Novel retinitis pigmentosa treatment

Abstract

To provide a novel retinitis pigmentosa treatment.SOLUTION: There is provided an isolated or purified antisense oligomer for modifying pre-mRNA splicing in the CNOT3 gene transcript or a part thereof. In one aspect of the invention, there is provided an antisense oligomer of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or within an intron of the CNOT3 gene transcript or the part thereof. In another aspect of the invention, there is provided an antisense oligomer of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or within an exon of the CNOT3 gene transcript or the part thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Technical Field The present invention relates to the use of antisense oligomers to treat, prevent, or ameliorate the effects of retinitis pigmentosa. [Background technology]

[0002] Background technology Retinitis pigmentosa (RP) is a degenerative eye disease that causes severe vision loss due to progressive degeneration of rod photoreceptor cells in the retina, and most cases of RP are hereditary. Early symptoms of this form of retinal dystrophy appear at any age; therefore, RP can be diagnosed at any age, from early infancy to late adulthood. RP is a rare disease that affects approximately 1 in 4000 people (more than 1.5 million worldwide). Of familial RP cases, 30%-40% show autosomal dominant inheritance (Hartong et al. 2006). There is currently no treatment for RP.

[0003] RP is caused by mutations in over 50 genes. Heterozygous mutations in the PRPF31 gene result in autosomal dominant retinitis pigmentosa (adRP). In some cases, such mutations show incomplete penetrance, with certain carriers developing retinal degeneration while others are completely asymptomatic. Asymptomatic carriers are protected from the disease by higher than average expression of the unmutated PRPF31 allele.

[0004] Expression of the PRPF31 gene is regulated by the Ccr4-Not deadenylase complex. Ccr4-Not is a nine-subunit protein complex that is a master regulator of translation and mRNA stability in eukaryotic cells. The core CCR4-Not complex consists of Ccr4p, Caf1p, five Not proteins (CNot1-CNot5), Caf40p, and Caf130p in yeast.

[0005] Currently, AAV-mediated gene replacement and CRISPR / Cas9 gene editing are being investigated as retinal disease treatments. Although the coding sequence of PRPF31 is within the capabilities of AAV vectors, the consequences of unregulated or excessive expression of PRPF31 are unknown. Furthermore, since seroconversion as a result of intraocular viral vector injection has been reported, it is not known whether virally mediated ophthalmic gene therapy drugs can be re-administered. Furthermore, CRISPR / Cas9 gene correction requires a different product for each family of PRPF31 mutations. Furthermore, both gene replacement and gene editing approaches require subretinal injection of viral vectors to achieve proper transfection.

[0006] There is a need to provide new treatments or preventative measures for retinitis pigmentosa, or at least provide methods that complement previously known treatments.

[0007] The present invention seeks to provide improved or alternative methods for treating, preventing, or ameliorating the effects of retinitis pigmentosa.

[0008] The preceding background discussion is intended only to facilitate an understanding of the present invention, and is not intended to be an admission or acknowledgement that any of the material cited is or has ever been part of the common general knowledge as of the priority date of this application. Summary of the Invention

[0009] Summary of the Invention Generally, according to one aspect of the present invention, there is provided an isolated or purified antisense oligomer for modifying pre-mRNA splicing in a CNOT3 gene transcript or a portion thereof.Preferably, there is provided an isolated or purified antisense oligomer for inducing non-productive splicing in a CNOT3 gene transcript or a portion thereof.

[0010] For example, in one embodiment of the present invention, there is provided an antisense oligomer of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or inside an intron of the CNOT3 gene transcript or a portion thereof. In another embodiment of the present invention, there is provided an antisense oligomer of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or inside an exon of the CNOT3 gene transcript or a portion thereof.

[0011] Preferably, the antisense oligomer is a phosphorodiamidate morpholino oligomer. Preferably, the antisense oligomer has a modified backbone.

[0012] Preferably, the antisense oligomer is selected from the group comprising the sequences set out in Table 1.

[0013] Preferably, the antisense oligomer is selected from the list comprising SEQ ID NOs: 1 to 74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16 to 18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64.

[0014] The antisense oligomer preferably acts to induce the skipping of one or more exons of the CNOT3 gene transcript or a part thereof.For example, the antisense oligomer can induce the skipping of exons 3, 8, 9, 12 and / or 17.

[0015] The antisense oligomer of the present invention can be selected to be an antisense oligomer that can bind to a selected CNOT3 target site, where the target site is an mRNA splicing site selected from a splice donor site, a splice acceptor site, or an exon splicing element.The target site can also include some adjacent intronic sequences when the donor or acceptor splice site is targeted.

[0016] More specifically, the antisense oligomer may be selected from the group including any one or more of SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, and / or sequences listed in Table 1, and combinations or cocktails thereof. This includes sequences that can hybridize to such sequences under stringent hybridization conditions, their complements, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that possess or modulate pre-mRNA processing activity in CNOT3 gene transcripts. In certain embodiments, the antisense oligomer may be 100% complementary to the target sequence or may contain mismatches, for example, to accommodate variants, so long as the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to resist the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, certain oligonucleotides may have about or at least about 70% sequence complementarity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity between the oligonucleotide and the target sequence.

[0017] The present invention also encompasses combinations of two or more antisense oligomers capable of binding to a selected target and inducing exon exclusion in the CNOT3 gene transcript, including constructs comprising two or more such antisense oligomers. The constructs may be used for antisense oligomer-based therapy.

[0018] According to a still further aspect thereof, the present invention extends to cDNA or cloned copies of the antisense oligomer sequences of the invention and to vectors comprising the antisense oligomer sequences of the invention. The present invention also extends to cells comprising such sequences and / or vectors.

[0019] 1. A method for manipulating splicing of a CNOT3 gene transcript, comprising: a) providing one or more of the antisense oligomers described herein and binding said oligomer(s) to a target nucleic acid site. A method is also provided, including:

[0020] A pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or ameliorating the effects of a disease related to CNOT3 expression in a patient, comprising: a) one or more antisense oligomers as described herein, and b) one or more pharma- ceutically acceptable carriers and / or diluents Also provided is a pharmaceutical, prophylactic, or therapeutic composition comprising:

[0021] The compositions may contain about 1 nM to 1000 nM of each desired antisense oligomer(s) of the invention. Preferably, the compositions may contain about 10 nM to 500 nM, most preferably between 1 nM and 10 nM, of each antisense oligomer(s) of the invention.

[0022] 1. A method of treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression, comprising: a) administering to a patient an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein. A method is also provided, including:

[0023] Also provided is the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression.

[0024] Also provided are kits for treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression in a patient, comprising at least one antisense oligomer described herein, and combinations or cocktails thereof, packaged in a suitable container together with instructions for use.

[0025] Preferably, the disease associated with CNOT3 expression in the patient is retinitis pigmentosa. The subject having a disease associated with CNOT3 expression may be a mammal, including a human.

[0026] Further aspects of the present invention are described herein with reference to the accompanying non-limiting examples and drawings. [Brief description of the drawings]

