WO2017198704A1

WO2017198704A1 - Yif1b for the diagnosis, prevention and / or treatment of ciliopathies

Info

Publication number: WO2017198704A1
Application number: PCT/EP2017/061834
Authority: WO
Inventors: Justine MASSON; Jorge Luis DIAZ; Michèle DARMON
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Paris Descartes
Priority date: 2016-05-18
Filing date: 2017-05-17
Publication date: 2017-11-23

Abstract

The present invention relates to the diagnosis, prevention and/or treatment of ciliopathies. The Inventors showed that Yif1B protein is involved in cilia integrity and function and that a lack of Yif1B protein in an animal model leads to alterations similar to some of clinical feature of human ciliopathies. The present invention thus particularly relates to an agent for Yif1B protein expression, preferably a nucleic acid encoding Yif1B protein or a fragment thereof or cells expressing Yif1B protein, for use in the prevention and/or treatment of a ciliopathy, for example characterized by ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy and/or male infertility. The present invention also relates to a method for diagnosing a ciliopathy based on the detection of the Yif1B gene or protein and to a method for selecting a compound useful in the prevention and/or treatment of a ciliopathy.

Description

YIF1 B FOR THE DIAGNOSIS, PREVENTION AND / OR TREATMENT OF

CILIOPATHIES

Field of the invention

The present invention relates to methods for diagnosing, preventing and/or treating ciliopathies.

Background

Ciliopathies are genetic diseases caused by dysfunction of primary cilia and/or motile cilia. Primary cilia are sensory organelles. Motile cilia for example encompass the flagellum of spermatozoids.

Primary cilia and flagella project from the apical side of the cells and emerge from the basal body, a modified centriole structure anchored to the plasma membrane. Their structural core is a microtubule-based cytoskeleton called the axonema, which is surrounded by a membrane contiguous with the cell plasma membrane, but expressing specific signaling molecules. The axonema of primary cilia is composed of nine doublets of microtubules, whereas the axonema of motile cilia usually comprises two additional central microtubule singlets.

Many genes are now known to be involved in ciliopathies, such as for example PKD1 , PKD2 and PKHD1 genes in polycystic kidney disease, ALMS1 gene in Alstrom syndrome or NHP1 gene in Joubert syndrome, nephronophthisis and Senior-Loken syndrome.

However, the whole genes and proteins involved in ciliopathies are far from being known and some ciliopathies are certainly not yet diagnosed as such.

Yif 1 B protein is an intracellular membrane-bound protein belonging to the Yip family. Yif 1 B protein was first identified as an intracellular protein interacting with 5-HT_1A serotonin receptor and controlling its targeting to neuronal dendrites (Carrel et ai, 2008, The Journal of Neuroscience, 28(32): 8063-8073). Yif 1 B protein was then shown to shuttle between several intracellular compartments (the endoplasmic reticulum (ER), the intermediate compartment (IC) and the Golgi apparatus) and to be involved in anterograde traffic from the ER to the plasma membrane, as well as in the maintenance of the Golgi structure; these results were obtained in vitro using Yif 1 B depleted Hela cells and hippocampal neurons cultured from Yif 1 B KO mice (Alterio et at., 2015, Traffic, 16: 978-993).

There is therefore still a need to provide means for the diagnosis of ciliopathies, in particular of ciliopathies characterized by ataxia, intellectual deficiency, visual dysfunction kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy and/or male infertility, as well as appropriate drugs useful for the prevention and/or treatment of said diseases. Description of the invention

The Inventors have surprisingly shown that Yif 1 B protein is involved in cilia integrity and function and that a lack of Yif 1 B protein in animal model leads to alterations similar to some of clinical feature of human ciliopathies. More particularly, the Inventors have studied the phenotype of Yif 1 B KO (knock-out) mice and have unexpectedly found: i) a substantial progressive Purkinje cell degeneration and loss of motor coordination learning abilities, ii) an altered visual perception involving a dysfunction of the photoreceptors, iii) altered olfactory performances and iv) the absence of progeny when mating with homozygote male Yif 1 B KO mice, which was shown to result from the absence of spermatozoids in the epididymis but not from an alteration of male hormone levels. Finally, the Inventors have shown that these different altered phenotypes of Yif 1 B KO mice result from a common alteration of the primary cilia and flagella structure.

Consequently, Yif 1 B protein and/or gene can be efficiently used for the diagnostic of ciliopathies, more particularly of ciliopathies involving a defect in Yif 1 B protein, as well as for the prevention and/or treatment of ciliopathies, more particularly of ciliopathies involving a defect in YifI B protein, said ciliopathies being for example characterized by ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy and/or male infertility. The prevention and/or treatment of said ciliopathies may be achieved by gene therapy, for example using a nucleic acid encoding Yif 1 B protein or a system of gene correction of a mutated Yif 1 B gene or a mutated Yif 1 B promoter, or by cellular therapy, for example using cells expressing Yif 1 B protein, such as stem cells expressing a Yif 1 B protein.

One object of the invention is thus an agent for Yif 1 B protein expression for use in the prevention and/or treatment of a ciliopathy, preferably a ciliopathy involving a defect in the Yif 1 B gene and/or protein. Said agent is preferably selected from the group consisting of a nucleic acid encoding Yif 1 B protein, a nucleic acid encoding a fragment of a Yif 1 B protein, a nucleic acid encoding a Yif 1 B promoter or a fragment of Yif 1 B promoter, cells expressing Yif 1 B protein and their combinations.

Another object of the invention is a method for preventing and/or treating a ciliopathy in a subject in need thereof, said method comprising a step of administering to said subject an effective amount of an agent for Yif 1 B protein expression, preferably of a nucleic acid encoding Yif 1 B protein, a nucleic acid encoding a fragment of a Yif 1 B protein, a nucleic acid encoding a Yif1 B promoter or a fragment of Yif1 B promoter and/or of cells expressing Yif 1 B protein.

Said ciliopathy may be characterized by ataxia, intellectual deficiency, visual dysfunction, male infertility, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy or their combinations.

Said ciliopathy may be selected in the group consisting of Alstrom syndrome, Joubert syndrome, Meckel syndrome, nephronophthisis, Bardet-Biedl syndrome, oral- facialdigital syndrome type 1 , Senior-Loken syndrome, polycystic kidney disease, polycystic liver disease, primary ciliary dyskinesia, asphyxiating thoracic dysplasia, Marden-Walker syndrome, situs inversus/isomerism, retinal degeneration, cerebello- oculo-renal syndrome, Ellis-van Creveld syndrome, Jeune asphyxiating thoracic dystrophy, Leber congenital maurosis and their combinations.

The Yif 1 B protein preferably comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 2.

The nucleic acid encoding Yif 1 B protein preferably comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 .

The nucleic acid encoding Yif 1 B protein, the nucleic acid encoding a fragment of Yif 1 B protein or the nucleic acid encoding a YifI B promoter or a fragment of Yif 1 B promoter may be provided in the form of a vector comprising said nucleic acid. Said vector may be a non-viral vector or a viral vector.

The nucleic acid encoding Yif 1 B protein, the nucleic acid encoding a fragment of Yif 1 B protein and/or the nucleic acid encoding a YifI B promoter or a fragment of Yif 1 B promoter may be used in combination with at least one nucleic acid encoding an endonuclease CAS and at least one nucleic acid encoding a guide RNA.

The agent for Yif 1 B protein expression, preferably the nucleic acid encoding Yif 1 B protein, the nucleic acid encoding a fragment of a Yif 1 B protein, the nucleic acid encoding a YifI B promoter or a fragment of YifI B promoter and/or the cells expressing YifI B protein, for use as defined above, may be used or administered by the arterial route, venous route, nasal route, intra-tissue route, for example intra-vitreal, subretinal, intra- testicular and/or intracerebral route, preferably in at least one affected tissue, and/or intraperitoneal route.

Still another object of the invention is a vector comprising a nucleic acid encoding Yif 1 B protein, wherein said nucleic acid comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 . Still another object of the invention is a method for diagnosing whether a subject suffers or is at risk of suffering from a ciliopathy, wherein said method comprises detecting in a biological sample of said subject (i) if at least one mutation leading to a non-functional Yif1 B protein or a Yif1 B protein with an altered function is present in the Yif1 B gene and/or (i) if a functional Yif 1 B protein is expressed by the cells, wherein the presence of said at least one mutation or the absence of a functional Yif 1 B protein indicates that said subject suffers from or is at risk of suffering from said ciliopathy.

Still another object of the invention is a method for selecting a compound useful in the prevention and/or treatment of a ciliopathy, wherein said method comprises:

a) administering a test compound to a Yif 1 B knock-out animal, to obtain a treated Yif 1 B knock-out animal,

b) assessing at least one symptom of said ciliopathy in the treated Yif 1 B knockout animal,

c) selecting said test compound as being useful in the prevention and/or treatment of said ciliopathy when at least one symptom of the ciliopathy is improved or absent in the treated Yif 1 B knock-out animal.

a) adding a test compound in a Yif 1 B knock-out ciliated cell culture, to obtain a treated cell culture,

b) assessing the structure of the cilia of the cells in the treated cell culture, c) selecting said test compound as being useful in the prevention and/or treatment when the structure of the cilia of the cells in the treated cell culture is not altered.

Yif 1 B protein

Yif 1 B protein is an intracellular membrane-bound protein, also known as YIP1 - interacting factor homolog B.

Yif 1 B protein is encoded by the Yif 1 B gene.

The human Yif 1 B gene is located on chromosome 19 and comprises 8 exons. The human Yif 1 B gene ID is for example 90522.

The sequence of the human Yif 1 B gene is for example the sequence SEQ ID NO: 3 of access number NC_000019.9 (NCBI database), as available on January 14, 2016. Several isoforms are encoded by the Yif 1 B gene, said isoforms resulting from an alternative splicing.

The human Yif 1 B protein may be the isoform of 314 amino acids or a variant thereof.

The Yif 1 B protein for the therapeutic use according to the invention is a functional Yif 1 B protein.

By "functional Yif 1 B protein" or "Yif 1 B protein with a functional activity", it is herein meant that, when expressed in a cell, this YifI B protein allows a normal kinetics of anterograde intracellular traffic and an unaltered primary cilia structure.

Determining whether a Yif 1 B protein is functional may be assessed by any method well-known by the skilled person, for example by assessing the kinetics of anterograde intracellular traffic and/or by analyzing the structure of primary cilia in the cells expressing said Yif 1 B protein.

