAGCCA CTGAGAAGGTGGCGCGTGTCT CCCTGGCCTAGGGAGCACTCA AAAGACTTTCAGTGGGACAGT GCGCGTGTCTTCATGCAGCAA CCCTAGAAGAATGATTGGACA ACCCGCAGGATTCTGCCTGAG CAGACTTTGGTTTCGCACAAC CAAGGCCTACGCCAAGAAGGA AGGCTGACGCCAAGAATGAAG TGGGACAGTGACAATATCTAC TTGCACCCTTCAGTGACCCTA CGCGTGTCTTCATGCAGCAAT CCTCATGGCCCGAGCTGAATA GTCGTTCTCGGAGCTGGAAGA GGGATTCAGCAGCTGCCTTAT CAGCGGGTGTTGGTGATTTGT CTGGGTCCTGTGCCTGTCCGT ATCCTGTATGAAGCCCTCTTC TGGAAGAGAAGATCCGTAGCA CCCAGCCTCCCTCAGGTTACT CCCACAGCATGGGCACTTATG AGGGACTGTCGGAATCTGTTC CACCCTTCAGTGACCCTAGAA AGAATGAAGTGGCCCACTGCA GACCTTTGTTACTCGGTGGTT ACCTGTCTCGCTTCATCCATA GGAGGGTTCCTTGGTCCTCAT AAAGAGGGACTGTCGGAATCT CGGAATATCTCTCACCTGGAT GCGGGCCTTGTGCTGGAGTTT GAGGCGGGAGCTGCTTCACAC TCAGTGGGACAGTGACAATAT CTGGACAAAGAGGGACTGTCG CAGTGGGACAGTGACAATATC GAAGTGGTAGCCATAAAGTGT CGGGCCTTGTGCTGGAGTTTG AATACTTGAAGGAGCAGGTCA GCGGGTGTTGGTGATTTGTAG CACTGAGGTTCAGAACCTCAT AATCTGTTCGTAGCTCTTGCA CCCTGCAATTCCTGCATGAAC CCGAGACCTGCTCAGAGAGAT CGGAATCTGTTCGTAGCTCTT AGTGACCCTAGAAGAATGATT GTACAAGGCCTACGCCAAGAA TCAGCGGGTGTTGGTGATTTG ACTGAGGTTCAGAACCTCATG CAAGAATGAAGTGGCCCACTG AAGAAGGACACTCGTGAAGTG CAGAGAGATGGCCCGGGACAA TTCAGTGGGACAGTGACAATA CCCGAGCTGAATACTTGAAGG CGGGACAAGCCACGCCTCCTA GGCCATCGTCTCCTCTTCCAA AGGCTGAGTGTAAAGAGTATC CTGCTTCACACTGAGGTTCAG GAAGCTGACACCCTGGACAAA AAGACTTTCAGTGGGACAGTG CTCAAGCATGAAAGCTGGATC CAAGCATGAAAGCTGGATCTT GCACTATGAAGTGGATGCCCA ACCCTAGAAGAATGATTGGAC CCAGTCTTTCTGGGAACTCAC TGGTACTTTAGGTTAATCCCT AGCTGCCTTATCACTCTACTG TCCCTCAAGCATGAAAGCTGG CGCCAAGAAGGACACTCGTGA CAAGAAGGACACTCGTGAAGT TTCTTGGATGGACAGTGCTGA GTACTTTAGGTTAATCCCTCA TGCCTCCAGGTCGTTCTCGGA TCTTCATGCAGCAATTAGCTA ATCACTCTACTGCAAGGCTCT GAGGCAATTAAGGCAAAGGTG CTGGAAGAGAAGATCCGTAGC GGGCACTTATGAGGACAAAGT CTGGACTTCTTTGTACCTGCC CATGGGCACTTATGAGGACAA AACCTCATGGCCCGAGCTGAA AGGGAGCACTCAGGAGGGTTC CACACTGAGGTTCAGAACCTC AAGAGGGACTGTCGGAATCTG ATTGTGCAGCTGAAAGACTTT TCAGCTCCTCTTTGCACTGGA GAAGGTGGCGCGTGTCTTCAT TATCTCTCACCTGGATCTGAA AAGAATGATTGGACAGATGTG ACAGATGTGAGCCATCTGGAG GGCTGATACCTTCACAGCCCA CACCCTGGACAAAGAGGGACT TGCCCTGCACTATGAAGTGGA GCGCCTGGACGGCTTCATCCT CCACAGAACCCACCAGTCTTT TGGGCACTTATGAGGACAAAG AACAGCACTTCTTTCATGAGA TGTGCAGCTGAAAGACTTTCA TTCTACTGAGCTCCTTGGAGA AGCTGAATACTTGAAGGAGCA TAGAAGAATGATTGGACAGAT CAGGAGGGTTCCTTGGTCCTC TAGCCACAGCGCGGGCCTTGT CGCCCTGCAATTCCTGCATGA CGTGAAGTGGTAGCCATAAAG CGCCAAGAATGAAGTGGCCCA CCCACATTGTGCAGCTGAAAG ATCTTGGTACTTTAGGTTAAT ATGTGAGCCATCTGGAGCAGA TACAAGGCCTACGCCAAGAAG TGCCCTCTAGGCCACCCACTT CCGGAGGCGGGAGCTGCTTCA CCGAGCTGAATACTTGAAGGA GCCATCTTGGTACTTTAGGTT GTCCCGGGCTGAGGAGCTCAA CAGCAGCTGCCTTATCACTCT TACCCGCAGGATTCTGCCTGA CTGCCCTTAAATGCTTCCTTG GAGCAGGTCAAGATGAGGGAA ACCCTTCAGTGACCCTAGAAG AGGGAGGCTTGGAGACAGTCT TAAAGAGTATCATTCATTGTT AGGAGGGTTCCTTGGTCCTCA AGCGCCCTGCAATTCCTGCAT TCTACTGAGCTCCTTGGAGAA AAGCATGAAAGCTGGATCTTT TAAATGCTTCCTTGGGCAAGA TGCAAGGCTCTGGACTTCTTT GTGACAATATCTACCTCATCA GGAAGCTGACACCCTGGACAA GGCTTCAGCTGCCATGGCCAA GACAGTGACAATATCTACCTC GACCCAGTGCCCTCTAGGCCA GAAGGAGGCAATTAAGGCAAA CCCTCTAGGCCACCCACTTCT CCTGCAGGAGGCTGAGTGTAA AGAATGATTGGACAGATGTGA CGGTGGTTACTGGGTCCTGTG GCCCACAGCATGGGCACTTAT GTAGCCCTTCTGCCCTTAAAT CCCGCAGGGTACTTCTGTGCT GCGTGTCTTCATGCAGCAATT GAAGAATGATTGGACAGATGT ACAGAACATTCTACTGAGCTC TTCAGTGACCCTAGAAGAATG CCCAGGCTGACGCCAAGAATG CGAGACCTGCTCAGAGAGATG TTCCTTGGGCAAGAGCTGTCT CTCACCTGGATCTGAAGCCAC CCACCAGTCTTTCTGGGAACT TAGGGAGCACTCAGGAGGGTT TTAAGAAGCCCAAGCCTTGTT TCAGTGACCCTAGAAGAATGA ATCCCTCAAGCATGAAAGCTG ATGAGGGAATCTCGCTGGGAA GGAAGAGAAGATCCGTAGCAA TGTAGCCCTTCTGCCCTTAAA GAGTGTAAAGAGTATCATTCA ACAGAACCCACCAGTCTTTCT CCCGCAGGATTCTGCCTGAGA TACCTTCACAGCCCACAGCAT CACATTGTGCAGCTGAAAGAC CCGCAGGGTACTTCTGTGCTT ACCTGGATCTGAAGCCACAGA CAGTCTTTCTGGGAACTCACA AAGGAGGCAATTAAGGCAAAG CGAGCTGAATACTTGAAGGAG GCGGGAGCTGCTTCACACTGA CTTCAGTGACCCTAGAAGAAT CGCCTCCTAGCTGCCCTGGAA CCGGGACAAGCCACGCCTCCT GTTAATCCCTCAAGCATGAAA CACAGATGGTTTGATGGCTGT CTACTGCAAGGCTCTGGACTT ACGGAGATTGAGATCCTCAAG GCGCCTGGGCAGCGGCACGTA CCGTCGCATCTCCTTCCAGGA TGCCCTGGACCTGTACCAGCA TCGCTGGGAAGCTGACACCCT CATCCTGTATGAAGCCCTCTT CGAGCAGGCTTCTTGGATGGA GCACTTCTTTCATGAGACCTT GACAGATGTGAGCCATCTGGA CACAGAACATTCTACTGAGCT AGCCCACAGCATGGGCACTTA GGAGGCTTGGAGACAGTCTGA ATCTCTCACCTGGATCTGAAG CGGAAGGAGGCAATTAAGGCA GTACCTGCCCTGCACTATGAA ATGCTTCCTTGGGCAAGAGCT CTCTGGGAGAACAGCACTTCT ^y GAACATTCTACTGAGCTCCTT TGAGGGAATCTCGCTGGGAAG CCCTGCAGGAGGCTGAGTGTA CAGGATTCTGCCTGAGAAGGT GAGCTGCTTCACACTGAGGTT TCTGGGAGAACAGCACTTCTT AGCTCTTGCACCCTTCAGTGA CTCCCTCAGGTTACTCTGCAC TCTGCCCTTAAATGCTTCCTT TTTCAGTGGGACAGTGACAAT CAATCAGGCCCTGCTGAGGCA CCTGGATCTGAAGCCACAGAA CAGCCTCCCTCAGGTTACTCT GCACTTATGAGGACAAAGTGA CACCTGGATCTGAAGCCACAG TCACACTGAGGTTCAGAACCT CATTGTGCAGCTGAAAGACTT CCAACTCAGCTCCTCTTTGCA ACAGTGACAATATCTACCTCA TGGGCAAGAGCTGTCTGTCCT AGGCCATCGTCTCCTCTTCCA CCACAGATGGTTTGATGGCTG CACCAGTCTTTCTGGGAACTC GACACCCTGGACAAAGAGGGA CCCACAGATGGTTTGATGGCT CCAGGCTGACGCCAAGAATGA ACCCAGGCTGACGCCAAGAAT GACCTGTCTCGCTTCATCCAT CGTGTCTTCATGCAGCAATTA TCAGGAGGGTTCCTTGGTCCT CGCAGGGTACTTCTGTGCTTG TGGCTTCAGCTGCCATGGCCA GTAAAGAGTATCATTCATTGT CCCTTCAGTGACCCTAGAAGA TTGGACAGATGTGAGCCATCT AGGACAAAGTGAATTTAACCT CGGGAGCTGCTTCACACTGAG CCTTCAGTGACCCTAGAAGAA TGACAATATCTACCTCATCAT CGCAGGATTCTGCCTGAGAAG ACCAGTCTTTCTGGGAACTCA AGCCACAGAACATTCTACTGA TTGGATGGACAGTGCTGAGAC GGAGGCTGAGTGTAAAGAGTA TCACCTGGATCTGAAGCCACA AGCAGGCTTCTTGGATGGACA CCCACCAGTCTTTCTGGGAAC TGGACAGATGTGAGCCATCTG GGGCCTTGTGCTGGAGTTTGA CAGTGACAATATCTACCTCAT AAGAGTATCATTCATTGTTTC CAGCCGTCGCATCTCCTTCCA CCACATTGTGCAGCTGAAAGA TACCTGCCCTGCACTATGAAG GGCTGAGGAGCTCAAGGCCAT ATCATTCATTGTTTCTCTATT TCTCGGAGCTGGAAGAGAAGA TAGCCCTTCTGCCCTTAAATG AGGAGGCTGAGTGTAAAGAGT TCGCATCTCCTTCCAGGACTT CCGCCGTGGAGCCCTGGGCAA CGCCCTGGTGGTGCAGGCTGT GGAGCAGGTCAAGATGAGGGA AGAACAGCACTTCTTTCATGA GTATCATTCATTGTTTCTCTA ATGCCCAGCGGAAGGAGGCAA