[0027] Further features of the present invention will be described in more detail in the following description of some non-limiting embodiments of the present invention. This description is for the purpose of illustrating the present invention only and is not to be understood as limiting the above summary, disclosure, or description of the invention. The following description is made with reference to the accompanying drawings: [Figure 1] Figure 1 is a schematic diagram of the CNOT3 open reading frame. CNOT3 consists of 18 exons and encodes a protein with 753 amino acids. Figure 1(a) In-frame exons are shown as boxes, while exons with junctions that interrupt the codons are shown as chevrons (blue, red). Figure 1(b) represents the CNOT3 protein, showing the functional domains and amino acid positions corresponding to the exons mentioned above. NAR: NOT anchoring region. CS: linking sequence. NOT box: TATA box negative. Removal of any of exons 2, 3, 8, 9, 11, 12, 13, 14, 15, and 16 disrupts the open reading frame. [Diagram 2]Figure 2 is a gel image showing CNOT3 transcripts from RP11 patient fibroblasts 48 hours after transfection with 2'O-methyl phosphorothioate AO (50, 25, and 12.5 nM). A control AO not targeting a known sequence (-ve control AO) was included for comparison. [Diagram 3] FIG. 3 is a gel image showing CNOT3 transcripts from fibroblasts 48 hours after transfection with 2′O-methyl phosphorothioate AO, designed to induce terminal intron retention at concentrations of 50 and 25 nM. [Figure 4] Figure 4A is pedigree 0255: 11 members with PRPF31 mutation (c.267delA). Figure 4B is pedigree 0080: 13 affected members (c.1205 G>A). Arrows = donated skin fibroblasts, black = patients currently undergoing CRE natural history studies. [Diagram 5]Figure 5A-H shows dermal fibroblasts from a patient with a CLN3 mutation that were reprogrammed to pluripotency. Patient-iPSCs (CLN3- / -) showed typical iPSC morphology and expressed pluripotency markers, including OCT4, NANOG, SOX2, and SSEA4 (A). Comparison of pluripotency gene expression in six iPSC lines by quantitative RT-PCR demonstrated similar expression patterns for all lines (B). Patient iPSCs were demonstrated to be capable of differentiation into three lineages. Patient iPSCs (CLN3-iPS-EBs), genetically corrected control iPSCs (CLN3HDR-iPS-EBs), and control human iPSCs (WT-iPS-EBs, ThermoFisher) were differentiated into embryoid bodies for 2 weeks and then screened by quantitative RT-PCR for expression of markers of ectodermal (PAX6, OTX1), mesodermal (SOX17, GATA4, FOX2A), and endodermal (BRACHYURY, FDGFR) lineages, as well as pluripotency genes (OCT4, NANOG, SOX2) (C). D-H: Retinal differentiation of iPSCs. Retinal organoids exhibited an optic cup-like morphology surrounded by clear, stratified retinal tissue (D), which contained an organized apical layer of photoreceptors expressing recoverin (E) and basolaterally residing retinal ganglion cells expressing Smi32 (F). Electron microscopy of photoreceptor outer segments in day 170 retinal organoids (G) demonstrated morphology similar to outer segments of the developing (E120) human retina (H). [Figure 6A] Figure 6 shows the screening of AO-induced CNOT3 exon skipping using 2'-O-methyl chemistry on the PS backbone. AOs are designed to target splice enhancer motifs in CNOT3 exons to mediate the exclusion of the targeted exon(s) during pre-mRNA splicing to knockdown CNOT3 or disrupt protein function. Dermal fibroblasts were transfected with CNOT3 AOs (2'OMe-PS chemistry) targeting exons (as indicated) for 48 hours. RT-PCR products were resolved on a 2% agarose gel. [Figure 6B]Figure 6 shows the screening of AO-induced CNOT3 exon skipping using 2'-O-methyl chemistry on the PS backbone. AOs are designed to target splice enhancer motifs in CNOT3 exons to mediate the exclusion of the targeted exon(s) during pre-mRNA splicing to knockdown CNOT3 or disrupt protein function. Dermal fibroblasts were transfected with CNOT3 AOs (2'OMe-PS chemistry) targeting exons (as indicated) for 48 hours. RT-PCR products were resolved on a 2% agarose gel. [Figure 7A] Figure 7 shows the screening of AO-induced CNOT3 exon skipping using 2'-O-methyl chemistry on the PS backbone. AOs are designed to target splice enhancer motifs in CNOT3 exons to mediate the exclusion of the targeted exon(s) during pre-mRNA splicing to knockdown CNOT3 or disrupt protein function. Dermal fibroblasts were transfected with CNOT3 AOs (2'OMe-PS chemistry) targeting exons (as indicated) for 48 hours. RT-PCR products were resolved on a 2% agarose gel. [Figure 7B] Figure 7 shows the screening of AO-induced CNOT3 exon skipping using 2'-O-methyl chemistry on the PS backbone. AOs are designed to target splice enhancer motifs in CNOT3 exons to mediate the exclusion of the targeted exon(s) during pre-mRNA splicing to knockdown CNOT3 or disrupt protein function. Dermal fibroblasts were transfected with CNOT3 AOs (2'OMe-PS chemistry) targeting exons (as indicated) for 48 hours. RT-PCR products were resolved on a 2% agarose gel. [Figure 8a]Figure 8 shows the inverse correlation of mRNA expression between CNOT3 and PRPF31. Figure 8(a) qRT-PCR analysis of PRPF31 and CNOT3 mRNA expression normalized to TATA-binding protein (TBP) expression in iPSC-derived retinal pigment epithelium from RP11, asymptomatic, and healthy (WT) individuals. RP11 and asymptomatic subjects are from the same family tree, both heterozygous for the PRPF31 c.1205C>A (Ser402*) mutation. Healthy subjects are from unrelated families. CNOT3 and PRPF31 mRNA expression in healthy controls was set to 1 (n≧2). Figure 8(b) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS) targeting exons 4, 6, 7, or 10 for 48 h. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression. PRPF31 expression in cells treated with 25 nM control AO was set to 1 (n=1). Figure 8(c) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS chemistry) targeting exons 3, 8, 9, 16, or 17 for 48 hours. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression and shown relative to PRPF31 expression in cells treated with 25 nM mock control AO (control PRPF31 expression set to 1) (n=1). [Figure 8b]Figure 8 shows the inverse correlation of mRNA expression between CNOT3 and PRPF31. Figure 8(a) qRT-PCR analysis of PRPF31 and CNOT3 mRNA expression normalized to TATA-binding protein (TBP) expression in iPSC-derived retinal pigment epithelium from RP11, asymptomatic, and healthy (WT) individuals. RP11 and asymptomatic subjects are from the same family tree, both heterozygous for the PRPF31 c.1205C>A (Ser402*) mutation. Healthy subjects are from unrelated families. CNOT3 and PRPF31 mRNA expression in healthy controls was set to 1 (n≧2). Figure 8(b) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS) targeting exons 4, 6, 7, or 10 for 48 h. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression. PRPF31 expression in cells treated with 25 nM control AO was set to 1 (n=1). Figure 8(c) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS chemistry) targeting exons 3, 8, 9, 16, or 17 for 48 hours. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression and shown relative to PRPF31 expression in cells treated with 25 nM mock control AO (control PRPF31 expression set to 1) (n=1). [Figure 8c]Figure 8 shows the inverse correlation of mRNA expression between CNOT3 and PRPF31. Figure 8(a) qRT-PCR analysis of PRPF31 and CNOT3 mRNA expression normalized to TATA-binding protein (TBP) expression in iPSC-derived retinal pigment epithelium from RP11, asymptomatic, and healthy (WT) individuals. RP11 and asymptomatic subjects are from the same family tree, both heterozygous for the PRPF31 c.1205C>A (Ser402*) mutation. Healthy subjects are from unrelated families. CNOT3 and PRPF31 mRNA expression in healthy controls was set to 1 (n≧2). Figure 8(b) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS) targeting exons 4, 6, 7, or 10 for 48 h. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression. PRPF31 expression in cells treated with 25 nM control AO was set to 1 (n=1). Figure 8(c) Dermal fibroblasts from RP11 patients were transfected with CNOT3 AO (2'OMe-PS chemistry) targeting exons 3, 8, 9, 16, or 17 for 48 hours. PRPF31 transcript levels were analyzed using qRT-PCR and normalized to TBP expression and shown relative to PRPF31 expression in cells treated with 25 nM mock control AO (control PRPF31 expression set to 1) (n=1). [Figure 9] FIG. 9 shows the effect of ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17) synthesized as a phosphorodiamidate morpholino oligomer (PMO) and transfected into RP11 iPSC-derived RPE. (A) CNOT3 exon 17 skipping in cells treated with PMO alone or cell-permeable peptide-tagged PMO (PPMO) at a concentration of 5 μM. (B) PRPF31 upregulation as a result of CNOT3 knockdown determined by qRT-PCR and normalized to TATA-binding protein (TBP) expression. PRPF31 expression in untreated controls was set to 1. [Figure 10A]FIG. 10 shows (A) immunostaining of cilia (red) and basal bodies (green) in wild-type and RP11 RPE with or without treatment with antisense oligomers with ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17). (B) Percentage of RPE cells expressing cilia counted from over 1,000 cells. (C) Cilia length measurements using NIS-Elements imaging software. Bar graphs represent the mean ± SEM for approximately 300 ciliated cells. Scale bar = 10 μm. Student's t-test. ***p<0.001. [Figure 10B] FIG. 10 shows (A) immunostaining of cilia (red) and basal bodies (green) in wild-type and RP11 RPE with or without treatment with antisense oligomers with ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17). (B) Percentage of RPE cells expressing cilia counted from over 1,000 cells. (C) Cilia length measurements using NIS-Elements imaging software. Bar graphs represent the mean ± SEM for approximately 300 ciliated cells. Scale bar = 10 μm. Student's t-test. ***p<0.001. [Figure 10C] FIG. 10 shows (A) immunostaining of cilia (red) and basal bodies (green) in wild-type and RP11 RPE with or without treatment with antisense oligomers with ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17). (B) Percentage of RPE cells expressing cilia counted from over 1,000 cells. (C) Cilia length measurements using NIS-Elements imaging software. Bar graphs represent the mean ± SEM for approximately 300 ciliated cells. Scale bar = 10 μm. Student's t-test. ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Description of the invention Detailed Description of the Invention Antisense oligonucleotides Photoreceptors are highly vulnerable to a general reduction in splicing activity. Although mutations in splicing factors can cause splicing defects in various tissues, photoreceptor cells are highly affected due to the high demand for mRNA production, including mRNAs encoding rhodopsin and other phototransduction proteins. This model predicts that the amount of mRNA produced by the retina greatly exceeds the levels of mRNA production by other tissues.

[0029] PRPF31 (also known as hPRP31) encodes an essential pre-mRNA splicing factor required for the assembly and recycling of the U4 / U6 RNA complex. Several studies have demonstrated reduced total PRPF31 mRNA levels in RP11 patients, as well as delayed spliceosome assembly and pre-mRNA processing, indicating that the disease occurs via a haploinsufficient mechanism. Compared to other tissues, the retina expresses seven times more of the major spliceosomal small nuclear RNAs (snRNAs).

[0030] CCR4-Not transcription complex subunit 3 (CNOT3, one of the five Not proteins in the Ccr4-Not deadenylase complex) has been identified as the master modifier gene that determines the penetrance of PRPF31 mutations through a transcriptional repression mechanism and regulates PRPF31 transcription by direct binding to its promoter. The expression level of the wild-type PRPF31 allele determines whether carriers of mutant PRPF31 alleles are symptomatic. In asymptomatic carriers, CNOT3 is expressed at low levels, preventing the development of retinal degeneration due to the production of greater amounts of wild-type PRPF31 transcripts.

[0031] Human CNOT3 is an 18-exon gene that encodes a protein with 753 amino acids. Elimination of exon 2 removes the translation initiation codon from the transcript. Elimination of exons 4, 5, 6, 7, 10, or 17 can disrupt functional domains of CNOT3. Elimination of exons 3, 8, 9, and any of 11-16 disrupts the open reading frame.

[0032] Since the CNOT3 gene is crucial for controlling mRNA turnover and cell cycle progression by controlling mRNA degradation in various physiological processes, knockout of the CNOT3 gene is embryonic lethal. However, if the levels of CNOT3 in the eyes of subjects at risk or affected by RP can be reduced or locally lost, it could affect disease progression if the amount of PRPF31 protein could be increased, mimicking the incomplete penetrance model in which the higher average expression of PRPF31 from the unaltered gene protects asymptomatic carriers from the disease.

[0033] Thus, the present invention provides antisense oligonucleotides to induce non-productive splicing of dysfunctional protein of CNOT3 (a negative regulator of PRPF31) to reduce (but preferably not eliminate) CNOT3 levels, thereby increasing transcription and translation from normal PRPF31 alleles.

[0034] In contrast to other antisense oligomer-based therapies, the present invention does not induce increased RNA degradation via recruitment of RNase H, where RNase H preferentially binds to and degrades duplex RNA bound to the DNA of the CNOT3 gene, nor does the present invention rely on hybridization of antisense oligomers to CNOT3 genomic DNA or binding of antisense oligomers to mRNA to modulate the amount of CNOT3 protein produced by interfering with normal functions such as replication, transcription, translocation, and translation.

[0035] Rather, antisense oligomers are used to alter pre-mRNA splicing in the CNOT3 gene transcript or a portion thereof, inducing exon "skipping" and / or terminal intron retention. This strategy preferably reduces total protein expression or produces a protein lacking a functional domain, thereby reducing protein function.

[0036] According to a first aspect of the present invention, there is provided an antisense oligomer capable of binding to a selected target on the CNOT3 gene transcript and modifying pre-mRNA splicing in the CNOT3 gene transcript or a portion thereof. In general, there is provided an isolated or purified antisense oligomer for inducing targeted exon exclusion and / or terminal intron retention in the CNOT3 gene transcript or a portion thereof.

[0037] In the general population, the expression of PRPF31 is highly variable, and the expression level follows a continuous distribution. The arbitrary unit of expression level varies from 0.53 to 2.48, which represents a 5-fold variation between the lowest and highest expression. Thirty percent of PRPF31 mutation carriers are asymptomatic, and these individuals have wild-type alleles that show nearly twice the expression of individuals with symptomatic PRPF31 mutations. Therefore, preferably, by adjusting CNOT3 expression, it is possible to express PRPF31 at least twice as much as individuals with symptomatic PRPF31 mutations. Preferably, the expression of PRPF31 resulting from the AO of the present invention that reduces CNOT3 expression is between 1.5 and 5 times that of individuals with symptomatic PRPF31 mutations. For example, the expression of PRPF31 can be between 2 and 4 times.

[0038] "Isolated" means a material that is substantially or essentially free from components that normally accompany the material in its native state. For example, "isolated polynucleotide" or "isolated oligonucleotide" as used herein can refer to a polynucleotide that has been purified or removed from adjacent sequences in its natural state (e.g., a DNA fragment that has been removed from adjacent sequences to the fragment in a genome). When referring to a cell, the term "isolation" refers to the purification of the cell (e.g., fibroblast, lymphoblast) from the subject from which the cell originated (e.g., a subject with a polynucleotide repeat disease). In the context of mRNA or protein, "isolation" refers to the recovery of the mRNA or protein from a source (e.g., a cell).