Determining whether a Yif 1 B protein is functional by assessing the anterograde intracellular traffic may be carried out as disclosed in Aterio et al., 2015, Traffic, 16: 978- 993). Briefly, test cells expressing said Yif 1 B protein are transfected with a plasmid encoding the thermosensitive GFP-tagged VSVG-ts045 protein (see for example Sciaky N. et al., 1997, J Cell Biol, 139: 1 137-1 155). The transfected cells are then cultured at 39,5°C for 24h, in order to allow accumulation of the protein within the ER, before being cultured at 32°C (permissive conditions for the protein trafficking) and in the presence of 50 μg/ml cycloheximide to prevent any further synthesis. The kinetics of synchronous transport in the different compartments (from endoplasmic reticulum to Golgi apparatus, followed by plasma membrane) is then assessed and may be compared to a control cell expressing a functional Yif 1 B protein and/or a control cell knock-out for the Yif 1 B protein. The test cells used for transfection are for example HeLa cells, or primary cell cultures derived from the Yif 1 B KO mouse (mouse embryonic fibroblasts, neurons, ependymocytes, spermatozoids, retinal cells or renal cells). In one embodiment, the test cells may be Yif 1 B KO cells transfected with a nucleic acid encoding the Yif 1 B protein, the function activity of which is assessed. In one other embodiment, the test cells are cells from a subject, preferably a subject suffering or being likely to suffer from a ciliopathy. The test cells used for analyzing the primary cilia structure are preferably mammal cells, for example human or non-human mammal cells. An accelerated kinetics leading to an early appearance at the cell membrane by comparison to those in a control cell expressing a functional Yif 1 B protein indicates a non-functional Yif 1 B protein. Determining whether a Yif 1 B protein is functional by analyzing the primary cilia structure in cells expressing said protein may be performed by confocal microscopy analysis, after axonema immunostaining, for example using anti-Arl13B, anti-gamma- tubulin, anti-adenylate cyclase III and/or anti-acetylated tubulin antibodies. Briefly, cells expressing said Yif 1 B protein are cultured in a medium poor in serum, in order to induce appearance of primary cilia. The number of primary cilia by cell and/or the length of their axonema may be then measured and/or the internal structure of the primary cilia may be assessed. On that purpose, image analysis software such as Volocity may be used. An unaltered primary cilium is particularly characterized by the presence of a single axonema by primary cilium, by a normal ciliary axonema length and a normal number of primary cilia by cell, whereas an altered primary cilium is particularly characterized by the presence of a double axonema by primary cilium and/or by an increased or decreased ciliary axonema length and/or optionally an abnormal number of primary cilia by cell. For example, an altered primary cilium in fibroblasts is characterized by a decreased axonema length, whereas an altered primary cilium in neurons, in particular in the cerebellum and/or olfactory bulb (OB) is characterized by an increased axonema length. The structure of the primary cilia may be compared to those of control cells expressing a functional YifI B protein and/or of control cells knock-out for the Yif 1 B protein. The cells used for analyzing the primary cilia structure may be fibroblasts (for example embryonic fibroblasts, or skin fibroblasts), ependymocytes, retinal cells, renal cells, neuronal cells or spermatozoids. In one embodiment, the cells used for analyzing the primary cilia structure may be Yif 1 B KO cells transfected with a nucleic acid encoding the Yif 1 B protein, the function activity of which is assessed. In one other embodiment, the cells are cells from a subject, preferably a subject suffering or being likely to suffer from a ciliopathy. The cells used for analyzing the primary cilia structure are preferably mammal cells, for example human or non-human mammal cells.

The decrease and/or increase in the axonema length in an altered primary cilium is preferably of at least 5 %, more preferably at least 8 %, more preferably at least 10 %, by comparison to the axonema length in an unaltered primary cilium, i.e. in cells expressing a functional Yif 1 B protein.

The axonema length in an altered primary cilium may for example be increased by at least 10% in the neurons of the cerebellum and/or at least 20% in the neurons of the olfactory bulb, by comparison to the axonema length in an unaltered primary cilium.

The axonema length in an altered primary cilium may for example be decreased by at least 25% in the skin fibroblasts, by comparison to the axonema length in an unaltered primary cilium. By "a medium poor in serum", it is herein meant a medium containing at most 0.05% of serum.

The Yif1 B protein is preferably of mammalian origin.

The expression "YifI B protein of mammalian origin" includes a mammalian YifI B protein or a variant thereof.

A variant of a mammalian Yif 1 B protein is a functional Yif 1 B protein that has a sequence at least 80% identical to said mammalian YifI B protein, preferably at least 85% identical to said mammalian Yif 1 B protein, more preferably at least 90% identical to said mammalian Yif 1 B protein, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to said mammalian Yif 1 B protein.

The term "mammalian" includes human and non-human mammalian.

The expression "non-human mammalian" for example includes rat, murine, pig, cat, dog, rabbit or primate.

In a preferred embodiment, the Yif 1 B protein is a human Yif 1 B protein or a variant thereof.

A functional human YifI B protein for use according to the invention is the isoform of 314 amino acids or a variant thereof.

A functional human YifI B protein is for example of sequence SEQ ID NO: 2 and corresponds to the sequence of access number Q5BJH7.1 (isoform 1 ) in the Swiss-Prot database, as available on January 12, 2016. This entry includes five additional isoforms generated through possible alternative splicing.

For example, the YifI B protein may comprise or consist of a sequence at least 80% identical to sequence SEQ ID NO: 2, preferably at least 85% identical to sequence SEQ ID NO: 2, more preferably at least 90% identical to sequence SEQ ID NO: 2, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 2, provided that said Yif 1 B protein is functional.

A preferred human YifI B protein comprises or consists of sequence SEQ ID NO: 2.

As defined herein, an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutation(s), such as deletion(s), insertion(s) and/or substitution(s) compared to the reference sequence.

In case of substitution, the substitution preferably corresponds to a conservative substitution as indicated in the Table 1 below. In a preferred embodiment, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by conservative substitutions.

Table 1

In another preferred embodiment, the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a naturally-occurring variant of the reference sequence.

In another preferred embodiment, the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a homologous sequence derived from another mammalian species than the reference sequence.

In a preferred embodiment, the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence differs from the reference sequence by conservative substitution(s) and/or corresponds to a naturally-occurring variant of the reference sequence and/or corresponds to a homologous sequence derived from another mammalian species than the reference sequence.

By "a sequence at least x% identical to a reference sequence", it is intended that the sequence is identical to the reference sequence or differ from the reference sequence by up to 100-x amino acid alterations per each 100 amino acids of the reference sequence.

The alignment and the determination of the percentage of identity may be carried out manually or automatically using for instance the Needle program which is based on the Needleman and Wunsch algorithm, described in Needleman and Wunsch (1970) J. Mol Biol. 48:443-453, with for example the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62, gap open penalty: 10 and gap extend penalty: 0.5, end gap penalty: false, end gap open penalty = 10, end gap extend penalty = 0.5; and the following parameters for polynucleotide sequence comparison: comparison matrix: DNAFULL; gap open penalty = 10, gap extend penalty = 0.5, end gap penalty: false, end gap open penalty = 10, end gap extend penalty = 0.5. Agent for Yif 1 B protein expression

By "agent for Yif 1 B protein expression", it is herein meant a compound allowing the expression of a functional Yif 1 B protein, preferably by increasing and/or restoring the expression of a functional Yif 1 B protein in the cells and/or providing cells expressing a functional Yif 1 B protein.

Said agent may result (i) directly in the expression of a functional Yif 1 B protein, for example when using a nucleic acid encoding Yif 1 B protein and/or cells expressing Yif 1 B protein, and/or (ii) indirectly, for example by correcting and/or replacing the endogenous Yif 1 B gene and/or correcting a defect in the transcription and/or expression machinery.

Said agent is preferably selected from the group consisting of a nucleic acid encoding Yif 1 B protein, a nucleic acid encoding a fragment of a Yif 1 B protein, a nucleic acid encoding a Yif 1 B promoter or a fragment of Yif 1 B promoter, cells expressing Yif 1 B protein and their combinations.

Nucleic acid encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter

The present invention particularly relates to a nucleic acid encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter. The Yif 1 B protein is a functional Yif 1 B protein as defined above in the section "Yif1B protein".

The Yif 1 B promoter is a functional Yif 1 B promoter.

In the context of the present invention, a "nucleic acid" refers to the phosphate ester polymeric form of ribonucleosides (also called "RNA molecule"), deoxyribonucleosides (also called "DNA molecule") or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form or a double- stranded form.

The nucleic acid is preferably an isolated nucleic acid.

The term "isolated" in reference to a biological component (such as a nucleic acid, a vector or a protein) refers to a biological component that has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, cells, and organelles. "Isolated nucleic acids" or "isolated vectors" include nucleic acid molecules purified by standard purification methods. These terms also encompass nucleic acids and vectors prepared by amplification and/or cloning, as well as chemically synthesized nucleic acids and vectors.

The nucleic acid encoding human Yif1 B protein for example comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 , preferably at least 85% identical to sequence SEQ ID NO: 1 , more preferably at least 90% identical to sequence SEQ ID NO: 1 , still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 1 . A definition of the percentage of sequence identity is provided above in the section

"Yif1B protein".

A nucleic sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutation(s), such as deletion(s), insertion(s) and/or substitution(s) compared to the reference sequence.

In case of substitution, the substitution preferably corresponds to a silent substitution or a substitution leading to a conservative substitution in the translated amino acid sequence, by comparison to the reference sequence, for example as indicated in the Table 1 above.

In a preferred embodiment, the nucleic sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by silent substitution(s) and/or substitution(s) leading to a conservative amino- acid substitution.

The nucleic acid is preferably cloned into a vector. Vector comprising a nucleic acid encoding YifI B protein, a fragment of YifI B protein, Yif1 B promoter and/or a fragment of Yif1 B promoter

The nucleic acid encoding YifI B protein, a fragment of YifI B protein, YifI B promoter and/or a fragment of YifI B promoter is preferably provided in the form of a vector comprising said nucleic acid.

The expressions "YifI B protein" and "nucleic acid encoding YifI B protein, a fragment of YifI B protein, YifI B promoter and/or a fragment of YifI B promoter" are as defined above in the sections of the same name.

The present invention thus also relates to a vector comprising a nucleic acid encoding Yif1 B protein, a fragment of Yif1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter. The "vector comprising a nucleic acid encoding Yif1 B protein, a fragment of Yif1 B protein, Yif 1 B promoter and/or a fragment of Yif1 B promoter" is thereafter also referred to as "vector".

The vector is preferably an isolated vector.

A preferred vector is a vector comprising a nucleic acid encoding Yif 1 B protein, wherein said Yif 1 B protein comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 2, more preferably at least 85% identical to sequence SEQ ID NO: 2, more preferably at least 90% identical to sequence SEQ ID NO: 2, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 2.

A preferred vector is a vector comprising a nucleic acid encoding Yif 1 B protein, wherein said nucleic acid comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 , more preferably at least 85% identical to sequence SEQ ID NO: 1 , more preferably at least 90% identical to sequence SEQ ID NO: 1 , still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 1 .

The vector may have a tropism for a specific tissue, for example a brain, kidney, liver, testes, olfactory and/or retina tropism.

The expression "a vector having a tropism for a specific tissue" means a vector that is preferentially expressed in said tissue by comparison to the other parts of a body, for example after intravenous administration.

The tropism for a specific tissue may result from an intrinsic tropism, for example in the case of a viral vector, and/or may result from the use of a tissue specific promoter and/or from the use of a tissue targeting molecule, in particularly in the case of a non-viral vector.

The vector preferably comprises a Yif 1 B protein expression cassette, i.e. a nucleic acid encoding Yif 1 B protein placed under the control of at least one expression signal allowing its expression.

The expression signal is particularly selected among a promoter, a terminator, an enhancer and their combinations.

Suitable promoters, terminators and enhancers are well-known by the skilled person. Said promoter may be CMV promoter, EF-1 alpha promoter or a tissue-specific promoter. Example of brain-specific promoter is synapsin. Examples of testis-specific promoter are Gata4, E1 b or Stra8. Examples of retinal-specific promoter are PGK (phosphoglycerate kinase 1 ) promoter, EFS (elongation factor-1 ) promoter, rhodopsin (Rho) promoter or the chimeric IRBPe/GNAT2 promoter.

The vector comprising a nucleic acid encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter may be a non-viral vector or a viral vector.

In one embodiment, the vector comprising a nucleic acid encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter is a non- viral vector.

A non-viral vector may be selected in the group consisting of a naked DNA, a plasmid, a liposomal nucleic acid complex and a carrier-associated nucleic acid.