CTAGAAGAATGATTGGACAGA AGGGAGAGGGAGGCTTGGAGA CTGGATCTGAAGCCACAGAAC TGGAGCCCTGGGCAAGCTCTT GTCCTCCCTGCAGGAGGCTGA ACAGATGGTTTGATGGCTGTG CAAGGCTCTGGACTTCTTTGT GTGCTTGCCCTCGCCCTGCCA TTCAGCAGCTGCCTTATCACT TGGAGACAGTCTGACCCAGTG ACAGGGAGAGGGAGGCTTGGA CTGGAGTTTGAGCCTGGGACA TCTCACCTGGATCTGAAGCCA CAGGGAGAGGGAGGCTTGGAG CAGCCCACAGCATGGGCACTT GACTCTGGGAGAACAGCACTT TCATTCATTGTTTCTCTATTA GCTGAAAGACTTTCAGTGGGA CTGGGAGAACAGCACTTCTTT AGCCTGGGACAGGGAGAGGGA GCAGGCTGTGAAGAAAGACCA CCCACAGAACCCACCAGTCTT ATTGTTTCTCTATTAAATTAT CGCATCTCCTTCCAGGACTTT CTTAAATGCTTCCTTGGGCAA TGCAGGAGGCTGAGTGTAAAG GGGCAGGACTCTGGGAGAACA CAAGGCCATCGTCTCCTCTTC TCATTGTTTCTCTATTAAATT CCACTGCAGCCCTGGCGAGCA CGCCTGGGCAGCGGCACGTAC TGTTTCTCTATTAAATTATTT GAGGACAAAGTGAATTTAACC TTGGAGACAGTCTGACCCAGT GGACGGCTTCATCCTCACCGA ATGGTTTGATGGCTGTGCTGT GCCCTTCTGCCCTTAAATGCT GGTGTACAAGGCCTACGCCAA TGGATCTGAAGCCACAGAACA CTCAAGGCCATCGTCTCCTCT GCCCAGCGGAAGGAGGCAATT GGTGGTGCAGGCTGTGAAGAA CTTCTTTGTACCTGCCCTGCA CCGCCGGAACCGCCGCCCGCA AGAAGAATGATTGGACAGATG AGCAGGTCAAGATGAGGGAAT AACCGCCGCCCGCAGCGAGGA CTTGAAGGAGCAGGTCAAGAT TGGGAGAACAGCACTTCTTTC CCGAGCGCCTGGGCAGCGGCA TGGTTTGATGGCTGTGCTGTA TAACCCAGGCTGACGCCAAGA CAATTCCTGCATGAACGGAAT GCCACAGAACATTCTACTGAG . GTGGTGCAGGCTGTGAAGAAA AATTCCTGCATGAACGGAATA GGCTGTGAAGAAAGACCAGGA ACAAAGTGAATTTAACCTGGA TCTGAACAAGGCATCGGTGGA CAGCTGAAAGACTTTCAGTGG AACCCAGGCTGACGCCAAGAA TGCCCAGCGGAAGGAGGCAAT AGCTGCCATGGCCAAGGAGGA AGTGTAAAGAGTATCATTCAT TGGAGAACCTCCTCACGGAGA TTGAAGGAGCAGGTCAAGATG GGGCTGAGGAGCTCAAGGCCA GCAATTCCTGCATGAACGGAA CTGGTGGTGCAGGCTGTGAAG CCCTTCTGCCCTTAAATGCTT GAGCTGGAAGAGAAGATCCGT CAGTGCCCTCTAGGCCACCCA AACCCACCAGTCTTTCTGGGA CTGCTGTTGCTGGCAGCGGAG TGAAGGAGCAGGTCAAGATGA CGCTGGGAAGCTGACACCCTG CAGCTGCCTTATCACTCTACT ACTTATGAGGACAAAGTGAAT CCTGGGTGGACCTGGAGCACA ATCTACCTCATCATGGAGTTT AAGAATGTATTTGAGAGTTTC AAAGAGTATCATTCATTGTTT TGCCCGAGACCTGCTCAGAGA ACCCACCAGTCTTTCTGGGAA AAGGAGCAGGTCAAGATGAGG TTTGTAGCCCTTCTGCCCTTA TGGAAGTGGCTTCAGCTGCCA GGGAGAACAGCACTTCTTTCA TCAGAGAGATGGCCCGGGACA TGCTGGAGTTTGAGCCTGGGA GGAAGGAGGCAATTAAGGCAA AGCTGACACCCTGGACAAAGA TCCCTGCAGGAGGCTGAGTGT TTCAGCTGCCATGGCCAAGGA AACATTCTACTGAGCTCCTTG AAAGAATGTATTTGAGAGTTT CACTTCTTTCATGAGACCTTT GCCCTGCAATTCCTGCATGAA CCCTGGTGGTGCAGGCTGTGA GGAGAACAGCACTTCTTTCAT CTGCAAGGCTCTGGACTTCTT CTGGGAAGCTGACACCCTGGA TATGAGGACAAAGTGAATTTA CCCTGGACCTGTACCAGCACA CTGTCTGTCCTCCCTGCAGGA CAAGAGCTGTCTGTCCTCCCT AGAGGGACTGTCGGAATCTGT CCTGGTGGTGCAGGCTGTGAA GTGTAAAGAGTATCATTCATT TGGACCTGGAGCACATGCCCA CCAAGCCTTGTTCCTGCCCTG TTCTTTCATGAGACCTTTGTT CCCGAGACCTGCTCAGAGAGA GAGCTGTCTGTCCTCCCTGCA TGCAGCTGAAAGACTTTCAGT ATGGCTGTGCTGTATACTGGA CAGGAGGCTGAGTGTAAAGAG AGAGTATCATTCATTGTTTCT GAACAAGGCATCGGTGGAGAA GAGGGCAGGACTCTGGGAGAA TGAGGACAAAGTGAATTTAAC AGCACTTCTTTCATGAGACCT GGAGGGCAGGACTCTGGGAGA AGAGGGAGGCTTGGAGACAGT ACACTGAGGTTCAGAACCTCA AATGTATTTGAGAGTTTCTTT TCTTTCATGAGACCTTTGTTA TTCTCGGAGCTGGAAGAGAAG TTGTTTCTCTATTAAATTATT ACGCCTCCTAGCTGCCCTGGA AAGCCACAGAACATTCTACTG TTGGTTTCGCACAACACATGT GGTGCAGGCTGTGAAGAAAGA CTCACACCTGCCCGCCATCTT TAGCCATAAAGTGTGTAGCCA CCGTGGGATGAGAAGCACGTG CAGGACTCTGGGAGAACAGCA GTGGTAGCCATAAAGTGTGTA TTATGAGGACAAAGTGAATTT CAGGCTTCTTGGATGGACAGT AGTGGTAGCCATAAAGTGTGT CATTCATTGTTTCTCTATTAA TGAAAGACTTTCAGTGGGACA GCCATAAAGTGTGTAGCCAAG AGAATGTATTTGAGAGTTTCT AGACTTTCAGTGGGACAGTGA ACTTTGGTTTCGCACAACACA TATCATTCATTGTTTCTCTAT CACTGCAGCCCTGGCGAGCAG CTTTGGTTTCGCACAACACAT CCATAAAGTGTGTAGCCAAGA CAGGATGCCCTGGACCTGTAC CTCGGAGCTGGAAGAGAAGAT
CATAAAGTGTGTAGCCAAGAA GCAGGATGCCCTGGACCTGTA ATCTGAAGCCACAGAACATTC
ATAAAGTGTGTAGCCAAGAAA TGGCTGTGCTGTATACTGGAG TCGGAGCTGGAAGAGAAGATC
CGCGGGCGCAGGCGGCCGGAA CAGGCTGTGAAGAAAGACCAG AGCGTGGAGCCACCCAGTGCT
and complementary strands.
Other examples of siRNA of the present invention are selected in the group consisting in the siRNA (sense and antisense) sequences disclosed in Figure 1 (ULKl), Figure 2 (ULK2) and Figure 3 (ULK3).
In another embodiment, the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase miRNA (microRNA) . While the term "miRNA" is usually used to refer to endogenous RNAs that are naturally expressed, similar molecules or precursors thereof that either mimic the sequence of naturally occurring miRNAs or are specifically designed to hybridize to a target transcript so as to result in a duplex structure containing one or more bulges can be introduced into, and expressed within, cells and can cause translational repression. Thus either double-stranded duplex molecule structurally similar or identical to siRNAs, or hairpin precursors that can be processed intracellularly in a similar manner to naturally occurring miRNA precursors, can be introduced into cells and can mediate RNAi via translational repression (see, Doench, J., et al., 2003, Genes and Dev., 17, 438-442) . An RNAi-inducing entity that mediates RNAi by repressing translation of a target transcript, and that consists of or comprises a strand that binds to a target transcript to form a duplex containing one or more bulges, is said herein to act via an miRNA translational repression pathway, and the strand that binds to the target may be referred to as an miRNA-like molecule. A binding site with which a small, single-stranded RNA can hybridize to form a duplex structure containing a bulge, such that the transcript containing the binding site (or multiple copies thereof) , is subject to RNAi via translational repression, is referred to herein as a miRNA binding site. Endogenous miRNAs can also mediate cleavage of RNA targets (i.e., they can act in an siRNA-like manner) if they have sufficient complementarity to the target. Further description of miRNAs and the mechanism by which they 'are believed to mediate silencing is found in Bartel, 2004, Cell, 116, 281-297.
