DE102022124232A1 - Antisense oligonucleotides for the treatment of Joubert syndrome - Google Patents

Abstract

The present invention relates to an exon skipping molecule that is linked to a polynucleotide with the nucleotide sequence shown in SEQ ID NO: 29, preferably SEQ ID NO: 30, more preferably SEQ ID NO: 31 or SEQ ID NO: 32, or a part binds to it and/or is complementary to it; a viral vector expressing the exon skipping molecule; a pharmaceutical composition comprising the exon skipping molecule or viral vector and a pharmaceutically acceptable excipient, and uses and methods related thereto.

Description

BACKGROUND

The present invention relates to an exon skipping molecule that is linked to a polynucleotide with the nucleotide sequence shown in SEQ ID NO: 29, preferably SEQ ID NO: 30, more preferably SEQ ID NO: 31 or SEQ ID NO: 32, or a part of which binds and/or is complementary thereto; a viral vector expressing the exon skipping molecule; a pharmaceutical composition comprising the exon skipping molecule or viral vector and a pharmaceutically acceptable excipient, and uses and methods related thereto.

Joubert syndrome (JS, OMIM #616490) is a rare genetic disorder. The diagnosis of JS can be confirmed by axial magnetic resonance imaging (MRI), which reveals hypoplasia or agenesis of the cerebellum in combination with malformations of the brainstem and cerebellum, resulting in the so-called “molar tooth sign” (MTS) (Maria et al . 1997). Other important diagnostic criteria include general developmental delay with hypotonia in infants. Later in development, the disease can progress to ataxia. In addition, a wide range of phenotypic findings have been described in JS, including retinal phenotypes, respiratory disorders, renal or hepatic changes, and polydactyly or reduced chest volume (Alby et al. 2015; Bachmann-Gagescu et al. 2015; Parisi and Glass 2003; Brancati et al. 2010).

JS is caused by dysfunction of the primary cilia and/or basal bodies (BB), so JS is classified as a ciliopathy. Indeed, ciliopathies include a growing number of genetic diseases caused by mutations in at least 200 genes relevant to ciliogenesis, cilia structure and function (Reiter and Leroux 2017).

Primary cilia are non-motile subcellular organelles found on the surface of almost all cell types. The primary cilium is an elongated structure anchored to the plasma membrane and contains a BB and an axoneme with a transition zone. The axoneme consists of nine paired microtubules and forms the structural core surrounded by a ciliary membrane (Carvalho-Santos et al. 2011; Fisch and Dupuis-Williams 2011). Primary cilia play an important role in signaling pathways such as Hedgehog, PDGF and Wnt. It is known that cilia sense and modulate extracellular signals and transmit signals, e.g. B. to regulate the proliferation, development or polarity of tissues (Singla and Reiter 2006; D'Angelo and Franco 2009; Park et al. 2019). The properties of cilia are important for sensory cells, particularly the cilium that connects the photoreceptors. Therefore, an increasing number of ciliopathies are experiencing symptoms associated with degenerative diseases of the retina (Adams et al. 2007).

More than 40 genes associated with primary cilia formation or maintenance have been described as carriers of pathogenic variants that cause JS. The JS-associated mutations are predominantly inherited in an autosomal recessive manner. Only variants in OFD1 are inherited in an X-linked manner (Coene et al. 2009; Reiter and Leroux 2017). Dominant inheritance patterns have not yet been identified in JS.

The human KIAA0586/TALPID3 gene (14q23.1) is the ortholog of the Talpid3 gene in chicken and mouse and has 13 known transcript variants with up to 34 exons (UCSC Genome Browser on human GRCh37/hg19).

KIAA0586/TALPID3 encodes a conserved protein at the distal end of the centriole that plays an important role in centrosomal migration, cilia formation, and hedgehog signaling (Yin et al. 2009; Ben et al. 2011; Davey et al. 2006) . It forms a ring-shaped structure at the distal end of the centrioles. The absence of KIAA0586/TALPID3 results in aberrant distribution of centriolar satellites important for protein trafficking. As a result, defects in ciliogenesis have been identified in KIAA0586/TALPID3-deficient cell lines (Yin et al. 2009; Ben et al. 2011; Bangs et al. 2011; Stephen et al. 2013; Alby et al. 2015). KIAA0586/TALPID3 appears to be an interaction partner of proteins important for cilia assembly, centriole maturation, and cell polarity, i.e., CP110, CEP120, and Rab8a (Kobayashi et al. 2014; Tsai et al. 2019; Wu et al . 2014).

KIAA0586/TALPID3 null mutations in various animal models, such as mice, chickens, and zebrafish, resulted in disruption of BB docking and loss of cilia, resulting in early embryonic death.

In 2015, KIAA0586/TALPID3 was described as a new JS gene. Biallelic likely pathogenic variants have been found to cause a relatively mild JS phenotype in several independent families (Bachmann-Gagescu et al. 2015b). In addition, some cases with single heterozygous mutations have been described in which a second mutation in KIAA0586/TALPID3 was not detected (Bachmann-Gagescu et al. 2015b; Roosing et al. 2015). These cases remained unexplained at the genetic level.

Due to the genetic heterogeneity and clinical variability of JS, treating patients is challenging. So far, the only therapeutic option is to treat the symptoms. Therefore, it is of great interest to develop new therapeutic approaches to treat JS symptoms caused by KIAA0586/TALPID3 mutations.

Accordingly, there is a need for new therapeutic approaches to treat the JS symptoms caused particularly by KIAA0586/TALPID3 mutations. Furthermore, there is a need for approaches to modulate splicing of KIAA0586/TALPID3 mutations.

The object underlying the present invention is therefore to provide products and/or methods that make it possible to treat the JS symptoms caused in particular by KIAA0586/TALPID3 mutations.

Another problem underlying the present invention is the provision of products and/or methods that make it possible to modulate the splicing of KIAA0586/TALPID3 mutations.

DESCRIPTION OF THE INVENTION

These requirements are met by the present invention with the subject matter specified in the independent and dependent claims. Surprisingly, it has now been shown that specific antisense oligonucleotides (AONs) are able to block the aberrant splicing of KIAA0586/TALPID3 caused by the intronic mutation.

In a first aspect, the object on which the present invention is based is achieved by an exon skipping molecule which is attached to a polynucleotide with the type in SEQ ID NO: 29, preferably SEQ ID NO: 30, more preferably SEQ ID NO: 31 or SEQ ID NO: 32 shown nucleotide sequence or a part thereof binds and / or is complementary thereto.

In a preferred embodiment, the exon skipping molecule is a nucleic acid molecule.

In another embodiment, the exon skipping molecule is a nucleic acid molecule comprising an antisense oligonucleotide, the antisense oligonucleotide having a length of from about 8 to about 179 nucleotides.

In another preferred embodiment, the antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.

In a further preferred embodiment, the exon skipping molecule comprises a 2'-O-alkylphosphorothioate antisense oligonucleotide, preferably 2'-O-methyl modified ribose (RNA), 2'-O-ethyl modified ribose, 2' -O-Propyl-modified ribose and/or substituted derivatives of these modifications, particularly preferably as halogenated derivatives, or a part thereof.

In a second aspect, the object on which the present invention is based is achieved by a viral vector which expresses an exon skipping molecule as defined by the present invention, preferably when placed under conditions that promote the expression of the molecule favor.

In a preferred embodiment, the viral vector is an AAV vector or a lentivirus vector, preferably an AAV2/5, AAV2/8, AAV2/9, AAV2/2 or a lentivirus vector.

In a third aspect, the object on which the present invention is based is achieved by a pharmaceutical composition which contains an exon skipping molecule according to the present invention or a viral vector according to the present invention and a pharmaceutically acceptable excipient.

In a fourth aspect, the object on which the present invention is based is achieved by the exon skipping molecule according to the present invention, the viral vector according to the present invention or the pharmaceutical composition according to the present invention for use as a drug.

In a fifth aspect, the object underlying the present invention is achieved by the exon skipping molecule according to the present invention, the viral vector according to the present invention or the pharmaceutical composition according to the present invention for use in the treatment of KIAA0586/TALPID3-related Disease or condition requiring modulation of KIAA0586/TALPID3 splicing, preferably for use in the treatment of Joubert syndrome.

In a sixth aspect, the object on which the present invention is based is achieved by a method for modulating the splicing of KIAA0586/TALPID3 in a cell, the method comprising the step of contacting the cell with an exon skipping molecule according to the present invention, the viral vector according to the present invention or the pharmaceutical composition according to the present invention.

The inventors of the present invention have analyzed two genetic variants in the KIAA0586/TALPID3 gene in all available relatives of two families using sequencing technologies. In the genetic changes in KIAA0586/TALPID3 (Chr. 14q23.1; 58,427,385-58,551,297; GRCh38:CM000676.2) are shown. The variant c.[1446delA],[=];p.[p.Val483*] is located in exon 12. The mutation c.[2512C>T],[=];p.[Arg838*] is located in exon 18. The deep intronic change c.[4149+3186G>A],[=]; p.[Asp1384Arg.fs*15] is located in intron 28. The nomenclature is based on the KiAA0586/TALPiD3 transcript variant NM_001244189.2.

In all embodiments of the present invention, the terms “modulatory splicing” and “exon skipping” are synonymous. With respect to KIAA0586/TALPID3, “modulatory splicing” or “exon skipping” is to be understood as the exclusion of the aberrant 132-nucleotide exon (SEQ ID NO: 28) from the KIAA0586/TALPID3 mRNA. The term “exon skipping” is defined here as the induction of a mature mRNA in a cell that does not contain a particular exon that would be present in the aberrant mRNA without exon skipping. Exon skipping is achieved by supplying a cell that expresses the pre-mRNA of the mature mRNA with a molecule capable of deleting sequences such as the (cryptic) splice donor or (cryptic) splice to disrupt the acceptor sequence required to enable the enzymatic process of splicing, or with a molecule capable of disrupting an exon inclusion signal required for the recognition of a stretch of nucleotides as one in the mature mRNA to be included is required; such molecules are referred to here as “exon skipping molecules”. The term “pre-mRNA” refers to an unprocessed or only partially processed precursor mRNA that is synthesized by transcription from a DNA template in the cell nucleus.

The term “antisense oligonucleotide” in all embodiments of the present invention refers to a nucleotide sequence that is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, an hrRNA (heterogeneous nuclear RNA), or an mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule containing the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. Preferably, the term refers to a nucleotide sequence that is completely complementary to a target nucleotide sequence in a pre-mRNA molecule, an hrRNA (heterogeneous nuclear RNA) or an mRNA molecule.

The terms “antisense oligonucleotide” and “oligonucleotide” within the meaning of the present invention are used interchangeably here and refer to an oligonucleotide with an antisense sequence.