By "plasmid", it is herein meant a double-stranded circular DNA. The plasmid may include a marker gene enabling to select the cells comprising said plasmid, an origin of replication to allow the cell to replicate the plasmid and/or a multiple cloning site allowing the insertion of a DNA fragment, in particular the nucleic acid encoding Yif 1 B protein according to the invention.

The liposomes used in liposomal DNA complexes are well-known in the art. Said liposomes may be cationic, anionic or neutral liposomes.

Non-limitative examples of carrier-associated nucleic acid are polymer-carried DNA and cationic lipids.

Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin Tm also known as DOTMA (N- [I- (2, 3-dioleyloxy) propyls N, N, N-trimethylammonium chloride), DOTAP (1 , 2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl- ammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (3 beta- (N- (Ν', Ν'- dimethyl aminomethane)-carbamoyl) cholesterol). Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine).

In another embodiment, the vector comprising a nucleic acid encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter is a viral vector.

By "viral vector", it is herein meant a recombinant viral vector. The viral vector is preferably selected in the group consisting of a retrovirus vector (preferably a lentivirus vector), an adenovirus vector and an adeno-associated virus vector.

A retrovirus vector is a single-stranded positive-sense RNA that encodes a transcriptase enabling to generate double-stranded DNA.

A lentivirus vector is a single-stranded RNA that integrates in the genome of the host, thereby allowing long term expression of the nucleic acid.

An adenovirus vector is a double-stranded DNA that does not integrate in the genome of the host.

An adeno-associated virus vector, also called AAV, is a single-stranded DNA virus.

Suitable viral vectors are well-known by the skilled person.

Preferred AAV vectors include AAV2, AAV5, AAV9 or a hybrid AAV vector.

A "hybrid AAV vector" is a vector comprising the rep gene of one AAV vector and the cap gene of another AAV vector.

Examples of brain-specific AAV vector are AAV2, AAV5, AAV9, rAAVrh.8, rAAVrh.9 or rAAVrh.10.

In one embodiment, the AAV vector is AAV9 comprising the synapsin promoter, preferably the synapsin 1 promoter.

Cells expressing Yif 1 B protein

The present invention also relates to cells expressing Yif 1 B protein.

The "YifI B protein" is a functional Yif 1 B protein as defined above in the section of the same name.

The "cells expressing Yif 1 B protein" are thereafter also referred to as "cells".

The "cells expressing Yif 1 B protein" are preferably isolated cells.

The cells expressing Yif 1 B protein for use according to the invention are healthy cells. Heathy cells do not comprise any oncogene mutation; they preferably do not comprise any disease involved mutation.

The cells may be autologous cells, i.e. cells originating from the subject to be treated. Alternatively, the cells may be cells from a cell line.

The cells are preferably stem cells, for example embryonic stem cells, adult stem cells or cells from a stem cell line.

By "stem cells", it is herein meant undifferentiated multipotent cells that have the ability to divide for indefinite periods and that, under specific conditions, can give differentiate to many different cell types. Adult stem cells are non-embryonic stem cells.

Adult stem cells may originate from bone marrow, adipose tissue and/or blood.

By "embryonic stem cells", it is herein meant pluripotent stem cells derived from the epiblast tissue of the inner cell mass of a blastocyst or earlier morula stage embryos.

The stem cells for use according to the invention are preferably not directly derived from a human embryo or preferably did not necessitate the destruction of a human embryo.

The stem cell line may be an embryonic stem cell line, an adult stem cell line or an Induced pluripotent stem-cell (iPSC) line.

The embryonic stem cell line is preferably a publicly available and previously established stem cell line which did not necessitate the destruction of a human embryo, as for example described in Chung et al. (2008) Cell Stem Cell 2:1 13-1 17).

Induced pluripotent stem-cell (iPSC) lines are pluripotent stem cells generated from adult or somatic cells.

The stem cell line is preferably an adult stem cell line or an Induced pluripotent stem-cell (iPSC) line.

Pharmaceutical composition

The agent for YifI B protein expression, in particular the nucleic acid encoding YifI B protein, a fragment of YifI B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter, the vector comprising said nucleic acid or the cells expressing Yif 1 B protein, may be formulated into a pharmaceutical composition. The invention thus also contemplates a pharmaceutical composition comprising an agent for YifI B protein expression, preferably a nucleic acid encoding YifI B protein, a fragment of YifI B protein, Yif 1 B promoter and/or a fragment of Yif 1 B promoter, a vector comprising said nucleic acid or cells expressing Yif 1 B protein, and a pharmaceutically acceptable vehicle.

The agent for YifI B protein expression, in particular the nucleic acid encoding YifI B protein, a fragment of YifI B protein, YifI B promoter and/or a fragment of YifI B promoter, the vector comprising said nucleic acid and the cells expressing Yif 1 B protein are as defined above in the sections of the same names.

Pharmaceutical compositions comprising an agent for YifI B protein expression, preferably a nucleic acid encoding YifI B protein, a fragment of YifI B protein, YifI B promoter and/or a fragment of YifI B promoter, a vector comprising said nucleic acid and/or cells expressing YifI B protein, include all compositions, wherein said agent, preferably said nucleic acid, said vector and/or said cells, is contained in an amount effective to achieve the intended purpose. The expression "pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is preferably not toxic to the host to which is administered.

Pharmaceutically acceptable vehicles can be prepared by any method known by those skilled in the art.

Suitable pharmaceutically acceptable vehicles may comprise excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable pharmaceutically acceptable vehicles are described for example in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985), which is a standard reference text in this field. Pharmaceutically acceptable vehicles can be routinely selected in accordance with the mode of administration, solubility and stability of the agent, in particular nucleic acid, vector and/or cells. For example, formulations for intravenous administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature.

The agent for Yif 1 B protein expression, in particular nucleic acid, vector and/or cells according to the invention, is preferably formulated as liquid (e.g. solutions, suspensions).

The pharmaceutical composition comprising cells expressing YifI B protein may comprise a solution of phosphate buffered saline (PBS) or lactated Ringer's solution containing a mixture of salts in physiologic concentrations. The amount of agent for Yif 1 B protein expression, in particular nucleic acid, vector and/or cells, to be used in a pharmaceutical composition depends, for example, on the strength of the promoter used in the DNA construct for the nucleic acid or vector, the immunogenicity, the condition of the mammal intended for administration (e.g., weight, age, sex, health, concurrent treatment, if any, and frequency of treatment), the mode of administration and the type of formulation.

For example, the pharmaceutical composition may comprise from 1 μg to 8 mg, preferably from 1 μg to 1 mg, preferably from 10 μg to 800 μg, more preferably from 25 μg to 250 μg, of the nucleic acid or vector, in particular when using a non-viral vector.

For example, the pharmaceutical composition may comprise a quantity of a viral vector according to the invention ranging from 10¹⁰ to 10¹⁸ viral genomes, preferably from 10¹⁰ to 10¹⁶ viral genomes, more preferably from 10¹⁰ to 10¹³ viral genomes. For example, the pharmaceutical composition may comprise from 10⁶ to 10¹⁰ cells expressing YifI B, preferably from 10⁶ to 10⁹ cells expressing YifI B, more preferably from 10⁶ to 10⁸ cells expressing Yif 1 B, for example 10^s, 5.10⁶, 10⁷, 5.10⁷ or 10⁸ cells expressing Yif 1 B.

In one embodiment, the pharmaceutical compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a pre-determined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions. In such compositions, the nucleic acid, vector and/or cells is/are usually a minor component, with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

The invention further provides kits comprising a pharmaceutical composition comprising an agent for YifI B protein expression, in particular nucleic acid, vector and/or cells according to the invention, and instructions regarding the mode of administration. These instructions may e.g. indicate the medical indication, the route of administration, the dosage and/or the group of patients to be treated.

Ciliopathv

A ciliopathy is a genetic syndrome resulting from defect(s) in cilia, more particularly in defect(s) in the cilia structure and/or function.

The term "cilia" herein encompasses the vibratile cilia, primary cilia or the flagella, for example the flagella of spermatozoids.

A ciliopathy according to the invention preferably involves a defect in the Yif 1 B protein.

A defect in the YifI B protein may consist in the absence or a low level of YifI B expression and/or in the expression of a non-functional YifI B protein or a YifI B protein with an altered function.

A YifI B protein with an altered function for example has a decreased activity by comparison to a functional YM fB protein.

The absence of Yif 1 B expression lead to the same phenotype as those obtained with a non-functional YifI B protein, a low level of YifI B expression and/or the expression of a Yif 1 B protein with an altered function. In a ciliopathy involving a defect in the Yif 1 B protein, the decreased level of Yif 1 B protein expression or the absence of Yif 1 B protein expression and/or the expression of a non-functional Yif 1 B protein or of a Yif 1 B protein with an altered function induce an accelerated kinetics of the anterograde intracellular traffic and an altered primary cilia structure, more particularly a double axonema instead of a single axonema and/or an increased or decreased ciliary axonema length and/or change(s) in the expression and/or localization of ciliary proteins, such as Arl13B.

The expression of a non-functional YifI B protein or of a YiF1 B protein with an altered function may for example result from at least one mutation in the Yif 1 B gene and/or a defect in the transcription and/or expression machinery.

The decreased level or the absence of Yif 1 B protein expression may for example result from at least one mutation in the Yif 1 B gene, a defect in the Yif 1 B promoter and/or a defect in the activation of Yif 1 B transcription and/or expression. In one embodiment, the ciliopathy is characterized by ataxia, intellectual deficiency, visual dysfunction, male infertility, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy or their combinations.

An intellectual deficiency could be associated with agenesis of the corpus callosum, clinical features, for example, comprises learning abilities dysfunctions, mental retardation, deficits in "higher" language function, complex information processing abilities, complex attention and/or memory skills, subtle social differences, cognitive difficulties and/or development delay.

A visual dysfunction includes defects in retinitis pigmentosa, maculopathy, retinal dystrophy, photophobia, hyperopia, keratoconus, blindness (at birth or developing during infancy or later during adulthood) and/or retinal defects, such as retinal degenerationoptometric, retinogram alterations abnormalities, optometric and/or retinogram alterations.

Defects in retinitis pigmentosa, maculopathy and retinal dystrophy may be assessed by for example coarse nystagmus, sluggish or near-absent pupillary responses.

The expression "kidney dysfunction" for example comprises cystic kidney or kidney fibrosis, dilatation of the renal collecting ducts, renal insufficiency, bilateral renal enlargement with microcystic dilatation, glomerulosclerosis cystic renal dystrophy, renal dysplasia, nephronophthisis and/or focal segmental glomerulosclerosis.

The expression "liver dysfunction » for example comprises hepatic fibrosis, intrahepatic biliary dysgenesis, non-obstructive dilation of the intrahepatic bile ducts in the liver and/or fibrocystic disease (for example biliary dysgenesis that includes congenital hepatic fibrosis, bile duct dilatation and cyst formation).

The expression "olfactory dysfunction" for example comprises anosmia, hyposmia and/or parosmia.

By "encephalopathy", it is herein meant a syndrome wherein at least one function and/or structure of the brain is altered, such as hypoplasia of the corpus callosum, cerebellar hypoplasia, delayed myelination and/or progressive parenchymal volume loss.

In one embodiment, the ciliopathy is selected in the group consisting of Alstrom syndrome, Joubert syndrome, Meckel syndrome, nephronophthisis, Bardet-Biedl syndrome, oral-facialdigital syndrome type 1 , Senior-Loken syndrome, polycystic kidney disease, polycystic liver disease, primary ciliary dyskinesia, asphyxiating thoracic dysplasia, Marden-Walker syndrome, situs inversus / isomerism, retinal degeneration, cerebrello-oculo-renal syndrome, Ellis-van Creveld syndrome, Jeune asphyxiating thoracic dystrophy, Leber congenital maurosis and their combinations.