According to special embodiment, miRNA of the present invention are defined by using the sequences provided above for siRNA.
In another embodiment, the said Unc51-Like Kinase inhibitor or partial inhibitor may have a short hairpin structure having a sticky portion at the 3' terminus and is an Unc51-Like Kinase shRNA (short hairpin RNA). As used herein, the term "shRNA" refers to short hairpin RNA which functions as RNAi and/or siRNA species but differs in that shRNA species are double stranded hairpin-like structure for increased stability. shRNA are molecule of about 20 or more base pairs in which a single-stranded RNA partially contains a palindromic base sequence and forms a double-strand structure therein (i.e., a hairpin structure). Such palindromic sequence is composed of a antisense sequence (that anneals to the target mRNA) in 5' to 3' (sequence a) orientation and an sense sequence in reverse orientation (3' to 5') (sequence b) separated by a linker sequence or loop sequence (that is unrelated to the target mRNA). shRNA may preferably have a 31 protruding end. The length of the double-stranded portion is not particularly limited, but is preferably about 10 or more nucleotides, and more preferably about 20 or more nucleotides. The 3? protruding end may be preferably DNA, more preferably DNA of at least 2 nucleotides in length, and even more preferably DNA of 2-4 nucleotides in length. According to special embodiment, the "sequence a" and "sequence b" are selected in the group consisting in the siRNA sequences listed above.
According to special embodiment, the said hairpin loop sequence of the present invention is selected in the group consisting in :
AUG (Sui et al. 2002 Proc. Natl. Acad. Sci. US A 99(8): 5515-5520.), CCC or CCACC or CCACACC (Paul et al., 2002, Nature Biotechnology, 20, 505-508), UUCG (Lee et al., 2002, Nature Biotechnology, 20, 500-505), CTCGAG or AAGCUU (Editors of Nature Cell Biology 2003 Nat Cell Biol. 5:489-490), UUCAAGAGA (Yu et al . 2002 Proc. Natl. Acad. Sci., 99, 6047- 6052), TTCAAGAGA (Brummelkamp et al. 2002 Science 296, 550- 553), AAGTTCTCT ( Promega), TTTGTGTAG (Scherr et al . , 2003, Cell Cycle, 2, 251-7), CTTCCTGTCA (Schwarz et al. 2003 Cell. 17; 115 (2) : 199-208. ), GUUUUGGCCACUGACUGAC (Invitrogen) .
According to special embodiment, shRNA of the present invention is selected in the group consisting in all combinations of antisense sequences "a" with each hairpin loop sequence described above and sense sequence "b" corresponding to the antisenses, therefore forming a group of palindromic sequences .
According to another embodiment of the present invention, the said Unc51-Like Kinase inhibitor or partial inhibitor is an Unc51-Like Kinase-specific ribozyme having specificity for a Unc51-Like Kinase-encoding nucleic acid. It can include one or more sequences complementary to the nucleotide sequence of a Unc51-Like Kinase cDNA and a sequence having known catalytic sequence responsible for mRNA cleavage (see US 5,093,246 or Haselhoff and Gerlach, 1988, Nature, 334, 585-591). Unc51-Like Kinase gene expression can further be inhibited by targeting nucleotide sequences complementary to the regulatory region of the Unc51-Like Kinase (e.g. the Unc51-Like Kinase promoter and/or enhancers) to form triple helical structures that prevent transcription of the Unc51-Like Kinase gene in target cells (Helene, 1991, Anticancer Drug Des., 6, 569-84; Maher, 1992, Bioassays, 14, 807-15).
The nucleic acid sequences encoding Unc51-Like Kinase are known in the art and thus are available to the skilled man : see for example references (Kuroyanagi, H. et al. Genomics 51 (1), 76-85 (1998), Yan,J et al. Oncogene 18 (43), 5850-5859 (1999) ) .
More specifically, cDNA nucleic acid sequence encoding ULKl (NM_003565.1) is :
GGATCCGGATTCGGATTAGCAGCCCGGGAAGAGTGCCGTGGCACAGGCGCCGGAGGGAGCGC
GACCCTCG
GACCCCGCCTGGCCCGCGGGGCTGGGACCCGGCCCCGGCCTGCCCGATGGGGCGCGCGGCCC
CGGAGATG
CGCCCTCGCCCGGCCCCGCGCCCCCGGCCCCGCGCCCCCGGCCCGCCCGCCCCGGCCCGCGC
CTCCGCCT
GAGTCCCCCGCGCCTTGGCCCGCCACCCCCCGCCCCGCGCCCCCGGCCCGCCTGCGCCATGG
AGCCCGGC
CGCGGCGGCACAGAGACCGTGGGCAAGTTCGAGTTCTCCCGCAAGGACCTGATCGGCCACGG
CGCCTTCG
CGGTGGTCTTCAAGGGCCGCCACCGCGAGAAGCACGATTTGGAGGTCGCCGTCAAGTGCATT
AACAAGAA
GAACCTCGCCAAGTCTCAGACGCTGCTGGGGAAGGAAATCAAAATCCTGAAGGAACTGAAAC
ATGAAAAC
ATCGTGGCCCTGTACGACTTCCAGGAAATGGCTAATTCTGTCTACCTGGTTATGGAGTACTG
CAACGGTG
GGGACCTGGCCGACTACCTGCACGCCATGCGCACGCTGAGCGAGGACACCATCAGGCTCTTC
CTGCAGCA
GATCGCGGGCGCCATGCGGCTTCTGCACAGCAAAGGCATCATCCACCGCGACCTGAAACCGC
AGAACATC
CTGCTGTCCAACCCCGCCGGCCGCCGCGCCAACCCCAACAGCATCCGCGTCAAGATCGCTGA
CTTCGGCT TCGCGCGGTACCTCCAGAGCAACATGATGGCGGCCACACTCTGCGGCTCCCCCATGTACATG
GCCCCCGA
GGTCATCATGTCCCAGCACTACGACGGGAAGGCGGACCTGTGGAGCATCGGCACCATCGTCT
ACCAGTGC
CTGACGGGGAAGGCGCCCTTCCAGGCCAGCAGCCCCCAGGACCTGCGCCTGTTCTACGAGAA
GAACAAGA
CGTTGGTCCCCACCATCCCCCGGGAGACCTCGGCCCCGCTGCGGCAGCTGCTCCTGGCCCTA
CTGCAACG
CAACCACAAGGACCGCATGGACTTCGATGAGTTTTTTCATCACCCTTTCCTCGATGCCAGCC
CCTCGGTC
AGGAAATCCCCACCCGTGCCTGTGCCCTCGTACCCAAGCTCGGGGTCCGGCAGCAGCTCCAG
CAGCAGCT
CCACCTCCCACCTGGCCTCCCCGCCGTCCCTGGGCGAGATGCAGCAGCTGCAGAAGACCCTG
GCCTCCCC
GGCTGACACCGCTGGCTTCCTGCACAGCTCCCGGGACTCTGGTGGCAGCAAGGACTCTTCCT
GTGACACA
GACGACTTCGTCATGGTCCCCGCGCAGTTTCCAGGTGACCTGGTGGCTGAGGCGCCCAGTGC
CAAACCCC
CGCCAGACAGCCTGATGTGCAGTGGGAGCTCACTGGTGGCCTCTGCGGGCTTGGAGAGCCAC
GGCCGGAC
CCCATCTCCATCCCCACCCTGCAGCAGCTCCCCCAGTCCCTCAGGCCGGGCTGGCCCGTTCT
CCAGCAGC
AGGTGCGGCGCCTCTGTCCCCATCCCAGTCCCCACGCAGGTGCAGAACTACCAGCGCATTGA
GCGAAACC
TGCAGTCACCCACCCAGTTCCAAACACCTCGGTCCTCTGCCATCCGCAGGTCAGGCAGCACC
AGCCCCCT
GGGCTTTGCAAGGGCCAGCCCCTCGCCCCCTGCCCACGCTGAGCATGGAGGCGTCCTGGCCA
GGAAGATG
TCTCTGGGTGGAGGCCGGCCCTACACGCCATCTCCTCAAGTTGGAACCATCCCTGAGCGGCC
AGGCTGGA
GCGGGACGCCCTCCCCACAGGGAGCTGAGATGCGGGGTGGCAGGTCCCCTCGTCCAGGCTCC
TCTGCACC
CGAGCACTCTCCCCGCACTTCCGGGCTGGGCTGCCGCCTGCACAGCGCCCCCAACCTGTCTG
ACTTGCAC GTCGTCCGCCCCAAGCTGCCCAAACCCCCCACGGACCCCCTGGGAGCTGTGTTCAGCCCACC
ACAGGCCA
GCCCTCCCCAGCCGTCCCACGGCCTGCAGTCCTGCCGGAACCTGCGGGGCTCACCCAAGCTG
CCCGACTT
CCTGCAGCGAAACCCCCTGCCCCCCATCCTGGGCTCCCCCACCAAGGCTGTGCCCTCCTTTG
ACTTCCCG
AAGACCCCCAGCTCCCAGAACCTGCTGGCCCTCCTAGCCCGGCAGGGCGTGGTGATGACGCC
CCCTCGAA
ACCGGACGCTGCCCGACCTCTCGGAGGTGGGACCCTTCCATGGTCAGCCGTTGGGCCCTGGC
CTGCGGCC
AGGCGAGGACCCCAAGGGCCCCTTTGGCCGGTCTTTCAGCACCAGCCGCCTCACTGACCTGC
TCCTTAAG
GCGGCGTTTGGGACACAAGCCCCGGACCCGGGCAGCACGGAGAGCCTGCAGGAGAAGCCCAT
GGAGATCG
CACCCTCAGCTGGCTTTGGAGGGAGCCTGCACCCAGGAGCCCGTGCTGGGGGCACCAGCAGC
CCCTCCCC
GGTGGTCTTCACCGTGGGCTCTCCCCCGAGCGGGAGCACGCCCCCCCAGGGCCCCCGCACCA
GGATGTTC
TCAGCGGGCCCCACTGGCTCTGCCAGCTCTTCTGCCCGCCACCTGGTGCCTGGGCCCTGCAG
CGAGGCCC
CAGCCCCTGAGCTCCCTGCTCCAGGACACGGCTGCAGCTTTGCCGACCCCATTGCTGCGAAC