In one embodiment according to the present invention, an exon skipping molecule as defined herein may be a composite molecule that binds to and/or is complementary to the specified sequence, or a protein such as an RNA binding protein or not -natural zinc finger protein modified to bind to the specified nucleotide sequence on an RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, in PCT/NL01/00697 and US patent 6,875,736 disclosed. Methods for designing RNA-binding zinc finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998). Binding to any of the specified sequences SEQ ID NO: 29, 30, 31 or 32, preferably in the context of the aberrant 132 nucleotide KIAA0586/TALPID3 exon (SEQ ID NO: 28), can be assessed using techniques similar to are known to a person skilled in the art. A preferred technique is the gel mobility shift assay, as in EP 1 619 249 described. In a preferred embodiment, an exon skipping molecule is said to bind to one of the specified sequences when the molecule binds to a marked sequence SEQ ID NO: 29, 30, 31 or 32 in a gel mobility shift assay is detectable.

In embodiments of the invention, an exon skipping molecule is preferably a nucleic acid molecule, preferably an oligonucleotide. Preferably, an exon skipping molecule according to the invention is a nucleic acid molecule, preferably an oligonucleotide, which is complementary or substantially complementary to a nucleotide sequence as shown in SEQ ID NO: 29, preferably SEQ ID NO: 30, more preferably SEQ ID NO : 31 or SEQ ID NO: 32, or a portion thereof, as later defined herein.

The term “substantially complementary” as used in the context of the present invention means that some mismatches in the antisense sequence are permitted as long as the functionality, i.e. H. skipping the aberrant 132 nucleotide KIAA0586ITALPID3 exon (SEQ ID NO: 28) is still acceptable. Preferably the complementarity is between 90% and 100%. This generally allows 1 or 2 mismatches in a 20 nucleotide oligonucleotide, or 1, 2, 3, or 4 mismatches in a 40 nucleotide oligonucleotide, or 1, 2, 3, 4, 5, or 6 mismatches in a 60 nucleotide oligonucleotide, etc .

• - Das Exon-Skipping-Molekül enthält vorzugsweise kein CpG oder einen CpG-Abschnitt,
• - Das Exon-Skipping-Molekül hat eine akzeptable RNA-Bindungskinetik und/oder thermodynamische Eigenschaften.

The present invention provides a method for designing an exon skipping molecule, preferably an oligonucleotide, capable of inducing skipping of the aberrant 132-nucleotide KIAA0586/TALPID3 exon (SEQ ID NO: 28). First, the oligonucleotide is selected to bind to one of SEQ ID NO: 29, 30, 31 or 32 or a portion thereof, as later defined herein. Subsequently, in a preferred method, at least one of the following aspects must be taken into account in order to further develop and improve the exon skipping molecule:

• - The exon skipping molecule preferably contains no CpG or a CpG section,
• - The exon skipping molecule has acceptable RNA binding kinetics and/or thermodynamic properties.

The presence of a CpG or a CpG section in an oligonucleotide is usually associated with increased immunogenicity of the oligonucleotide (Dorn and Kippenberger, 2008). This increased immunogenicity is undesirable because it can lead to damage to the tissue to be treated, i.e. H. the eye, retina, brain, lung, kidney, liver, extremities or parts thereof, oral hamartomas, muscles, peripheral nerves, endocrine tissue or other affected tissues. Immunogenicity can be assessed in an animal model by the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration. Immunogenicity can also be assessed in the blood of an animal or human treated with an oligonucleotide according to the invention by detecting the presence of a neutralizing antibody and/or an antibody that recognizes the oligonucleotide using a standard immunoassay known to those skilled in the art.

An increase in immunogenicity can be assessed by detecting the presence or increasing amount of a neutralizing antibody or an antibody that recognizes the oligonucleotide using a standard immunoassay.

The invention enables the design of an oligonucleotide with acceptable RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or the thermodynamic properties are determined at least in part by the melting temperature of an oligonucleotide (Tm; calc net with the Oligonucleotide Property Calculator (www.unc.edul-caillbiotool/oligo/index.html) for single-stranded RNA using the base Tm and nearest neighbor model) and/or the free energy of the AON target exon Complex determined (using RNA structure version 4.5). If the Tm value is too high, the oligonucleotide is likely to be less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to specify preferred ranges for each of these parameters. An acceptable Tm can be between 35 and 70°C and an acceptable free energy can be between 15 and 45 kcal/mol.

The person skilled in the art may therefore initially select as a potential therapeutic compound an oligonucleotide that binds to and/or is complementary to SEQ ID NO: 29, 30, 31 or 32 or a portion thereof, as later defined herein. The person skilled in the art can check whether the oligonucleotide is able to bind to the sequences defined above. Optionally, in a second step, he can use the invention to further optimize the oligonucleotide by checking the absence of CpG and/or optimizing its Tm and/or the free energy of the AON-target complex. He may attempt to design an oligonucleotide in which CpG is preferentially absent and/or in which a more acceptable Tm and/or free energy is obtained by using a different sequence of KIAA0586/TALPID3 (including SEQ ID NO: 29, 30, 31 or 32) is chosen, to which the oligonucleotide is complementary. Alternatively, if an oligonucleotide complementary to a particular portion within SEQ ID NO: 29, 30, 31 or 32 includes a CpG and/or does not have an acceptable Tm and/or free energy, one skilled in the art may improve any of these parameters, by reducing the length of the oligonucleotide and/or selecting a different portion within one of SEQ ID NO: 29, 30, 31 or 32 to which the oligonucleotide is complementary and/or by changing the chemistry of the oligonucleotide.

In each step of the process, an oligonucleotide of the invention is preferably an oligonucleotide that can still have an acceptable level of functional activity. A functional activity of the oligonucleotide is preferably to induce skipping of the aberrant 132-nucleotide KIAA0586/TALPID3 exon (SEQ ID NO: 28) to some extent to provide an individual with a functional KIAA0586/TALPID3 protein and/or or a functional mRNA and/or to at least partially reduce the production of an aberrant KIAA0586/TALPID3 protein and/or an aberrant mRNA. In a preferred embodiment, an oligonucleotide is said to induce skipping of the aberrant 132-nucleotide KIAA0586/TALPID3 exon (SEQ ID NO: 28) when the aberrant 132-nucleotide KIAA0586/TALPID3 exon (SEQ ID NO: 28) Skipping percentage measured by real-time quantitative RT-PCR analysis, at least 1%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30 %, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80 %, or at least 85%, or at least 90%, or at least 95%, or 100%.

Preferably, a nucleic acid molecule according to the invention, preferably an oligonucleotide, which comprises a sequence that is complementary or substantially complementary to a nucleotide sequence as shown in SEQ ID NO: 29, preferably SEQ ID NO: 30, particularly preferably SEQ ID NO: 31 or SEQ ID NO: 32, or a part thereof of KIAA0586/TALPID3 is such that the (substantially) complementary part makes up at least 50% of the length of the oligonucleotide according to the invention, more preferably at least 60%, more preferably at least 70%, more preferably at least 80% , more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99% or even more preferably 100%. Preferably, an oligonucleotide according to the invention comprises or consists of a sequence that is complementary to a part of SEQ ID NO: 29, 30, 31 or 32. For example, an oligonucleotide may include a sequence complementary to a portion of SEQ ID NO: 29, 30, 31 or 32, as well as additional flanking sequences. In a more preferred embodiment, the length of the complementary part of the oligonucleotide is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides. Additional flanking sequences can be used to modify the binding of a protein to the oligonucleotide or to modify a thermodynamic property of the oligonucleotide, preferably to modify the target RNA binding affinity.

It is therefore not essential that all bases in the region of complementarity can pair with bases on the opposite strand. For example, when designing the oligonucleotide, one may want to incorporate a residue that does not pair with the base of the complementary strand. Mismatches can nen may be permissible to a certain extent if the nucleotide section is sufficiently capable of hybridizing with the complementary part under the conditions in the cell. In this context, "sufficient" preferably means that the binding of an oligonucleotide with a gel mobility shift test, as in Example 1 of EP1619249 described, can be proven.

Optionally, the oligonucleotide can also be tested by transfection into cells derived from the patient, preferably fibroblasts, lymphoblastoids, retinal cells, stem cells or differentiated cells thereof. Skipping a targeted exon can be done by RT-PCR (as in EP1619249 described). The complementary regions are preferably designed so that in combination they are specific for the exon in the pre-mRNA. Such specificity can be established with different lengths of the complementary regions, as this depends on the actual sequences in other (pre-)mRNA molecules in the system. The risk that the oligonucleotide can also hybridize to one or more other pre-mRNA molecules decreases as the size of the oligonucleotide increases. It is clear that oligonucleotides that have mismatches in the region of complementarity but retain the ability to hybridize and/or bind to the target region(s) in the pre-mRNA can be used in the present invention. Preferably, however, at least the complementary parts do not contain such mismatches, since these typically have a higher efficiency and a higher specificity than oligonucleotides with such mismatches in one or more complementary regions. Higher hybridization strengths (i.e., increasing number of interactions with the opposite strand) are thought to increase the efficiency of the process of disrupting the system's splicing machinery. Preferably the complementarity is between 90% and 100%. This generally allows 1 or 2 mismatches in a 20 nucleotide oligonucleotide, or 1, 2, 3, or 4 mismatches in a 40 nucleotide oligonucleotide, or 1, 2, 3, 4, 5, or 6 mismatches in a 60 nucleotide oligonucleotide, etc .

An exon skipping molecule in the sense of the invention is preferably an isolated molecule.

An exon skipping molecule in the context of the invention is preferably a nucleic acid molecule or a nucleotide-based molecule, preferably an (antisense) oligonucleotide, which is complementary to a sequence selected from SEQ ID NO: 29, 30, 31 and 32 .

A preferred exon skipping molecule according to the invention is a nucleic acid molecule comprising an antisense oligonucleotide, the antisense oligonucleotide having a length of from about 8 to about 179 nucleotides, more preferably from about 8 to 70, more preferably from about 10 to 50 nucleotides , more preferably from about 10 to about 40 nucleotides, more preferably from about 12 to about 30 nucleotides, more preferably from about 14 to about 28 nucleotides, nucleotides, most preferably about 20 nucleotides, such as 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 45 nucleotides, 47 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides or 52 nucleotides kleotides.

A preferred exon skipping molecule of the invention is an antisense oligonucleotide comprising or consisting of 8 to 179 nucleotides, more preferably about 10 to 50 nucleotides, more preferably about 10 to 40 nucleotides, more preferably 12 to 30 nucleotides, more preferably 14 to 22 nucleotides , or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145 , 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides or consists of them.

In all embodiments of the present invention, in which an exon skipping molecule comprises or consists of an antisense oligonucleotide that binds to or is complementary to at least the portion of SEQ ID NO: 29 that contains the c.4149+3186G>A mutation, the exon skipping molecule preferably comprises a “T/U” nucleotide at the position complementary to the mutated “G” nucleotide in SEQ ID NO: 29.

In certain embodiments, the invention provides an exon skipping molecule comprising, or preferably consisting of, an antisense oligonucleotide selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.

In a preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 33. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 33 preferably comprises or preferably consists of 24 to 179 nucleotides, more preferably 24 to 40 nucleotides, even more preferably 24 to 30 nucleotides, even more preferably 24 to 22 nucleotides 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides.

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 34. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 34 preferably comprises or preferably comprises or consists of 19 to 143 nucleotides, more preferably 19 to 40 nucleotides, even more preferably 19 to 30 nucleotides, even more preferably 19 to 20 nucleotides 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70,75,80,85,90,95,100,110,115,120,125,130,135,140,145,150,155,160,165,170,175,176,177,178 or 179 nucleotides.

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 35. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 35 preferably comprises or preferably comprises or consists of 20 to 143 nucleotides, more preferably 20 to 40 nucleotides, even more preferably 20 to 30 nucleotides, even more preferably 20 to 21 nucleotides 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides .

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 36. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 36 preferably comprises or preferably comprises or consists of 21 to 143 nucleotides, more preferably 21 to 40 nucleotides, even more preferably 21 to 30 nucleotides, even more preferably 21 to 20 nucleotides 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides.

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 37. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586ITALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 37 preferably comprises or preferably consists of 20 to 143 nucleotides, more preferably 20 to 40 nucleotides, even more preferably 20 to 30 nucleotides, even more preferably 20 to 21 nucleotides 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides .

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 38. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 38 preferably comprises or preferably comprises or consists of 21 to 143 nucleotides, more preferably 21 to 40 nucleotides, even more preferably 21 to 30 nucleotides, even more preferably 21 to 22 nucleotides 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides.

In a further preferred embodiment, the invention provides an exon skipping molecule which comprises or preferably consists of the antisense oligonucleotide SEQ ID NO: 39. This molecule was found to be very efficient in modulating splicing of the aberrant 132-nucleotide KIAA0586/TALPID3 exon. This preferred exon skipping molecule of the invention, SEQ ID NO: 39 preferably comprises or preferably comprises or consists of 19 to 143 nucleotides, more preferably 22 to 40 nucleotides, even more preferably 22 to 30 nucleotides, even more preferably 22 to 23 nucleotides 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 176, 177, 178 or 179 nucleotides.

An exon skipping molecule according to the invention can have one or more RNA residues (consequently a "t" residue is a "u" residue as an RNA counterpart) or one or more DNA residues and / or one or more nucleotide analogues or - equivalents, as explained in more detail below.

It is preferred that an exon skipping molecule of the invention comprises one or more residues modified to increase nuclease resistance and/or increase the affinity of the antisense oligonucleotide for the target sequence. In a preferred embodiment, the antisense nucleotide sequence therefore comprises at least one nucleotide analogue or equivalent, where a nucleotide analogue or equivalent is defined as a residue with a modified base and/or a modified backbone and/or an unnatural internucleoside bond or a combination of these modifications . In a preferred embodiment, the nucleotide analog or equivalent comprises a modified backbone. Examples of such backbones are morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate, sulfonate backbones. and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are backbone-modified oligonucleotides that have been studied as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six-membered ring and the phosphodiester bond is replaced by a phosphodiamidate bond. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by stopping translation or interfering with pre-mRNA splicing rather than activating RNase H. Morpholino oligonucleotides have been successfully delivered into tissue culture cells by methods that physically disrupt the cell membrane, and a study comparing several of these methods found that scraping was the most efficient delivery method; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake into cells. In a recent report, triplex formation was demonstrated by a morpholino oligonucleotide, and due to the nonionic backbone, these studies demonstrated that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

It is further preferred that the bond between residues in a backbone does not contain a phosphorus atom, such as: B. a bond formed by short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatom and alkyl or cycloalkyl internucleoside bonds, or one or more short chain heteroatomic or heterocyclic internucleoside bonds.

A preferred nucleotide analog or equivalent is a peptide nucleic acid (PNA) with a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base pair recognition. The backbone of PNA consists of N-(2-aminoethyl)-glycine units linked by peptide bonds, with the nucleobases linked to the backbone by methylenecarbonyl bonds. An alternative backbone consists of a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule does not contain any charged phosphate groups, PNA-RNA hybrids are generally more stable than RNA-RNA or RNA-DNA hybrids (Egholm et al. (1993) Nature 365, 566-568).

Another preferred backbone comprises a morpholino nucleotide analog or equivalent in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A particularly preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO) in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring and the anionic phosphodiester bond between adjacent morpholino rings is replaced by a nonionic phosphorodiamidate bond is.

In a further embodiment, a nucleotide analog or equivalent according to the invention comprises a substitution of one of the non-bridging oxygen atoms in the phosphodiester bond. This modification slightly destabilizes base pairing but significantly increases resistance to nuclease degradation. A preferred nucleotide analog or equivalent includes phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonates including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkylphosphorame idat , thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

Another preferred nucleotide analog or equivalent of the invention comprises one or more sugar moieties mono- or di-substituted at the 2', 3' and/or 5' positions, such as: B. -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl or aralkyl, which may be interrupted by one or more heteroatoms; 0-, S- or N-alkyl; 0-, S- or N-alkenyl; 0-, S- or N-alkynyl; 0-, S- or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; - Dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar component may be a pyranose or a derivative thereof or a deoxypyranose or a derivative thereof, preferably ribose or a derivative thereof or deoxyribose or a derivative thereof. A preferred derivatized sugar component comprises a locked nucleic acid (LNA) in which the 2' carbon atom is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar component. A preferred LNA includes 2'-0,4'-C-ethylene bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions make the nucleotide analog or equivalent resistant to RNase H and nuclease and increase the affinity for the target RNA.

In another embodiment, a nucleotide analog or equivalent of the invention comprises one or more base modifications or substitutions. Modified bases include synthetic and natural bases such as inosine, xanthine, hypoxanthine and others - aza-, -deaza-, -hydroxy-, -halo-, -thio-, -thiol-, -alkyl-, -alkenyl-, -alkynyl- and thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.

One skilled in the art will understand that not all positions in an antisense oligonucleotide need to be uniformly modified. Furthermore, more than one of the above analogues or equivalents can be incorporated into a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogs or equivalents.

A preferred exon skipping molecule according to the invention comprises a 2'-O-alkyl phosphorothioate antisense oligonucleotide, such as. B. 2'-O-methyl-modified ribose (RNA), 2'-O-ethyl-modified ribose, 2'-O-propyl-modified ribose and / or substituted derivatives of these modifications such as halogenated derivatives. An effective antisense oligonucleotide according to the invention comprises a 2'-O-methyl ribose with a phosphorothioate backbone.

Those skilled in the art will also understand that various antisense oligonucleotides can be combined to efficiently skip the aberrant 132-nucleotide exon of KIAA0586/TALPID3. In a preferred embodiment, a combination of at least two antisense oligonucleotides is used in a method according to the invention, for example two different antisense oligonucleotides, three different antisense oligonucleotides, four different antisense oligonucleotides or five different antisense oligonucleotides.

An antisense oligonucleotide can be linked to a component that improves the uptake of the antisense oligonucleotide into cells. Examples of such entities are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including, but not limited to, antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, polyarginine, oligolysine or polylysine, antigen binding domains such as those from an antibody, a Fab fragment of an antibody, or a single-chain antigen-binding domain such as a cameloid single-chain antigen-binding domain.

An exon skipping molecule according to the invention can be administered indirectly using suitable means known in the art. If the exon skipping molecule is an oligonucleotide, it can, for example, be assigned to an individual or a cell, a tissue or an organ Individuals are administered in the form of an expression vector, the expression vector encoding a transcript comprising the oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, a viral-based expression vector is provided comprising an expression cassette or a transcription cassette that directs expression or transcription of an exon skipping molecule as identified herein. Accordingly, the present invention provides a viral vector that expresses an exon skipping molecule according to the invention when placed under conditions that favor expression of the exon skipping molecule. A cell can be provided with an exon skipping molecule capable of interfering with essential sequences that result in highly efficient skipping of the aberrant 132-nucleotide KIAA0586/TALPID3 exon through plasmid-derived antisense Oligonucleotide expression or viral expression provided by adenovirus or adeno-associated virus-based vectors. Expression can be controlled by a polymerase III promoter such as a U1, a U6 or a U7 RNA promoter. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV) or a retroviral vector such as a lentivirus vector and the like. Plasmids, artificial chromosomes and plasmids that are suitable for targeted homologous recombination and integration into the human genome of cells can also be used for the transfer of an oligonucleotide defined here. In the context of the present invention, preference is given to vectors in which transcription is driven by PollII promoters and/or in which the transcripts are in the form of fusions with U1 or U7 transcripts, which provide good results in providing small transcripts. The development of suitable transcripts is in the hands of the expert. PollII-controlled transcripts are preferred. Preferably in the form of a fusion transcript with a U1 or U7 transcript. Such fusions can be created as described (Gorman L et al, 1998 or Suter D et al, 1999).

The exon skipping molecule according to the invention, preferably an antisense oligonucleotide, can be supplied as such. However, the exon skipping molecule can also be encoded by the viral vector. Typically this occurs in the form of an RNA transcript which contains the sequence of an oligonucleotide according to the invention in part of the transcript.

A preferred system for expressing antisense oligonucleotides is an adenovirus-associated virus (AAV)-based vector. Single-chain and double-chain AAV-based vectors have been developed that can be used for extended expression of small antisense nucleotide sequences for highly efficient skipping of the aberrant 132-nucleotide KIAA0586/TALPID3 exon.

An AAV vector according to the present invention is a recombinant AAV vector and refers to an AAV vector comprising a portion of an AAV genome comprising an encoded exon skipping molecule according to the invention contained in a protein coat Capsid protein derived from an AAV serotype as described elsewhere herein.

Part of the AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9 and others. The protein coat of capsid protein can be from an AAV serotype such as AAV1, 2, 3, 4, 5, 8, 9 and others. A protein coat can also be called a capsid protein coat. An AAV vector may have one or preferably all wild-type AAV genes deleted, but may still contain functional ITR nucleic acid sequences. Functional ITR sequences are required for replication, rescue, and packaging of AAV virions. The ITR sequences can be wild-type sequences or have a sequence identity of at least 80%, 85%, 90%, 95% or 100% with wild-type sequences or e.g. B. can be changed by insertion, mutation, deletion or substitution of nucleotides as long as they remain functional. In this context, functionality refers to the ability to package the genome directly into the capsid shell and then enable expression in the host cell or target cell to be infected. Within the scope of the present invention, a capsid protein coat may be of a different serotype than the AAV vector genome ITR. An AAV vector according to the present invention can therefore consist of a capsid protein coat, i.e. H. the icosahedral capsid, which contains capsid proteins (VP1, VP2 and/or VP3) of an AAV serotype, e.g. B. AAV serotype 2, while the ITR sequences contained in this AAVS vector can be from any of the AAV serotypes described above, including an AAV2 vector. An “AAV2 vector” therefore comprises a capsid protein coat of serotype 2, while e.g. B. an “AAV5 vector” comprises an AAV serotype 5 capsid protein coat, both of which can encapsulate any AAV vector genome ITR according to the invention.

Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein coat of AAV serotype 2, 5, 8 or AAV serotype 9, wherein the AAV genome or the ITRs of AAV serotype 2, 5 present in the AAV vector , 8 or AAV serotype 9; such an AAV vector is called AAV2/2, AAV2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV5/9, AAV8/2 -, AAV 8/5, AAV8/8, AAV8/9, AAV9/2, AAV9/5, AAV9/8 or AAV9/9 vector.

Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein coat of AAV serotype 2, and the AAV genome or ITRs present in this vector are of AAV serotype 5; such a vector is referred to as an AAV2/5 vector.

Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein coat of AAV serotype 2, and the AAV genome or ITRs present in this vector are of AAV serotype 8; such a vector is referred to as an AAV2/8 vector.

Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein coat of AAV serotype 2, and the AAV genome or ITRs present in this vector are of AAV serotype 9; such a vector is referred to as an AAV2/9 vector.

Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein coat of AAV serotype 2, and the AAV genome or ITRs present in this vector are of AAV serotype 2; such a vector is referred to as an AAV2/2 vector.

A nucleic acid molecule encoding an exon skipping molecule according to the present invention and represented by any nucleic acid sequence is preferably inserted between the AAV genome or the above-mentioned ITR sequences, e.g. B. an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3' termination sequence.

“AAV helper functions” generally refer to the corresponding AAV functions required for AAV replication and packaging and provided to the AAV vector in trans. AAV helper functions complement the AAV functions that are missing in the AAV vector, but they lack the AAV ITRs (which are provided by the AAV vector genome). The AAV helper functions include the two major ORFs of AAV, namely the repcoding region and the cap coding region, or functionally substantially identical sequences thereof. Rep and cap regions are well known in the art, see e.g. B. Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or US 5,139,941 , which is referred to here. The AAV helper functions can be provided on an AAV helper construct, which can be a plasmid. The introduction of the helper construct into the host cell can e.g. B. by transformation, transfection or transduction before or simultaneously with the introduction of the AAV genome that is present in the AAV vector described here. The AAV auxiliary constructs according to the invention can therefore be selected so that they produce the desired combination of serotypes for the capsid protein shell of the AAV vector on the one hand and for the AAV genome present in the replication and packaging of the AAV vector on the other hand. “AAV helper viruses” provide additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2), and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as in US 6,531,456 described.

Preferably, an AAV genome, as present in a recombinant AAV vector according to the present invention, does not contain nucleotide sequences encoding viral proteins, such as: B. the rep (replication) or cap (capsid) genes of AAV. An AAV genome may also contain a marker or reporter gene, such as B. a gene encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) coded, which is known in the art.

A preferred AAV vector according to the present invention is an AAV vector, preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, which expresses an exon skipping molecule according to the present invention , which comprises an antisense oligonucleotide, wherein the antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39. More preferably, the antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of : SEQ ID NO: 33 and SEQ ID NO: 34.

In a preferred embodiment, a viral vector according to the present invention expresses two or more exon skipping molecules according to the present invention, preferably when placed under conditions that favor expression of the molecules.

In view of the progress already made, improvements can be expected in terms of providing an individual or a cell, a tissue or an organ of this individual with an exon skipping molecule according to the invention. Such future improvements can of course be incorporated to achieve the mentioned effect on the restructuring of mRNA using a method according to the invention. An exon skipping molecule according to the invention can be administered to an individual, a cell, a tissue or an organ of that individual as it is. When administering an exon skipping molecule according to the invention, the molecule is preferably dissolved in a solution that is compatible with the method of administration. Cells can be provided with a plasmid for the expression of antisense oligonucleotides by providing the plasmid in an aqueous solution. Alternatively, a plasmid can also be provided by transfection with known transfection agents. For intravenous, subcutaneous, intramuscular, subretinal, intravitreal, intrathecal and/or intraventricular administration, it is preferred that the solution is a physiological saline solution. Particularly preferred in the invention is the use of an adjuvant or transfection agent which supports the delivery of each of the components defined here to a cell and/or into a cell. Preferred are adjuvants or transfection agents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that transport any component defined herein, complexed or enclosed in a vesicle or liposome, through a cell membrane. Many of these excipients are known in the professional world. Suitable adjuvants or transfection agents include polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropylenimine or polyethyleneimine copolymers (PECs) and derivatives, synthetic amphiphiles (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins capable of self-assembling into particles capable of delivering any component defined herein into a cell. It has been shown that such adjuvants can efficiently deliver an oligonucleotide such as antisense nucleic acids to a variety of cultured cells. Their high transfection potential is associated with low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be exploited to enable further modifications and analysis of their further (in vivo) nucleic acid transfer properties and toxicity.

Lipofectin is an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cf. DOTAP, the methyl sulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine ( DOPE). The neutral component mediates intracellular release.

Another group of carrier systems are polymeric nanoparticles. Polycations such as diethylaminoethylaminoethyl (DEAE)-dextran, known as a DNA transfection reagent, can be combined with butyl cyanoacrylate (PBCA) and hexyl cyanoacrylate (PHCA) to formulate cationic nanoparticles that carry any component defined herein, preferably an oligonucleotide, across cell membranes cells can be introduced.

In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulating an oligonucleotide with colloids. This colloidal nanoparticle system can form so-called proticules, which can be prepared through a simple self-assembly process to package an oligonucleotide and mediate its intracellular release. One skilled in the art may select and adapt any of the above or other commercially available alternative adjuvants and delivery systems to package and deliver an exon skipping molecule for use in the present invention to prevent, treat or delay KIAA0586/TALPID3-related disease to administer an illness or condition. “Prevention, treatment or delay of a KIAA0586/TALPID3-related disease or condition” is preferably defined herein as preventing, stopping, terminating the progression or reversing partial or complete Joubert syndrome caused by a genetic defect in the KIAA0586/TALPID3 gene is caused.

In addition, an exon skipping molecule according to the invention could be covalently or noncovalently linked to a targeting ligand that is specifically designed to facilitate uptake into the cell, cytoplasm and/or nucleus. Such a ligand could be (i) a compound (including, but not limited to, peptide (like) structures) that has cell, tissue or organ-specific Ele ments that facilitate cellular uptake, and/or (ii) comprise a chemical compound that facilitates uptake into cells and/or intracellular release of an oligonucleotide from vesicles, e.g. B. endosomes or lysosomes, can facilitate.

Therefore, in a preferred embodiment, an exon skipping molecule according to the invention is formulated in a composition or a drug or a composition containing at least one adjuvant and/or a targeting ligand for delivery and/or a delivery device to a cell and/or for improving the intracellular delivery.

If a composition contains an additional ingredient, such as: B. an additional compound as defined later herein, not each component of the composition need be formulated in a single combination or composition or preparation. Depending on their identity, the person skilled in the art will know which type of formulation is most suitable for each ingredient defined here. In a preferred embodiment, the invention provides a composition or preparation which is in the form of a kit containing an exon skipping molecule according to the invention and a further additional compound as defined later herein.

If necessary, an exon skipping molecule according to the invention or a vector, preferably a viral vector, which expresses an exon skipping molecule according to the invention, can be introduced into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier. Accordingly, the invention also provides a composition, preferably a pharmaceutical composition, which contains an exon skipping molecule according to the invention or a viral vector according to the invention and a pharmaceutically acceptable excipient. Such a composition may comprise a single exon skipping molecule according to the invention, but may also comprise several different exon skipping molecules according to the invention. Such a pharmaceutical composition may contain any pharmaceutically acceptable excipient, including a carrier, a capsule, a filler, a preservative, an adjuvant, a solubilizer and/or a diluent. Such pharmaceutically acceptable carriers, fillers, preservatives, excipients, solubilizers and/or diluents can be found, for example, in Remington, 2000. Each feature of the composition has already been defined herein. In a preferred embodiment, a composition according to the present invention comprises two or more exon skipping molecules according to the present invention, or a viral vector according to the present invention expressing two or more exon skipping molecules according to the present invention, or two or more viral vectors according to the present invention, wherein the two or more viral vectors are both or all according to the present invention but are different from each other.

When several different exon skipping molecules are used according to the invention, the concentration or dose defined herein may refer to the total concentration or dose of all oligonucleotides used or to the concentration or dose of each exon skipping molecule used or added. Therefore, in one embodiment, a composition is provided in which each or the total amount of the exon skipping molecules used according to the invention is dosed in an amount between 0.1 and 20 mg/kg, preferably between 0.5 and 20 mg/kg.

A preferred exon skipping molecule according to the invention is intended for the treatment of a KIAA0586/TALPID3-related disease or condition of an individual. In all embodiments of the present invention, the term “treatment” is intended to include preventing and/or delaying the KIAA0586/TALPID3-related disease or condition. An individual who can be treated with an exon skipping molecule of the invention may have already been diagnosed with a KIAA0586/TALPID3-related disease or condition. Alternatively, an individual who can be treated with an exon skipping molecule according to the invention may not have yet been diagnosed with a KIAA0586/TALPID3-related disease or condition, but may be an individual who is at increased risk due to their genetic background , to develop a KIAA0586/TALPID3-related disease or condition in the future. A preferred individual is a human or an animal, particularly preferably a human. In a preferred embodiment, the disease or condition associated with KIAA0586/TALPID3 is Joubert syndrome.

Accordingly, the present invention further provides an exon skipping molecule or viral vector or composition of the invention for use as a medicament for treating a KIAA0586/TALPID3-related disease or a condition requiring modulation of KIAA0586/TALPID3 splicing and for use as a drug to prevent, treat or delay a KIAA0586/TALPID3-related disease or condition. A preferred KIAA0586/TALPID3-related disease or condition is Joubert syndrome. Each feature of this use has already been defined here.

The invention further contemplates the use of an exon skipping molecule or a viral vector or composition of the invention for treating a KIAA0586/TALPID3-related disease or condition that requires modulation of KIAA0586/TALPID3 splicing. In a preferred embodiment, the KIAA0586/TALPID3-related disease or condition is Joubert syndrome.

The present invention further provides the use of an exon skipping molecule according to the invention or a viral vector or a composition according to the invention for the manufacture of a medicament, for the manufacture of a medicament for the treatment of a KIAA0586/TALPID3-related disease or condition, the or the one modulation of KIAA0586/TALPID3 splicing and for the manufacture of a medicament for the prevention, treatment or delay of a KIAA0586/TALPID3-related disease or condition. A preferred KIAA0586/TALPID3-related disease or condition is Joubert syndrome. Therefore, in a further aspect, the use of an exon skipping molecule, a viral vector or a composition as defined herein for the manufacture of a medicament for the manufacture of a medicament for treating a condition which involves the modulation of splicing of KIAA0586/ TALPID3 requires and is provided for the manufacture of a medicament to prevent, treat or delay a KIAA0586/TALPID3-related disease or condition. A preferred KIAA0586/TALPID3-related disease or condition is Joubert syndrome. Each feature of this use has already been defined here. An exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention for producing a medicament according to the invention is preferably administered systemically, in utero, intravitreal, subretinal, intrathecal or intraventricular.

A treatment in an application according to the invention or in a method according to the invention lasts at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or longer. Any exon skipping molecule or exon skipping oligonucleotide or an equivalent thereof, as defined herein for use according to the invention, may be suitable for direct administration to a cell, tissue and/or organ in vivo from individuals who are already affected or are at risk of developing a KIAA0586/TALPID3-related disease or condition and can be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an oligonucleotide according to the invention, a composition according to the invention, a compound according to the invention or an additive according to the invention may depend on various parameters such as the age of the patient, the mutation of the patient, the number of exon skipping molecules (i.e. the dose) and the formulation of the molecule. The frequency can range from at least once in two weeks, or three weeks, or four weeks, or five weeks, or a longer period.

The dose ranges of an exon skipping molecule according to the invention, preferably an oligonucleotide, are preferably determined on the basis of increasing dose studies in clinical trials (in vivo use) for which there are strict protocol requirements. An exon skipping molecule or an oligonucleotide according to the present definition can be used at a dose between 0.1 and 20 mg/kg, preferably between 0.5 and 20 mg/kg. In a preferred embodiment, a concentration of an oligonucleotide defined here that is between 0.1 nM and 1 μM is used. Preferably, this region is intended for in vitro use in a cellular model such as cells or tissue. Preferably the concentration used is in the range of 1 to 400 nM, more preferably in the range of 10 to 200 nM, even more preferably in the range of 50 to 100 nM. If multiple oligonucleotides are used, this concentration or dose may refer to the total concentration or dose of the oligonucleotides or to the concentration or dose of each individual oligonucleotide added.

In a preferred embodiment, a viral vector, preferably an AAV vector, as described above, is used as a carrier for a molecule according to the invention in a dose of 1×10 ⁹ - 1×10 ¹⁷ virus particles per injection, more preferably from 1x10 ¹⁰ - 1x10 ¹⁴ and most preferably 1x10 ¹⁰ - 1x10 ¹² virus particles administered per injection.

The concentration or dose ranges of the oligonucleotides given above are preferred concentrations or doses for in vitro or ex vivo applications. The person skilled in the art will understand that depending on the oligonucleotide or oligonucleotides used, the target cell to be treated, the target gene and its expression level, the medium used and the transfection and incubation conditions, the concentration or dose of the oligonucleotide(s) used will vary further can and may need to be further optimized.

An exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention for use according to the invention may be suitable for administration to a cell, a tissue and / or an organ in vivo of individuals already suffering from a KIAA0586 / TALPID3-related disease or a related condition or who are at risk of developing one and may be administered in vivo, ex vivo or in vitro. The exon skipping molecule or a viral vector or composition according to the invention can be administered directly or indirectly to a cell, a tissue and / or an organ in vivo of an individual already suffering from a KIAA0586 / TALPID3-related disease or a related condition is affected or at risk and can be administered directly or indirectly in vivo, ex vivo or in vitro. An exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention for use according to the invention is preferably administered systemically, in utero, intravitreal, subretinal, intrathecal or intraventricular.

The invention further provides a method for modulating splicing of KIAA0586/TALPID3 in a cell, comprising contacting the cell with an exon skipping molecule or a viral vector or composition according to the invention. The features of this aspect are preferably those previously defined herein. Bringing the cell into contact with an exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention can be carried out using any method known to those skilled in the art. The use of the methods described herein for administering exon skipping molecules, viral vectors and compositions is included. The contacting can take place directly or indirectly and in vivo, ex vivo or in vitro. In a preferred method for modulating splicing of KIAA0586/TALPID3 in a cell, the method comprises contacting the cell with two or more exon skipping molecules according to the present invention, or two or more viral vectors according to the present invention , or two or more compositions according to the present invention.

The invention further provides a method for treating a KIAA0586/TALPID3-related disease or condition that requires modulating the splicing of KIAA0586/TALPID3 of an individual in need thereof, the method comprising contacting a cell of the individual with a Exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention. The features of this aspect are preferably those previously defined herein. Bringing the cell into contact with an exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention can be carried out using any method known to those skilled in the art. The use of the methods described herein for delivery of molecules, viral vectors and compositions is included. The contacting can take place directly or indirectly and in vivo, ex vivo or in vitro. A preferred KIAA0586/TALPID3-related disease or condition is Joubert syndrome. Accordingly, an exon skipping molecule according to the invention or a viral vector according to the invention or a composition according to the invention is preferably administered systemically, in utero, intravitreal, subretinal, intrathecally or intraventricularly in a treatment method according to the invention. In a preferred method for treating a KIAA0586/TALPID3-related disease or condition that requires modulation of KIAA0586/TALPID3 splicing of an individual in need thereof, the method comprises contacting the cell with two or more exons -Skipping molecules according to the present invention or two or more viral vectors according to the present invention or two or more compositions according to the present invention.

Unless otherwise stated, each embodiment described herein may be combined with another embodiment described herein.

In this document and in the claims, the verb "comprise" and its conjugations are used in their non-limiting meaning, meaning that the elements following the word are included, but elements not expressly mentioned are not excluded. Furthermore, reference to an element by the indefinite article “a” or “an” does not preclude the presence of more than one of the elements, unless the context clearly requires that it be a single element. The indefinite article “a” or “an” therefore usually means “at least one”. The word “about” or “approximately,” when used in conjunction with a numerical value (e.g., about 10), preferably means that the value is more or less than 0.1% of the stated value (of 10). value can be.

The sequence information provided here should not be construed so narrowly as to require the inclusion of incorrectly identified bases. The person skilled in the art is able to recognize such incorrectly identified bases and knows how to correct such errors. In the event of sequence errors, the sequence of the polypeptide obtainable by expression of the gene in SEQ ID NO: 1 containing the nucleic acid sequence encoding the polypeptide should prevail.

The present invention is further illustrated by the following figures and examples, from which additional features, embodiments, aspects and advantages of the present invention can be seen.

DESCRIPTION OF THE FIGURES

• shows Joubert syndrome families carrying a novel deep intronic sequence alteration in KIAA0586/TALPID3.
• In A family tree of family 1 is shown including the co-segregation of genotypes among family members. The index patient is indicated by an arrow. The MRI scans of the index patient and the sibling are shown below the family tree. Axial T1-weighted MRI images documented the molar sign (arrowhead) characteristic of JS with elongated and thickened superior cerebellar peduncles and a deepened interpeduncular fossa in both siblings of family 1. In Sanger sequencing of the two genetic variants in the KIAA0586/TALPID3 gene in all available relatives of family 1 is shown. In an RT-PCR analysis of patient and control cells (family 1, index patient) of the coding region from exon 28 to exon 30 is shown. A 454 bp long fragment was identified that was exclusively expressed in the patient. The 322 bp RT-PCR fragment was identified in control and patient samples. In A family tree of Family 2 is shown with the genotypes of the available family members. In Sanger sequencing of the two genetic variants in the KIAA0586/TALPID3 gene in all available relatives of family 2 is shown. In a representation of the genetic changes in KIAA0586/TALPID3 (Chr. 14q23.1; 58.427.385-58.551.297; GRCh38:CM000676.2) can be seen. The variant c.[1446delA],[=];p.[ p.Val483*] is located in exon 12. The mutation c.[2512C>T],[=];p.[Arg838*] is located in exon 18. The deep intronic change c.[4149+3186G>A],[=]; p.[Asp1384Arg.fs*15] is located in intron 28. The nomenclature is based on the KIAA0586/TALPID3 transcript variant NM_001244189.2. NTC: non-template control; Ref. seq.: reference sequence; BP: branch point; SAS: splice acceptor site; SDS: splice donor site; II: father; II.I: mother; II.I; first child; II.II: second child. Female and male people are represented by circles and squares, respectively. Solid symbols indicate affected family members.
• shows the position of the AONs in and around the cryptic exon in intron 28 of KIAA0586/TALPID3.
• demonstrates the therapeutic effect of AON treatments to correct mutational cryptic splicing of patient-derived KIAA0586/TALPID3 transcripts.
• In the region of intron 28 is shown, which carries the deep intronic alteration KIAA0586: c.4149+3186G>A (NM_001244189.2). The mutation creates a branch point (red) that leads to a splicing defect of parts of intron 28 and a cryptic exon (blue bar) in patients. The early in-frame stop of the cryptic exon is shown in bold and asterisks. The binding sites of AON_+26 and AON_+57 are marked by green brackets above the cryptic exon. In are RT-PCR analyzes of patient and control fi broblasts presented under different treatment conditions. The fibroblasts were incubated with AON_+26 and/or AON_+57 to correct the splicing defect caused by the mutation. The larger PCR product (454 bp) corresponds to the mis-spliced transcript carrying the cryptic exon. The lower PCR fragment (322 bp) represents the reference transcript. Control PCR reactions without reverse transcriptase (-RT) or template (NTC) are shown on the right. In semiquantitative analyzes using fragment intensity measurements confirm that the cryptic exon (blue) is reduced relative to the correctly spliced product by AON treatment. *: p-value <0.05; ***: p-value <0.001; ns: not significant; NTC: non-template control; PBS: cells treated with PBS only; BP: branch point; SAS: splice acceptor site; SDS: splice donor site.
• shows the measurement of cilia length.
• In , images of primary cilia from patient fibroblasts compared to two untreated controls are shown (scale bars: 5 µm, 1 µm for inlet). In The length of the primary cilia of patient-derived fibroblasts and unrelated controls was quantified. A total of 750 cilia per cell line were measured in three independent experiments (250 cilia per experiment). The median of the boxblots was 2.6 µm in the controls and 1.4 µm in the patient. Statistical analysis was performed using the Kruskal-Wallis test. ***: p-value < 0.001. ns: not significant.

SEQUENCES

All sequences are shown here from 5'->3'. Table 1. Sequences as given in the sequence listing SEQ ID NO: SEQ type Description/sequence 1 Genomic DNA KIAA0586/TALPID3 2 cDNA KIAA0586/TALPID3, transcript variant 3 3 cDNA KIAA0586/TALPID3, transcript variant 4 4 cDNA KIAA0586/TALPID3, transcript variant 2 5 cDNA KIAA0586/TALPID3, transcript variant 1 6 cDNA KIAA0586/TALPID3, transcript variant 12 7 cDNA KIAA0586/TALPID3, transcript variant 9 8th cDNA KIAA0586/TALPID3, transcript variant 13 9 cDNA KIAA0586/TALPID3, transcript variant 5 10 cDNA KIAA0586/TALPID3, transcript variant 11 11 cDNA KIAA0586/TALPID3, transcript variant 10 12 cDNA KIAA0586/TALPID3, transcript variant 7 13 cDNA KIAA0586/TALPID3, transcript variant 8 14 cDNA KIAA0586/TALPID3, transcript variant 6 15 PRT KIAA0586/TALPID3 protein, isoform 3 16 PRT KIAA0586/TALPID3 protein, isoform 4 17 PRT KIAA0586/TALPID3 protein, isoform 2 18 PRT KIAA0586/TALPID3 protein, isoform 1 19 PRT KIAA0586/TALPID3 protein, isoform 12 20 PRT KIAA0586/TALPID3 protein, isoform 9 21 PRT KIAA0586/TALPID3 protein, isoform 3 22 PRT KIAA0586/TALPID3 protein, isoform 5 23 PRT KIAA0586/TALPID3 protein, isoform 11 24 PRT KIAA0586/TALPID3 protein, isoform 10 25 PRT KIAA0586/TALPID3 protein, isoform 7 26 PRT KIAA0586/TALPID3 protein, isoform 8 27 PRT KIAA0586ITALPID3 protein, isoform 6 28 DNA 132 nucleotide aberrant KIAA0586/TALPID3 Exon: agacggggtctcactatgttggctaggctgatctccaacttctgac cgcaagtgatcctcctgcctcctcagccttccaaagtcctgggatt ataggcatgagccaccatttctggcttagtatataattttgag 29 Polynucleotide 179 nucleotide motif: taggcgtgtaccatacctagctaattttttttttgcagagacggggt ctcactatgttggctaggctgatctccaacttctgaccgcaagtga tcctgcctcagccttccaaagtcctgattataggcatgagccacc atttctggcttagtatataattttgaggtgtgtgt 30 Polynucleotide 50 nucleotide motif: ggctgatctccaacttctgaccgcaagtgatcctcctgcctcagc cttcc 31 Polynucleotide 24 nucleotide motif: ggctgatctccaacttctgaccgc 32 Polynucleotide 19 nucleotide motif: cctcctgcctcagccttcc 33 AON-1 gcggucagaaguuggagaucagcc 34 AON-2 ggaaggcugaggcaggagg 35 AON-3 gcuagguaugguacacgccu 36 AON-4 auuagcuagguaugguacacg 37 AON-5 gagaccccgucucugcaaaa 38 AON-6 acacaccucaaaaauuauauac 39 AON-7 gccagaaaugguggcucaugcc

According to WIPO ST.26, uracil and thymine in a sequence of the sequence listing are both represented by “t” and without further annotation; “t” stands for uracil in RNA and thymine in DNA. Since the sequences according to SEQ ID NOs 33 to 39 in Table 1 are RNA sequences and therefore contain “u” for uracil, the corresponding sequences SEQ ID NOs 33 to 39 each contain a “t” in the required sequence listing according to WIPO ST.26. if the RNA sequence contains a “u” according to SEQ ID NOs 33 to 39 in Table 1. Table 2. Sequences of the primers as given in the sequence listing Primers SEQ ID NO: sequence Length [bp] KIAA0586_ex1-2_fwd 40 gtcgggagctcttcaagtct 20 KIAA0586_ex1-2_rev 41 gaggaacacagggcaactca 20 KIAA0586_ex2-6_fwd 42 tgttcctccggcattgtctg 20 KIAA0586_ex2-6_rev 43 ccactgtgatcactccagcc 20 KIAA0586_ex6-8_fwd 44 actgcagtctctacctcctca 21 KIAA0586_ex6-8_rev 45 gctgctctgtaacacgttgc 20 KIAA0586_ex8-10_fwd 46 gcaacgtgttacagagcagc 20 KIAA0586_ex8-10_rev 47 ccttgaaacaggtacaggagca 22 KIAA0586_ex10-13_fwd 48 tttgctcctgtacctgtttcaag 23 KIAA0586_ex-10-13_rev 49 tctgtggaagatctggaagctt 22 KIAA0586_ex13-16_fwd 50 gagctgccatgtattcgctt 20 KIAA0586_ex13-16_rev 51 tggtctctgtggtctggact 20 KIAA0586_ex16-18_fwd 52 tatcagggccatcgaagcac 20 KIAA0586_ex16-18_rev 53 ttacagggtgtggcttgctg 20 KIAA0586_ex17-19_fwd 54 aggtcatctgattcctatggcaa 23 KIAA0586_ex17-19_rev 55 tgcacaggaggaagagatgc 20 KIAA0586_ex19-22_fwd 56 acccagtgtagatattgacagca 23 KIAA0586_ex19-22_rev 57 ttgctggactggaaagagcc 20 KIAA0586_ex22-24_fwd 58 ggctctttccagtccagcaa 20 KIAA0586_ex22-24_rev 59 atgaaggtgagcaaggaggc 20 KIAA0586_ex24-26-fwd 60 gctcaccttcatcacctgct 20 KIAA0586_ex24-26_rev 61 gaggctgagcagggaaatcc 20 KIAA0586_ex26-28_fwd 62 cctcacagatgccaggttct 20 KIAA0586_ex26-28_rev 63 ctgaatgatagactgcttgctgg 23 KIAA0586_ex28-30_fwd 64 agtcagcagtttcccagcaa 20 KIAA0586_ex28-30_rev 65 gctgtcagaagtgttgtggg 20 KIAA0586_ex30-33_fwd 66 cccacaacacttctgacagc 20 KIAA0586_ex30-33_rev 67 atgacaaacctccacagccc 20 KIAA0586_ex32-34_fwd 68 ttgagcttaatccgtacctcaca 23 KIAA0586_ex32-34_rev 69 aaacactgtgctggctgtct 20 KIAA0586_cryE28_fwd 70 gtttcagtctgttgttcaggc 21 KIAA0586_cryE28_rev 71 gggatctcatacacagctagggg 21 KIAA0586_Exon18_fwd 72 ggggtgattgtcagataggg 20 KIAA0586_Exon18_rev 73 ggatttccgtactaacgttaagc 23

“KIAA0586_ex1-2_fwd” means that the forward primer in the KIAA0586 gene binds in exon 1 and “KIAA0586_ex1-2_rev” means that the reverse primer binds in exon 2. The same applies to the other primers up to and including exon 34.

The primers according to SEQ ID NOs 69 to 73 are genomic primers, i.e. H. they bind in the intron flanking the sequence to be analyzed by Sanger sequencing.

Unless otherwise stated, conventional standard methods of molecular biology, virology, microbiology or biochemistry are used in carrying out the invention. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

The present invention will be described in more detail using the following examples, which should not be construed as limiting the scope of the invention.

EXAMPLES

EXAMPLE 1

Materials and methods

DNA extraction

EDTA blood samples from available family members or cell pellets from patient-derived fibroblasts were collected and submitted for molecular genetic analysis. The DNA samples were prepared using the Gentra Puregene Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Quantity and quality of the DNA samples were analyzed using UV spectrometry (Biospectrometer, Eppendorf, Hamburg, Germany).

Primers, PCR amplification and Sanger sequencing

Forward and reverse primers (SEQ ID NOs: 40 - 73) located in the regions of interest of human KIAA0586/TALPID3 (NM_001244189.2) were designed using Primer Input3 (http://primer3.ut.ee/). Additionally, the primer binding sites in the human genome (GRCh37/hg19) were checked for known single nucleotide polymorphisms using SNPCheck (https://secure.ngrl.org.uk/SNPCheck/). PCR amplification of target regions was performed with 20 ng of genomic DNA using HotFirePol DNA polymerase (Solis BioDyne, Tartu, Estonia) according to standard protocols. For Sanger sequencing, the PCR amplicons were enzymatically purified with Exo-SAP (New England Biolabs (NEB), Frankfurt, Germany) and analyzed using the BigDye® Terminator v3.1 Cycle Sequencing Kit on the ABI Prism 3130x) Genetic Analyzer (Applied Biosystem, Carlsbad, California, USA). Sequencing data were analyzed using SnapGene software (GLS Biotech, Chicago, Illinois, USA).

AON design

Two different AONs complementary to portions of the cryptic exon were designed (AON_+26:5'-GCGGUCAGAAGUUGGAGAUCAGCC-3', = SEQ ID NO: 33, further described herein as “AON_+26”; AON_+57:5 '-GGAAGGCUGAGGCAGGAGG-3', = SEQ ID NO: 34, further described herein as “AON_+57”). The AONs were modified with a 2'-O-methyl group and with a phosphorothioate bond to aid uptake and stability (Beltinger et al. 1995; Geary R. S. et al. 2000; Eckstein 2014). After synthesis (Eurogentec, Cologne, Germany), the AONs were dissolved in phosphate-buffered saline (PBS; Chemsolute, Renningen, Germany) and stored at −80 °C.

Cell culture and AON treatment

Primary fibroblasts from patients and unrelated controls were obtained from a skin biopsy as previously described (Glaus et al. 2011). Human dermal fibroblasts were grown at 37 °C and 5% CO ₂ in Minimum Essential Medium (MEM; L0440-500, Biowest, Nuaillé, France) with 20% fetal bovine serum (Biowest), 1.4% -L-glutamine (Biowest). and 1% penicillin-streptomycin solution (Biowest). 1.6 or 2.5 x 10 ⁵ cells per well were placed in a twelve or six well plate. The AONs were added to the medium without transfection reagent at a concentration of 10 nM.

After an incubation period of 48 hours, the cells were washed with PBS and used for further analysis.

RNA isolation and RT-PCR analyses

After 48 hours of incubation with or without AONs, the fibroblasts were washed with ice-cold PBS and then lysed with 350 μl of lysis buffer RA1 (Macherey & Nagel, Düren, Germany) containing 3.5 μl of β-mercaptoethanol (Serva, Heidelberg, Germany). . The lysates were obtained by scraping with a pipette tip in combination with pipetting up and down at least 5 times. Total RNA was purified according to the manufacturer's instructions (Nucleospin RNA Isolation Kit, Macherey & Nagel). The cDNA first-strand sequence was prepared using 500 ng RNA and random primers (Metabion, Planegg, Germany) and reverse transcriptase (SuperScript III, Invitrogen). Reverse transcription was performed following standard protocols. To amplify the desired 1 µl cDNA was used to create the transcript. Amplification was performed using HotFire Taq Polymerase (Solis Biodyne, Tartu, Estonia) according to the manufacturer's recommendations. The PCR products were analyzed using agarose gel electrophoresis (1 to 1.5% agarose gels in TAE buffer). The primers used are listed in Table 2.

Immunocytochemistry

Fibroblasts were placed in 12-well plates on coverslips (12 mm) in supplemented MEM with AONs and incubated for 24 h at 37 °C and 5% CO ₂ , followed by 48 h of nutrient deprivation at 37 °C, 5% CO ₂ in MEM without FBS, but with 1.25% penicillin-streptomycin solution and 1.75% L-glutamine and AONs). After 48 hours of incubation, cells were fixed with 4% paraformaldehyde (Carl Roth) and blocked in PBS containing 2.5% bovine serum albumin (BSA, fraction V, Carl Roth) and 0.1% Tween-20 (AppliChem, Darmstadt, Germany). . Immunocytochemical staining was performed with primary antibodies against D-tubulin (1:1000; Merck, Darmstadt, Germany) and γ-tubulin (1:500; Abcam, Cambridge, UK). Anti-mouse AF 488 (1:2000; Life Technologies, Carlsbad, CA, USA) and anti-rabbit AF 568 or AF 647 (both 1:1000; Life Technologies) were used as secondary antibodies. The coverslips were mounted with a mounting agent containing 4′,6-diamidine-2-phenylindole (DAPI) (Fluoromount-G, SouthernBiotech, Birmingham, Alabama, USA). The slides were analyzed using a Zeiss Axio Observer 7 microscope (Carl Zeiss, Oberkochen, Germany) with a 40x/0.6 corr Ph2 M27 plan apochromat objective. All images were taken with the same illumination intensity and exposure time. Image processing was carried out using the software ZEN 3.4 (blue edition) (Zeiss) and Fiji-lmageJ (ImageJ 1.53c; https://fiji.sc/).

Measurements of primary cilia

To analyze the length of primary cilia, 250 cilia per cell line were measured in three independent experiments (a total of 750 cilia per cell line) as previously described (Da Costa et al. 2015). For each experiment, cells were introduced independently on different days at different passage numbers between 6 and 12. The length of the axoneme of the primary cilia was determined using Fiji-ImageJ (Segmented Line Tool).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics Software, version 27 and GraphPad PRISM9 (2020). Statistical significance was analyzed using the nonparametric Kruskal-Wallis test. A p value of <0.05 was considered statistically significant.

Results

Clinical findings

The two independent families described here had only a single heterozygous likely pathogenic variant in KIAA0586/TALPID3 but showed clear symptoms of Joubert syndrome. The index patient (II.I) of family 1 was born to unrelated healthy parents ( . He was diagnosed with JS with epilepsy and a central respiratory regulation disorder. Symptomatic epilepsy with tonic seizures first appeared at the age of ten months. He also suffers from retrognathia and a combined developmental disorder. Please refer to Table 3 for further clinical information. The diagnosis of JS was confirmed by magnetic resonance imaging, which showed typical MTS with an abnormally deep interpeduncular fossa and elongated and thick superior cerebellar peduncles ( . A second child in the family was recently born and diagnosed with generalized respiratory regulation disorder due to a ciliary defect after birth, suggesting that the second child is also affected by JS. The suspicion was confirmed by an MRI scan, which also revealed the characteristic MTS ( . Other symptoms included chronic bronchitis, atelectasis, hypotension, ataxia and intellectual disability. Kidney and liver cysts were not detected (Table 3). Using Sanger sequencing, we confirmed that the second child of family 1, like the brother, was a carrier of the heterozygous likely pathogenic mutation. Table 3: Bi-allelic variants in KIAA0586/TALPID3 cause a neurodevelopmental disorder in children from two unrelated families. Family 1 Family 2 first child (index patient) second child first child cDNA (GenBank; NM_001244189.2) c.[4149+3186G>A];c.[2512C>T] c.[4149+3186G>A];c.[2512C>T] c.[4149+3186G>A]; c.[1446delA] Protein (GenBank; NP_001231118.1) p.Asp1384Arg.fs*15 p.Arg838* p.Asp1384Arg.fs*15 p.Arg838* p.Asp1384Arg.fs*15 p.Val483* Ethnicity German German sex masculine female Pregnancy complications/drug use ICSI, ventricles borderline enlarged spontaneous Estimated gestational age (weeks) 40+2 40+6 Birth weight (g, z-score) 3375 (-0.6z) 3790 (0.5z) Birth length (cm, z-score) 53 (0.14z) 55 (1.31z) Head circumference at birth (cm, z-score) 35 (-0.5z) 35 (-0.1z) Apgar 2/5/8 3/9/10 newborn period 5 weeks in the Meppen Children's Hospital; 6 days of CPAP, then high-flow ventilation; Start of CPAP home ventilation on day 37 postpartum short mask ventilation; Age 48 hours. Meppen Children's Clinic; 3 weeks in Meppen and Bochum; Atelectasis right pulmonary upper lobe Age at diagnosis (years, months) 6 weeks 3 weeks Age at last contact (years, months) 4y, 2m 1y, 1m Weight (kg and z-score) 13 (-2.3z) 8.6 (1.4z) Length (cm and z-score) 94 (-2.6z) 74 (-1.3z) Head circumference (cm and z-score) 49.5 (-1.4z) 43.8 (-3.0z) Dysmorphic features Retrognathia Development Outdoor space (months) n/a n/a Shuffling (months) 30 m no Crawling (months) 50 m no Free walking (months) no (only with rollator) no Linguistic development active 2 words, better understanding difficult active 3 words, better understanding moderate Neurological system hypotension? Ataxia? moderate moderate Other Mov Dis no no Epilepsy? Type? tonic and generalized no Onset of epilepsy (months) 8 months therapy (LEV), (OXC), VPA, LTG Mental disability? Yes probably cMRI findings 6 weeks: MTS 16 months: MTS Visual system oculomotor apraxia no oculomotor apraxia Hearing system n/a n/a The respiratory system clinical symptoms? Chronic bronchitis chronic bronchitis, colonization of the respiratory tract with Pseudomonas aeruginosa Chest x-ray? peri-bronchial cuff Persistent segmental atelectasis of the right upper lobe Nasal NO 154 ppb 39 ppb High frequency video microscopy nasal brush not yet carried out Stiff cilia with reduced amplitude and reduced frequency (8-12/s) Electron microscopy nose brush n/a n/a Neonatal course Not eventful? respiratory failure; Atelectasis of the right upper lobe Kidney: cysts? no no Liver: cysts? no no

Using panel diagnostics that examined more than 30 different JS genes at the exome level, the heterozygous genetic alteration KIAA0586/TALPID3: c.[2512C>T],[=];p.Arg838* (NM_001244189.2) was identified in the index patient and identified as the father of family 1. Sanger sequencing confirmed the change ( , left panel), which should lead to an early translational stop. The same sequence variant was previously described as a likely pathogenic variant (Stephen et al. 2015). Thus, the heterozygous mutation KIAA0586/TALPID3 results in: c.[2512C>T],[=]; p.Arg838* (NM_001244189.2) probably results in a loss of function of one allele of the KIAA/TALPID3 gene. During the genetic diagnosis of the JS-associated gene, no mutation on the second allele was identified, a prerequisite for explaining the recessively inherited JS phenotype in the patients. Copy number variations on the second allele were not identified in the high-throughput data set.

Similarly, in family 2, the heterozygous sequence alteration KIAA0586/TALPID3: c.[1446delA]; p.Val483* (NM_001244189.1) in the patient and in the father ( and E) identified using panel diagnostics. A sequence change on the second allele was not identified.

In conclusion, the results of genetic diagnostics in both families were inconclusive to confirm the JS phenotype of the affected children and only a single heterozygous stop mutation was detected on one allele of the KIAA0586/TALPID3 gene.

Transcriptional analysis in patient-derived cells revealed a novel deep intronic sequence change

We speculated that the missing mutation(s) on the second allele may have occurred in a deep intronic region of the KIAA0586/TALPID3 gene, as genetic diagnostics would normally miss such types of mutations. To continue the search for a deep intronic mutation in KIAA0586/TALPID3, we generated skin fibroblast cell lines from biopsies of Family 1 patients. Transcriptional analysis was performed using reverse transcription-PCR (RT-PCR) and patient-derived cell lines were compared with healthy, unrelated controls. We analyzed the KIAA0586/TALPID3 transcripts with primers that amplify all coding exons of KIAA0586/TALPID3. The RT-PCR analyzes between exon 28 and exon 30 revealed an additional transcript in the patient samples that was absent in the controls ( ). In addition to the expected product of 322 bp, a larger product of 454 bp was detected. This suggests the presence of a cryptic exon resulting from a defective splicing process in introns 28 or 29. Sequencing of the larger product confirmed the presence of 132 additional base pairs identical to parts of intron 28. Targeted sequencing of the genomic region flanking the cryptic exon in the index patient's DNA revealed a previously unknown deep intronic variant: NM_001244189.2: c.[4149+3186G>A],[=]; ( . Using the bioinformatics prediction program ESEFinder 3.0 (http://krainer01.cshl.edu/cgibin/tools/ESE3/esefinder.cgi?process=home), it was determined that the base shift from guanine to adenine likely stimulates the splicing process by activating a new branch point influenced. The score assigned by ESEfinder for the suspected branch point increases from 0.17 to 5.36. The base change to adenine at this position creates the 5mer motif CTRAY, which matches the known highly conserved branch point sequence in humans (Lim and Burge 2001). The prediction was confirmed using SVMBPfinder (http://regulatorygenomics.upf.edu/Software/SVM_BP/). Together with the presence of a nearby splice acceptor and splice donor, 132 bp of intron 28 was spliced into the KIAA0586/TALPID3 transcript as a cryptic exon. This cryptic exon has an early stop codon that likely causes premature termination of translation at amino acid 15. When the family members of Family 1 were analyzed, the deep intronic genetic alteration c.[4149+3186G>A] was detected in both the patient and his unaffected mother ( . In support of this, targeted sequencing of the genomic DNA of the sister affected by JS revealed the same two genetic changes as the older brother ( .

Targeted Sanger sequencing of the corresponding intronic region in the available family members of Family 2 revealed the same deep intronic change. The affected child and the mother of family 2 carried the deep intronic mutation c.[4149+3186G>A]. A skin biopsy of the affected child of family 2 was not available.

Ciliary defects in patient-derived fibroblasts

To analyze the ciliary properties of patient-derived fibroblasts compared to unrelated controls, the BBs and axonemes of primary cilia were stained using immunocytochemistry ( . We found that the index patient (II.I) had a defect in cilia formation and/or stability. The length of patient-derived primary cilia was approximately 1 μm shorter than controls ( .

Treatments with small nucleic acids correct the consequences of the deep intronic mutation

The deep intronic mutation KIAA0586: c.4149+3186G>A (NM_001244189.2) causes a splicing defect that creates a cryptic frameshift exon in KIAA0586/TALPID3 transcripts. We employed two modified small nucleic acids, i.e., antisense oligonucleotides (AON), to correct the splicing defect caused by the deep intronic mutation. Both AONs are designed to bind to the cryptic exon ( ). The binding positions of the AONs were predicted to affect the enhancer sites of the exon during splicing. Patient-derived fibroblasts were treated with either single AONs or a combination of both. Additional AONs were tested (data not shown).

Each of the two in presented AONs showed the effectiveness of partially correcting the splicing of the cryptic exon ( ). AON_+26 showed a significant reduction in transcripts carrying the cryptic exon (p ≤ 0.05) ( ). The effectiveness of AON_+57 to reduce the expression of the cryptic exon was higher (p ≤ 0.001) ( ). Furthermore, we applied an equimolar ratio of AON_+26 and AON_+57, a combination that conferred the strongest therapeutic potential ( ). We did not detect any AON-associated change in KIAA0586/TALPID3 transcripts in control cells ( .

AON treatments improve the ciliary defect in patient-derived cells

The ciliary properties of AON-treated fibroblasts were examined using immunofluorescence imaging. We measured cilia length of patient and control cell lines and found that AON treatment combining both AON_+26 and AON_+57 significantly increased cilia length in patient fibroblasts (p≤0.001) ( and B). The therapeutic approach showed no adverse effects on cilia length in the control cell lines ( and B).

Taken together, our data demonstrate that the small nucleic acids/AONs are efficient therapeutic options for correcting disease-related splicing defects. The treatment is able to improve the functional correlates of ciliopathies, i.e. cilia length as a measure of cilia stability or maintenance.

discussion

Ciliopathies are often caused by genetic changes in highly conserved genes. An increasing number of genes encode proteins that act and interact at the cilium, the BB or the centrosomes, e.g. B. by forming complexes that are important for the formation, maintenance or function of cilia. Mutations in such genes can affect cilia functions in various organs and tissues (Hildebrandt et al. 2011). Since cilia are involved in a variety of different processes, patients with ciliary dysfunction often suffer from combinations of different symptoms, i.e. H. of syndromes. Well-described examples include Bardet-Biedl syndrome, Orofaciodigital syndrome 1, Meckel-Gruber syndrome, and JS. Features commonly seen in ciliopathies include retinopathy, kidney and lung disease, and brain abnormalities/malformations. Other manifestations may include skeletal dysplasia, diabetes, mental retardation, and/or polydactyly (Waters and Beales 2011; Gerdes et al. 2009). Therefore, ciliopathies represent an enormous burden for patients and families. Therapeutic applications are hardly known.

Mutations in the KIAA0586/TALPID3 gene have been associated with severe ciliary defects (Yin et al. 2009; Stephen et al. 2013). The gene's role in centriolar organization and formation of a germ cell for axonome growth has been linked to the symptoms of ciliopathies in patients. Although the exact function of KIAA0586/TALPID3 is still unclear, it appears to interact with several ciliary proteins and localize to the distal end of centrioles sier (Wu et al. 2014; Kobayashi et al. 2014; Stephen et al. 2015; Wang et al. 2016; Wang et al. 2018; Ojeda Naharros et al. 2018; Tsai et al. 2019; Wang et al. 2020 ; Yan et al. 2020). Loss of functional KIAA0586/TALPID3 proteins has been shown to prevent the dissociation of centriolar proteins involved in distal process assembly, centriole maturation, and cilia formation in various models and cell lines (Wang et al. 2018; Yan et al. 2020; Kobayashi et al. 2014). The KIAA0586/TALPID3 protein has been associated with proper cilia gating, intraflagellar transport (IFT), and primary cilia assembly. Taken together, the KIAA0586/TALPID3 protein is likely involved in multiple processes relevant to cilia stability and/or maintenance and provides the molecular basis for understanding the devastating diseases associated with KIAA0586ITALPID3 mutations in patients.

JS can be diagnosed by a malformation of the cerebellum called MTS, which is often found in MRI analyses. It is also characterized by psychomotor delay and impaired respiratory regulation, symptoms that are present in the patients described here. JS is inherited in an autosomal recessive manner, and mutations in over 40 genes associated with JS have been described to date (Reiter and Leroux 2017). Together with AHI1, CC2D2A, CEP290, CPLANE1 and TMEM67, KIAA0586/TALPID3 is one of the most frequently mutated JS genes (Parisi and Glass 2003; Mitchison and Valente 2017). Biallelic mutations in KIAA0586/TALPID3 were first linked to JS in 2015 (Bachmann-Gagescu et al. 2015b).

Since the diagnosis of JS can be accompanied by a variety of different symptoms - most of which are serious diagnoses - it is particularly desirable to develop therapeutic approaches that target the cause of the disease and thus open up the possibility of treating many of the different JS symptoms treat. Here, we investigated a small nucleic acid/AON-based treatment capable of restoring a significant amount of correctly spliced KIAA0586/TALPID3 transcripts. Treatment results in partial correction of the ciliary defect and subsequent increase in ciliary length. The recessive pathomechanism underlying the disease suggests that efficient therapies do not require complete restoration of KIAA0586/TAPLID3 function. With recessively inherited genes, loss of function of both alleles is required to cause disease. Heterozygous carriers of KIAA0586/TALPID3 mutations are not affected. Therefore, partial correction of cilia length might be sufficient to treat the physiological consequences of KIAA0586/TALPID3 mutations. In support of this, a KIAA0586/TALPID3 knockdown of approximately 80% resulted in no or only minor defects in centriole maturation and ciliogenesis in human retinal pigment epithelial cells (Yan et al. 2020). Both Wang et al. as well as Yan et al. described that the role of KIAA0586/TALPID3 is dose-dependent (Wang et al. 2018; Yan et al. 2020). Although the exact amount of KIAA0586/TALPID3 required to prevent progression of disease symptoms is not known, it seems likely that less than 50% restoration of KIAA0586/TALPID3 function is sufficient to prevent centriole maturation and the ciliary properties and thus improve or prevent the symptoms of the disease.

The enormous advances in sequencing technologies used in genetic research and diagnostics have simplified the identification of disease-associated mutations and genes. In 2009, mutations in approximately 40 genes affecting cilia were known (Gerdes et al. 2009). In recent years, a large number of new cilia genes have been identified. In total, over 200 genes are known to date that are associated with ciliopathies (Focşa et al. 2021). However, it may happen that standardized genetic diagnostic methods do not detect deep variants. Although whole genome or transcriptome sequencing promises to overcome these limitations, intensive research approaches are required to translate knowledge into routine diagnostics.

We describe two unrelated families in which the same deep intronic mutation co-segregated in affected family members. The families came from different geographical regions (Germany and Sweden). Consequently, the mutation c.[4149+3186G>A] could be a founder mutation, a speculation that requires further analysis of additional, as yet unexplained cases with a single heterozygous mutation in KIAA0586/TALPID3.

Together, we developed an efficient AON-mediated treatment option for KIAA0586/TALPID3 mutations and successfully used the treatment to correct ciliary phenotypes in patient-derived cell lines. Our findings will contribute to the development of therapeutics to help patients with genetic diseases.

A sequence listing follows as an electronic document. This can be accessed both in DEPATISnet and in the DPMAregister.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• NL 0100697 PCT [0030]
• US 6875736 [0030]
• EP 1619249 [0030, 0040, 0041]
• US 5139941 [0078]
• US 6531456 [0078]

Claims (10)

Exon skipping molecule that binds to a polynucleotide with the nucleotide sequence shown in SEQ ID NO: 29, preferably SEQ ID NO: 30, more preferably SEQ ID NO: 31 or SEQ ID NO: 32, or a part thereof and/or is complementary to this. Exon skipping molecule Claim 1 , where the molecule is a nucleic acid molecule. Exon skipping molecule Claim 2 , wherein the nucleic acid molecule comprises an antisense oligonucleotide, the antisense oligonucleotide having a length of from about 8 to about 179 nucleotides. Exon skipping molecule Claim 3 , wherein the antisense oligonucleotide comprises a sequence or consists of a sequence selected from the group consisting of: - SEQ ID NO: 33, - SEQ ID NO: 34, - SEQ ID NO: 35, - SEQ ID NO: 36 - SEQ ID NO: 37 - SEQ ID NO: 38 and - SEQ ID NO: 39. Exon skipping molecule according to one of the Claims 1 until 4 , comprising a 2'-O-alkylphosphorothioate antisense oligonucleotide, such as 2'-O-methyl modified ribose (RNA), 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose and/or substituted derivatives of these modifications, such as halogenated derivatives. Viral vector containing an exon skipping molecule, as in one of the Claims 1 until 5 defined, expressed, preferably when placed under conditions that favor the expression of the molecule. Pharmaceutical composition comprising an exon skipping molecule according to one of Claims 1 until 5 or a viral vector Claim 6 and a pharmaceutically acceptable excipient, wherein the viral vector is preferably an AAV vector or a lentivirus vector, particularly preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 or a lentivirus vector. The exon skipping molecule according to one of the Claims 1 until 5 , the vector after Claim 6 or the composition Claim 7 for use as medication. The exon skipping molecule according to one of the Claims 1 until 5 , the vector after Claim 6 or the composition Claim 7 for use in the treatment of a KIAA0586-related disease or condition that requires modulation of KIAA0586/TALPID3 splicing, preferably for use in the treatment of Joubert syndrome. A method for modulating splicing of KIAA0586/TALPID3 in a cell, the method comprising contacting the cell with an exon skipping molecule, as in one of Claims 1 until 5 defined, according to the vector Claim 6 or according to the composition Claim 7 includes.

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