Polycystic kidney disease (also called PKD) includes ADPKD (Autosomal Dominant Polycystic Kidney Disease) and ARPKD (Autosomal Recessive Polycystic Kidney Disease).

Primary ciliary dyskinesia is also referred to as Kartagener Syndrome.

Senior-Loken syndrome is also referred to as SLSN.

Bardet-Biedl syndrome is also referred to as BBS.

Cerebrello-oculo-renal syndrome is also referred to as CORS.

Ellis-van Creveld syndrome is also referred to as EVC.

Joubert syndrome is also referred to as JBTS.

Jeune asphyxiating thoracic dystrophy is also referred to as JATD.

Leber congenital amaurosis is also referred to as LCA.

Meckel syndrome is also referred to as MKS.

Nephronophthisis is also referred to as NPHP.

Oral-facialdigital syndrome type 1 is also referred to as OFD1 .

Primary ciliary dyskinesia is also referred to PCD.

Subject to be treated or to be diagnosed

A subject in need of a treatment for preventing and/or treating a ciliopathy and/or to be diagnosed whether he suffers or is at risk of suffering from a ciliopathy may be a mammal, for example a human being or a non-human mammal.

A human being is also referred to as an "individual" or a "patient". Said human being may be of any age, for example an infant, child, adolescent, adult, elderly people, and of any sex.

Alternatively, said human being may be an embryo or a foetus.

A non-human mammal is preferably a mouse, rat, cat, dog, rabbit or primate.

The subject to be treated may suffer from a ciliopathy or may be likely to be affected by a ciliopathy.

A subject suffering from a ciliopathy or likely to be affected by a ciliopathy, in particular of a ciliopathy involving the Yif 1 B protein, may be identified by the diagnostic method defined below.

Prevention and/or treatment of a ciliopathy

The ciliopathy is particularly as defined above in the section of same name.

By the expression "treatment of a ciliopathy", it is herein meant to eliminate or reduce the symptom(s) of said ciliopathy and/or to slow down the progression of said ciliopathy, in particular in a subject suffering from said ciliopathy, in particularly by restoring Yif 1 B expression and/or function.

Desirable effects of treatment for example include:

reducing and/or eliminating ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, olfactory dysfunction and/or skeletal dysplasia,

stopping or slowing down the progression of ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, olfactory dysfunction and/or skeletal dysplasia, and/or

- improving or restoring male fertility.

By the expression "prevention of a ciliopathy", it is herein meant to prevent, at least partially, the appearance of the symptom(s) of said ciliopathy, in particular in a subject likely to be affected by a ciliopathy.

For example, the prevention of a ciliopathy may prevent, at least partially, the appearance of ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, olfactory dysfunction, skeletal dysplasia and/or male infertility.

Ataxia, intellectual deficiency, visual dysfunction, kidney dysfunction, liver dysfunction, olfactory dysfunction, skeletal dysplasia and male fertility may be assessed by any method well known by the skilled person. Agent for Yif 1 B protein expression for use in the prevention and/or treatment of a ciliopathv and method of prevention and/or treatment of a ciliopathv

The present invention advantageously allows preventing and/or treating ciliopathies, in particular ciliopathies involving a defect in Yif 1 B protein, by increasing and/or restoring the expression of a functional Yif 1 B protein, in particular in the cells of the tissues affected by the disease and/or providing cells expressing a functional Yif 1 B protein, in particular in the tissues affected by the disease.

The prevention and/or treatment of said ciliopathies may be achieved by gene therapy, for example using a nucleic acid encoding Yif 1 B protein or using a system of gene correction, or by cellular therapy, for example using cells expressing Yif 1 B protein, such as stem cells expressing a Yif 1 B protein.

Gene therapy is a therapy using gene(s) as a medicament, which may for example be obtained by delivering in a cell a gene of interest (for example a nucleic acid encoding a functional Yif 1 B protein) and/or correcting gene(s) of interest at the endogenous site (for example correcting a Yif 1 B gene carrying mutation(s) leading to the absence of Yif 1 B protein, a non-functional Yif 1 B protein or a Yif 1 B protein with an altered function and/or correcting a mutated Yif 1 B promoter).

The correction of gene(s) may be carried out by the CRISPR/CAS system, for example using a CRISPR/CAS vector and optionally a DNA template for gene correction.

The DNA template for Yif 1 B gene correction may be a nucleic acid encoding a functional Yif 1 B protein as defined above (for example a nucleic acid comprising or consisting of a sequence at least 80% identical to sequence SEQ ID NO: 1 or SEQ ID NO: 3) or a nucleic acid encoding a fragment of a functional Yif 1 B protein, wherein said fragment comprises the amino acid(s) to be corrected.

A nucleic acid encoding a fragment of a functional Yif 1 B may comprise or consist of a fragment of a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein said fragment comprises the nucleotide(s) to be corrected.

The DNA template for Yif 1 B promoter correction may be a nucleic acid encoding a functional Yif 1 B promoter or a nucleic acid encoding a fragment of a functional Yif 1 B promoter, wherein said fragment comprises the amino acid(s) to be corrected.

The length of the nucleic acid encoding a fragment of a functional Yif 1 B protein or of a functional Yif 1 B promoter is preferably at least 40, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1 100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900 or at least 2000 nucleotides and/or at most 50, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, at most 500, at most 550, at most 600, at most 650, at most 700, at most 750, at most 800, at most 850, at most 900, at most 950, at most 1000, at most 1 100, at most 1200, at most 1300, at most 1400, at most 1500, at most 1600, at most 1700, at most 1800, at most 1900, at most 2000 or at most 2100 nucleotides.

The DNA template for gene correction may be a single-stranded oligonucleotide, a double-stranded oligonucleotide or a double-stranded DNA plasmid.

The CRISPR/CAS vector for example comprises at least one nucleic acid encoding an endonuclease CAS (for example CAS9) and at least one nucleic acid encoding the guide RNA (gRNA) specific to the targeted mutated YifI B gene and/or the targeted mutated Yif 1 B promoter.

The present invention thus relates to an agent for Yif 1 B protein expression, preferably a nucleic acid encoding Yif 1 B protein, a nucleic acid encoding a fragment of a YifI B protein, a nucleic acid encoding Yif 1 B promoter or a fragment of Yif 1 B promoter and/or cells expressing YifI B protein, for use in the prevention and/or treatment of a ciliopathy, in particular in a subject in need thereof.

The present invention is also directed to a method for preventing and/or treating a ciliopathy in a subject in need thereof, said method comprising a step of administering to said subject an effective amount of an agent for Yif 1 B protein expression, preferably of a nucleic acid encoding Yif 1 B protein, a nucleic acid encoding a fragment of a Yif 1 B protein a nucleic acid encoding YifI B promoter or a fragment of YifI B promoter and/or of cells expressing Yif 1 B protein.

The nucleic acid encoding YifI B protein, the nucleic acid encoding a fragment of a YifI B protein and/or the nucleic acid encoding YifI B promoter or a fragment of YifI B promoter is preferably provided in the form of a vector.

The present invention thus also relates to a vector comprising a nucleic acid encoding Yif 1 B protein, a vector comprising a nucleic acid encoding a fragment of a Yif 1 B protein and/or a vector comprising a nucleic acid encoding Yif 1 B promoter or a fragment of Yif 1 B promoter for use in the prevention and/or treatment of a ciliopathy, in particular in a subject in need thereof. The present invention also relates to a method for preventing and/or a ciliopathy in a subject in need thereof, said method comprising a step of administering an effective amount of a vector comprising a nucleic acid encoding Yif 1 B protein, a vector comprising a nucleic acid encoding a fragment of a Yif 1 B protein and/or a vector comprising a nucleic acid encoding Yif 1 B promoter or a fragment of Yif 1 B promoter to said subject.

The agent for Yif 1 B protein expression, preferably the nucleic acid, vector and/or the cells expressing Yif 1 B protein of the invention, may be provided in the form of a pharmaceutical composition.

The expressions "Yif 1 B protein", "agent for Yif 1 B protein expression", "acid nucleic encoding Yif 1 B protein, a fragment of Yif 1 B protein, Yif 1 B promoter or a fragment of Yif 1 B promoter", "vector", "cells expressing YifI B protein", "pharmaceutical composition", "ciliopathy" and "prevention and/or treatment of a ciliopathy" are as defined above in the corresponding sections of the same name.

The subject in need thereof is as defined above in the section "subject to be treated".

By "effective amount" or "therapeutically effective amount", it is herein meant an amount sufficient to achieve a Yif 1 B protein expression, which is capable of preventing and/or treating the ciliopathy. Such effective amounts can be routinely determined by those of skilled in the art. The amount of the agent actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual agent administered, the age, sex, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered agent.

The effective amount may also vary according to the drug or prodrug with which the agent, in particular the nucleic acid, vector and/or cells, may be co-administered.

A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the Yif 1 B protein are outweighed by the therapeutically beneficial effects.

The agent for Yif 1 B protein expression, preferably the nucleic acid, vector and/or cells expressing Yif 1 B protein, or the pharmaceutical composition comprising said agent is preferably used or administered by arterial route, venous route, nasal route, intra-tissue route, - for example intravitreal, subretinal, intra-testicular and/or intracerebral route -, and/or intraperitoneal route. When the intra-tissue route is used, the agent for YifI B protein expression, in particular nucleic acid, vector and/or cells, or the pharmaceutical composition comprising said agent is used or administered in at least one affected tissue.

The administration of the agent for Yif 1 B protein expression, in particular nucleic acid, vector and/or cells, or the pharmaceutical composition comprising said agent may be achieved in a single dose or several doses of the pharmaceutical composition according to the invention, said several doses being injected simultaneously, separately or sequentially. In a preferred embodiment of the invention, the agent for Yif 1 B protein expression, in particular the nucleic acid, vector and/or cells, or the pharmaceutical composition comprising said agent is used or administered as a single dose, said single dose being for example efficient for the prevention and/or treatment of the ciliopathy for several months or years.

The agent for Yif 1 B protein expression, in particular the nucleic acid encoding

Yif 1 B protein, the nucleic acid encoding a fragment of a Yif 1 B protein, the nucleic acid encoding Yif 1 B promoter or a fragment of Yif 1 B promoter and/or cells expressing Yif 1 B protein, may be used or administered in combination with at least another compound useful for the prevention and/or treatment of the ciliopathy.

Prevention and/or treatment of a ciliopathy characterized by visual dysfunction may for example comprise injection into at least one eye, preferably the two eyes, with a vector comprising a nucleic acid encoding Yif 1 B protein under the control of a ubiquitous promoter, such as PGK, EF-1 or CMV promoter. Said vector is preferably provided in the form of viral particles, for example adenovirus or lentivirus particles.

Prevention and/or treatment of a ciliopathy characterized by infertility may for example comprise injection into testes with a vector comprising a nucleic acid encoding Yif 1 B under the control of a ubiquitous promoter, such as PGK, EF-1 or CMV promoter. Said vector is preferably provided in the form of viral particles, for example adenovirus or lentivirus particles.

Method for diagnosing a ciliopathy

The present invention also provides a method for diagnosing whether a subject suffers or is at risk of suffering from a ciliopathy, in particular from a ciliopathy involving a defect in the Yif 1 B protein.