CTGGAGGG
GGCTGTGACCTTCGAGGCCCCCGACCTCCCTGAGGAGACCCTCATGGAGCAAGAGCACACGG
AGATCCTG
CGTGGCCTGCGCTTCACGCTGCTGTTCGTGCAGCACGTCCTGGAGATCGCAGCCCTGAAGGG
CAGCGCCA
GTGAGGCGGCGGGGGGCCCTGAGTACCAGCTGCAGGAGAGTGTGGTGGCCGACCAGATCAGC
CTGCTGAG
CCGAGAATGGGGCTTCGCGGAACAGCTGGTGCTGTACCTGAAGGTGGCCGAGCTACTGTCCT
CCGGCCTG
CAAAGTGCCATCGACCAGATCCGGGCCGGCAAGCTCTGCCTGTCGTCCACTGTGAAGCAGGT
GGTGCGCA
GGCTGAATGAGCTGTACAAGGCCAGCGTGGTGTCCTGCCAGGGCCTGAGCCTGCGGCTGCAG
CGCTTCTT CCTGGACAAGCAGCGGCTCCTGGACCGCATTCACAGCATCACTGCCGAGAGGCTCATCTTCA
GCCACGCT
GTGCAGATGGTGCAGTCGGCTGCCCTGGACGAGATGTTCCAGCACCGTGAGGGCTGCGTCCC
ACGCTACC
ACAAGGCCCTGCTGCTCCTGGAGGGGCTGCAGCACATGCTCTCGGACCAGGCCGACATCGAG
AACGTCAC
CAAGTGCAAGCTGTGCATTGAGCGGAGACTCTCGGCGCTGCTGACTGGCATCTGTGCCTGAC
CTTTCTGG
CCTGGCTGGGCCCCCCGTCCTGCCGAGCCCTGCAGAGTGGGCTCTGTGTGCTGGCTGGACTC
CTCGGGAC
AAGCCCATGGCGCTGATCGCTGGTGCTGAGCCCTGCCCTGGGCCCCACGGACAGTCAGCCTG
CCGGCCTC
CCTGCAGCTCACGGGGCAGAACCAGCACATCTGGAGCCACACAGCTTGGGGGGTGTCTCCCA
TCTTTTAC
AGGTGGGGATCACAGAATTTCTGCCCCTCCAGCTGCCTGGCTCAGCAGGCGTGGGTGCCACC
ACCCTCTA
GCCCCAGGGCAGCCCCGGAGGACAGGCAAGGGCCTGAGACCACTGCCGACTCAAAGCCAAAG
CGAGCTCC
TGCTTAGGGCAGGTCAGCAGGCACTGTGCCCAGGAAGAGCCTGCGGCCTCGGCGTCCCCCAG
TCTCCAGG
AGCCTCTCCCTCCGAGATACCCACCCAGCTTTGTCAATCACCCAAGCACTTTATGCATATAG
AGACAGAA
CCTGGACCTCACCAGGGACTGCTGGGCAGCGATTCCTGGCAGTGGCCTGGTGTTTGTACATA
CACATATG
CAGACACATGCCAGGGCCCCCCAAGCCCGAGCACCGGACCACGTTGCTGCCCAGGTCTGGAC
CTCAGCGG
GAGAACTGGCTCCGGGGGGAGTGGGGCCCTGCGCTAGAGGCAGAGGCAGTTCTTTGTTCAAG
CGTTCCTC
TGGGGACCGGCAGCAGAGGCACCGTGTTCTCTCAGCCCTGGATACGTCTTGTAATCTTTCAC
ACTTTATT
CCTAAAACGTGTCTTATTTTTATGCAGCTCATTTTTTCTTTAAAGGAGAAAACTTGTAGGTG
TTTAAGAA
TTGGTTTTGGGAGGGCGAGGACTGGGCCAGGTTAGAGGCAGATGGCACAGGGGCGTGTGGCG
GGCGGGTG AGGCTGCTTTGCACACCTGTGTTGGTGGCTGTCCCCTGCCGCCCCTCCCTGTGGCAGCAGCA
GGACAGGT
GTGTGCCCAGCACCCTCCCTACCTGGGCCTGGAAGCAGATGAGGGGAATACTTCATGCAAAG
AAAAAAGT
AACATGTGCAAAAGCTCCCCGTCCAGCTTTGACAGTCAGTTTTGATGTCAGCTCCTCGGCAG
GGTAGGCC
TGATGACAGCCCTGTCCCTCCCTGCCTCCGCCTTGCCCAAGGCCACGGAGGGCGTCTGCAGA
GAGGCCTG
CCTTCCGGATTCCAGGCGGGCATGCCCTGCAAACCCCGCCTGGGCCTCCCTTGGTCTGCCCA
GCCCTCGG
TTAGCCCTGCCTGAATCAGTAGATACTTGAACGAGTCCCCAGTCTGCGGGAGGCAGTGGTGG
GGCCATGG
ACCCATGCGGGGGGTTCCAGGGTCACACGCCACATAACAGACAAAAATACACACACGTGTGT
TTTTCTTT
GCAATACTTGAAATATTGCCACTGTGCTTGGACTTAGAAGAAGAAAATCCCCGTGACTTCTT
CCTCATCA
CCTTGATGGCTTTATTCTCACCTTGTGGGGCATGTTTGAATTTATTGCTTCATGGCCGACTG
GAATCCTG
AGTCCTGGGAAGCTGGCACTGCGGGGATCTTGCCCGGTGTCCTGGTCCTCTTGCTTCCGTCG
CGGCCGCA
TGTGCGTGTGTCCAAGCAGGTCCTGGGCGCCTCAACTGCTGCCCCTGGTTGAATGTTCTCTT
GATAGTGC
TGGACCCTTTGTCTATTTTAAAGCGAATTTTGTGTGATTTCCTGCCCTTTGCGTTATATTGT
ATAATACC
AACGTAAGGAAATAAACCTTTGGAATTGTTGAAAAAAAAAAAAAAAAA cDNA nucleic acid sequence encoding ULK2 (NM_014683.2 ) is :
GCGCGCGCGAGGGCGTTGGGCGCCGCCGCGAGGCGGGGAAGCGCGGGGCCGCGGCGGTGCGG
GTTCTAGG
GCGGCGGCCGTCGCCGTCGCAGCAGCGCCCCGAGCGGGGAGGGCCGAGGAGGCCCGACGAGC
TGGGGATG
GAGAGTACCGGGCCCCTCACTGCCTCAGAGCGCGTGTGCGGCTCTGGGCGCGCACAGTGACG
GTGACGGC ACCCCTGGCCCGGCAGCGCCGAGGCCGCTTCGCCAGACAGCCAGCGGCCGGCGGCAGGCCGG
GCCATGAG
CGGCAGGGGCCGGGCCGGGCCTCGCTGACCCTGGCTCCGCGCGGCAGCTTCCCCAGTTTCCG
CTCCGGTC
TCTCGGCATGAGAGTCCGCCCGGGCCCGGGGCTGCGGCTGCCCCAGACCCGCCGCACGCTGG
CGCGCTCC
GGGCCCGCGGAGCCGCGGTGCTGATACCTGCGCCGCACTGCGCCGCCCGCCCGTCCGCTGTG
TGCCCCGG
GGGCGCGGCCATGGAGGTGGTGGGTGACTTCGAGTACAGCAAGAGGGATCTCGTGGGACACG
GGGCCTTC
GCCGTGGTCTTCCGGGGGCGGCACCGCCAGAAAACTGATTGGGAGGTAGCTATTAAAAGTAT
TAATAAAA
AGAACTTGTCAAAATCACAAATACTGCTTGGAAAGGAAATTAAAATCTTAAAGGAACTTCAG
CATGAAAA
TATTGTAGCACTCTATGATGTTCAGGAATTACCCAACTCTGTCTTTTTGGTGATGGAGTATT
GCAATGGT
GGAGACCTCGCAGATTATTTGCAAGCGAAAGGGACTCTCAGTGAAGACACGATCAGAGTGTT
TCTGCATC
AGATTGCTGCTGCCATGCGAATCCTGCACAGCAAAGGAATCATCCACAGAGATCTCAAACCA
CAGAACAT
CTTGCTGTCCTATGCCAATCGCAGAAAATCAAGTGTCAGTGGTATTCGCATCAAAATAGCGG
ATTTTGGT
TTTGCTCGTTACCTACATAGTAACATGATGGCTGCAACACTGTGTGGATCCCCGATGTACAT
GGCTCCTG
AGGTTATTATGTCTCAACATTATGATGCTAAGGCTGACTTGTGGAGCATAGGAACAGTGATA
TACCAATG
CCTAGTTGGAAAACCACCTTTTCAGGCCAATAGTCCTCAAGACTTAAGGATGTTTTATGAAA
AAAACAGG
AGCTTAATGCCTAGTATTCCCAGAGAAACATCACCTTATTTGGCTAATCTCCTTTTGGGTTT
GCTTCAGA
GAAACCAAAAAGATAGAATGGACTTTGAAGCGTTTTTTAGCCATCCTTTTCTTGAGCAAGGT
CCAGTAAA
AAAATCTTGCCCAGTTCCAGTGCCCATGTATTCTGGTTCTGTCTCTGGAAGCTCCTGTGGCA
GCTCTCCA TCTTGTCGTTTTGCTTCTCCACCATCCCTTCCAGATATGCAGCATATTCAGGΔAGAAAACTT
ATCTTCCC
CACCATTGGGTCCTCCCAACTATCTACAAGTTTCCAAAGATTCTGCCAGTACTAGTAGCAAG
AACTCTTC
TTGTGACACGGATGACTTTGTTTTGGTGCCACACAACATCTCGTCAGACCACTCATGTGATA
TGCCAATG
GGGACTGCTGGCAGACGTGCTTCAAATGAATTCTTGGTGTGTGGAGGGCAGTGTCAGCCTAC
TGTGTCAC
CTCACAGCGAAACAGCACCAATTCCAGTTCCTACTCAAATAAGGAATTATCAGCGCATAGAG
CAGAATCT
TACATCTACTGCCAGCTCAGGCACAAATGTACATGGTTCTCCAAGATCTGCAGTGGTACGAA
GGTCCAAC
ACCAGCCCCATGGGCTTCCTCCGGCCGGGATCATGCTCCCCAGTACCAGCAGACACAGCACA
GACAGTTG
GACGAAGGCTCTCCACTGGGTCTTCTAGGCCTTACTCACCTTCCCCTTTGGTTGGTACCATT
CCTGAGCA
ATTCAGTCAGTGCTGCTGTGGGCATCCTCAGGGCCATGACTCCAGGAGTAGAAACTCCTCAG
GTTCTCCA
GTGCCACAAGCTCAGTCCCCACAGTCTCTCTTATCGGGTGCTAGACTGCAGAGCGCCCCCAC
CCTCACTG
ACATCTATCAGAACAAGCAGAAGCTCAGAAAACAGCACTCTGACCCCGTGTGCCCATCCCAT
ACTGGGGC
TGGGTACAGCTACTCGCCTCAGCCCAGTCGGCCTGGCAGCCTTGGAACTTCTCCCACCAAGC
ACTTGGGG
TCCTCTCCACGGAGTTCTGACTGGTTCTTTAAAACTCCTTTGCCAACAATCATTGGCTCTCC
TACTAAGA
CCACAGCTCCTTTCAAAATCCCTAAAACTCAAGCATCTTCCAACCTGTTAGCCTTGGTTACT
CGTCATGG
GCCTGCTGAAGAACAGTCGAAAGATGGGAATGAGCCACGGGAATGTGCCCATTGCCTCTTAG
TGCAAGGA
AGTGAGAGGCAGCGGGCCGAGCAGCAGAGCAAGGCAGTGTTTGGCAGATCTGTCAGTACCGG
GAAGTTAT
CAGATCAACAAGGAAAGACTCCTATATGTCGACATCAGGGCAGCACAGACAGTTTAAATACA
GAACGACC AATGGATATAGCTCCGGCAGGAGCCTGTGGTGGTGTTCTGGCACCTCCTGCAGGTACAGCAG
CAAGTTCC
AAGGCTGTCCTCTTCACTGTAGGGTCTCCTCCACACAGTGCGGCAGCCCCCACTTGTACCCA
CATGTTCC
TTCGAACAAGAACAACCTCAGTGGGGCCCAGCAACTCCGGGGGCTCTCTTTGTGCCATGAGT
GGCCGCGT
GTGCGTGGGGTCCCCGCCTGGCCCAGGCTTCGGCTCTTCCCCTCCAGGAGCAGAGGCAGCTC
CCAGCCTG
AGATACGTGCCTTACGGTGCTTCACCCCCCAGCCTAGAGGGGCTCATCACCTTTGAAGCCCC
TGAACTGC
CGGAGGAGACGCTGATGGAGCGGGAACACACAGACACCTTACGCCATCTGAATGTGATGCTG
ATGTTCAC
TGAGTGTGTGCTGGACCTGACAGCCATGAGGGGAGGAAACCCTGAGCTGTGCACATCTGCTG
TGTCCTTG
TACCAGATCCAGGAGAGTGTGGTGGTGGACCAGATCAGTCAGCTGAGCAAAGACTGGGGGTG
GGTGGAGC
AGCTGGTGTTGTACATGAAAGCAGCACAGCTGCTTGCGGCTTCTCTGCATCTTGCCAAAGCC
CAGATCAA
GTCCGGGAAACTGAGCCCATCCACAGCTGTGAAACAAGTTGTCAAGAATCTGAACGAACGAT
ATAAATTC
TGCATCACCATGTGCAAGAAACTTACAGAAAAGCTGAATCGATTCTTCTCTGACAAACAGAG
GTTTATTG
ATGAAATCAACAGTGTGACTGCAGAGAAACTCATCTATAATTGTGCTGTAGAAATGGTTCAG
TCTGCAGC
CCTGGATGAGATGTTTCAGCAGACCGAAGATATTGTTTATCGCTATCATAAGGCAGCCCTTC
TTTTGGAA
GGCCTAAGTAGGATTCTACAGGACCCTGCAGATATTGAAAATGTGCATAAATATAAATGTAG
TATTGAGA
GAAGACTGTCGGCGCTCTGCCATAGCACCGCAACCGTGTGAGCAGCAGGCTCATCCCGTGGA
CCGGTGGT
GGGAACGTGAGGTGATGCCTTTGGGATTACAGCTTGAGTTCTGTCACCCCATCCCCAGGAAA
CTGTAGCT
TCTTAACTGGTGACTACCAAAGAACAAGCAGTGATTTGAAAAAGGAAAAACAATCCAAAAAC
TACATATT TGTAGGAAATCTGCCTTATTGGAGAAAATCACCCTTTCCCTTTTTCTTTGTAGAAGCAGGAG
CAAGAGTG