[0039] Antisense oligomers can be said to be "directed to" or "targeted" the target sequence to which they hybridize. In certain embodiments, the target sequence includes regions of preprocessed mRNA that contain the 3' or 5' splice sites, branch points, or other sequences involved in the control of splicing. The target sequence can be within an exon, within an intron, or span an intron / exon junction.

[0040] In certain embodiments, the antisense oligomer has sufficient sequence complementarity to block a region of the target RNA (e.g., pre-mRNA) in an effective manner to the target RNA (i.e., the RNA for which splice site selection is regulated). In exemplary embodiments, such blocking of CNOT3 pre-mRNA acts to modulate splicing by masking the binding site of a native protein that would normally regulate splicing and / or by changing the structure of the targeted RNA. In some embodiments, the target RNA is a target pre-mRNA (e.g., CNOT3 gene pre-mRNA).

[0041] An antisense oligomer having sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense oligomer has a sequence sufficient to induce masking of a binding site of a native protein that would normally modulate splicing of the targeted RNA and / or alter its three-dimensional structure.

[0042] The antisense oligomer selected can be shorter (e.g., about 12 bases) or longer (e.g., about 50 bases) and can contain a small number of mismatches, as long as its sequence is sufficiently complementary to modulate splicing upon hybridization to the target sequence and, if desired, forms a heteroduplex with the RNA with a Tm of 45°C or greater.

[0043] Preferably, the antisense oligomer is selected from the group comprising the sequences set out in Table 1. Preferably, the antisense oligomer is selected from the group comprising the sequences in SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64.

[0044] In certain embodiments, the degree of complementarity between the target sequence and the antisense oligomer is sufficient to form a stable duplex. The region of complementarity of the antisense oligomer with the target RNA sequence can be as short as 8-11 bases, but can be 12-15 bases or more (e.g., 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases (including all integers between these ranges). An antisense oligomer of about 16-17 bases is generally long enough to have a unique complementary sequence. In certain embodiments, the shortest complementary bases may be necessary to obtain the requisite binding Tm, as discussed herein.

[0045] In certain embodiments, oligonucleotides 50 bases in length may be appropriate if at least a minimum number of bases (e.g., 10-12 bases) are complementary to the target sequence. However, in general, oligonucleotides less than about 30 bases in length are optimal for easy or active uptake into cells. For phosphorodiamidate morpholino oligomer (PMO) antisense oligomers, a length of 18-25 bases generally provides the optimal balance between binding stability and uptake. Included are antisense oligomers consisting of about 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, or 50 bases (e.g., CPPMO, PPMO, PMO, PMO-X, PNA, LNA, 2'-OMe).

[0046] In certain embodiments, antisense oligomers can be 100% complementary to the target sequence, or can include mismatches, for example, to accommodate variants, so long as the heteroduplex formed between the oligonucleotide and the target sequence is stable enough to resist the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, certain oligonucleotides can have about or at least about 70% sequence complementarity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity between the oligonucleotide and the target sequence.

[0047] Mismatches, if present, are typically less unstable toward the terminal regions of the hybrid duplex than toward the center. The number of mismatches tolerated depends on the length of the oligonucleotide, the ratio of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well-understood principles of duplex stability. Such antisense oligomers do not necessarily have to be 100% complementary to the target sequence, but are effective for stably and specifically binding to the target sequence so that splicing of the target pre-RNA is modulated.

[0048] The stability of the duplex formed between the antisense oligomer and the target sequence correlates with the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of the oligonucleotide with respect to the complementary sequence RNA may be measured by conventional methods (such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107). In certain embodiments, the antisense oligomer may have a binding Tm with respect to the complementary sequence RNA that is greater than body temperature, preferably greater than about 45°C or 50°C. Tms in the range of 60-80°C or higher are also included.

[0049] Further examples of variants include antisense oligomers having about or at least about 70% sequence identity or homology (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology to any of SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, or the full length of the sequences provided in Table 1.

[0050] More specifically, antisense oligomers capable of binding to selected target sites and altering pre-mRNA splicing in the CNOT3 gene transcript or a portion thereof are provided. The antisense oligomers are preferably selected from the antisense oligomers provided in Table 1 (i.e., SEQ ID NOs: 1-74), more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64.

[0051] The pre-mRNA splicing modification preferably induces "skipping" (i.e., removal of one or more exons or introns of the mRNA) and / or retention of terminal introns. The resulting protein may be shorter due to either internal truncation or premature tailing, or longer due to retention of terminal introns, when compared to the parent full-length CNOT3 protein. These CNOT3 proteins may be designated as isoforms of the unmodified CNOT3 protein.

[0052] The remaining exons of the generated mRNA may be in frame and produce a shorter sequence with a sequence similar to the parent full-length protein, except for an internal truncation in the region between the original 3' and 5' ends. Another possibility is that exon skipping may induce a frameshift, resulting in a protein in which a first portion of the protein is substantially identical to the parent full-length protein, but a second portion of the protein has a different sequence (e.g., a nonsense sequence) due to the frameshift. Alternatively, exon skipping may induce the production of a prematurely terminated protein due to the destruction of the reading frame and premature termination of translation. Additionally, antisense oligomers may produce artificially extended proteins due to the retention of terminal introns in frame.

[0053] The functional domains of CNOT3 include the coiled-coil, linker, NAR (Not anchor region), CS (connector sequence), and NOT box. Exclusion of exon 4 removes part of the coiled-coil domain, and loss of exon 9 disrupts the open reading frame, leading to mRNA degradation and truncation of any encoded protein. On the other hand, skipping of exon 17 alters the NOT box, which is predicted to affect complex formation and therefore CNOT3 function.

[0054] Removal of one or more exons may further lead to misfolding of the CNOT3 protein, reducing the ability of the protein to be successfully transported across membranes.

[0055] It is preferable that there is an internal truncated protein (i.e., a protein that lacks the amino acids encoded by one or more exons).If CNOT3 protein is knocked out, the body may have a problem of increasing CNOT3 transcription to counterbalance the reduction in the total amount of CNOT3 protein.In contrast, it should be sufficient to prevent the increase in transcription by the presence of an internal truncated protein (preferably lacking one or more of the characteristics of complete CNOT3 protein), but also obtain therapeutic benefits due to the reduction in the total amount of functional CNOT3 protein.

[0056] The antisense oligomer-induced exon skipping of the present invention need not completely or even substantially disrupt the function of the CNOT3 protein. Preferably, the exon skipping process reduces or impairs the functionality of the CNOT3 protein.

[0057] The skipping process of the present invention using antisense oligomers may skip individual exons or may skip two or more exons at once.

[0058] The antisense oligomer of the present invention can be a combination of two or more antisense oligomers that can bind to a selected target and induce exon exclusion in the CNOT3 gene transcript. The combination can be a cocktail of two or more antisense oligomers and / or a construct that includes two or more antisense oligomers linked together. [Table 1-1] [Table 1-2]

[0059] 1. A method for manipulating splicing of a CNOT3 gene transcript, comprising: a) providing one or more of the antisense oligomers described herein and binding said oligomer(s) to a target nucleic acid site. A method is also provided, including:

[0060] According to yet another aspect of the present invention, there is provided a target nucleic acid sequence for splice engineering of CNOT3 comprising a DNA equivalent of a nucleic acid sequence selected from Table 1 (i.e., the group consisting of SEQ ID NOs: 1 to 74), more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16 to 18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, and sequences complementary thereto.

[0061] Even if antisense oligomers are designed to completely mask consensus splice sites, they may not necessarily change the splicing of targeted exons. Furthermore, the present inventors have found that the size or length of the antisense oligomer itself is not always the main factor when designing antisense oligomers. For some targets, such as IGTA4 exon 3, antisense oligomers as short as 20 bases can induce some exon skipping more efficiently than other longer (e.g., 25 bases) oligomers directed to the same exon in certain cases.

[0062] The inventors have also found that there do not appear to be any canonical motifs that can be blocked or masked by antisense oligomers to redirect splicing, and it has been found that antisense oligomers must be designed and their individual effectiveness empirically evaluated.

[0063] More specifically, the antisense oligomer may be selected from those set forth in Table 1. The sequence is preferably selected from the group consisting of any one or more of SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, and any one or more of combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions that possess or modulate mRNA processing activity in the pre-CNOT3 gene transcript, complements thereof, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof.

[0064] An oligomer and a DNA, cDNA, or RNA are mutually complementary when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or pairing between an oligomer and a DNA, cDNA, or RNA target for stable and specific binding to occur. It is understood in the art that an antisense oligomer sequence does not need to be 100% complementary to its target sequence to be specifically hybridizable. An antisense oligomer can specifically bind if the binding of the compound to the target DNA or RNA molecule does not interfere with the normal function of the target DNA or target RNA product, and the degree of complementarity is sufficient to avoid non-specific binding of the antisense oligomer to non-target sequences under the conditions where specific binding is desired (i.e., under physiological conditions for in vivo assays or therapeutic treatments, and under assay conditions for in vitro assays).

[0065] Selective hybridization is possible under low, medium, or high stringency, but high stringency is preferred. Those skilled in the art will recognize that the stringency of hybridization is influenced by conditions such as salt concentration, temperature, or organic solvent, in addition to base composition, length of complementary strand, and number of nucleotide base mismatches between hybridizing nucleic acids. Stringent temperature conditions generally include temperatures above 30°C, typically above 37°C, preferably above 45°C, preferably at least 50°C, typically 60°C to 80°C, or higher. Stringent salt conditions are usually less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the criteria of any single parameter. An example of stringent hybridization conditions is 65°C and 0.1×SSC (1×SSC=0.15M NaCl, 0.015M sodium citrate (pH 7.0)). Thus, antisense oligomers of the present invention may include oligomers that selectively hybridize to the sequences provided in Table 1 (i.e., SEQ ID NOs: 1-74), more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, and even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64.

[0066] It is recognized that the arrangement of the codons at the ends of exons in structural proteins does not always allow the codon ends to be destroyed, so it may be necessary to delete more than one exon from pre-mRNA to ensure in-frame reading of mRNA.In such a situation, multiple antisense oligomers may need to be selected by the method of the present invention, each antisense oligomer directed to a different region that induces the inclusion of the desired exon and / or intron.At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to a complementary polynucleotide.Such hybridization can occur when the antisense oligomer has exact complementarity to the target sequence, but also when it has "close" or "substantial" complementarity.

[0067] Typically, selective hybridization occurs when the nucleotide of the antisense oligomer is at least about 55% identical, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98%, or 99% identical over a stretch of at least about 14 nucleotides.The length for homology comparison may be over a longer stretch as described, and in certain embodiments, it often over a stretch of at least about 9 nucleotides, usually at least about 12 nucleotides, more usually at least about 20, often at least about 21, 22, 23, or 24 nucleotides, at least about 25, 26, 27, or 28 nucleotides, at least about 29, 30, 31, or 32 nucleotides, at least about 36 or more nucleotides.