The present invention thus particularly relates to a method, preferably an in vitro method, for diagnosing whether a subject suffers or is at risk of suffering from a ciliopathy, wherein said method comprises detecting in a biological sample of said subject (i) if at least one mutation leading to a non-functional Yif1 B protein or a Yif1 B protein with an altered function, for example by detecting an alternative splicing, is present in the Yif1 B gene and/or (ii) if a functional Yif 1 B protein is expressed by the cells, wherein the presence of said at least one mutation or the absence of a functional Yif 1 protein indicates that said subject suffers from or is at risk of suffering from said ciliopathy.

The "YifI B protein", "functional Yif 1 B protein", "YifI B protein with an altered function", "subject", "ciliopathy" are as defined above.

The biological sample may be a blood sample, a plasma sample or a tissue sample, for example a skin biopsy. In the case of the alternative (i), the biological sample may be a nucleic acid sample. In the case of alternative (ii), the biological sample comprises cells or a protein cell extract.

The biological sample may be submitted to at least one treatment step before being analyzed in the detection step. Non-limitative examples of treatment step include dilution, centrifugation, heat treatment, cell lysis, solubilization, denaturation, extraction, PCR (Polymerase Chain Reaction), RT (Reverse Transcription)-PCR and/or cell culturing.

(i) Detecting if at least one mutation leading to a non-functional Yif 1 B protein or a Yif 1 B protein with an altered function is present in the Yif 1 B gene in a biological sample may be performed by any method well-known by the skilled person.

More particularly, said step of detecting the presence or not of at least one mutation leading to a non-functional YifI B protein or a YifI B protein with an altered function may comprise:

sequencing the YifI B gene from the DNA present in the biological sample or sequencing the YifI B cDNA corresponding to the mRNA present in the biological sample, and

- comparing the obtained sequence to a reference sequence encoding a functional Yif 1 B protein or comparing the amino acid sequence encoded by the obtained sequence to a reference sequence of a functional Yif 1 B protein.

A reference sequence encoding a functional Yif 1 B protein is for example sequence

SEQ ID NO: 3 when comparing the YifI B gene sequence or sequence SEQ ID NO: 1 when comparing the Yif 1 B cDNA sequence.

A reference sequence of a functional YifI B protein is for example sequence SEQ ID NO: 2.

The sequence comparison may consist in a sequence alignment, for example carried out as disclosed above. Said step of detecting the presence or not of at least one mutation leading to a non-functional YifI B protein or a YifI B protein with an altered function may for example comprise or consist in detecting an alternative splicing. If at least one nucleotide mutation is detected in the obtained gene or cDNA sequence, but without changing the encoded amino acid sequence by comparison to reference sequence of a functional YifI B protein or by comparison to the sequence encoded by the reference sequence encoding a functional YifI B protein, said mutation does not lead to a non-functional YifI B protein or to a YifI B protein with an altered function.

If at least one amino acid mutation is detected, it is then examined whether said mutation leads to a non-functional Yif1 B protein or a Yif1 B protein with an altered function. For example, the following mutations lead to a non-functional YifI B protein or a YifI B protein with an altered function:

- mutation(s) resulting in the absence of the five transmembrane segments of the Yif1 B protein, for example mutation(s) leading to a truncated protein lacking at least amino acids 157 to 314 of sequence SEQ ID NO: 2,

- mutation(s) in the N-terminal part of the YifI B amino acid sequence that prevent, at least partially, the interaction of the YifI B protein with rab6, Yipl A and/or receptor 5-HT1 A, said mutations being preferably comprised in the region spanning from amino acid 1 to amino acid 152 of sequence SEQ ID NO: 2 .

The five transmembrane segments are located in C-terminal part of the YifI B protein. For example, in the YifI B protein of sequence SEQ ID NO: 2, the first transmembrane segment consists of amino acid 158 to amino acid 175 of sequence SEQ ID NO: 2; the second transmembrane segment consists of amino acid 193 to amino acid 210; the third transmembrane segment consists of amino acid 227 to amino acid 252; the fourth transmembrane segment consists of amino acid 256 to amino acid 273; and the fifth transmembrane segment consists of amino acid 293 to amino acid 310.

Tests for assessing the interaction between two proteins, for example between (i) the YifI B protein and (ii) rab6, Yipl A and/or receptor 5-HT1 A are well known by the skilled person. For example, the interaction may be measured by plasmon surface resonance between His-tagged YifI B N-terminus (His-Yif 1 BNt, as described in Al awabdh, 2012) and the second protein. When at least one mutation leading to a non-functional Yif1 B protein or a Yif1 B protein with an altered function is present in the Yif 1 B gene, then said subject suffers from or is at risk of suffering from said ciliopathy.

(ii) Detecting if a functional Yif 1 B protein is expressed by the cells in a biological sample may be performed by any method well-known by the skilled person.

More particularly, said step of detecting the expression or not of a functional Yif 1 B protein by the cells may comprise:

culturing the cells in a medium poor in serum, in order to induce the appearance of primary cilia,

- determining a) the presence or absence of a YifI B protein of the expected molecular weight, for example by western blot, for example in a plasma sample and/or b) the kinetics of the anterograde intracellular traffic and/or the primary cilia structure and/or c) the Yif 1 B mRNA levels, for example by quantitative RT- PCR and/or the length of mRNA.

The antibody used for detecting the presence or absence of a Yif 1 B protein may be a monoclonal or polyclonal antibody specific for the wild-type Yif 1 B protein. For example, the antibody ab188127 from Abeam^® or the antibody disclosed in Carrel et al. (2008, The Journal of Neuroscience, 28(32): 8063-8073) may be used for detecting the presence or absence of human Yif 1 B protein.

The excepted molecular weight for a functional human Yif 1 B protein is 32 kDa.

The absence of a Yif 1 B protein or the absence of a Yif 1 B protein of the expected molecular weight indicates the absence of a functional Yif 1 B protein expressed by the cells in the biological sample.

A kinetics of anterograde intracellular traffic corresponding to a non-functional Yif 1 B protein (for example an accelerated kinetics by comparison to a control cell expressing a functional Yif 1 B protein and/or a kinetics similar to those of a control Yif 1 B protein KO cell) indicates the absence of a functional Yif 1 B protein expressed by the cells in the biological sample.

A primary cilia structure corresponding to a non-functional Yif 1 B protein (for example double axonema and/or increased or decreased axonema length) indicates the absence of a functional Yif 1 B protein in the biological sample. The absence of Yif 1 B mRNA or YifI B mRNA levels lower than those in control cells expressing a functional YifI B protein indicates the absence of a functional Yif 1 B protein in the biological sample.

A YifI B mRNA length lower than those in control cells expressing a functional Yif 1 B protein indicates the absence of a functional Yif 1 B protein in the biological sample.

When no functional Yif 1 protein is present in the biological sample, then said subject suffers from or is at risk of suffering from said ciliopathy.

Method for selecting a compound useful in the prevention and/or treatment of a ciliopathy

The invention also provides a method for selecting a compound useful in the prevention and/or treatment of a ciliopathy, in particular of a ciliopathy involving a defect in the YifI B protein, by assessing the effect of a test compound in a cell or an animal deficient in the YifI B protein, for example a YifI B KO cell or animal. If said compound is able to restore the phenotype corresponding to a cell or animal expressing a functional Yif 1 B protein, then said compound may be used in the prevention and/or treatment of said ciliopathy.

The present invention thus relates to a method for selecting a compound useful in the prevention and/or treatment of a ciliopathy, wherein said method comprises:

a) administering a test compound to a YifI B knock-out animal, to obtain a treated Yif 1 B knock-out animal,

c) selecting said test compound as being useful in the prevention and/or treatment of said ciliopathy when at least one symptom of said ciliopathy is improved or absent in the treated Yif 1 B knock-out animal.

The animal is for example a mouse, rat, rabbit or primate, preferably a mouse. The Yif 1 B knock-out animal thus suffers from a ciliopathy.

The test compound may be a peptide, protein, a chemically modified protein, a fusion protein, a peptidomimetic, a nucleic acid encoding said peptide, protein or fusion protein, a cell expressing said peptide, protein or fusion protein.

The test compound may be administered by any appropriate route, such as the arterial route, venous route, nasal route, intra-tissue route, for example intra-vitreal, subretinal, intracerebral and/or intra-testicular route, and/or intraperitoneal route. The symptom of said ciliopathy may be selected in the group consisting of ataxia, intellectual deficiency, visual dysfunction, male infertility, kidney dysfunction, liver dysfunction, olfactory dysfunction, skeletal dysplasia, an altered cilia structure (for example double axonema and/or increased or decreased axonema length) and their combinations.

In step c), said at least one symptom is improved or absent in the treated Yif 1 B knock-out animal, for example by comparison to those in a non-treated Yif 1 B knock-out animal and/or to those before treatment.

The method may thus comprise, between step b) and step c), a step b1 ) of comparing at least one symptom of said ciliopathy in the treated Yif 1 B knock-out animal to those in a control non-treated Yif 1 B knock-out animal and/or to those in said Yif 1 B knockout animal before treatment.

The Yif 1 B knock-out ciliated cell culture may be obtained by culturing the Yif 1 B knock-out cells in a medium poor in serum.

The structure of the primary cilia is altered in the YifI B knock-out ciliated cell culture. In particular, the cilia of the cells in the Yif 1 B knock-out ciliated cell culture have a double axonema and an increased axonema length.

The cells of the cell culture may be neuronal cells, fibroblasts (for example embryonic fibroblasts or skin fibroblasts), retinal cells, renal cells, ependymocytes or spermatozoids.

The test compound is for example as defined above.

The structure of the cilia may be assessed as disclosed above, by confocal microscopy, in particular after axonema immunostaining, for example using anti-Arl13B antibodies.

When the structure of the cilia is not altered after treatment, for example no more altered by comparison to the structure before treatment, said test compound is selected as being useful in the prevention and/or treatment. The method optionally comprises, between step b) and step c): a step b1 ) of comparing the structure of the cilia in the treated cell culture by comparison to those before treatment and/or to those of a non- treated cell culture.

The present invention will be further illustrated in view of the following examples and figures.

All references cited herein, including journal articles or abstracts, published or unpublished patent application, issued patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references.

Brief description of the sequences

SEQ ID NO: 1 corresponds to the nucleic acid coding sequence of human Yif 1 B protein.

SEQ ID NO: 2 corresponds to the amino acid sequence of the human Yif 1 B protein encoded by sequence SEQ ID NO: 1 .

SEQ ID NO: 3 corresponds to the nucleic sequence of the human Yif 1 B gene of access number NC_000019.9 (NCBI database), as available on January 14, 2016.

Brief description of the figures

Figure 1 : Loss of Purkinje cells in the Yif 1 B-KO cerebellum.

The time course of neurodegeneration of Purkinje cells in the Yif 1 B-KO mice. Parasagittal cerebellar sections of WT and Yif 1 B-KO mice at 3 and 12 months were immunostained with the calbindin antibody and the number of immunoreactive-Purkinje cells was counted along the Purkinje cell layer in confocal images. Ordinate: Purkinje cell/1 ΟΟμηι; "WT": wild-type mice; "KO": Yif 1 B KO mice; "M": Months. The values represent the mean ± SEM of three slices. ^*p < 0.05 ^**p < 0.0001 (t-test).

Figure 2: Behavioral analysis of WT and Yif 1 B KO littermates.

Locomotor activity was recorded every 5 min over a 60 min period in an actimeter. Ordinate: locomotor activity (cm); abscissa: time in minutes; "WT": wild-type mice; "Yif 1 B KO": Yif 1 B KO mice.