TTTGGCTCCCAGTTTGGACTTGGTGAATAAATGTACCTTAGAACTAGGATAATCGGTACAGT
TATTCTTA
AAGATAATTAAAAATGAAACAAAGTGAGTGCTCGTCACTGGGTTCATCAGAGCAGTGTGTGA
AATTCCAT
GTGTTTGCTGAGGTGTAAAGGTAAATGTATTCACCCCTCATCCAGGCAGTTTGATATTTGGA
GTAAGTTT
GTTTAAATCTGAGCATGCATCTTTAAACAGCTCAGGAAGAAATAGCTTAAGAAGAAGTGAAA
CATGGATC
TTGGAAGAAATTTTGAAATCTTCAATTTGATCCTAATATGGATACATGTTAATCTTCCAAAA
TCTTTCAT
ATTGCACTAATTTATTAAAACAACTGTGTATTGGATTTTGTAATTTAACTAAGGCACAATGG
ACTTGTTT
AAAATATTTTACTTGATTGTATACATAGACCCTTTCCAGAATTCACATGTAATCTCCAGTGA
ACTTTTAA
GTGGTTAAAACTTGTATTCATGTGAACCTTTGCACATTTTTTTTTTTTACTTCTTTATCTAC
ACCTACAG
ATTTTCTCAGTAATGTTTTTGTTAGCTTTTGGTTCCATTTTTTATTGTGCATGCAGAATGTA
CATTGATG
CCTGTGACCTTAGGTTTATTAAAGGCTAGGTTTATTTGGGCAGTATTAGAAACAAAATCATG
GATCAAGA
GATACTCTTGATAATTTGAATAGGGCCAAAACAAAGTTGGTGACCTAAAGGCTTGTTAGTGA
TGTGGAGT
TCCTACATGCAGTGAGTGGAAAATGAAGTTCGTTTTCTCTTAGGAAAATGGGCAGCTGTCTT
CTGCCTAA
TGTGTATTTTTCATGTTAATTCTGACAGTTCACCAAATAGCTAGTCATGGAGAATGCAGGCA
GTTAACTT
AATATCCCTCCAGGAATGGTTCCTACGTTGTGTATTATTTGGTTTCTTTTACTTACCTGCTT
GAATACTT
GAATAAACCATTCACCAATTTTAATCCTTTTATTTTAATCCTTTTACATAAAATAATCTGAA
CTCTTTGA
CAAATTGCACAGAGCTCTTTGGCATTAATCTAATTTTAATGTACTGATAAAAACAAACATGG
TTGTCCTT TACTTTGACAAAGTAATGTAATTTTTACCTTATTTATCTGTATGAAATTCCAGTAGTTAATT
TGAACATT
TATTTATATGACGTTTGTATTTTTAGGTCTTTAATACAGTGTTTCTACCTCTCATTTGTAAC
TGCATGCA
TTATTCTTGAAACTAGGTAAAACTCACTGAATTGTTGTGTAATAGCCTTTTTATTATTGCCT
GTACAAAT
GTATATTAAGGTAAAATAAAACTGACAAAGTGTTTCTAGGGTGTAGCTGGGTACATATTAAG
TGGCTTGT
TGAGCCAGGTACTTCCTTAGTGAGTTTAGAGACTTGGCCATGAATATCCTTTGTCCTGCCCC
AGGATTTA
GATCTTGGCTACTGTCATGCAGGCTTCCAGGAACATAGACTGTTTTACCTCCACAACCCTAT
TTGTTATT
AGTGATACTTTATTTTATATAATATTTTTTATTCACAGTGAAATTTCATTCATGTTCTTTCA
GTTATCAC
CTGTGTTATCTCAGTTGTAGGTTTATTCTATCCTCTCCTCTTCCTCTCCCATTTCTTTTTTA
ACACAGGA
TGAAACAGGTTCAGAGAGGGGAAGTGATTGGCCTAAAGTCAGGAACTAGGCAAGTGGTCAAG
CCATGCTT
TGTGACTTTCAAGTTAATTCTTCTTGTTCTTGTATATTAAAGGTCTTGGGGTAGATGGTGTG
TGTGAAAC
AGTGAAGTCTCAACAGCAGAAAAGAACAAAATGTAAATTCATGAATAATGGTTCTGGTTATA
CTTCCATT
ATCAAGGCTAATTAAGAGATTTTGCCTTGAGTATAGCAATAATAAACAAATGCTTTATGTTT
CCCTG cDNA nucleic acid sequence encoding ULK3 (XM_001134013.1) is :
CGTGGAGCCACCCAGTGCTCGGCCCGCCCCGCAACCCGCCGGAACCGCCGCCCGCAGCGAGG AAGCGCCC
GCGCGGGCGCAGGCGGCCGGAATGGCGGGGCCCGGCTGGGGTCCCCCGCGCCTGGACGGCTT CATCCTCA
CCGAGCGCCTGGGCAGCGGCACGTACGCCACGGTGTACAAGGCCTACGCCAAGAAGGACACT CGTGAAGT
GGTAGCCATAAAGTGTGTAGCCAAGAAAAGTCTGAACAAGGCATCGGTGGAGAACCTCCTCA CGGAGATT GAGATCCTCAAGGGCATTCGACATCCCCACATTGTGCAGCTGAAAGACTTTCAGTGGGACAG
TGACAATA
TCTACCTCATCATGGAGTTTTGCGCAGGGGGCGACCTGTCTCGCTTCATCCATACCCGCAGG
ATTCTGCC
TGAGAAGGTGGCGCGTGTCTTCATGCAGCAATTAGCTAGCGCCCTGCAATTCCTGCATGAAC
GGAATATC
TCTCACCTGGATCTGAAGCCACAGAACATTCTACTGAGCTCCTTGGAGAAGCCCCACCTAAA
ACTGGCAG
ACTTTGGTTTCGCACAACACATGTCCCCGTGGGATGAGAAGCACGTGCTCCGTGGCTCCCCC
CTCTACAT
GGCCCCCGAGATGGTGTGCCAGCGGCAGTATGACGCCCGCGTGGACCTCTGGTCCATGGGGG
TCATCCTG
TATGAAGCCCTCTTCGGGCAGCCCCCCTTTGCCTCCAGGTCGTTCTCGGAGCTGGAAGAGAA
GATCCGTA
GCAACCGGGTCATCGAGCTCCCCTTGCGGCCCCTGCTCTCCCGAGACTGCCGGGACCTACTG
CAGCGGCT
CCTGGAGCGGGACCCCAGCCGTCGCATCTCCTTCCAGGACTTTTTTGCGCACCCCTGGGTGG
ACCTGGAG
CACATGCCCAGTGGGGAGAGTCTGGGGCGAGCAACCGCCCTGGTGGTGCAGGCTGTGAAGAA
AGACCAGG
AGGGGGATTCAGCAGCTGCCTTATCACTCTACTGCAAGGCTCTGGACTTCTTTGTACCTGCC
CTGCACTA
TGAAGTGGATGCCCAGCGGAAGGAGGCAATTAAGGCAAAGGTGGGGCAGTACGTGTCCCGGG
CTGAGGAG
CTCAAGGCCATCGTCTCCTCTTCCAATCAGGCCCTGCTGAGGCAGGGGACCTCTGCCCGAGA
CCTGCTCA
GAGAGATGGCCCGGGACAAGCCACGCCTCCTAGCTGCCCTGGAAGTGGCTTCAGCTGCCATG
GCCAAGGA
GGAGGCCGCCGGCGGGGAGCAGGATGCCCTGGACCTGTACCAGCACAGCCTGGGGGAGCTAC
TGCTGTTG
CTGGCAGCGGAGCCCCCGGGCCGGAGGCGGGAGCTGCTTCACACTGAGGTTCAGAACCTCAT
GGCCCGAG
CTGAATACTTGAAGGAGCAGGTCAAGATGAGGGAATCTCGCTGGGAAGCTGACACCCTGGAC
AAAGAGGG ACTGTCGGAATCTGTTCGTAGCTCTTGCACCCTTCAGTGACCCTAGAAGAATGATTGGACAG
ATGTGAGC
CATCTGGAGCAGAGGGGCACTAACCCAGGCTGACGCCAAGAATGAAGTGGCCCACTGCAGCC
CTGGCGAG
CAGGCTTCTTGGATGGACAGTGCTGAGACCCCCATATCCCAGAGTCCCCAGCCTCCCTCAGG
TTACTCTG
CACCCCACAGATGGTTTGATGGCTGTGCTGTATACTGGAGGGGAGGGCAGGACTCTGGGAGA
ACAGCACT
TCTTTCATGAGACCTTTGTTACTCGGTGGTTACTGGGTCCTGTGCCTGTCCGTTTTGGGGCA
TGCAGCCC
TCTATCATTTTTGGCTCCGAGAAGAGGGCAAGGGGCCCCCGCAGGGTACTTCTGTGCTTGCC
CTCGCCCT
GCCAGCAGGCAGCTGTGCCCCTGGCCTGCCCTTCCCGGGACCCCTTATTCCAACTCAGCTCC
TCTTTGCA
CTGGAATGGGGCACTCCAACACCCCTCAGGGACCACCCTCCCCACAGTATGCACTCAGCCCC
ACAGAACC
CACCAGTCTTTCTGGGAACTCACACCTGCCCGCCATCTTGGTACTTTAGGTTAATCCCTCAA
GCATGAAA
GCTGGATCTTTTGGGGTTTAAGAAGCCCAAGCCTTGTTCCTGCCCTGGCCTAGGGAGCACTC
AGGAGGGT
TCCTTGGTCCTCATCTCTCCCACCTCCGTTCCCTCTGGGCCCCACACTAGCCACAGCGCGGG
CCTTGTGC
TGGAGTTTGAGCCTGGGACAGGGAGAGGGAGGCTTGGAGACAGTCTGACCCAGTGCCCTCTA
GGCCACCC
ACTTCTAGGCCTGCCCTGCCGCCGTGGAGCCCTGGGCAAGCTCTTTCCCCTTTCTGGGCCTG
GGTCTCCC
CATCTCTTCAATGGGGCTGATACCTTCACAGCCCACAGCATGGGCACTTATGAGGACAAAGT
GAATTTAA
CCTGGAAAAGAATGTATTTGAGAGTTTCTTTTAAATAATCAGCGGGTGTTGGTGATTTGTAG
CCCTTCTG
CCCTTAAATGCTTCCTTGGGCAAGAGCTGTCTGTCCTCCCTGCAGGAGGCTGAGTGTAAAGA
GTATCATT
CATTGTTTCTCTATTAAATTATTTTCTCTATT Figure 4 provides a table with correspondences between sequences from nucleic acid and corresponding polypeptides.
According to another embodiment, the term "indirect inhibitor" as used herein, refers to an anti-Unc51-Like Kinase antibody or an inhibitory peptide. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i. e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F (ab1) 2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively. The antibody can be a polyclonal, monoclonal, recombinant, e.g. a chimeric or humanized, fully human, non-human, e.g. murine or single chain antibody. The antibody can be coupled to a toxin or imaging agent. A full-length Unc51-Like Kinase protein or, antigenic peptide fragment of Unc51-Like Kinase can be used as an immunogen or can be used to identify anti- Unc51-Like Kinase antibodies made with other immunogens, e. g. , cells, membrane preparations, and the like. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of Unc51-Like Kinase located on the surface of the protein, e. g., hydrophilic regions, as well as regions with high antigenicity. Additionally, chimeric, humanized, and completely human antibodies are also within the scope of the invention. Chimeric, humanized, but most preferably, completely human antibodies are desirable for applications which include repeated administration, e. g., therapeutic treatment of human patients. These terms and methods for producing these antibodies by recombinant DNA techniques are widely known in the art (see for example EP184187, EP171496, EP173494, WO 86/01533, US4816567.
The invention provides methods (also referred to herein as "screening assays") for identifying modulators of the present invention (especially Unc51-Like Kinase inhibitors, including Unc51-Like Kinase partial inhibitors), i.e. candidate or test compounds or agents (e.g. proteins, peptides, peptidomimetics, peptoids, small molecules, nucleic acids, oligonucleotides or any other drugs) which have a modulating effect on Unc51-Like Kinase activity, and more specifically on Unc51-Like Kinase expression (indirect inhibitors). According to the present invention, "candidate or compounds or agents" are synonyms.
Selecting as modulator according to the invention a test substance that modulates (preferably inhibits) Unc51-Like Kinases activity. It can be evaluated as follows: quantification of Unc51-Like Kinases activity by liquid-phase electrophoretic differencial mobility of Unc51-Like Kinases fluorescent substrate with respect to its phosphorylated or dephosphorylated status (see US 7,105,304). Unc51-Like Kinases substrate used can be for example a peptide that upon phosphorylation by ULK, shows an electrophoretic migration advance as compared to dephosphorylated substrate, which shows a retardation. The substrate/product chromotagraphic separation is performed using a microfluidic device. Quantification of phospho- versus non-phospho substrate, using the fluorescence, gives a precise evaluation of Unc51-Like Kinases activity (NOVASCREEN-CALIPER LabChip 3000 (see US 7,105,304)
The following method is for example usefull and well known in the art for measuring these properties : total Unc51- Like Kinase quantification using a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) (Invitrogen) . A mouse monoclonal antibody specific for Unc51-Like Kinase is coated onto the wells of the microtiter strips provided. Samples, including a standard containing Unc51-Like Kinase, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the Unc51-Like Kinase antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for Unc51-Like Kinase is added to the wells.
In yet another embodiment, an in vivo assay is provided in which the capacity of test substance to reduce photoreceptor induced degeneration in vivo is quantified after intravitreal injection of the test substance. Mouse animal models can display a photoreceptor degeneration induced by different mutations, e . g. rdl mouse mutated in Phospho-di- esterase (Bowes et al., 1993, PNAS, 90, 2955-2959); Zaprinast induced photoreceptor degeneration (Vallaza-Deschamps et al., 2005, Eur. J. Neuroscience, 22, 1013-1022); Rhodopsin P23H mutated mouse or rat (Ollson, 1992, Neuron, 9, 815-830 and Lewin, 1998, Nat. Med., 4, 967-971). Photoreceptor survival quantification is assessed by histological section, co- labelling with photoreceptor specific marker (rhodopsin) , outer nuclear layer thickness quantification and cell counting. Electroretinogram is used to assess functionality of surviving photoreceptors (Frasson et al., 1999, Nat Med., 5, 1183-7) .
More specifically, modulators (more specifically inhibitors) of Unc51-Like Kinase expression are identified. For example, a cell or cell free mixture is contacted with a test substance and the expression of Unc51-Like Kinase mRNA or protein evaluated relative to the level of expression of Unc51-Like Kinase mRNA or protein in the absence of the test substance. When expression of Unc51-Like Kinase mRNA or protein is greater in the presence of the test substance than in its absence, the test substance is identified as a stimulator of Unc51-Like Kinase mRNA or protein expression. Alternatively, when expression of Unc51-Like Kinase mRNA or protein is less (statistically significantly less) in the presence of the test substance than in its absence, the candidate compound is identified as an inhibitor of Unc51-Like Kinase mRNA or protein expression. The level of Unc51-Like Kinase mRNA or protein expression can be determined by quantitative RTPCR or Western-blotting respectively using an antibody specific to Unc51-Like Kinase (Abnova, Evertest Biotech) .
The sources for test substances to be screened include natural sources, such as a cell extract (e.g., invertebrate cells including, but not limited to, bacterial, fungal, algal, and plant cells) and synthetic sources, such as chemical compound libraries or biological libraries such as antibody substance or peptide libraries. Agents are screened for the ability to either stimulate or inhibit the activity. There are a number of different libraries used for the identification of small molecule modulators, including: (i) chemical libraries, (ii) natural product libraries, and (iii) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules Chemical libraries consist of random chemical structures, or analogs of known compounds, or analogs of compounds that have been identified as "hits" or "leads" in prior drug discovery screens, some of which may be derived from natural products or from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to modulate activity.
In one aspect, the methods of the invention comprise steps of culturing a neuronal cell in the presence and absence of the modulators as set out above; measuring and comparing cell growth or survival or differentiation in the presence and absence of the modulator; and selecting a modulator that promotes increased survival or growth or differentiation of said neuronal cell. In a certain embodiment, the cell is selected from the group consisting of a hippocampal neuron or neural stem cell, a subventricular neuron or neuron stem cell, a cortical neuron or neuron stem cell, and a neuroblastoma cell. In another embodiment, the neuronal cell is a retinal cell selected from the group consisting of photoreceptors (rods and cones), ganglion cells, horizontal cells, amacrine cells, bipolar cells. Alternatively, the methods of the invention comprise steps of culturing a non neuronal retinal cell in the presence and absence of the modulators as set out above; measuring and comparing cell growth or survival or differentiation in the presence and absence of the modulator; and selecting a modulator that promotes increased survival or growth or differentiation of said cell. In this special embodiment, the cell is selected from the group consisting Muller cells and pigmented epithelial cells. In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating (activator or inhibitor) agent of the invention can be identified using a cell-based or a cell free assay, and the ability of the said agent to (i) inhibit the expression of a Unc51-Like Kinase protein and/or (ii) reduce the Unc51-Like Kinase mRNA levels can be confirmed in vivo, e. g. , in an animal such as a mouse model (e.g., animal models of retinal degeneration disclosed above such as P23H rat, P23H mouse or rdl mouse) .