[0068] Thus, the antisense oligomer sequences of the present invention preferably have at least 75% homology to the sequences shown in the sequence listing herein, more preferably at least 85%, more preferably at least 86, 87, 88, 89, or 90% homology. More preferably, the homology is at least 91, 92, 93, 94, or 95%, more preferably at least 96, 97, 98%, or 99%. In general, the shorter the length of the antisense oligomer, the higher the homology required for selective hybridization. Thus, when the antisense oligomer of the present invention consists of less than about 30 nucleotides, it is preferred that the percent identity is greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96, 97, 98%, or 99%, compared to the antisense oligomers listed in the sequence listing herein. Nucleotide homology comparisons are performed using the GCG This may be done by sequence comparison programs such as the Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or substantially different length to the sequences cited herein can be compared by inserting gaps in the alignment (such gaps being determined, for example, by the comparison algorithm used by GAP).

[0069] The antisense oligomer of the present invention may have regions of low homology and regions of exact homology with the target sequence. The oligomer does not need to have exact homology over its entire length. For example, the oligomer may have a continuous stretch of at least 4 or 5 bases identical to the target sequence, preferably a continuous stretch of at least 6 or 7 bases identical to the target sequence, more preferably a continuous stretch of at least 8 or 9 bases identical to the target sequence. The oligomer may have a stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases identical to the target sequence. The remaining stretch of the oligomer sequence may be intermittently identical to the target sequence; for example, the remaining sequence may have identical bases, followed by non-identical bases, followed by identical bases. Alternatively (or similarly), the oligomeric sequence may have several stretches of identical sequence (e.g., 3, 4, 5, or 6 bases) interspersed with stretches of less than perfect homology. Such sequence mismatches preferably result in no or very little loss of splice switching activity.

[0070] The term "modulate" or "modulates" includes "increasing" or "decreasing" one or more quantifiable parameters by a defined amount and / or a statistically significant amount, as appropriate. The terms "increase" or "increasing", "enhance" or "enhancing", or "stimulate" or "stimulating" generally refer to the ability of one or more antisense oligomers or compositions to produce or cause a higher physiological response (i.e., downstream effects) in a cell or subject compared to the physiological response (i.e., downstream effects) produced by either no antisense oligomer or a control compound. The term "decreasing" or "decrease" generally refers to the ability of one or more antisense oligomers or compositions to produce or cause a lower physiological response (i.e., downstream effects) in a cell or subject compared to the physiological response (i.e., downstream effects) produced by either no antisense oligomer or a control compound.

[0071] The relevant physiological or cellular response (in vivo or in vitro) is clear to one skilled in the art and may increase the exclusion of specific exons in CNOT3-encoding pre-mRNA, decrease the amount of CNOT3-encoding pre-mRNA, or decrease the expression of functional CNOT3 protein in cells, tissues, or subjects in need. The "decreased" or "reduced" amount is typically a statistically significant amount and may be 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or less (e.g., 500, 1000 times) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8)) than the amount produced when the antisense oligomer is not present (absence of agent) or when a control compound is used.

[0072] The terms "reduce" or "inhibit" may generally refer to the ability of one or more antisense oligomers or compositions to "reduce" a relevant physiological or cellular response (such as a symptom of a disease or condition described herein) as measured according to routine techniques in the diagnostic field. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and may include alleviation of symptoms or pathology of a disease such as retinitis pigmentosa.

[0073] A "decrease" in response may be statistically significant compared to the response obtained without the antisense oligomer or with a control composition and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers in between).

[0074] The length of the antisense oligomer may vary so long as it is capable of selectively binding to the intended location within the pre-mRNA molecule. The length of such sequences may be determined according to the selection procedures described herein. Generally, the antisense oligomer is from about 10 to about 50 nucleotides in length. However, it is recognized that any length of nucleotide within this range may be used in the method. Preferably, the length of the antisense oligomer is between 10 and 40, between 10 and 35, 15-30, or 20-30 nucleotides in length, most preferably about 25-30 nucleotides in length. For example, the oligomer may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0075] As used herein, "antisense oligomer" refers to a linear nucleotide sequence or nucleotide analog sequence whose nucleobases can hybridize to a target sequence in RNA by Watson-Crick base pairing to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms "antisense oligomer", "antisense oligonucleotide", "oligomer" and "antisense compound" may be used interchangeably to refer to an oligonucleotide. The cyclic subunits may be based on ribose or another pentose sugar or, in certain embodiments, morpholino groups (see description of morpholino oligonucleotides below). Among the numerous antisense agents known in the art, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and 2'-O-methyl oligonucleotides are also contemplated.

[0076] Non-naturally occurring antisense oligomers or "oligonucleotide analogs" include antisense oligomers or oligonucleotides having (i) modified backbone structures (e.g., backbones other than the standard phosphodiester linkages found in naturally occurring oligonucleotides and polynucleotides), and / or (ii) modified sugar moieties (e.g., morpholino moieties rather than ribose or deoxyribose moieties). Oligonucleotide analogs bear bases that can hydrogen bond to standard polynucleotide bases by Watson-Crick base pairing, where the backbone of the analog provides bases capable of sequence-specific hydrogen bonding between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those that have a substantially unchanged phosphorus-containing backbone.

[0077] One method of production of antisense oligomers is methylation of the 2' hydroxyribose position; although incorporation of a phosphorothioate backbone produces molecules that appear similar to RNA but are much more resistant to nuclease degradation, those of skill in the art will be aware of other suitable backbone forms that may be used for the purposes of the present invention.

[0078] To avoid degradation of pre-mRNA during duplex formation with antisense oligomer, antisense oligomer used in the method can be adapted to minimize or prevent cleavage by endogenous RNase H. This property is highly desirable, since treatment of RNA with unmethylated oligomer either in cells or in crude extracts containing RNase H will degrade pre-mRNA:antisense oligomer duplex. Any form of modified antisense oligomer that can bypass or not induce such degradation can be used in the present invention. Antisense oligomer of the present invention can be modified to include partially unsaturated aliphatic hydrocarbon chains and one or more polar or charged groups, including carboxylic acid, ester, or alcohol groups, to confer nuclease resistance.

[0079] Antisense oligomers that do not activate RNase H can be made according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense oligomers, which can be deoxyribonucleotide or ribonucleotide sequences, simply include any structural modification that sterically hinders or prevents RNase H from binding to the double-stranded molecule that contains the oligomer as one of its members, and the structural modification does not substantially hinder or destroy duplex formation. Since the oligomer portion that participates in duplex formation is substantially different from the portion that participates in RNase H binding to the oligomer portion, a large number of antisense oligomers that do not activate RNase H are available. For example, such antisense oligomers can be oligomers in which at least one or all of the internucleotide bridging phosphate residues are modified phosphates (such as methylphosphonates, methylphosphorothioates, phosphoromorpholidates, phosphoropiperazidates, boranophosphates, amide bonds, and phosphoramidates). For example, every other internucleotide bridging phosphate residue may be modified as described. In another non-limiting example, such an antisense oligomer may have at least one or all of the nucleotides with a 2' lower alkyl moiety (e.g., C 1 ~C 4 , straight or branched, saturated or unsaturated alkyl, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl. For example, every other nucleotide may be modified as described.

[0080] An example of an antisense oligomer that is not cleaved by cellular RNase H when duplexed with RNA is a 2'-O-methyl derivative. Such 2'-O-methyl-oligoribonucleotides are stable in cellular environments and animal tissues, and the Tm value of their duplexes with RNA is higher than that of their ribo or deoxyribo counterparts. Alternatively, the nuclease-resistant antisense oligomer of the present invention may have at least one of the last 3' terminal nucleotides fluorinated. Furthermore, the nuclease-resistant antisense oligomer of the present invention may have phosphorothioate bonds linking at least two of the last three terminal nucleotide bases, and preferably has phosphorothioate bonds linking the last four 3' terminal nucleotide bases.

[0081] Alternative oligonucleotide chemistries may be used to increase splice switching. For example, the antisense oligomer may be selected from the list including: phosphoramidate or phosphorodiamidate morpholino oligomers (PMO); PMO-X; PPMO; peptide nucleic acid (PNA); locked nucleic acid (LNA) and derivatives, including α-L-LNA, 2'-amino LNA, 4'-methyl LNA, and 4'-O-methyl LNA; ethylene bridged nucleic acid (ENA) and derivatives; phosphorothioate oligomers; tricyclo-DNA oligomers (tcDNA); tricyclophosphorothioate oligomers; 2'O-methyl modified oligomers (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabino (FANA); unlocked nucleic acid (UNA); hexitol nucleic acid (HNA); cyclohexenyl nucleic acid (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine, or any combination of the above as a mixture or gapmer. To further improve delivery efficacy, the aforementioned modified nucleotides are often conjugated to the sugar or nucleobase moieties with fatty acids / lipids / cholesterol / amino acids / carbohydrates / polysaccharides / nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct exon skipping antisense oligomers. Antisense oligomers induced splice alterations of human CNOT3 gene transcripts generally use either 2OMe or MOE modified bases on oligoribonucleotides, PNA, phosphorothioate backbones. 2OMeAO has been shown to be effective in in vivo delivery when delivered as cationic lipoplexes. Although used for oligo design because they are efficiently incorporated in vitro, these compounds are susceptible to nuclease degradation and are not considered ideal for in vivo or clinical applications. When alternative chemistries are used to generate the antisense oligomers of the invention, uracil (U) in the sequences provided herein may be substituted with thymine (T).

[0082] While the antisense oligomers described above are preferred forms of antisense oligomers of the invention, the invention includes other oligomeric antisense molecules, including but not limited to oligomeric mimetics such as those described below.

[0083] Specific examples of preferred antisense oligomers useful in the present invention include oligomers that contain modified backbones or non-natural internucleoside linkages.As defined herein, oligomers with modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone.For the purposes of this specification, modified oligomers that do not have a phosphorus atom in the internucleoside backbone, as sometimes referred to in the art, can also be considered antisense oligomers.

[0084] In other preferred oligomeric mimetics, both the sugar and the internucleoside linkage (i.e., backbone) of the nucleotide unit are replaced with novel groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligomeric mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligomer is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0085] Another preferred chemistry is phosphorodiamidate morpholino oligomer (PMO) oligomeric compounds, which are not degraded by any known nucleases or proteases. These compounds are invariant, do not activate RNase H activity when bound to RNA strands, and have been shown to maintain splice modifications after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0086] Modified oligomers may also include one or more substituted sugar moieties. Oligomers may also include modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions, even more specifically when combined with 2'-O-methoxyethyl sugar modifications, have been shown to increase nucleic acid duplex stability by 0.6-1.2°C.

[0087] Another modification of the oligomers of the invention involves chemically linking to the oligomer one or more moieties or conjugates that improve the activity, cellular distribution, or cellular uptake of the oligomer. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, myristyl, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0088] To enhance cellular uptake and nuclear localization, cell-penetrating peptides have been added to phosphorodiamidate morpholino oligomers. As shown by et al. (2008), Mol. Ther. 16 9, 1624-1629, different peptide tags have been shown to affect uptake efficiency and target tissue specificity.

[0089] Not all positions in a given compound need to be uniformly modified, and in fact more than one of the above modifications can be incorporated into a single compound or into a single nucleoside in an oligomer.The present invention also includes antisense oligomers that are chimeric compounds.A "chimeric" antisense oligomer or "chimera" in the context of the present invention is an antisense oligomer, particularly an oligomer that comprises two or more chemically distinct regions, each of the above regions being composed of at least one monomer unit (i.e., nucleotide in the case of an oligomeric compound).These oligomers typically comprise at least one region in which the oligomer or antisense oligomer is modified to increase nuclease degradation resistance, cellular uptake, and an additional region to increase binding affinity to target nucleic acid.

[0090] The activity of antisense oligomers and their variants can be assayed according to routine techniques in the art.For example, the splice forms and expression levels of the RNA and protein being investigated can be assessed by any of a wide variety of well-known methods for detecting the splice forms and / or expression of transcribed nucleic acid or protein.Non-limiting examples of such methods include RT-PCR of spliced ​​forms of RNA followed by size separation of PCR products, nucleic acid hybridization methods (e.g., using Northern blots and / or nucleic acid arrays); nucleic acid amplification methods; immunological detection methods of protein; protein purification methods; and protein function or activity assays.

[0091] RNA expression levels can be assessed by preparing mRNA / cDNA (i.e., transcribed polynucleotides) from cells, tissues, or organisms, and hybridizing the mRNA / cDNA with a reference polynucleotide that is the complement of the nucleic acid to be assayed or a fragment thereof. The cDNA can be amplified, if desired, by any of a variety of polymerase chain reaction or in vitro transcription methods before hybridization with the complementary polynucleotide, but is preferably not amplified. The expression of one or more transcripts can also be detected using quantitative PCR to assess the expression level of the transcript(s).

[0092] The present invention provides antisense oligomer-induced splice switching of CNOT3 gene transcripts, clinically relevant oligomer chemistry, and a delivery system for directing CNOT3 splice engineering to clinical levels. Substantial reduction in the amount of full-length CNOT3 mRNA, and therefore CNOT3 protein derived from CNOT3 gene transcripts, is achieved by: 1) in vitro refinement of oligomers using fibroblast cell lines through experimental assessment of (i) intron-enhancer targeting motifs, (ii) antisense oligomer length and oligomer cocktail development, (iii) chemistry selection, and (iv) addition of cell penetrating peptides (CPPs) to improve oligomer delivery; and 2) detailed evaluation of novel approaches to generate CNOT3 transcripts with the loss of one or more exons.

[0093] As such, it is demonstrated herein that specific antisense oligomers can be used to manipulate the processing of CNOT3 pre-mRNA.In this way, the amount of CNOT3 protein can be functionally significantly reduced, thereby alleviating the severe pathology associated with retinitis pigmentosa.

[0094] The antisense oligomers used according to the present invention can be conveniently produced by well-known solid phase synthesis techniques. Such synthesis equipment is commercially available from several vendors, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligomers on modified solid supports is described in U.S. Patent No. 4,458,066.

[0095] Any other means of such synthesis known in the art may be used in addition or instead.It is well known to use similar techniques to prepare oligomers such as phosphorothioates and alkylated derivatives.In one such automated embodiment, diethyl-phosphoramidite is used as starting material and may be synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0096] The antisense oligomers of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or genetic vector constructs designed for the in vivo synthesis of antisense oligomers. The molecules of the present invention may also be mixed, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, e.g., as liposomes, receptor targeting molecules, etc.

[0097] Antisense oligomers can be formulated for oral, topical, parenteral or other delivery, particularly for local ocular delivery and ocular delivery by injection.Preparation can be formulated to aid uptake, distribution and / or absorption at delivery site or active site.Preferably, antisense oligomers of the present invention are formulated for local ocular delivery or intraocular injection or intraocular implant, so that the effect of CNOT3 production is spatially limited and not systemic.

[0098] Treatment According to yet another aspect of the present invention, there is provided one or more antisense oligomers as described herein for use in antisense oligomer-based therapy.Preferably, the therapy is for a condition related to CNOT3 expression.More preferably, the therapy for a condition related to CNOT3 expression is for retinitis pigmentosa.

[0099] More specifically, the antisense oligomer may be selected from Table 1 (i.e., the group consisting of any one or more of SEQ ID NOs: 1-74), more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, and combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions that possess or modulate mRNA processing activity in the pre-CNOT3 gene transcript, complements thereof, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof.

[0100] The present invention also extends to combinations of two or more antisense oligomers capable of binding to a selected target and inducing exon exclusion in the CNOT3 gene transcript. The combinations can be cocktails of two or more antisense oligomers for use in antisense oligomer-based therapy, constructs containing two or more antisense oligomers linked together.

[0101] Thus, a method of treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression, comprising: a) administering to a patient an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein. The present invention provides a method comprising:

[0102] Preferably, the disease associated with CNOT3 expression in the patient is retinitis pigmentosa.

[0103] Accordingly, the present invention provides a method of treating, preventing, or ameliorating the effects of retinitis pigmentosa, comprising: a) administering to a patient an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein. The present invention provides a method comprising:

[0104] Preferably, the above-mentioned treatment is used to reduce the level of functional CNOT4 protein through exon skipping strategy.Preferably, the CNOT3 level is reduced by reducing the level of transcript by modifying pre-mRNA splicing in CNOT3 gene transcript or part thereof.

[0105] Preferably, reducing CNOT3 reduces the amount, duration, or severity of symptoms of a condition or pathology associated with CNOT3 (such as retinitis pigmentosa).

[0106] As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention used to change the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and can be administered either prophylactically, after the onset of a pathological event, or after contact with a pathogen. Also included are "prophylactic" treatments that can be directed to slow the progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0107] The subject having a disease associated with CNOT3 expression can be a mammal, including a human.

[0108] The antisense oligomers of the invention may also be used in conjunction with alternative therapies (such as drug therapies).

[0109] Thus, the present invention provides a method of treating, preventing, or ameliorating the effects of a disease or condition associated with CNOT3 expression, comprising administering an antisense oligomer of the present invention sequentially or simultaneously with another alternative therapy associated with treating, preventing, or ameliorating the effects of a disease or condition associated with CNOT3 expression, preferably, the disease or condition being retinitis pigmentosa.

[0110] delivery The antisense oligomer of the present invention can also be used as a prophylactic or therapeutic agent that can be used to treat disease.Thus, in one embodiment, the present invention provides a therapeutically effective amount of an antisense oligomer that binds to a selected target in CNOT3 pre-mRNA and induces efficient and consistent exon skipping as described herein, mixed with a pharma-ceutically acceptable carrier, diluent, or excipient.

[0111] A pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or ameliorating the effects of a disease related to CNOT3 expression in a patient, comprising: a) one or more antisense oligomers described herein; and b) one or more pharma- ceutically acceptable carriers and / or diluents Also provided is a pharmaceutical, prophylactic, or therapeutic composition comprising:

[0112] Preferably, the antisense oligomer of the present invention is delivered via local ophthalmic route to avoid systemic effects.Administration routes include, but are not limited to, intravitreal, intracameral, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, or topical (eye drops, eye washes, creams, etc.).Delivery methods include, for example, injection by syringe and drug delivery device (such as implanted vitreous delivery device (i.e. VITRASERT®)).

[0113] In one embodiment, the antisense oligomer is administered intravenously at a dose of 20 mg / kg. For example, the antisense oligomer may be administered intravenously at a dose of 20 mg / kg in mice.

[0114] Preferably, the antisense oligomer is administered by intravitreal injection at 0.01-1.5 mg / kg body weight, 0.1-0.1 mg / kg body weight, 0.2-0.8 mg / kg body weight, 0.4-0.7 mg / kg body weight, or more preferably 0.4-0.6 mg / kg body weight. The antisense oligomer may be administered by intravitreal injection at, for example, about 0.05 mg / kg body weight, 0.1 mg / kg body weight, 0.2 mg / kg body weight, 0.3 mg / kg body weight, 0.4 mg / kg body weight, 0.5 mg / kg body weight, 0.6 mg / kg body weight, 0.7 mg / kg body weight, 0.8 mg / kg body weight, 0.9 mg / kg body weight, 1.0 mg / kg body weight, 1.1 mg / kg body weight, 1.2 mg / kg body weight, 1.3 mg / kg body weight, 1.4 mg / kg body weight, or 1.5 mg / kg body weight. Preferably, the antisense oligomer is administered by intravitreal injection at about 0.5 mg / kg body weight. For example, the antisense oligomer may be administered by intravitreal injection to mice at about 0.5 mg / kg body weight.

[0115] More preferably, the antisense oligomer is administered by intravitreal injection at 0.5 to 50 mg / eye, 0.5 to 40 mg / eye, 0.5 to 30 mg / eye, 2 to 30 mg / eye, 2 to 20 mg / eye, 0.5 to 20 mg / eye, or more preferably, 5 to 20 mg / eye. Antisense oligomers may be administered by intravitreal injection at, for example, about 0.5 mg / eye, 1.0 mg / eye, 2.0 mg / eye, 3.0 mg / eye, 4.0 mg / eye, 5.0 mg / eye, 6.0 mg / eye, 7.0 mg / eye, 8.0 mg / eye, 9.0 mg / eye, 10.0 mg / eye, 11.0 mg / eye, 12.0 mg / eye, 13.0 mg / eye, 14.0 mg / eye, 15.0 mg / eye, 16.0 mg / eye, 17.0 mg / eye, 18.0 mg / eye, 19.0 mg / eye, 20.0 mg / eye, 21 mg / eye, 22 mg / eye, 23 mg / eye, 24 mg / eye, 25 mg / eye, 30 mg / eye, 35 mg / eye, 40 mg / eye, 45 mg / eye, or 50 mg / eye. Preferably, the antisense oligomer is administered by intravitreal injection at about 5-20 mg / eye.

[0116] Antisense oligomers may be administered at regular intervals for a short period (e.g., daily for 2 weeks or less). In many cases, however, oligomers are administered intermittently over a long period. Antibiotics or other therapeutic treatments may be administered after or at the same time. Treatment regimens may be adjusted (dosage, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests, and physiological tests of the subject undergoing treatment.

[0117] Dosage may depend on the severity and responsiveness of the condition to be treated, and a course of treatment may continue for a few days to a few months, or until cured or the condition is alleviated. Alternatively, dosing may be titrated according to the rate of progression of the disease. A baseline progression is established. The rate of progression after the administration of the first dose is then monitored to check for a decrease in rate. Preferably, there is no progression after dosing. Re-dosing is preferably only necessary if the rate of progression remains unchanged. Treatment is preferably successful and the disease does not further progress, or even some vision is restored. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body. Those skilled in the art can easily determine the optimal dosage, method of administration, and number of repetitions.

[0118] Optimal dosages may vary depending on the relative potency of individual oligomers, and generally can be estimated based on the EC50 found to be effective in in vitro and in vivo animal models.

[0119] In general, the dosage is 0.01-1.5 mg / kg body weight or 0.5-50 mg / eye via intravitreal injection, and may be administered once or more times per day, week, month, or year, or even once every 2-20 years. The repetition rate of dosing depends on the rate of progression of the disease. Those skilled in the art can easily estimate the number of repetitions of dosing based on measurements of residence time and concentration of the drug in bodily fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease, where the oligomer is administered in a maintenance dose, and the dose may be 0.01-1.5 mg / kg body weight or 0.5-50 mg / eye via intravitreal injection, and may be administered once or more times per day, week, month, or year, or even once every 2-20 years.

[0120] Effective in vivo treatment regimens using the antisense oligomers of the present invention may vary in duration, dosage, frequency, and route of administration, as well as the condition of the subject being treated (i.e., prophylactic administration versus administration in response to localized or systemic infection). Thus, such in vivo treatments often require monitoring with appropriate tests and adjustment of dosage or treatment regimens to accommodate the particular type of disorder being treated in order to obtain optimal therapeutic results.

[0121] Treatment can be monitored, for example, by general indicators of disease known in the art. The effectiveness of the antisense oligomer of the present invention administered in vivo can be determined from biological samples (tissue, blood, urine, etc.) taken from the subject before, during, and after administration of the antisense oligomer. Assay of such samples includes: (1) monitoring the presence or absence of heteroduplex formation with target sequence and non-target sequence using procedures known to those skilled in the art (e.g., electrophoretic gel mobility assay); (2) monitoring the amount of mutant mRNA compared to standard normal mRNA or protein, as determined by standard techniques (e.g., RT-PCR, Northern blotting, ELISA, or Western blotting).

[0122] Nuclear oligomer delivery is a major challenge for antisense oligomers. Different cell-penetrating peptides (CPPs) localize to varying degrees in PMOs under different conditions and cell lines, and we are evaluating novel CPPs for their ability to deliver PMOs to target cells. The term CPP or "cell-uptake enhancing peptide moiety" is used interchangeably and refers to cationic cell-penetrating peptides, also called "transport peptides", "carrier peptides", or "peptide permeation domains". The peptides, as shown herein, have the ability to induce cell permeation of about or at least within about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of a given cell culture population, and are capable of translocating macromolecules into multiple tissues in vivo when administered systemically. CPPs are well known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2010 / 0016215, which is incorporated by reference in its entirety.

[0123] Thus, the present invention provides an antisense oligomer of the invention in combination with a cell penetrating peptide for the preparation of a therapeutic pharmaceutical composition.

[0124] Excipients The antisense oligomers of the present invention are preferably delivered in a pharma- ceutically acceptable composition. The composition may contain about 1 nM to 1000 nM of each desired antisense oligomer(s) of the present invention. Preferably, the composition may contain about 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 10 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, and most preferably between 1 nM and 10 nM of each antisense oligomer(s) of the present invention.

[0125] The compositions may contain about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm of each desired antisense oligomer(s) of the invention.

[0126] The present invention further provides one or more antisense oligomers adapted to be useful in the prophylactic or therapeutic treatment of a disease, such as a disease or condition associated with CNOT3 expression, preventing or ameliorating symptoms, in a form suitable for delivery to a patient.

[0127] The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically tolerated when administered to a patient and typically do not cause allergic or similar adverse reactions (such as stomach upset). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with a compound. Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils derived from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or saline and aqueous solutions of dextrose and glycerol are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, PA (2013).

[0128] In a more specific form of the present invention, a pharmaceutical composition is provided that includes a therapeutically effective amount of one or more antisense oligomers of the present invention together with pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include various buffer components (e.g., Tris-HCI, acetate, phosphate), pH, and ionic strength diluents and additives (surfactants and solubilizers (e.g., Tween® 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol), etc.). Materials can be incorporated into particle preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, etc.) or liposomes. Hyaluronic acid can also be used. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the present invention. See, for example, Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, PA (2013). The compositions may be prepared in liquid form or may be a dry powder (such as a lyophilized form).

[0129] It is recognized that the pharmaceutical composition provided according to the present invention can be administered by any means known in the art.The pharmaceutical composition for administration is administered by injection, or orally, topically, or by pulmonary or nasal route.For example, antisense oligomer can be delivered by administration via intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous route.Those skilled in the art can appropriately determine the appropriate route according to the condition of the subject being treated.Preferably, antisense oligomer is delivered locally to the eye, or by intraocular injection or intraocular implant, so that the effect of CNOT3 production is spatially limited and does not extend to the whole body.

[0130] Formulations for topical administration include those in which the disclosed oligomers are in admixture with topical delivery agents (such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants). Lipids and liposomes include neutral (e.g., dioleoyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), anionic (e.g., dimyristoyl phosphatidyl glycerol DMPG), and cationic (e.g., dioleoyl tetramethylaminopropyl DOTAP and dioleoyl phosphatidyl ethanolamine DOTMA). For topical or other administration, the disclosed oligomers can be encapsulated in liposomes or complexed with, in particular, cationic liposomes. Alternatively, the oligomers can be complexed with, in particular, cationic lipids. Fatty acids and esters, their pharma- ceutically acceptable salts, and their uses are further described in US Patent No. 6,287,860 and / or US patent application Ser. No. 09 / 315,298 (filed May 20, 1999).

[0131] In certain embodiments, the disclosed antisense oligomers can be delivered by topical or transdermal methods (e.g., via incorporation of the antisense oligomer into an emulsion, optionally packaged in a liposome), including delivery to the ocular surface. Such topical or transdermal and emulsion / liposome-mediated delivery methods have been described in the art, e.g., in U.S. Pat. No. 6,965,025, for delivery of antisense oligomers. Preferably, the topical delivery is to the eye.

[0132] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is an art-recognized process, for example, using the synthetic implant designs described in U.S. Patent No. 6,969,400. Preferably, the implants can be implanted into the eye for sustained delivery of the antisense oligomers.

[0133] Compositions and formulations for ocular administration (including ocular injections, topical ocular delivery, and ocular implants) may comprise sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharma- ceutically acceptable carriers or excipients.

[0134] The therapeutically useful amount of antisense oligomers can be delivered by previously published methods. For example, antisense oligomers can be delivered via a composition that includes a mixture of antisense oligomers and an effective amount of block copolymer. An example of this method is described in US Patent Publication No. 20040248833. Other methods for delivering antisense oligomers to the nucleus are described in Mann CJ et al. (2001) Proc, Natl. Acad. Science, 98(1) 42-47 and Gebski et al. (2003) Human Molecular Genetics, 12(15): 1801-1811. Methods for introducing nucleic acid molecules into cells by expression vectors, either as naked DNA or DNA complexed with lipid carriers, are described in US Patent No. 6,806,084.

[0135] Antisense oligomers can be introduced into cells using art-recognized techniques (e.g., transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and non-viral vectors and other means known in the art).The choice of delivery method depends at least on the cells to be treated and the location of the cells, and this choice is obvious to those skilled in the art.For example, localization can be achieved by liposomes with specific markers to direct liposomes on the surface, direct injection into tissues containing target cells, specific receptor-mediated uptake, etc.

[0136] It may be desirable to deliver antisense oligomer in colloidal dispersion system.Colloidal dispersion system includes macromolecule complex, nanocapsule, microsphere, bead, and lipid-based system, including oil-in-water emulsion, micelle, mixed micelle, and liposome or liposome preparation.These colloidal dispersion systems can be used in the manufacture of therapeutic pharmaceutical composition.

[0137] Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. These formulations can have cationic, anionic, or neutral final charge characteristics, making them useful for in vitro, in vivo, and ex vivo delivery methods. It has been shown that large unilamellar vesicles can encapsulate a significant proportion of aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated in the aqueous interior and delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).

[0138] For liposomes to be efficient gene transfer vehicles, they should exhibit the following characteristics: (1) encapsulation of antisense oligomers of interest with high efficiency without compromising the biological activity of said antisense oligomers; (2) preferential and substantial binding to target cells compared to non-target cells; (3) efficient delivery of the aqueous contents of the vesicles to the target cytoplasm; and (4) accurate and efficient expression of genetic information (Mannino, et al., 2003). al., Biotechniques, 6:682, 1988). The composition of liposomes is usually a combination of phospholipids, especially phospholipids with high phase transition temperatures, usually in combination with steroids, specifically cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are believed to entrap DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA into cells.

[0139] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes that contain one or more specialized lipids, which, when incorporated into liposomes, improve circulation life compared to liposomes that lack such specialized lipids. Examples of sterically stabilized liposomes are liposomes in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0140] As is known in the art, antisense oligomers may be delivered using methods including, for example, liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, and receptor-mediated endocytosis, as well as additional non-endocytic delivery modalities such as microinjection, permeabilization (e.g., streptolysin-O permeabilization, anionic peptide permeabilization), electroporation, and a variety of non-invasive, non-endocytic delivery methods known in the art (referenced in Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, which is incorporated by reference in its entirety).

[0141] Antisense oligomers can also be combined with other pharma- ceutically acceptable carriers or diluents to produce pharmaceutical compositions.Suitable carriers and diluents include isotonic saline (e.g., phosphate buffered saline).Compositions can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0142] The described routes of administration are intended as a guide only, as one of skill in the art will be able to readily determine the optimum route of administration and any dosage for any particular animal and condition.

[0143] Several approaches have been attempted to introduce novel genetic material into cells that is functional both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S); incorporation into nonretroviral vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or liposome-mediated delivery of a transgene linked to a heterologous promoter-enhancer element (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; ​​Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupling to ligand-specific cathine transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or using naked DNA, expression vectors (Nabel et al. (1990), supra; Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues results in only localized expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra).The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) reported in vivo transfection exclusively in mouse lungs after either intravenous or intratracheal administration of DNA-liposome complexes. Examples of reviews on human gene therapy procedures are: Anderson, Science (1992) 256:808-813; Barteau et al. (2008), Curr Gene Ther; 8(5): 313-23; Mueller et al. (2008). Clin Rev Allergy Immunol; 35(3): 164-78; Li et al. (2006) Gene Ther., 13(18): 1313-9; Simoes et al. (2005) Expert Opin Drug Deliv; 2(2): 237-54.

[0144] The antisense oligomers of the present invention include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds that can provide (directly or indirectly) a biologically active metabolite or residue thereof upon administration to an animal, including a human. Thus, by way of example, the present disclosure also draws upon prodrugs and pharma-ceutically acceptable salts of the compounds of the present invention, pharma-ceutically acceptable salts of such prodrugs, and other bioequivalents.

[0145] The term "pharmaceutically acceptable salt" refers to physiologically and pharma- ceutically acceptable salts of compounds of the present invention (i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects to the parent compound). For oligomers, preferred examples of pharma- ceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines (such as spermine and spermidine); (b) acid addition salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid); (c) salts formed with organic acids (such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid); and (d) salts formed from anionic elements (such as chlorine, bromine, and iodine). The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be via topical routes (including ocular or muscular membranes, and rectal delivery), pulmonary routes (e.g., inhalation or insufflation of powders or aerosols (including nebulizers), intratracheal, intranasal, epithelial and transdermal), oral, or parenteral routes. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intraocular, or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. Oligomers having at least one 2'-O-methoxyethyl modification are believed to be particularly useful for intraocular administration. Preferably, the antisense oligomer is delivered via an intraocular route.

[0146] The pharmaceutical preparation of the present invention, which can be conveniently provided in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with pharmaceutical carrier(s) or excipient(s). In general, the preparation is prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0147] Swiss Style According to another aspect of the present invention, there is provided the use of one or more of the antisense oligomers described herein in the manufacture of a medicament for modulating or inhibiting a disease associated with CNOT3 expression.

[0148] The present invention also provides the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for the treatment of a disease associated with CNOT3 expression.

[0149] Also provided is the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression.

[0150] Preferably, the condition or disease associated with CNOT3 is retinitis pigmentosa.

[0151] According to a still further aspect thereof, the present invention extends to cDNA or cloned copies of the antisense oligomer sequences of the invention and to vectors comprising the antisense oligomer sequences of the invention. The present invention also extends to cells comprising such sequences and / or vectors.

[0152] kit Also provided are kits for treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression in a patient, comprising at least one antisense oligomer described herein, and combinations or cocktails thereof, packaged in a suitable container together with instructions for use.

[0153] In a preferred embodiment, the kit comprises at least one antisense oligomer described herein or set forth in Table 1 (i.e., SEQ ID NOs: 1-74), more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, or a cocktail of antisense oligomers described herein. The kit may also include auxiliary agents such as buffers, stabilizers, etc.

[0154] Thus, provided is a kit for treating, preventing, or ameliorating a disease or condition associated with CNOT3 expression in a subject, the kit comprising at least an antisense oligomer described herein or shown in Table 1, and a combination or cocktail thereof, packaged in a suitable container together with instructions for use.

[0155] Also provided is a kit for treating, preventing, or ameliorating a disease or condition associated with CNOT3 expression in a subject, the kit comprising at least an antisense oligomer selected from the group consisting of any one or more of SEQ ID NOs: 1-74, more preferably SEQ ID NOs: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64, and 67, and even more preferably SEQ ID NOs: 4, 7, 27, 30, 34, and 64, and combinations or cocktails thereof, packaged in a suitable container together with instructions for use.

[0156] Preferably, the disease or condition is retinitis pigmentosa.

[0157] The contents of the kit can be lyophilized and the kit can further include a suitable solvent for reconstitution of the lyophilized components. Each component of the kit is packaged in an individual container. Such container can bear a notice in a form approved by a government agency regulating the manufacture, use, or sale of drugs or biological products, reflecting approval by the government agency of the manufacture, use, or sale for administration to humans.

[0158] When the contents of the kit are provided in one or more solutions, the solution can be an aqueous solution, for example, a sterile aqueous solution.For in vivo use, the expression construct can be formulated into a pharma-ceutically acceptable injectable composition.In this case, the container means can itself be an inhaler, syringe, pipette, eye dropper, or other similar device, and the formulation from these means can be applied to a lesion in an animal, such as the lung, injected into an animal, or even applied to other components of the kit and mixed.

[0159] In one embodiment, the kit of the present invention comprises a composition comprising an effective amount of antisense oligomer that can bind to a selected target on CNOT3 gene transcript and modify pre-mRNA splicing in CNOT3 gene transcript or a part thereof.In another embodiment, the formulation is pre-measured, pre-mixed, and / or pre-packaged.Preferably, the intraocular solution is sterile.

[0160] The kit of the present invention may also include instructions designed to facilitate user compliance. Instructions, as used herein, refer to any label, insert, etc., and may be placed on one or more surfaces of the packaging material, the instructions may be provided on a separate sheet, or a combination of the above. For example, in one embodiment, the kit of the present invention includes instructions for administration of the formulation of the present invention. In one embodiment, the instructions indicate that the formulation of the present invention is suitable for treating retinitis pigmentosa. Such instructions may also include instructions regarding dosage, and instructions for administration via topical delivery to the eye or intraocular injection.

[0161] The antisense oligomer and suitable excipients can be packaged separately, so that the practitioner or user can formulate the components into a pharma- ceutically acceptable composition as needed. Alternatively, the antisense oligomer and suitable excipients can be packaged together, thereby minimizing the formulation required by the practitioner or user. In any case, the packaging material should maintain the chemical, physical, and aesthetic integrity of the active ingredient.

[0162] General rules Those skilled in the art will recognize that the invention described herein is capable of variations and modifications other than those specifically described. The present invention includes all such variations and modifications. The present invention also includes all steps, features, formulations, and compounds mentioned or shown herein, individually or collectively, and includes any and all combinations of steps or features, or any two or more steps or features.

[0163] Each document, reference, patent application, or patent cited herein is expressly incorporated herein by reference in its entirety, meaning that the reader should read and consider these documents to be part of this specification. Each document, reference, patent application, or patent cited herein is not repeated herein solely for the purpose of brevity of this specification.

[0164] Any manufacturer's instructions, manuals, product specifications, and product sheets referred to in this specification, or in any documentation for any product incorporated by reference herein, are incorporated by reference herein and may be used in the practice of this invention.

[0165] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended to be exemplary only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention described herein.

[0166] The invention described herein may include one or more ranges of values ​​(e.g., size, displacement, field strength, etc.). A range of values ​​is understood to include all values ​​within the range (including the values ​​defining the range) and values ​​adjacent to the aforementioned range (adjacent to the values ​​defining the boundaries of the aforementioned range, which will give the same or substantially the same results). Thus, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations and may vary depending on the desired properties sought to be obtained by the present invention. Thus, "about 80%" means "about 80%" and also means "80%". At the very least, each numerical parameter should be construed in light of significant digits and ordinary rounding approaches.

[0167] Throughout this specification, unless the context requires otherwise, the term "comprise" or variations thereof (such as "comprises" or "comprising") are understood to mean the inclusion of a reference integer or group of integers, but not the exclusion of any other integer or group of integers. In this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises", "comprised" and "comprising" can have the meaning ascribed to the term in U.S. patent law; for example, these terms can mean "includes", "included", "including", etc.; terms such as "consisting essentially of" and "consists essentially of" also indicate the meaning ascribed to the term in U.S. patent law (for example, these terms exclude elements found in the prior art or that affect the basic or novel characteristics of the invention, even if the elements are not specifically recited).

[0168] Other definitions for selected terms used herein can be found in the detailed description of the invention and can be applied throughout.Unless otherwise defined, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs.The term "active agent" can mean one active agent or can include two or more active agents.

[0169] The sequence identification numbers ("SEQ ID NOs") that contain the nucleotide and amino acid sequence information contained herein are summarized at the end of the description and were generated using the Patentln version 3.0 program. Each nucleotide or amino acid sequence is represented by a numerical designation in the sequence listing. <210> followed by a sequence identifier (e.g., <210> 1. <210> For each nucleotide or amino acid sequence, the length, type, and source organism of the sequence are entered in the numeric fields, respectively. <211> , <212> , and <213> The nucleotide and amino acid sequences referred to herein are represented by the information provided in the numerical designation fields. <400> The information provided in the sequence identifier following (e.g., <400> 1. <400> 2) is defined as follows.

[0170] An antisense oligomer nomenclature scheme has been proposed and published to distinguish between different antisense oligomers (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature scheme allows specific association when several slightly different antisense oligomers are tested, all directed to the same target region, as shown below: H#A / D(x:y) The first letter indicates the species (e.g., H: human, M: mouse) "#" indicates the target exon number "A / D" indicates the acceptor or donor splice sites at the beginning and end of the exon, respectively. (xy) represents the annealing coordinates, where "-" or "+" indicates the intron or exon sequence, respectively. As an example, A(-6+18) indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. These coordinates are preceded by an "A" because the closest splice site is considered to be the acceptor. The description of the annealing coordinates at the donor splice site can be D(+2-18), where the last 2 exon bases and the first 18 intron bases correspond to the annealing site of the antisense oligomer. The full exon annealing coordinate is represented as A(+65+85), which is the site between the 65th and 85th nucleotides (inclusive) from the beginning of the aforementioned exon.

[0171] The following examples not only serve to more fully describe the manner of using the above-described invention, but also to set forth the best modes contemplated for carrying out various aspects of the invention, it being understood that these methods in no way limit the true scope of the invention, but rather are presented for illustrative purposes. EXAMPLES

[0172] Example 1 AO-mediated exon skipping to induce frameshift of CNOT3 The exon structure of CNOT3 is shown in Figure 1. The splice-switching AO was designed to target enhancer sites within the frame-shifted exons in CNOT3 pre-mRNA (Figure 1) to induce exon skipping of CNOT3, resulting in loss of the open reading frame and knockdown.

[0173] Normal human fibroblasts and fibroblasts from RP11 patients (PRPF31c.1205 C>A Ser402 *) were taken from skin biopsies and cultured in DMEM supplemented with 10% FBS. Antisense sequences (Table 1), synthesized in the laboratory as 2'O-methyl phosphorothioate oligomers, were transfected into fibroblasts in 24-well plates as lipoplexes using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. Sequences that were effective in selecting the altered target exons were then synthesized as phosphorodiamidate morpholino oligomers (PMOs) and the PMOs were transfected into fibroblasts: i) in the uncomplexed state, ii) annealed to the sense ODN leash and delivered using Lipofectamine 3000 (as above), or iii) nucleofected using P2 primary cell 4-D NucleofectorX Kit S (Lonza) according to the manufacturer's instructions.

[0174] Total RNA was extracted from transfected and control cells using the MagMax RNA extraction system (Life Technologies). Transcripts of interest were evaluated first by semi-quantitative RT-PCR and then by qRT-PCR. cDNA (300 ng RNA) was synthesized using the SuperScriptIV reverse transcriptase kit (Life Technologies) according to the manufacturer's instructions. RT-PCR was performed using LA Taq DNA polymerase (TAKARA) with GC buffer I according to the manufacturer's instructions.

[0175] After cells were transfected with CNOT3 exon skipping AO, three different primer sets were used to evaluate CNOT3 transcripts: 1. For AO targeting exon 3, a forward primer annealing to exon 2 was paired with a reverse primer in exon 6. 2. For AO targeting exons 8 and 9, a forward primer in exon 7 was paired with an exon 11 reverse primer. 3. For AO targeting exons 16 and 17, a forward primer in exon 15 was paired with a reverse primer in exon 18.

[0176] qRT-PCR was used to quantify PRPF31 transcript levels following transfection with CNOT3 exon skipping AO. A forward primer spanning the exon 2 and 3 junction of PRPF31 was paired with a reverse primer spanning the exon 3 and 4 junction (Table 2). Results from all CNOT3 AO-treated cells were normalized to the expression of two housekeeping genes, TBP and GAPDH, and the fold change in PRPF31 transcript was calculated compared to cells treated with anti-ISS-N1 and mock AO. [Table 2]

[0177] Sequencing of the induced (smaller) CNOT3 transcripts predicted to result from induction of exon skipping revealed that AO3697 and AO3698 mediated exon 3 skipping, AO3702 induced both partial and complete skipping of exon 8, and AO3703 mediated dose-dependent exon 8 skipping (data not shown). AOs that mediated efficient CNOT3 exon 3, 8, or 9 skipping were then transfected into RP patient fibroblasts for 48 h, and CNOT3 transcripts were analyzed by RT-PCR (Figure 2). CNOT3 exon skipping was evident 48 h after transfection, with some reduction in full-length transcript levels. Exon 3 was excluded by AO3697, exon 8 was skipped by AO3702 and AO3703, and exon 9 was skipped by AO3706.

[0178] Previously, we have shown that phosphorodiamidate morpholino (PMO) chemistry mediates splice alteration more effectively than the same sequences synthesized as 2'O-methyl PS chemistry. Therefore, the AO sequences most promising for knocking down CNOT3 transcripts are synthesized as PMOs and transfected into normal unconjugated fibroblasts either unconjugated, with leash / lipoplexes, or via nucleofection (Nucleofector, Lonza). Evaluation and optimization of AO sequences targeting CNOT3 is ongoing.

[0179] SH-SY5Y cells are often used as an in vitro model of neuronal function and differentiation. The cells are phenotypically adrenergic, but also express dopaminergic markers. AO, which modifies CNOT3 transcripts in fibroblasts, is evaluated for its ability to downregulate CNOT3 and increase PRPF31 expression (transcripts and protein) in differentiated SH-SY5Y cells.

[0180] Example 2 AO-mediated terminal intron retention to knockdown CNOT3 Antisense sequences were designed to target the terminal exons of CNOT3 to induce terminal intron retention and knockdown of CNOT3.

[0181] 2'O-methyl AOs targeting the terminal exons of CNOT3 were transfected into adRP11 patient fibroblasts for 48 h. RT-PCR analysis of CNOT3 transcripts showed that AOs 3887, 3888, and 3889 (Table 1) induced terminal intron retention and a reduction in full-length transcript levels in a dose-dependent manner (Figure 3).

[0182] Example 3 Recruitment and review of families with RP We studied three generations of two WA families with PRPF31 mutations (one Caucasian and one Aboriginal) (Figure 4). We harvested skin fibroblasts from seven patients and began monitoring disease progression in 10 of the 24 affected individuals in these families. [Table 3]

[0183] Example 4 Generation of induced pluripotent stem cells from RP11 patients and control fibroblasts and assessment of gene expression as a result of CNOT3 knockdown Induced pluripotent stem cells are generated from patient and control fibroblasts. Patient fibroblasts are transfected with reprogramming episomes (ThermoFisher®) using the NEON® electroporation system. In a typical reprogramming experiment, 12-15 iPSC-ready colonies are picked 3-4 weeks after transfection and passaged for assessment of pluripotency gene expression by immunostaining and RT-PCR analysis (Figure 5A-C). Three clones are then selected for further testing, including gene expression profiling by TaqMan Arrays (Human Stem Cell Pluripotency Arrays, ThermoFisher) and virtual karyotyping by chromosomal G-banding analysis using QuantiSNP analysis (AGRF).

[0184] To demonstrate tri-lineage differentiation potential, iPSCs are cultured as embryoid bodies for 2-4 weeks and tested by RT-PCR for expression of markers of ectoderm, mesoderm, and endoderm differentiation, as well as downregulation of pluripotency markers (Figure 5C).iPSCs are differentiated into retinal organoids using CIF's published protocol (Mellough et al.,Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells.Stem Cells,2012.30(4):p.673-86.) (Figure 5D-H).

[0185] Differentiated cells are transfected with lead CNOT3-targeting AOs synthesized as morpholino compounds in triplicate for each analysis.

[0186] Analyze the transcripts of CNOT3, PRPF31, and other splicing factors. CNOT3, PRPF31, and selected paraspeckle proteins and splicing factors are assessed by Western blot and immunofluorescence to reflect the integrity of the splicing machinery and splicing pathways.

[0187] Example 5 Dose-dependency study of AO AO sequences were designed to skip selected exons from CNOT3 messenger RNA and transfected into fibroblasts as 2'O-methyl phosphorothioate AOs after complexation with cationic liposomes for efficient transfection. Skipping of the targeted exon results in shorter messenger RNA RT-PCR products, as identified by separation and staining of the products on a 2% agarose gel (Figures 6 and 7).

[0188] Preferred sequences show dose-dependent skipping of the targeted exon. Lead sequences were then synthesized as phosphorodiamidate morpholino oligomers (PPMOs) for testing by transfection into patient fibroblasts.

[0189] RP11 patients show PRPF31 levels that are approximately 50% of the healthy population. CNOT3 and PRPF31 messenger RNA were quantified by reverse transcription and quantitative PCR (qRT-PCR) in RP11 family members and healthy controls. Figure 8a shows that higher CNOT3 expression correlates with reduced PRPF31 and clinical outcome in heterozygous PRPF31 mutation carriers. Skipping of the targeted CNOT3 exon was assessed by qRT-PCR (Figures 8b, 8c), and PRPF31 expression after AO treatment is shown compared to PRPF31 expression in cells treated with a control AO sequence (transfection value set to 1 at 25 nM control AO) that does not target any region of the healthy human genome (no effect expected - negative control). Data from untreated cells are included for comparison.

[0190] RP11 patients exhibit PRPF31 levels that are approximately 50% of the healthy population, whereas symptomatic PRPF31 mutation carriers exhibit levels that are at least 70% or greater than healthy levels. A 1.5-fold increase in PRPF31 expression (e.g., skipping of CNOT3 exons 3 or 10 (Figure 8b) and skipping of CNOT3 exons 9, 16, or 17 (Figure 8c)) is expected to rescue splicing function in RP11 retinal pigment epithelium.

[0191] Example 6 Antisense oligomer-mediated CNOT3 exon skipping upregulates PRPF31 expression and rescues primary cilia length and number in iPSC-derived retinal pigment epithelium (RPE) from RP11 patients ASO6 (sequence number 64, CNOT3_H17A(+83+107), targeting CNOT3 exon 17) was synthesized as a phosphorodiamidate morpholino oligomer (PMO) and transfected into RP11 iPSC-derived RPE by direct transfection using 5 uM ASO in culture medium.

[0192] Immunocytochemistry was used for immunostaining of cilia and basal bodies in wild type and RP11 RPE with or without treatment with antisense oligomers.

[0193] Cilia Immunostaining Protocol RPE cells on chamber slides were fixed with ice-cold acetone-methanol (1:1) for 4 min and then air-dried. Cells were blocked in PBS containing 10% filtered goat serum for 30 min. For basal body staining, cells were incubated with mouse anti-pericentrin antibody (1:100) for 1 h at room temperature. Primary antibodies were detected using Alexafluor 488 anti-mouse (1:400). For cilia staining, cells were incubated with rabbit anti-ARL13B antibody (1:500) and incubated overnight at 4°C. Secondary primary antibodies were detected with Alexafluor 568 anti-rabbit (1:400) for 1 h at room temperature and counterstained with Hoechst (diluted 1:125) for nuclear detection. Coverslips were mounted on slides using Prolong Gold antifade medium.

[0194] Confocal microscopy analysis and quantification RPE primary cilia were assessed to evaluate PRPF31 function using confocal microscopy. Cilia length in approximately 300 RPE cells / sample was measured using NIS-Elements imaging software. In certain embodiments, for example, the following are provided: (Item 1) An isolated or purified antisense oligomer for altering pre-mRNA splicing in a CNOT3 gene transcript or a portion thereof. (Item 2) 2. The antisense oligomer according to item 1, which induces non-productive splicing or dysfunction in the CNOT3 gene transcript or a portion thereof. (Item 3) The antisense oligomer according to item 1, selected from the list comprising SEQ ID NOs: 1 to 74. (Item 4) 2. The antisense oligomer of claim 1, wherein the antisense oligomer comprises one or more nucleotide positions that have been subjected to an alternative chemistry or modification selected from the list comprising: (i) an altered backbone structure; (ii) an altered sugar moiety; (iii) RNase H resistance; (iv) an oligomer mimetic chemistry. (Item 5) 2. The antisense oligomer of claim 1, wherein the antisense oligomer is further modified by (i) chemical conjugation to a moiety; and / or (ii) tagging with a cell-penetrating peptide. (Item 6) 2. The antisense oligomer according to claim 1, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. (Item 7) 2. The antisense oligomer according to item 1, wherein any uracil (U) present in the nucleotide sequence is substituted with thymine (T). (Item 8) The antisense oligomer according to item 1, which acts to induce skipping of one or more exons of the CNOT3 gene transcript or a portion thereof. (Item 9) 2. The antisense oligomer of item 1, wherein expression of PRPF31 protein is between 1.5 and 5-fold higher than PRPF31 protein expression in a subject having a syndromic PRPF31 mutation. (Item 10) 1. A method for manipulating splicing of a CNOT3 gene transcript, comprising: a) providing one or more of the antisense oligomers according to any one of items 1 to 8 and binding said oligomer(s) to a target nucleic acid site; A method comprising: (Item 11) 1. A pharmaceutical, prophylactic or therapeutic composition for treating, preventing or ameliorating the effects of a disease related to CNOT3 expression in a patient, comprising: a) one or more antisense oligomers according to any one of items 1 to 8, and b) one or more pharma- ceutically acceptable carriers and / or diluents 20. A pharmaceutical, prophylactic, or therapeutic composition comprising: (Item 12) 1. A method of treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression, comprising: a) administering to a patient an effective amount of one or more antisense oligomers according to any one of items 1 to 9 or a pharmaceutical composition comprising one or more antisense oligomers. A method comprising: (Item 13) Use of the purified and isolated antisense oligomer according to any one of items 1 to 9 for the manufacture of a medicament for treating, preventing or ameliorating the effects of a disease associated with CNOT3 expression. (Item 14) A kit for treating, preventing, or ameliorating the effects of a disease associated with CNOT3 expression in a patient, comprising at least one antisense oligomer according to any one of items 1 to 9, and combinations or cocktails thereof, packaged in a suitable container together with instructions for use. (Item 15) The composition according to item 11, the method according to item 10 or 12, the use according to item 13, or the kit according to item 14, wherein the disease related to CNOT3 expression is retinitis pigmentosa. (Item 16) The composition of item 11, the method of item 10 or 12, the use of item 13, or the kit of item 14, wherein the subject having a disease associated with CNOT3 expression is a human. (Item 17) 15. The composition of claim 11, the method of claim 10 or 12, the use of claim 13, or the kit of claim 14, wherein the expression of PRPF31 protein is between 1.5 and 5-fold greater than the expression of PRPF31 protein in a subject with a symptomatic PRPF31 mutation. (Item 18) 2. The antisense oligomer according to item 1, selected from the list comprising SEQ ID NOs: 4, 7, 9, 11, 15, 16-18, 27, 30, 34, 35, and 64. (Item 19) 2. The antisense oligomer according to item 1, selected from the list comprising SEQ ID NOs: 4, 7, 27, 30, 34 and 64.

Claims

[Claim 1] The invention described in the specification.

Images