Figure 3: Behavioral analysis of WT and Yif 1 B KO littermates.

Rotarod test was performed on three consecutive days. Ordinate: Time to fall (% first day); abscissa: time in day; "WT": wild-type mice; "Yif 1 B KO": Yif 1 B KO mice. Figure 4: Microglial activation in the Yif1 B-KO cerebellum.

Quantification on immunofluorescent anti-lbal labelled cells on cerebellar sections of WT and Yif1 B-KO mice. Bar graphs represent number of microglia per 0.1 mm² of section ± SEM, n=16 on 4 sections. "WT": wild-type mice; "KO": Yif 1 B KO mice.

Figure 5: Visual performance of Yif 1 B KO mice in scotopic condition.

Optometer response at different spatial frequencies in control (WT) and Yif 1 B KO animals. Ordinate: animal tracking features; abscissa: grating frequency (cpd).

Figure 6: Visual performance of Yif 1 B KO mice in scotopic condition.

Electroretinogram (ERG) response of a- and b-wave amplitudes measured following a 0.16 cds/m2 scotopic flash in control (WT) and Yif 1 B KO animals. Ordinate: μν; "cpd": cycle per degree; "a": a-wave; "b": b-wave.

Figure 7: Alteration of Yif 1 B-KO male fertility: testis weight (in mg) per genotype.

Figure 8: Alteration of Yif 1 B-KO male fertility: spermatozoa number in epididymis per genotype

Figure 9: Alteration of Yif 1 B-KO male fertility: classification of seminiferous tubules at 2-3 months and 12 months per genotype in three categories: "n": normal, "v": vacuolated or "SCO": Sertoli cells only. Ordinate: tubule shapes in percentage; "m": months".

Figure 10: Alterations of primary cilia in neurons of Yif 1 B KO brain.

The length of primary cilia (in μηι) was quantified in confocal images of the WT and Yif1 B-KO cerebellar sections immunostained with the anti-Arl13B antibody. The values represent the mean ± SEM of three slices. ^***p < 0.001 (t-test).

EXAMPLES

Material and methods

Animals

Homozygous Yif 1 B (Yif 1 B KO) mice and WT littermates were obtained from the previously established colony (Alterio, J., J. Masson, J. Diaz, K. Chachlaki, H. Salman, J. Areias, S. Al Awabdh, M.B. Emerit, and M. Darmon, Traffic, 2015. 16(9): p. 978-93). Animals were maintained under controlled conditions (21 ±1 °C, 60% relative humidity, 12- h light-dark cycle with lights on at 7:00 AM, food and water ad libitum). Experiments were performed during the light phase on 2-3 month-old male mice, using matched WT littermates as controls. Experiments were performed in agreement with the institutional guidelines for the use of animals and their care, in compliance with national and international laws and policies {Council directives no. 87-848, October 19, 1987, Ministere de /Agriculture et de la Foret, Service Veterinaire de la Sante et de la Protection Animale, permissions no. 75-974 to M.D. and no. 75-805 to J.M.). All efforts were made to minimize animal suffering and to reduce the number of animals used in these experiments.

Immunofluorescence

Mice were anaesthetized deeply using pentobarbital (60 mg/kg,) and then perfused transcardially with 10 ml of saline solution (0.9% NaCI warmed at 37°C), followed by 150 ml of an ice-cooled fixative solution containing (2-4% paraformaldehyde). The brains and testis were removed, post-fixed in the same fixative at 4°C for 1 hr and rinsed in PB 0.1 M. Free floating coronal brain sections (40 μΓΤΐ-thick from telecephalic and cerebellar regions of the brain were made with a vibratome (LeicaVT1000E) and maintained at 4°C in PB 0.1 M, pH 7.4.

Free floating coronal sections of telencephalon and cerebellum were treated during 20 min with a solution containing 1 % NaBH4; Na2HP04 0.1 M; pH 8.0. Sections were rinsed five times in 0.05 M Tris buffer, pH 7.4, containing 150 mM NaCI (TBS) and blocked for 1 hr at 37° with 10% normal donkey serum, 0.4% BSA, 0.1 % gelatine, and 0.1 % Tween-20 in TBS. After incubation for 24h-36h at 4°C in the primary antibodies (rabbit anti-Calbindin D28K, Sigma, 1 /3000, rabbit anti-lbal , Wako, 1 /800; goat anti- rootletin 1/500, Santa-Cruz; and mouse anti-CTR433 1 /1600, Jasmin et al, 1989) diluted in TBS containing 0.4% BSA, 0.1 % gelatin and 0.1 % Tween-20), sections were rinsed three times for 10 min-each in TBS containing 0.1 % gelatine and 0.05% Tween-20 and incubated 1 h with the fluorophore-labeled secondary donkey-antibodies (Cy3-conjugated anti-rabbit 1/500; Alexa 488-conjugated anti-goat antibody 1/500; Cy3-conjugated anti- mouse 1 /500, Jackson ImmunoResearch) diluted in TBS, 0.05% Tween-20. They were washed in TBS; 0.1 % gelatine and 0.05% Tween-20, mounted on Super Frost Plus slides and coverslipped using Fluoromount (Clinisciences) mounting solution for fluorescence microscopy.

Electron microscopy

For electron microscopy section, perfusion was performed with 2.5% glutaraldehyde and 2% paraformaldehyde. Brains and testis were removed and post-fixed in the same fixative at 4°C for 4 hr and rinsed in PB 0.1 M. Free floating coronal sections taken throughout the dorsal hippocampus (150 μΓΤΐ-thick) and the testis (200 μΓΤΐ-thick) were made with a vibratome (LeicaVT1000E) and maintained at 4°C in PB 0.1 M, pH 7.4. Free floating coronal brain sections taken throughout the dorsal hippocampus (150 μηι- thick) and transversal sections of testis (200 μΓΤΐ-thick) were rinsed in PBS 50 mM, pH 7.4. Tiny pieces of 2mm x 1 mm from the CA1 region of the hippocampus and from the seminiferous tubules were dissected and post-fixed for 30 min in 1 % osmium tetroxide. The tiny pieces of CA1 -hippocampus and seminiferous tubules were rinsed and incubated 1 h in a solution of 1 % uranyl acetate in water. After dehydratation in a graded series of ethanol solutions, the pieces of CA1 -hippocampus and seminiferous tubules were flat- embedded in epoxy resin (Epon) and allowed to polymerize for 48 h at 60°C. The tiny tissue-pieces embedded in epon (from CA1 -hippocampus and seminiferous tubules) were then cut with a Reichert ultramicrotome. The ultrathin sections were mounted on mesh grids, stained with lead citrate and analysed on a JEOL 100 electron microscope equipped with a GATAN CCD camera Retinal electrophvsioloqy

Electroretinograms (ERG) were performed on 1 -2 months male mice. Animals were dark adapted for 12 h prior to the recording. They were anaesthetized with an intraperitoneal injection (10 μΙ/g) of a mixture of ketamine (10 %, Virbac, France) and xylazine (7.5 %, Bayer, Germany) diluted in a 0.9 % NaCI solution. Corneas were anaesthetized with oxybuproca^'ine chlorhydrate (0.4 %) (Thea Lab., France) and the pupil dilated with tropicamide (0.5 %, Thea Lab). Each animal was placed on a heating pad; eyelids were retracted to maintain eyes open during the recording. A gold electrode was placed on the cornea with a drop of methylcellulose (Ocry-gel, Therapeutique Veterinaire Moderne, France) while the neutral and the reference electrodes were placed on the tail and on the head of the animals, respectively. Light stimulations were delivered in a Ganzfeld with flash intensity at 0.16 cds/m2. Amplitudes of the scotopic ERG a- and b-waves were measured at the maximum negative and positive peaks of the recordings with respect to the baseline before the stimulation.

Behavior

(i) Locomotor activity test

Activity was measured using an actimeter, a computer-based photo-beam apparatus (Actisystem II, Panlab). Actimeter boxes (area: 30 x 15cm; height: 18cm; with grid floor) detected mouse movements by means of infrared light beams. Mice were placed in the boxes 60 min and horizontal activity (in cm) was monitored.

(ii) Rotarod test

Each mouse was placed onto the horizontal rod of an accelerating rotarod apparatus (model 7650 for mice, Ugo-Basile, Comerio, Italy) rotating at a speed increasing from 4 to 40 rpm over 5 min. The time required for the mouse to fall from the rod, expressed as seconds, was recorded. Mice were acclimatized to the apparatus by placing them for 2 min on the rod at 5 rpm, 3 min before the first test. Motor coordination performance was evaluated the first day just after acclimatization session of each mouse (with a 20-min resting period between two successive training). Mice were trained three times a day on the two consecutive days. Motor coordination performance of each mouse was determined by the longer time to fall on the three trials.

(iii) Optomotor response

Mice were placed on a platform in the form of a grid (1 1 .5 cm diameter, 19.0 cm above the bottom of the drum) surrounded by a motorized drum (29.0 cm diameter) that could be revolved clockwise or anticlockwise at two revolutions per minute, the optimal velocity for evoking an optokinetic response in the mouse. After 10 min of adaptation in the dark, vertical black and white stripes of a defined spatial frequency were presented to the animal. These stripes were rotated alternately clockwise and anticlockwise, for 2 min in each direction with an interval of 30 s between the two rotations. Various spatial frequencies subtending 0.06, 0.13, 0.25 and 0.5 cpd (cycles/degree) were tested individually on different days in a random sequence. Animals were videotaped with a digital video camera (Sony, DCR-TRV24E) for subsequent scoring of head tracking movements. Tests were performed in scotopic conditions, using the night shot position of the camera. Head movements were scored only if the angular speed of the head corresponded to that of the drum rotation. If the spatial frequency of the black and white stripes was increased, a threshold was reached beyond which no tracking movements of the head were detected.

(iv) Mnesic performance The fear conditioning paradigm was done according to Mongeau et al. (2007) protocol with modifications. The test was conducted in a chamber (length 26 cm ^χ wide 18 cm x high 22 cm), housed in a sound-attenuating box, possessing aluminum sidewalls and Plexiglas rear and front walls, and a stainless steel grid floor (MED Associates, St. Albans, VT). On day-1 , the mouse was placed into the chamber washed with a vanilla odor and allowed to acclimate for 3 min. Then, the animal was exposed to six episodes of a tone (conditioned stimulus, CS) of 2500 Hz frequency and 85 dB intensity for 30 s immediately followed by a foot shock of 0.75 mA for 2 s (unconditioned stimulus, US). The interval between the tone + shock pairings was 2 min (inter-trial interval, ITI). On day-2 (24 h later), mice were exposed to the same procedure as on day-1 , but without CS or US and allowed to explore for 20 min. On day-3 mice were tested for cued fear memory by returning to the chamber with various modifications (no vanilla odor, addition of hatch designs to the walls and lights on). Only the CS was presented 40 times separated by an ITI of 5 s. All sessions were recorded using infrared cameras and controlled by a computerized system interface (MED Associates, St. Albans, VT). Freezing behavior, defined as complete absence of voluntary movements except for respiration, was measured manually in the ANY-maze software (Stoelting) and expressed as percentage of time spent freezing.