This invention further pertains to novel compounds identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a Unc51-Like Kinase direct inhibitor, a Unc51-Like Kinase indirect inhibitor) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel compounds identified by the above-described screening assays can be used for treatments as described herein.
According to another special embodiment, said Unc51-Like Kinase inhibitor is an indirect inhibitor and is a nucleic acid.
The (Unc51-Like Kinase) modulator themselves also are aspects of the invention, as are mixtures of these modulators; mixtures of modulators with any other neurotropic factors; and pharmaceutical compositions comprising the modulators in pharmaceutically acceptable carriers. Non limiting examples of neurotropic factors according to the invention are NGF, BDNF, NT-3, 4, 5, or 6, CNTF, IGFI, IGFII, GDNF, GPA, bFGF, TGFB, and apolipoprotein E.
Pharmaceutical compositions are within the scope of the present invention. Such pharmaceutical compositions are compositions as disclosed above admixed with pharmaceutically or physiologically acceptable formulations. More particularly, the compositions of the present invention are comprising the Unc51-Like Kinase modulators of the Invention combined with further ingredients that are physiologically tolerable and do not typically produce adverse reactions when administered to a subject in need thereof (e.g. human). These ingredients are preferably "pharmaceutically or physiologically acceptable" as approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, and more particularly in humans. Further, the present invention relates to a composition containing an effective non-toxic dose of the compounds of the present invention (i.e. the Unc51-Like Kinase modulator) as well as pharmaceutically or physiologically acceptable carriers or solvents. The pharmaceutical compositions are obtained by blending a therapeutically active amount of at least one compound of the present invention with a pharmaceutically or physiologically acceptable carrier, which may have different forms depending on the desired administration route. The term "carrier" applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which an active compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition. According to one embodiment of the present invention carriers are suitable for immediate-release, i.e., release of most or all of the active ingredients over a short period of time, such as 60 minutes or less, and make rapid absorption of the drug possible.
According to preferred embodiment, the composition of the invention is delivered to areas within the eye, most preferably in the vicinity of the retina. It can be accomplished by injection, canula or other invasive device designed to introduce precisely metered amounts of a desired ophthalmic composition to a particular compartment or tissue within the eye (e.g. posterior chamber or retina) . An intraocular injection may be into the vitreous (intravitreal) , or under the conjunctiva (subconjunctival), or behind the eye (retrobulbar) , into the sclera, or under the Capsule of Tenon (sub- Tenon) , and may be in a depot form. Other intraocular routes of administration and injection sites and forms are also contemplated and are within the scope of the invention. In preferred embodiment the combination product of the invention will be delivered by sub-retinal or intra-vitreal injection.
In one embodiment, the ophthalmic composition is intraocularly injected (e.g. into the vitreous or sub retinal) to treat or prevent an ophthalmic condition related to cellular degenerative conditions, and more specifically retinal degenerative diseases. When administering the ophthalmic composition by intraocular injection, the active agents should be concentrated to minimise the volume for injection. Preferably, the volume for injection is less than about 5 ml. Volumes such as this may require compensatory drainage of the vitreous fluid to prevent increases in intraocular pressure and leakage of the injected fluid through the opening formed by the delivery needle. More preferably, the volume injected is between about 1.0 ml and 0.05 ml. Most preferably, the volume for injection is approximately 0.1 ml. For injection, a concentration of the compound of the present invention less than about 20 mg/ml may be injected. Preferably a dose of about 10 mg/ml is administered. Sample concentrations include, but are not limited to, about 5 μg/ml to about 50 μg/ml; about 25 μg/ml to about 100 μg/ml; about 100 μg/ml to about 200 μg/ml; about 200 μg/ml to about 500 μg/ml; about 500 μg/ml to about 750 μg/ml; about 500 μg/ml up to 1 mg/ml etc. preferred 50mg/ml. For viral injections with viruses expressing shRNA, sample concentrations include, but are not limited to, 103, lθ\ 105, 106, 107, 108, 109, 1010 1011 to 1012 genome copy/ml.
Intraocular injection may be achieved by a variety of methods well known in the art. For example, the eye may be washed with a sterilising agent such as Betadine® and the- pharmaceutical composition of the Invention is injected in an appropriate carrier with a fine gauge needle (e.g. 27 gauge) at a position in the eye such that the compound will settle to the posterior pole towards the ventral surface. It may be necessary to prepare the eye for injection by application of positive pressure prior to injection. In some cases, preliminary vitrectomy may be necessary. Local anaesthetic or general anaesthetic may be necessary.
The syringe used in practicing the method of this invention is suitably one which can accommodate a 21 to 40 gauge needle and is preferably of a small volume, for example 1.5 ml, or more preferably 0.1 ml. Although it is possible that the needle and syringe may be of the type where the needle is removable from the syringe, it is preferred that the arrangement is of a unitary syringe/needle construction. This would clearly limit the possibility of disengagement of the needle from the syringe. It is also preferred that the arrangement be tamper evident. The pharmaceutical composition of the present invention may therefore be provided in the form of a single unit dose, or separated unit doses each containing part of the combination product, in a pre-prepared syringe ready for administration.
A suitable style of syringe is, for example, sold under the name of Uniject® manufactured by Becton Dickinson and Company. In this style of syringe, the material is expelled through the needle into the eye by pressure applied to the sides of a pliable reservoir supplying the needle, rather than by a plunger. As the name implies, the construction of the reservoir and needle forms a single unit.
Topical application of composition of the invention for the treatment or prevention of disorders according to the present invention may be as ointment, gel or eye drops. The topical ophthalmic composition may further be an in situ gellable aqueous formulation. Such a formulation comprises a gelling agent in a concentration effective to promote gelling upon contact with the eye or with lacrimal fluid in the exterior of the eye. Suitable gelling agents include, but are not limited to, thermosetting polymers such as tetra- substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamine) ; polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa- carrageenan and iota-carrageenan) , chitosan and alginate gums.
The phrase "in situ gellable" as used herein embraces not only liquids of low viscosity that form gels upon contact with the eye or with lacrimal fluid in the exterior of the eye, but also more viscous liquids such as semi-fluid and thixotropic gels that exhibit substantially increased viscosity or gel stiffness upon administration to the eye.
To prepare a topical ophthalmic composition for the treatment of disorders as disclosed herein, a therapeutically effective amount of the composition of the invention is placed in an ophthalmological vehicle as is known in the art. For example, topical ophthalmic formulations containing steroids are disclosed in US 5,041,434, whilst sustained release ophthalmic formulations of an ophthalmic drug and a high molecular weight polymer to form a highly viscous gel have been described in US 4,271,143 and US 4,407,792. Further GB 2007091 describes an ophthalmic composition in the form of a gel comprising an aqueous solution of a carboxyvinyl polymer, a water-soluble basic substance and an ophthalmic drug. Alternatively, US 4,615,697, discloses a controlled release composition and method of use based on a bioadhesive and a treating agent, such as an anti- inflammatory agent.
The composition of the invention may be also administered as a slow release formulation, with a carrier formulation such as microspheres, microcapsules, liposomes, etc., as a topical ointment or solution, an intravenous solution or suspension, or in an intraocular injection, as known to one skilled in the art to treat or prevent ophthalmic disorders. By "slow release", "time-release", "sustained release" or "controlled release" is meant that the therapeutically active component is released from the formulation at a controlled rate such that therapeutically beneficial levels (but below toxic levels) of the component are maintained over an extended period of time ranging from e.g., about 12 to about 24 hours, thus, providing, for example, a 12 hour or a 24 hour dosage form. A time-release drug delivery system may be administered intraocularly to result in sustained release of the combination product over a period of time. The composition may be in the form of a vehicle, such as a micro- or macro-capsule or matrix of biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, 'polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and other polymers such as those disclosed in US Patents Nos. 6,667,371; 6,613,355; 6,596,296; 6,413,536; 5,968,543; 4,079, 038; 4,093,709; 4,131,648; 4,138,344; 4,180,646; 4,304,767; 4,946,931, each of which is expressly incorporated by reference herein in its entirety, or lipids that may be formulated as microspheres or liposomes. A microscopic or macroscopic ophthalmic composition may be administered through a needle, or may be implanted by suturing within the eye, eg intravitreal cavity or sub-retinal space. Delayed or extended release properties may be provided through various formulations of the vehicle (coated or uncoated microsphere, coated or uncoated capsule, lipid or polymer components, unilamellar or multilamellar structure, and combinations of the above, etc.). The formulation and loading of microspheres, microcapsules, liposomes, etc and their ocular implantation are standard techniques known by one skilled in the art.
The invention also provides a method for the treatment or prophylaxis of ophthalmic disorders, said method comprising the step of administering a composition of the Invention in a biocompatible, biodegradable matrix, for example in the form of a gel or polymer which is preferably suited for insertion into the retina or into a cavity of the eye, anterior or posterior, as an implant. In the case that the pharmaceutical composition is delivered as an implant, it may be incorporated in any known biocompatible biodegradable matrix as a liquid, or in the form, for example, of a micelle using known chemistry or as microparticles .
Slow or extended-release delivery systems include any of a number of biopolymers (biological-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, can be utilized with the compositions described herein to provide a continuous or long term source of therapeutic compound (i.e. the 4EBPs modulator) . In one form, implants used in the method of the present invention are formulated with composition of the invention entrapped within the bio-erodible polymer matrix. Release of the therapeutic compounds is achieved by erosion of the polymer followed by exposure of previously entrapped compound to the vitreous, and subsequent dissolution and release of compound. The release kinetics achieved by this form of drug release are different than that achieved through formulations which release drug through polymer swelling, such as with hydrogels such as methylcellulose . In that case, the therapeutic compound is not released through polymer erosion, but through polymer swelling, which releases active compound as liquid diffuses through the pathways exposed. The parameters which determine the release kinetics include the size of the therapeutic compound particles, the water solubility of the active compound, the ratio of active compound to polymer, the method of manufacture, the surface area exposed, and the erosion rate of the polymer.
Exemplary biocompatible, non-biodegradable polymers of particular interest include polycarbamates or polyureas, particularly polyurethanes, polymers which may be cross-linked to produce non- biodegradable polymers such as cross-linked poly (vinyl acetate) and the like. Also of particular interest are ethylene-vinyl ester copolymers having an ester content of 4% to 80% such as ethylene-vinyl acetate (EVA) copolymer, ethylene-vinyl hexanoate copolymer, ethylene-vinyl propionate copolymer, ethylene-vinyl butyrate copolymer, ethylene-vinyl pentantoate copolymer, ethylene-vinyl trimethyl acetate copolymer, ethylene-vinyl diethyl acetate copolymer, ethylene- vinyl 3-methyl butanoate copolymer, ethylene-vinyl 3-3- dimethyl butanoate copolymer, and ethylene-vinyl benzoate copolymer.
Additional exemplary naturally occurring or synthetic non- biodegradable polymeric materials include poly (methylmethacrylate) , poly (butylmethacrylate) , plasticized poly (vinylchloride) , plasticized poly (amides) , plasticized nylon, plasticized soft nylon, plasticized poly (ethylene terephthalate) , natural rubber, silicone, poly (isoprene) , poly (isobutylene) , poly (butadiene) , poly (ethylene) , poly (tetrafluoroethylene) , poly (vinylidene chloride), poly (acrylonitrile, cross-linked poly (vinylpyrrolidone) , poly (trifluorochloroethylene) , chlorinated poly (ethylene) , poly (4, 4'- isopropylidene 'diphenylene carbonate), vinylidene chloride-acrylonitrile copolymer, vinyl chloridediethyl fumarate copolymer, silicone, silicone rubbers (especially the medical grade), poly (dimethylsiloxanes) , ethylene- propylene rubber, silicone-carbonate copolymers, vinylidene chloride- vinyl chloride copolymer, vinyl chloride-acrylonitrile copolymer, vinylidene chloride-acrylonitrile copolymer, poly (olefins) , poly (vinyl- olefins), poly (styrene) , poly(halo- olefins) , poly (vinyls) , poly (acrylate) , poly (methacrylate) , poly (oxides) , poly (esters) , poly (amides) , and poly (carbonates) .
Diffusion of the composition from the implant may also be controlled by the structure of the implant. For example, diffusion of the pharmaceutical composition from the implant may be controlled by means of a membrane affixed to the polymer layer comprising the drug. The membrane layer will be positioned intermediate to the polymer layer comprising the pharmaceutical composition and the desired site of therapy.
The skilled reader will appreciate that the duration over which any of the composition used in the method of the invention will dwell in the ocular environment will depend, inter alia, on such factors as the physicochemical and/or pharmacological properties of the compounds employed in the formulation, the concentration of the compound employed, the bioavailability of the compound, the disease to be treated, the mode of administration and the preferred longevity of the treatment. Where that balance is struck will often depend on the longevity of the effect required in the eye and the ailment being treated.
The frequency of treatment according to the method of the invention is determined according to the disease being treated, the deliverable concentration of the composition and the method of delivery. If delivering the combination product by intravitreal injection, the dosage frequency may be monthly. Preferably, the dosage frequency is every three months. The frequency of dosage may also be determined by observation, with the dosage being delivered when the previously delivered combination product is visibly cleared. In general, an effective amount of the compound is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
Compositions prepared for used in the method of the present invention to prevent or treat ophthalmic disorders will preferably have dwell times from hours to many months and possibly years, although the latter time period requires special delivery systems to attain such duration and/or alternatively requires repetitive administrations. Most preferably the combination product for use in the method of the invention will have a dwell time (ie duration in the eye) of hours (i.e. 1 to 24 hours), days (i.e. 1, 2, 3, 4, 5, 6 or 7 days) or weeks (i.e. 1, 2, 3, 4 weeks). Alternatively, the combination product will have a dwell time of at least a few months such as, 1 month, 2 months, 3 months, with dwell times of greater than 4, 5, 6, 7 to 12 months being achievable.
If desired, the method or use of the invention can be carried out alone, or. in conjunction with one or more conventional therapeutic modalities (such as photodynamic therapy, laser surgery, laser photocoagulation or one or more biological or pharmaceutical treatments. These methods are well known from the skilled man in the art and widely disclosed in the literature) . The use of multiple therapeutic approaches provides the patient with a broader based intervention. In one embodiment, the method of the invention can be preceded or followed by a surgical intervention. In another embodiment, it can be preceded or followed by photodynamic therapy, laser surgery, laser photocoagulation. Those skilled in the art can readily formulate appropriate therapy protocols and parameters which can be used.
The present Invention further concerns a method for improving the treatment of a patient which is undergoing one or more conventional treatment as listed above, which comprises co-treatment of said patient along with at least one Unc51-Like Kinase modulator of the present invention.
The present invention further concerns a method for treating cell degenerative ophthalmic conditions, and related diseases, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one Unc51-Like Kinase modulator of the present ' invention.
The present Invention further concerns a method for improving the treatment of a patient which is undergoing one or more conventional treatment as listed above, which comprises co-treatment of said patient along with at least one Unc51-Like Kinase inhibitor of the present invention, and preferably said inhibitor is an indirect inhibitor.
The present invention further concerns a method for treating cell degenerative ophthalmic conditions, and related diseases, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one Unc51-Like Kinase modulator of the present invention (preferably an indirect Unc51-Like Kinase inhibitor) , effective to limit cellular degeneration in the eye, and more preferably retinal cell degeneration, and most preferably photoreceptor cell degeneration.
The term "therapeutically effective amount" in the context of degenerative diseases or conditions described herein refers to an amount effective to achieve measurable improvement (compared to an untreated control) as assessed by any relevant medical parameter used to evaluate subjects receiving treatment for the disease or condition.
According to a first embodiment, the term "cell degenerative ophthalmic conditions, and related diseases" relates to a disorder characterized by retinal cell degeneration, i.e. degeneration affecting one or more of retinal cells, and more preferably photoreceptors (rods and/or cones) .
According to a preferred embodiment, the term "cell degenerative ophthalmic conditions, and related diseases" relates more specially to a disorder characterized by photoreceptor cell degeneration. It is defined herein as any condition marked by a decrease in photoreceptor cell number and/or function. In one embodiment of the invention, the disorder is age-related macular degeneration. As used herein, "age-related macular degeneration" is defined as an age- related disorder which causes a decrease in visual acuity and possible loss of central vision. In another embodiment of the invention, the disorder is retinitis pigmentosa. Other disorders characterized by photoreceptor cell degeneration according to the invention include edema (e.g. macular and retinal edema), ischemic conditions and uveitis.
In a further aspect, a mammalian subject is treated with a therapeutically effective amount of a modulator of the invention, which is an amount that is sufficient to induce a desired response in the treated subject. Thus, a biologically or therapeutically effective amount of a modulator may be the amount that interferes with physiological activity of the treated mammal in a non-lethal manner. For the purpose of diagnosing the illness or of evaluating the treatment efficacy of disorder characterized by retinal cell degeneration, it is usual to rely on such tests as examination of the fundus of the eye, examination of the visual field, electroretinograms, fluorangiography, and visus examination:
examination of the fundus of the eye aims at assessing the condition of the retina and to look for the presence of the characteristic pigment spots on the retinal surface, which in the illness assume a characteristic "osteoblast-like" appearance; examination of the visual field makes it possible to evaluate the sensitivity of the various parts of the retina to light stimuli; visual fields 'can be measured by means of microperimetry, Goldmann dynamic perimetry, and photopic and scotopic (dark-adapted) automated static perimetry. electroretinogram (ERG) consists of recording the electrical activity of the retina in response to particular light stimuli, thus making possible distinct valuations of the functionality of the two different types of photoreceptors (-i.e. cone cells and rod cells). The electroretinogram is a very important examination for diagnosing retinitis pigmentosa, because, even when the illness is in its initial stages, the resulting trace is almost always either very flat or altogether absent; Even more importantly, multi-focal ERG make it possible to detect defects and benefits on few photoreceptors.
Retinal thickness is determined by optical coherence tomography. fluorangiography is performed by means of the intravenous injection of a fluorescent substance and subsequent photography of the retina at different times. Due to blood circulation, in fact, the fluorescent substance arrives at the retina, where it colours the arteries, the capillaries and the veins and thus renders them visible, as also the functional state of their walls; visus examination permits a valuation of visual acuity and consists of the patient reading letters of different sizes at a distance of three metres.
According to another embodiment, the compound or composition of the present invention allows to improve at least one disease related parameter selected in the group of parameters measured by examination of the fundus of the eye, examination of the visual field, electroretinograms, fluorangiography, and visus examination.
The term subject in need thereof as used herein refers to a mammal preferably a human.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.
The invention described herein may include one or more range of values (e.g. size, concentration, etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

Claims

1 - Composition comprising at least one Unc51-Like Kinase modulator in combination with a pharmaceutically acceptable carrier for treating and/ or preventing cellular degenerative disease in a subject in need thereof.
2 - The composition of claim 1 wherein said Unc51-Like Kinase modulator is a Unc51-Like Kinase inhibitor or partial inhibitor.
3 - The composition of claim 2 wherein said inhibited or partially inhibited Unc51-Like Kinase (ULK) is selected in the group consisting of ULKl, ULK2 and ULK3.
4 - The composition of claims 1 to 3 wherein said Unc51- Like Kinase modulator is selected in the group consisting in an antisense to ULKs-encoding nucleic acid molecules, a siRNA, a miRNA, a ribozyme, a shRNA, a nucleic acid capable of modulating ULKs nucleic acid expression, a vector more particularly an expression vector expressing at least one nucleic acid capable of modulating ULKs nucleic acid expression.
5 - Use of any of the composition of claims 1 to 4 for for treating cell degenerative conditions, and related diseases .
6 - The use of claim 5, wherein said cell degenerative conditions, and related diseases is a disorder characterized by photoreceptor cell degeneration.
7 - The use of claim 6, wherein said disorder is selected in the group consisting in age-related macular degeneration, retinitis pigmentosa, edema, macular and retinal edema, ischemic conditions and uveitis.
PCT/EP2008/010922 2007-12-28 2008-12-19 Compositions and methods for prophylaxis and treatment of cellular degenerative ophthalmic disorders WO2009083185A2 (en)

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WO2011042030A1 (en) * 2009-10-06 2011-04-14 Tallinn University Of Technology Inhibition or activation of serine/threonine ulk3 kinase activity
US20130040894A1 (en) * 2009-10-06 2013-02-14 Tallinn University Of Technology Inhibition or activation of serine/threonine ulk3 kinase activity
WO2016033100A1 (en) * 2014-08-25 2016-03-03 Salk Institute For Biological Studies Novel ulk1 inhibitors and methods using same
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