(v) Spontaneous odor exploration and discrimination task

Mice were tested in a well-ventilated room using a Plexiglas testing box with a grid floor allowing us to expose an odorized paper filter (47mm diameter, Whatman) at various locations without disturbing the animal (see Martel et al, 2015 for details). 10 μΙ of odorant solution (1 % in mineral oil, Sigma-Aldrich) dropped at the center of the paper was used as odorant source. Three different odors were used for this experiment: the habituation odor, pentanal (H), a similar odor with one more carbon atom in the molecular structure, hexanal (C1 ) and an odor with 3 more carbon atoms, octanal (C3). Grouped mice were handled daily for two weeks and individually habituated to the testing box for 20 min the two days before the experiment. On the day of the experiment, after 5 min period of habituation, the habituation odor was presented 4 consecutive times during 2 min (H1 to H4). Then, C+1 odor was presented for 2 min (discrimination 1 ) followed by another presentation of the habituation odor (H5) and finally mice were exposed to C+3 odor (discrimination 2). Each odor presentation was followed by a 3 min inter-trial interval (ITI). The whole experiment was videorecorded and the time spent actively sniffing (nose on the filter) was measured a posteriori for each 2 min-exposure by a experimenter blind to the animal genotype. Results are expressed as mean ± standard error of the mean. The degree of statistical significance was calculated using ANOVAs for repeated measures and Wilcoxon non parametric tests using STATVIEW 5.0.1 software (SAS Institute).

Brain electrophvsioloqy

Recording electrodes were pulled from borosilicate capillaries (approx. 2.5 ΜΩ with chloride-based internal solution). Electrophysiological recordings were performed with a Heka EPC-9 amplifier (Heka Elektronik, Darmstadt, Germany). mlPSCs (mlPSCs) were recorded in the presence of TTX (0.5 μΜ) using a cesium-based internal solution to optimize voltage-clamp in Purkinje neurons with following composition: 150 mM CsCI, 4.6 mM MgCI2, 0.1 mM CaCI2, 10 mM HEPES, 1 mM EGTA, 4 mM Na-ATP, and 0.4 mM Na- GTP. Holding potential was - 70 mV. After a control period longer than 5 min, long-term depression (LTD) was induced by a previously described pairing protocol (Casado et al., 2000 and Casado et al., 2002). Briefly, pairing was performed in voltage clamp using a cesium-based intracellular solution. Two PF stimuli with an interstimulus interval of 10 ms were paired at 0.5 Hz for 2 min with a depolarization of the Purkinje neuron to 0 mV for 100 ms. Somatic depolarization preceded parallel fiber stimulation by 20 ms.

Transverse hippocampal slices (400 μηι) were obtained as previously described (Potier et al, 2000) from mice anesthetized with halothane before decapitation. Slices were prepared in ice-cold artificial cerebrospinal fluid (aCSF) and placed in a holding chamber for at least 1 hr. The composition of aCSF was as follows (in mM): NaCI 124, KCI 3.5, MgS04 1 .5, CaCI2 2.3, NaHC03 26.2, NaH2P04 1 .2, and glucose 1 1 , pH 7.4. A single slice was transferred to the recording chamber at a time and continuously perfused with aCSF pre-gassed with 95% 02/5% C02.

Extracellular recordings were obtained at RT from the apical dendritic layer of the CA1 area using micropipettes filled with 2 M NaCI. Presynaptic fiber volleys (PFVs) and field excitatory postsynaptic potentials (fEPSPs) were evoked by electrical stimulation of the Schaffer collaterals and commissural fibers located in the stratum radiatum. The averaged slope of three successive PFVs and fEPSPs was measured using Win LTP software (Anderson WW and GL Collingridge 2001 ). To evaluate the level of receptor activation and compare between groups, the fEPSP/PFV ratio was calculated giving an index of synaptic efficacy (ISE) that was plotted against increased stimulus intensities (300, 400 and 500 μΑ). In order to investigate LTP of synaptic transmission, a test stimulus was applied every 10 sec in a control medium and adjusted to obtain a fEPSP with a baseline slope of 0.1 V/sec. In one set of experiments, the averaged slope of 3 fEPSPs was thus measured for 15 min before the delivery of theta-burst stimulation (TBS), consisting of 5 trains of four 100 Hz pulses each, separated by 200 ms and delivered at the test intensity. This sequence was repeated three times with an interburst interval of 10 s. In a second set of recordings, a high frequency stimulation (HFS) was delivered as a conditioning stimulation, consisting of 1 train at 100 Hz pulses for 1 sec. In all experiments, testing with a single pulse was then resumed for 60 min after the delivery of the conditioning stimulation to determine the level of LTP.

Rescue of the vision phenotype

Yif 1 B KO mice are treated by injection into eyes with an AAV expressing YifI B under the control of a ubiquitous promoter or with a control AAV. The protocol of injection is as described in Busskamp (2010, Science, 329, 413-417). The animals are anaesthetized using 3% isoflurane. A small incision is made with a sharp 30-gauge needle in the sclera near the lens. Through this hole, 2μΙ of AAV are injected slowly (in 20-30 s) into the subretinal space using a blunt 5 μΙ Hamilton syringe held in a micromanipulator. Mice older than P21 are used for injections. After a minimum incubation time of 21 days, vision is assessed in AAV-injected animals versus control animals.

Rescue of the unfertile phenotype

Male YifI B KO mice are treated by injection into testes with viral particles expressing Yif 1 B under the control of a ubiquitous, as described in Parrington et al. (201 1 , Systems Biology in Reproductive Medicine, 57: 35-42).

The male mice (8-10 days post-natal) are anaesthetized using 3% isoflurane. Surgery allows injecting the testis, outside of the abdominal cavity, with 5μΙ of a solution containing 10⁶ to 10⁷ TU/ml ( Transducing Unit/ml), using a glass micropipette. After suture of the peritoneum and the skin, animals are allowed to recover till adulthood. Rescue of the phenotype is then assessed by mating male Yif 1 B mice and observing if descendants are generated.

Results

(i) Structural and functional alterations in the cerebellum and olfactory bulb of Yif 1 B-KO mice

The external anatomy of the brain of WT and Yif 1 B-KO mice was examined in order to detect eventual structural alterations in the central nervous system. The morphological analysis revealed a reduction in size of the cerebellum without changed in total brain weight and length (Cerebellum/total brain, WT = 12.96 ± 0.2453, n=7; Yif 1 B KO = 1 1 .67 ± 0.1788, n=6; student's t-test, p=0.0017) but normal cerebellar foliation in three months Yif1 B-KO mice. The morphological analysis of the brain also revealed that Yif 1 B KO mice have a smaller olfactory bulb.

The shape of the cerebellum in Yif 1 B KO mice was flat compared to WT animal with a decrease of the molecular layer (data not shown).

In order to evaluate the origin of the reduction in the cerebellum size, the cerebellar cortex structure was examined using Calbindin-immunostaining of Purkinje cell. A confocal analysis of immunostained sections demonstrated a reduced calbindin- immunoreactivity in the molecular and Purkinje cell layers of mutant mice (confocal images of parasagittal sections of cerebellar cortex at P90 immunostained with anti- calbindin D28K antibodies not shown), suggesting a loss of Purkinje cells.

An evaluation of the density of calbindin -positive Purkinje cells in different cerebellar lobules from three months Yif1 B-KO and control WT mice, demonstrated a significant loss of about 25% of Purkinje cells in mutant mice which was also found in 12 months Yif 1 B-KO mice (see figure 1).

(ii) Motor coordination

Moreover, the three months Yif 1 B-KO mice showed poor motor coordination learning on the accelerated rotarod test. No differences were observed between Yif 1 B-KO and WT mice in spontaneous locomotor activity assessed in an actimeter during 60 min (see figure 2). The motor adaptation of mice was evaluated using rotarod learning during 3 consecutive days. Whereas no difference was observed the first day of rotarod test between genotype in motor coordination performance (WT=85.86±8.552 sec, n=14; Yif 1 B- KO=91 .25±9.022, n=12; student's t-test, p > 0.05), altered motor learning was observed in Yif 1 B KO during successive sessions (see figure 3). Two-way ANOVA indicated a significant effect of time [F(2,50) = 1 1 .4, p < 0.0001 ] and a significant time x genotype interaction [F(2,50) = 3.6, p = 0.05]. Bonferroni's post hoc test indicated a significant effect of genotype the third day of testing (^*, p<0.05). All these results suggest that the deletion of the Yif 1 B gene induces a substantial progressive Purkinje cells degeneration associated with loss of motor coordination learning abilities in the adult Yif 1 B-mutant mice.

(iii) Purkinie cell degeneration induced microglial activation in cerebellum of Yif 1 B- KO mice

A consequence of Purkinje cells degeneration could be an activation of the microglia. The distribution and the morphology of cerebellar microglia throughout the cerebellar cortex were examined using an antibody against a calcium-binding protein (anti-lbal ), specifically expressed in microglia. In WT mice, the resting microglia was homogenously distributed throughout all the layers of cerebellar cortex. The morphological features characterizing these Iba1 -positive glial cells are a small cell body with numerous thin, branched processes (pictures not shown).

In contrast, in the Yif1 B-KO cerebellum, the density of microglia was increased (see figure 4) indicating a proliferation of reactive microglia. Some foci of Iba1 -positive cells were seen mainly confined to the molecular and Purkinje cell layers (data not shown), suggesting a close spatial relationship between the sites of Purkinje cell degeneration and the presence of reactive microglial foci. Indeed, the glial cells observed in foci displayed severe morphological changes that are characteristic of reactive microglia, including hypertrophy of cell bodies and a shortening and thickening of their spine-like processes (data not shown). All these morphological data indicate that in the adult Yif1 B-KO cerebellum, there is a progressive Purkinje cell degeneration which maintains pro-inflammatory-mediated microglial activation. (iv) Disorganization of mitral cells in olfactory bulb

The olfactory bulb showed a structural disorganization of mitral cells. Reelin- positive volume density was evaluated along the antero-posterior axis on the OB (olfactory bulb) and showed a genotype effect [F (1 ,28)=5.605, P=0.0251 ]. Comparison between the most posterior level of the OB and the AOB (accessory olfactory bulb) level shows an increase in the mitral cell density [F(1 ,28)=17.912, P=0.0002], but no difference between genotypes (genotype x level interaction, F(1 ,28)= 0.486, P=0.4916). Comparison between dorsal and ventral mitral density showed a genotype effect (side x genotype interaction, F(1 ,28) =6.2, P=0.019], revealing that mitral cell density is increased significantly at the ventral side in Yif 1 B KO as compared to WT [F(1 ,14)=12.567, P=0.0032], but not at the dorsal side [(F(1 ,18)=0.12, P=0.733]. In the olfactory bulb, no change in the microglial activation was observed (data not shown).

(v) Visual dysfunction in Yif 1 B KO mice

Visual performances of KO mice were evaluated by the optomotor response measuring head turns following the rotation of a drum covered with black and white stripes at various spatial frequencies (cpd, cycle per degree). In scotopic conditions, WT mice show an increasing numbers of head movements when increasing the spatial frequencies with a maximum at 0.25 cpd. In contrast, this increase in the numbers of head movement was not observed with YifI B KO mice although they showed head movements demonstrating some visual perception. These head turns were as numerous as in WT animals at 0.06 and 0.5 cpd. However, the curve providing information on visual acuity did not show any increase between 0.06 and 0. 5 cpd, suggesting that visual perception in Yif 1 B KO was altered (see figure 5). Two-way ANOVA indicated significant distinction among spatial frequencies [F(3,36)=9.5; p<0.0001 ] and genotypes [F(1 ,36) = 4.5, p<0.05] with no significant optometer stripe x genotype interaction [F(3,36) = 1 .2, NS].

In order to determine the origin of the visual alteration in Yif 1 B KO mice, electroretinograms evoked with a light flash in scotopic conditions were recorded, a-wave and b-wave amplitudes were both reduced in Yif 1 B KO (n=7) when compared to those of WT animals (n=6). The differences were statistically significant (a-wave, P=0.01 16; fa- wave, P=0.0270) (see figure 6). Because ERG a- and b-waves provide information on photoreceptor and inner retinal neuron integrity, respectively, the decrease in a-wave amplitude indicates a dysfunction of photoreceptors in Yif 1 B KO mice. The decrease in fa- wave amplitude may simply reflect the decrease in inner neuronal activity due to the photoreceptor dysfunction. Therefore, perturbation of the visual performance in YifI B KO mice is likely due to photoreceptor dysfunction.

(vi) Olfactory performances

Olfactory performances were evaluated in WT and Yif 1 B KO mice using spontaneous odor exploration paradigm. Mice habituated normally to repeated presentation of the habituation odorant. Mice of both genotypes also discriminated changes of odorant, with responses differing according to the novel stimulus. For example, both genotypes discriminated C1 , whereas Yif 1 B KO mice discriminated less C3 showing a small defect in olfactory performances.

(vii) Learning memory

As YifI B is highly expressed in hippocampus, hippocampal function was investigated by behavioral and electrophysiological experiments. As most of behavioral memory tests use visual clues and Yif 1 B KO mice have impaired vision, fear conditioning experiments that do not depend on visual acuity were used. Progressive enhanced memory retention (training), context and tone extinction was not altered in Yif 1 B KO mice.

The basal neurotransmission and synaptic plasticity in CA1 was tested in hippocampus of WT and YifI B KO mice, by input/output curves and fEPSP slopes respectively. Comparison of input/output (I/O) curves showed that I_SE was not significantly altered in Yif 1 B KO as compared to WT animals whatever the stimulation intensity. These results indicate that YifI B deletion does not impacts basal glutamate synaptic transmission in hippocampal CA1 area. In slices from WT as well as from Yif 1 B KO mice, HFS induced a significant increase in the fEPSP slope, when compared to baseline levels, that persisted until the end of the recording. Comparison of the mean LTP magnitude determined for the last 15 min of recordings, eg between 45 and 60 min after the conditioning stimulation, was similar in WT and Yif 1 B KO mice. In the same way, no change in the amplitude of synaptic plasticity was found when TBS-induced LTP induced in slices of WT mice was compared to the potentiation induced in KO mice. These results therefore indicate that the loss of Yf 1 B does not affect either the electrophysiology of the hippocampus and related behavior.

(viii) Infertility and hypogonadism

When breeding the Yif 1 B KO line, no progeny was observed from mating with homozygote male KO mice (see Table 1). This led us to suspect a Yif 1 B KO male infertility. In order to analyze the origin of this infertility, testes from WT and Yif 1 B-KO male mice were first examined. The weight of KO testes is significantly decreased in Yif 1 B-KO versus WT mice (see figure 7, student's t-test, p<0.0001 ) and no spermatozoid could be visualized in the epididymis of Yif 1 B-KO (see figure 8). In addition, histological examination of seminiferous tubules revealed the presence of numerous vacuolized tubules (pictures not shown) reaching 50% of tubules at 3 months and nearly 80% at 12 months (see figure 9). No alteration of male hormone levels was detected between Yif 1 B KO and WT mice (data not shown).

Table 1 : Genotypic distribution of offspring of Yif 1 B mutants depending on parent genotypes (Animals were genotyped at 1 -3 weeks of age (n: number of animals); no offspring was ever observed with Yif1 B-KO male breeding).

(ix) The altered fine structure of neuronal primary cilia and the spermatozoid flaqella in Yif 1 B KO mice Because of the multi-organ alterations observed in the Yif1 B KO mouse including motor learning deficits, vision defects, defect in olfactory performances and infertility, the hypothesis was made that they could result from a common alteration affecting several organs. So ultrastructural analyses of brain and testes sections were performed to visualize alteration of primary cilia and flagella structure.

The different components were investigated in pyramidal CA1 hippocampus neurons, including axonema and basal bodies of primary cilia ultrastructure and their anchoring striated rootlets as well as the ultrastructure of spermatozoid flagella in the testis of WT and Yif1 B-KO mice by using transmission electron microscopy. For confocal microscopy analysis of components of primary cilia, the axonemas were selectively immunostained with anti-Arl13B antibodies and their associated striated rootlets were identified using anti-rootletin antibodies. Evidence for the spatial relationship between the basal bodies and the striated rootlets with the Golgi apparatus was provided by both electron microscopy and double-labeling with anti-rootletin and the anti-CTR433 antibodies, a specific marker of the Golgi complex (data not shown). This close spatial association between the striated rootlet and the Golgi apparatus was visualized in different cerebral regions including striatum, cerebellum (not shown) and nucleus accumbens (data not shown).

Whereas in WT mice the ultrastructure of primary cilium and the spermatozoid flagella exhibited a normal morphological structure, ultrastructural defects were evidenced in both primary cilia and spermatozoid flagella in Yif1 B-KO mice (data not shown). The fine structural alterations included abnormal arrangement of the axonemal-microtubules in primary cilium and spermatozoid flagella, the disorganization of basal bodies and of their connected striated rootlets. Confocal analysis revealed abnormal primary cilia with double axonema, as well as a significant increase of ciliary axonema length in neurons of the Yif1 B-KO cerebellum (data not shown). Moreover, striatal, cerebellar and nucleus accumbens mutant neurons, exhibited a marked reduction in the length of rootlet-profiles (data not shown).

More particularly, in a number of CA1 pyramidal neurons, where the basal bodies were ideally sectioned in the longitudinal axis, a significant reduction in the length of basal bodies was found in KO mice (basal bodies mean length = 303.9 nm in KO mice compared with basal bodies mean length = 382.3 nm in the control WT mice, Mann Whitney test, p = 0.0042). A significant increase in the average inter-basal body/centriole distance was also identified from 392.3 nm in YifI B WT to 760.15 nm in YifI B KO mice (Mann Whitney test, p = 0.0007). Striated rootlets were often applied against several electron-dense filamentary-tubular aggregates. The ultrastructural analysis of YifI B KO CA1 pyramidal neurons, also revealed the presence of thicker bundles of striated rootlets extending from the basal bodies-centrioles, compared to the control animals.

Immunofluorescent techniques were therefore used to examine the primary cilium on sections from WT and Yif 1 B KO brain at the level of hippocampus, olfactory bulb and cerebellum. Preliminary observations using Arl13B and AC3 antibodies as classical markers of cilia showed brain cilium heterogeneity in labelling and in mean cilium length. AC3 was not detected in the cerebellum, while Arl13B was not detected in the hippocampus and poorly in the olfactory bulb.

Cilium length was different in the three brain regions analyzed. Whereas, for example in WT brain sections, cilium length mean was 8,8 μηι in the mitral layer of olfactory bulb (OB) and 7,7 μηι in CA1 layer of the hippocampus, it was shorter in the Purkinje cell layer of the cerebellum with a mean of 3.7 μηι. Moreover, in OB and hippocampus, whereas means were slightly similar, the distribution was completely different (data not shown). Statistical analysis revealed that the length of AC3- immunostained cilium in the pyramidal layer of the CA1 in the hippocampus was not different between WT and Yif 1 B KO sections (Mann Whitney test, p=0.4828). On a contrary, a significant increase of the length of cilium labeled with Arl13B antibody in the cerebellum in Yif 1 B KO section compared to WT (1 1 .4 % increase; Mann Whitney test, p=0.0005) and also for those labeled with AC3 antibody in the mitral layer in the olfactory bulb (22.8 %; Mann Whitney test, p<0.0001 ; WT, n=514 on 2-4 pictures from 5 animals; Yif 1 B, n=806 on 2-4 sections from 4 animals). The cilium length was thus increased in the structures exhibiting a behavioral / functional defect, i.e. cerebellum and olfactory bulb.

These data demonstrated that in absence of Yif 1 B expression, anatomical and associated behavioral defects in brain structure correlate with increase in cilium length (like in the cerebellum), whereas change in basal body organization without modification in ciliary length has no consequences on cellular anatomy, electrophysiology and related structure dependent behavior (like in the hippocampus). Ciliary length was also assessed in a completely different type of cells, i.e. in skin fibroblasts, and was found to be reduced in fibroblasts isolated from skin of Yif 1 B KO mice compared to control mice.

Claims

1. Agent for Yif1 B protein expression for use in the prevention and/or treatment of a ciliopathy.

2. The agent for use according to claim 1 , wherein said agent is selected from the group consisting of a nucleic acid encoding YifI B protein, a nucleic acid encoding a fragment of Yif 1 B protein, a nucleic acid encoding a Yif1 B promoter or a fragment of Yif 1 B promoter, cells expressing Yif 1 B protein and their combinations.

3. The agent for use according to claim 1 or 2, wherein said ciliopathy is characterized by ataxia, intellectual deficiency, visual dysfunction, male infertility, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy or their combinations.

4. The agent for use according to any one of claims 1 to 3, wherein said ciliopathy is selected in the group consisting of Alstrom syndrome, Joubert syndrome, Meckel syndrome, nephronophthisis, Bardet-Biedl syndrome, oral-facialdigital syndrome type 1 , Senior-Loken syndrome, polycystic kidney disease, polycystic liver disease, primary ciliary dyskinesia, asphyxiating thoracic dysplasia, Marden-Walker syndrome, situs inversus/isomerism, retinal degeneration, cerebrello-oculo-renal syndrome, Ellis-van Creveld syndrome, Jeune asphyxiating thoracic dystrophy, Leber congenital maurosis and their combinations.

5. The agent for use according to any one of claims 1 to 4, wherein said Yif 1 B protein comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 2.

6. The agent for use according to any one of claims 2 to 5, wherein said nucleic acid encoding Yif 1 B protein comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 .

7. The agent for use according to any one of claims 2 to 6, wherein said nucleic acid encoding YifI B protein, said nucleic acid encoding a fragment of Yif 1 B protein and/or said nucleic acid encoding a Yif 1 B promoter or a fragment of Yif 1 B promoter is provided in the form of a vector comprising said nucleic acid.

The agent for use according to claim 7, wherein said vector is a non-viral vector or a viral vector.

9. The agent for use according to any one of claims 1 to 8, wherein said agent is administered by the arterial, venous, nasal, intra-tissue and/or intraperitoneal route.

10. The agent for use according to claim 9, wherein said agent is administered by the intravitreal, subretinal, intra-testicular and/or intracerebral route.

11. A vector comprising a nucleic acid encoding YifI B protein, wherein said nucleic acid comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1 .

12. A method for diagnosing whether a subject suffers or is at risk of suffering from a ciliopathy, wherein said method comprises detecting in a biological sample of said subject (i) if at least one mutation leading to a non-functional Yif 1 B protein or a Yif 1 B protein with an altered function is present in the Yif 1 B gene and/or (i) if a functional Yif 1 B protein is expressed by the cells, wherein the presence of said at least one mutation or the absence of a functional Yif 1 B protein indicates that said subject suffers from or is at risk of suffering from said ciliopathy.

13. The method according to claim 12, wherein said biological sample is a blood sample, a plasma sample, a tissue sample or a nucleic acid sample.

14. A method for selecting a compound useful in the prevention and/or treatment of a ciliopathy, wherein said method comprises:

b) assessing at least one symptom of said ciliopathy in the treated Yif 1 B knock- out animal, c) selecting said test compound as being useful in the prevention and/or treatment of said ciliopathy when at least one symptom of the ciliopathy is improved or absent in the treated Yif 1 B knock-out animal.

15. A method for selecting a compound useful in the prevention and/or treatment of a ciliopathy, wherein said method comprises: