RU2817702C2 - New method of treating retinitis pigmentosa - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: presents an isolated or purified antisense oligomer for modifying pre-mRNA splicing in a CNOT3 gene transcript, a pharmaceutical composition for treating the effects of a disease associated with CNOT3 expression in a patient, a method of treating a disease associated with CNOT3 expression, and a kit for treating the effects of a disease associated with CNOT3 expression in a patient. Kit contains an antisense oligomer and combinations or cocktails packed in a suitable container together with instructions for use thereof.

EFFECT: invention is aimed at obtaining an antisense oligomer for modifying pre-mRNA splicing in a transcript of the CNOT3 gene or part thereof.

12 cl, 3 tbl, 19 dwg, 6 ex

Description

TECHNICAL FIELD

[0001] This invention relates to the use of antisense oligomers for treating, preventing or reducing the effects of retinitis pigmentosa.

BACKGROUND OF THE ART

[0002] Retinitis pigmentosa (RP) is a degenerative eye disease that causes severe vision impairment due to the progressive degeneration of rod photoreceptor cells in the retina; Most cases of PR are inherited. This form of retinal dystrophy exhibits initial symptoms regardless of age; Thus, the diagnosis of PD occurs everywhere, from early childhood to old age. PR is a rare disease that affects approximately one in 4,000 individuals (more than 1.5 million people worldwide). 30%-40% of familial cases of PD show autosomal dominant genetic inheritance (Hartong et al. 2006). There is currently no treatment for PR.

[0003] PR is caused by mutations in more than 50 genes. Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adPR). In some cases, such mutations show incomplete penetrance, with some carriers developing retinal degeneration and others being completely asymptomatic. Asymptomatic carriers are protected from disease by above average expression of the unmutated PRPF31 allele.

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

[0005] AAV-mediated gene replacement and CRISPR/Cas9 gene editing are currently being studied as treatments for retinal diseases. Although the coding sequence of PRPF31 is within the capacity of AAV vectors, the consequences of unregulated expression or overexpression of PRPF31 are unknown. In addition, it is unknown whether virus-mediated gene therapy can be re-administered for ocular diseases, as seroconversion has been reported as a consequence of intraocular injection of the viral vector. In addition, CRISPR/Cas9 gene correction will require a separate product for the PRPF31 mutation in each family. Additionally, these approaches, such as gene replacement and gene editing, require subretinal injection of viral vectors to achieve adequate transfection.

[0006] There is a need to develop new methods for treating or preventing retinitis pigmentosa; or at least in proposing methods complementary to previously known methods of treatment.

[0007] This invention is directed to providing an improved or alternative method of treating, preventing or reducing the effects of retinitis pigmentosa

[0008] The previous discussion of the prior art is intended only to facilitate understanding of the present invention. This discussion is not an endorsement or admission that any of the material referred to is or was part of the general knowledge at the priority date of the application.

SUMMARY OF THE INVENTION

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

[0010] For example, in one aspect of the present invention there is provided an antisense oligomer of 10-50 nucleotides containing a targeting sequence complementary to a region adjacent to or within an intron of a CNOT3 gene transcript or a portion thereof. Another aspect of the invention provides an antisense oligomer of 10-50 nucleotides containing a targeting sequence complementary to a region adjacent to or within an exon of a 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 consisting of the sequences presented in Table 1.

[0013] Preferably, the antisense oligomer is selected from the list consisting of: SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67 even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64.

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

[0015] The antisense oligomer of the present invention may be selected as an antisense oligomer capable of binding to a selected CNOT3 target site, wherein the target site is an mRNA splice site selected from a splice donor site, splice acceptor sites, or exonic splice elements. The target site may also include some flanking intronic sequences when the oligomer is targeted to splice donor or acceptor sites.

[0016] More specifically, the antisense oligomer may be selected from the group consisting of any one or more of SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27 , 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64, and/or the sequences presented in Table 1, and combinations or cocktails thereof. The group includes sequences that can hybridize with such sequences under stringent hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones and functional truncations or extensions thereof, which have or modulate pre-mRNA processing activity in the CNOT3 gene transcript. In some embodiments, antisense oligomers may be 100% complementary to the target sequence or may include mismatches, such as to accommodate variants, as long as the heteroduplex formed between the oligonucleotide and the target sequence is stable enough to withstand the action of cellular nucleases and other degradation scenarios occurring in vivo. Therefore, certain oligonucleotides may have about or at least about 70% sequence complementarity, for example, 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 invention also extends to a combination of two or more antisense oligomers capable of binding to a selected target to induce exon exclusion in a CNOT3 gene transcript, including a construct containing two or more such antisense oligomers. The construct can be used for therapy based on antisense oligomers.

[0018] The invention extends, according to yet another aspect, to cDNAs or cloned copies of the antisense oligomeric sequences of the invention, as well as vectors containing the antisense oligomeric sequences of the invention. The invention also extends to cells containing such sequences and/or vectors.

[0019] A method for controlling splicing in a CNOT3 gene transcript is also provided, comprising the step of:

a) providing one or more antisense oligomers as described herein and causing the oligomer(s) to bind to the target nucleic acid site.

[0020] Also provided is a pharmaceutical, prophylactic or therapeutic composition for treating, preventing or reducing the effects of a disease associated with the expression of CNOT3 in a patient, wherein the composition contains:

a) one or more antisense oligomers as described herein, and

b) one or more pharmaceutically acceptable carriers and/or diluents.

[0021] The composition may contain from about 1 nM to 1000 nM of each of the desired antisense oligomers of this invention. Preferably, the composition may contain from about 10 nM to 500 nM, most preferably from 1 nM to 10 nM, of each of the antisense oligomers of this invention.

[0022] Also provided is a method of treating, preventing or reducing the effects of a disease associated with the expression of CNOT3, comprising the step of:

a) administering to the patient an effective amount of one or more antisense oligomers or a pharmaceutical composition containing one or more antisense oligomers, as described herein.

[0023] The use of purified and isolated antisense oligomers as described herein is also proposed for the production of a medicament for treating, preventing, or reducing the effects of a disease associated with CNOT3 expression.

[0024] Also provided is a kit for treating, preventing, or mitigating the effects of a disease associated with CNOT3 expression in a patient, which includes at least an antisense oligomer as described herein, and combinations or cocktails thereof, packaged in a suitable container along with instructions on its application.

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

[0026] Additional aspects of the present invention will now be described with reference to the accompanying non-limiting examples and drawings.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0027] Additional features of the present invention are more fully described in the following description of several non-limiting embodiments. This description is included solely for the purpose of illustrating the present invention. It should not be construed as limiting the general content, disclosure or description of the present invention set forth above. The description will be made with reference to the attached graphic materials, on which:

In FIG. Figure 1 shows a diagram of the CNOT3 reading frame. CNOT3 consists of 18 exons and encodes a protein with 753 amino acids. In FIG. In Figure 1(a), exons within the reading frame are shown as rectangles, while exons with junctions that interrupt codons are shown with corners (blue, red). In FIG. 1(b) shows the CNOT3 protein, showing the functional domains and amino acid positions corresponding to the above exons. NAR: NOT an assigned region. CS: binding sequence NOT box: Negative for TATA box. Exclusion of exons 2, 3, 8, 9, 11, 12, 13, 14, 15 and 16 will disrupt the open reading frame.

In FIG. Figure 2 is a gel image showing CNOT3 transcript products from patient PR11 fibroblasts 48 hours after transfection with 2'O-methylphosphorothioate AO (50, 25, and 12.5 nM). Control AOs targeting an unknown sequence (-ve control AO) were included for comparison.

In FIG. Figure 3 is a gel image showing CNOT3 transcription products from fibroblasts 48 hours after transfection with 2'O-methylphosphorothioate AO designed to induce terminal intron retention at concentrations of 50 and 25 nM.

Fig. 4A and 4B: in FIG. Figure 4A shows pedigree 0255: 11 members with the PRPF31 mutation (c.267delA). In FIG. 4B shows the pedigree of 0080: 13 members suffering from the disorder (about 1205 G>A). Arrow = donor dermal fibroblasts, black = patients currently participating in a natural history study of CRE disease.

In FIG. 5A-H show skin fibroblasts from a patient with a CLN3 mutation reprogrammed to pluripotency. Patient iPSC (CLN3−/−) exhibit typical iPSC morphology and strong markers of pluripotency, including the presence of OCT4, NANOG, SOX2, and SSEA4 (A). Comparison of pluripotent gene expression in 6 iPSC lines using qRT-PCR demonstrated similar expression patterns for all lines (B). Patient iPSCs demonstrated trilineage differentiation potential. Patient iPSCs (CLN3-iPS-EB), gene-corrected control iPSCs (CLN3HDR-iPS-EB) and control human iPSCs (WT-iPS-EB, ThermoFisher) were differentiated as embryoid bodies within 2 weeks and then screened for expression of ectoderm (PAX6, OTX1), mesoderm (SOX17, GATA4, FOX2A) and endoderm (BRACHYURY, FDGFR) markers, as well as pluripotency genes (OCT4, NANOG, SOX2) using quantitative RT-PCR (C). D-H: iPSC differentiation in the retina. The retinal organoids had a morphology similar to the optic cup, surrounded by clear laminated retinal tissues (D), which contained an organized apical layer of recoveryin-expressing photoreceptors (E), as well as basally located Smi32-expressing retinal ganglion cells (F). Electron microscopy of photoreceptor outer segments of retinal organoids (G) at day 170 demonstrated similar morphology to outer segments in the developing (E120) human retina (H).

In FIG. Figure 6 shows a screen for AO-induced CNOT3 exon skipping using a 2'-O-methyl compound on the PS backbone. AOs are designed to target CNOT3 exon splicing enhancer motifs to mediate exclusion of the target exon during pre-mRNA splicing to mediate CNOT3 knockdown or disrupt protein function. Dermal fibroblasts were transfected for 48 hours with CNOT3 AO (2'OMe-PS chemistry) targeting exons (as indicated). RT-PCR products were separated on 2% agarose gels.

In FIG. Figure 7 shows a screen for AO-induced CNOT3 exon skipping using a 2'-O-methyl compound on the PS backbone. AOs are designed to target CNOT3 exon splicing enhancer motifs to mediate exclusion of the target exon during pre-mRNA splicing to mediate CNOT3 knockdown or disrupt protein function. Dermal fibroblasts were transfected for 48 hours with CNOT3 AO (2'OMe-PS chemistry) targeting exons (as indicated). RT-PCR products were separated on 2% agarose gels.

In FIG. Figure 8 shows a negative correlation between CNOT3 and PRPF31 mRNA expression. In FIG. Figure 8(a) demonstrates RT-qPCR analysis of PRPF31 and CNOT3 mRNA expression normalized to TATA binding protein (TBP) expression in iPSC-derived retinal pigment epithelium from PR11 in asymptomatic and healthy (WT) individuals. PR11 and the asymptomatic subject are from the same pedigree, both heterozygous for the PRPF31 c.1205C>A (Ser402*) mutation. Healthy subject from an unrelated family. CNOT3 and PRPF31 mRNA expression in healthy controls was set to 1 (n ≥ 2). In FIG. 8(b) dermal fibroblasts from a PR11 patient were transfected for 48 hours with CNOT3 AO (2'OMe-PS) targeting exon 4, 6, 7, or 10. PRPF31 transcript levels were analyzed by RT-qPCR and normalized to TBP expression. PRPF31 expression in cells treated with 25 nM control AO was set to 1 (n=1). In FIG. 8(c): PR11 patient skin fibroblasts were transfected for 48 hours with CNOT3 AO (2'OMe-PS chemistry) targeting exon 3, 8, 9, 16, or 17. PRPF31 transcript levels were analyzed by RT-qPCR and normalized relative to TBP expression, and it is shown relative to PRPF31 expression in cells treated with AO placebo control at 25 nM (PRPF31 control expression set to 1 (n=1).

In FIG. 9 shows the effect of ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting exon 17 of CNOT3) synthesized as phosphorodiamidate morpholino oligomer (PMO) and transfected into RPE derived from PR11 iPSC. (A) CNOT3 exon 17 skipping in cells treated with PMO alone or cell-permeable peptide-tagged PMO (PPMO) at 5 μM. (B) Up-regulation of PRPF31 as a consequence of CNOT3 knockdown determined by RT-qPCR and normalized to TATA binding protein (TBP) expression. PRPF31 expression in the untreated control was set to 1.

In FIG. 10 shows (A) immunostaining of cilia (red) and basal body (green) in wild-type and PR11 RPE with or without treatment with an antisense oligomer with ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107) targeting exon 17 of CNOT3 ). (B) Percentage of RPE cells expressing cilia, calculated from ≥1000 cells. (C) Measurement of cilia length using NIS-Elements Imaging software. The histogram represents the mean ± SEM of ~300 ciliated cells. Scale = 10 µm. Student's t test. ***p <0.001.

DETAILED DESCRIPTION OF THIS INVENTION

DETAILED DESCRIPTION OF THE INVENTION

Antisense oligonucleotides

[0028] There is an increased vulnerability of photoreceptors to decreased splicing activity. While mutations in splicing factors can cause splicing deficiencies in various tissues, photoreceptor cells will be severely affected due to their high requirement for mRNA production (eg, mRNA encoding rhodopsin and other phototransduction proteins). This model suggests that the amount of mRNA produced by the retina greatly exceeds the levels of mRNA produced 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 a decrease in the amount of total PRPF31 mRNA in patients with PR11, as well as a slower rate of spliceosome assembly and pre-mRNA processing. This indicates that the disease occurs through the mechanism of haploinsufficiency. Compared to other tissues, the retina expresses seven times more major spliceosomal small nuclear RNAs (snRNAs).

[0030] CCR4-Not transcription complex subunit 3 (CNOT3, one of the five Not proteins of the Ccr4-Not deadenylase complex) has been identified as a major modifier gene determining the penetrance of PRPF31 mutations through a transcriptional repression mechanism; modulation of PRPF31 transcription by direct binding to its promoter. The expression level of the wild-type PRPF31 allele determines whether carriers of the mutated PRPF31 allele are symptomatic. In asymptomatic carriers, CNOT3 is expressed at low levels, which allows the production of more wild-type PRPF31 transcripts and prevents the manifestation of retinal degeneration.

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

[0032] Knockout of the CNOT3 gene is embryonic lethal because it is vital for cell cycle progression through regulation of mRNA turnover and regulation of mRNA decay in various physiological processes. However, if CNOT3 levels can be reduced or eliminated locally in the eyes of subjects at risk for or suffering from PR, then disease progression will be affected as increased amounts of PRPF31 protein may be produced, mimicking a model of incomplete penetrance, with asymptomatic carriers protected from disease by PRPF31 expression above the average level of the unmodified gene.

[0033] Thus, the present invention provides antisense oligonucleotides for inducing non-productive splicing or functionally impaired CNOT3 protein (a negative regulator of PRPF31) to reduce (but preferably not eliminate) CNOT3 levels and therefore increase transcription and translation of the normal PRPF31 allele.

[0034] Unlike other antisense oligomer based therapies, this invention does not cause increased RNA degradation by recruiting RNase H, but RNase H preferentially binds and the degraded RNA duplexes to the CNOT3 gene DNA. The present invention also does not rely on hybridizing an antisense oligomer to CNOT3 genomic DNA or linking 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 modify pre-mRNA splicing in the CNOT3 gene transcript or a portion thereof, and to induce exon skipping and/or terminal intron retention. This strategy preferentially reduces overall protein expression or produces proteins that lack functional domains, resulting in decreased protein function.

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

[0037] In the general population, PRPF31 expression is highly variable, with a continuous distribution of levels. Expression levels range from 0.53 to 2.48 arbitrary units, representing a 5-fold variation between the lower and higher expressors. Thirty percent of PRPF31 mutation carriers are asymptomatic, and these individuals have wild-type alleles that are expressed at nearly 2-fold higher levels than carriers with symptomatic PRPF31 mutations. Therefore, preferably, modulation of CNOT3 expression results in at least twofold higher expression of PRPF31 than is the case with symptomatic PRPF31 mutations. Preferably, PRPF31 expression due to the AOs of the present invention reducing CNOT3 expression is 1.5-5 times higher than expression with symptomatic PRPF31 mutations. For example, PRPF31 expression may be 2-4 times higher.

[0038] By “isolated” is meant a material that is substantially or substantially free of the components that would normally accompany it in its native state. For example, "isolated polynucleotide" or "isolated oligonucleotide", as used herein, may refer to a polynucleotide that has been purified or removed from sequences flanking it in its natural state, for example, a fragment of DNA that has been removed from sequences that flank fragment in the genome. The term "isolation" in relation to cells refers to the purification of cells (eg, fibroblasts, lymphoblasts) from a source subject (eg, a subject with polynucleotide repeat disease). In the context of mRNA or protein, the term "isolation" refers to the release of mRNA or protein from a source, such as cells.

[0039] An antisense oligomer can be said to be “directed toward” or “targeted against” the target sequence to which it hybridizes. In some embodiments, the target sequence comprises a region including a 3' or 5' splice site of a preprocessed mRNA, a branch point, or other sequences involved in the regulation of splicing. The target sequence may be within an exon or within an intron, or span an intron/exon junction.

[0040] In some embodiments, the antisense oligomer has sufficient sequence complementarity to the target RNA (ie, the RNA for which splice site selection is modulated) to effectively block a region of the target RNA (eg, pre-mRNA). In exemplary embodiments, such blocking of CNOT3 pre-mRNA serves to modulate splicing either by masking a binding site for a native protein that would otherwise modulate splicing and/or by altering the structure of the target RNA. In some embodiments, the target RNA is a target pre-mRNA (eg, 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 trigger masking of a binding site for a native protein that might otherwise modulate splicing and/or alter three-dimensional structure of the target RNA.

[0042] Selected antisense oligomers may be shorter, for example about 12 bases, or longer, for example about 50 bases, and include a small number of mismatches, provided that the sequence is sufficiently complementary to influence the modulation of splicing when hybridized with target sequence, and optionally forms a heteroduplex with RNA having Tm. 45°C or higher.

[0043] Preferably, the antisense oligomer is selected from the group consisting of the sequences presented in Table 1. Preferably, the antisense oligomer is selected from the group consisting of the sequences of SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64.

[0044] In some 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 antisense oligomers with the target RNA sequence may be as small as 8-11 bases, but may be 12-15 bases or more, for example 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 usually long enough to have a unique complementary sequence. In some embodiments, a minimum length of additional bases may be required to achieve the required Tm. binding as discussed in this document.

[0045] In some embodiments, oligonucleotides up to 50 bases in length may be suitable, where at least a minimum number of bases, for example 10-12 bases, are complementary to the target sequence. In general, however, facilitated or active uptake into cells is optimized for oligonucleotide lengths less than about 30 bases. For phosphorodiamidate morpholino oligomer (PMO) antisense oligomers, the optimal balance of binding stability and capture is typically achieved at a length of 18–25 bases. Included are antisense oligomers (e.g., CPPMO, PPMO, PMO, PMO-X, PNA, LNA, 2'-OMe), which consist 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.

[0046] In some embodiments, antisense oligomers may be 100% complementary to the target sequence or may include mismatches, for example, to accommodate variants, as 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 scenarios occurring in vivo. Therefore, certain oligonucleotides may have about or at least about 70% sequence complementarity, for example, 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] Mating errors, if present, typically have less of a destabilizing effect toward the end regions of the hybrid duplex than toward the middle. The number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatches in the duplex, according to well-understood principles of duplex stability. Although such an antisense oligomer is not necessarily 100% complementary to the target sequence, it is effective in 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 is a function of Tm. binding and duplex susceptibility to cellular enzymatic degradation. Melt. oligonucleotide to RNA of complementary sequence can be measured by conventional methods such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada C. G. and Wallace R. B., 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In some embodiments, antisense oligomers may have a Tm. binding to RNA with a complementary sequence above body temperature and preferably above about 45 °C or 50 °C. Also included Tmel. in the range of 60-80 °C or higher.

[0049] Additional examples of variants include antisense oligomers having about or at least about 70% sequence identity or homology, for example, 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 over the entire length of any of SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14 , 16-18, 27, 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64, or the sequences listed in Table 1.

[0050] More specifically, an antisense oligomer is provided that is capable of binding to a selected target site to modify pre-mRNA splicing in a CNOT3 gene transcript or a portion thereof. The antisense oligomer is preferably selected from the antisense oligomers presented in Table 1 or SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64.

[0051] Pre-mRNA splicing modification preferably causes skipping or deletion of one or more exons or introns of the mRNA and/or retention of a terminal intron. The resulting protein may be shorter in length compared to the original full-length CNOT3 protein due to internal truncation or premature termination, or may be longer due to terminal intron retention. These CNOT3 proteins can be called isoforms of the unmodified CNOT3 protein.

[0052] The remaining exons of the resulting mRNA may be in frame and produce a shorter protein with a sequence similar to that of the parent full-length protein, except that it has an internal truncation in the region between the original 3' and 5' ends. In another possibility, exon skipping may cause a frameshift, resulting in a protein in which the first part of the protein is essentially identical to the parent full-length protein, but where the second part of the protein has a different sequence (eg, a nonsense sequence) due to the frameshift. Alternatively, exon skipping may cause the production of a prematurely terminated protein due to the disruption of the reading frame and the presence of premature translation termination. Additionally, the antisense oligomer may produce an artificially extended protein due to retention of the terminal intron in frame.

[0053] The functional domains of CNOT3 include coiled coil, linker, NAR (Not anchor region), CS (junction sequence) and NOT box. Elimination of exon 4 removes part of the coiled-coil domain, loss of exon 9 will disrupt the open reading frame resulting in mRNA degradation and any encoded protein will be truncated, whereas skipping exon 17 alters the NOT box and is expected to affect complex formation and hence function CNOT3.

[0054] Removal of one or more exons may further result in misfolding of the CNOT3 protein and a decrease in the ability of the protein to be successfully transported across the membrane.

[0055] Preferably, internally truncated proteins (ie, proteins lacking the amino acids encoded by one or more exons) are present. If the CNOT3 protein is knocked out, there may be problems with increased CNOT3 transcription as the body tries to compensate for the decrease in the overall amount of CNOT3 protein. In contrast, the presence of an internally truncated protein (preferably lacking one or more features of the complete CNOT3 protein) should be sufficient to prevent increased transcription but still provide a therapeutic benefit due to the reduction in the total amount of functional CNOT3 protein.

[0056] The exon skipping-inducing antisense oligomer of the present invention does not necessarily need to completely or even significantly disrupt the function of the CNOT3 protein. Preferably, the exon skipping method results in reduced or impaired functionality of the CNOT3 protein.

[0057] The skipping method of this invention using antisense oligomers can skip a single exon or can skip two or more exons simultaneously.

[0058] Antisense oligomers of this invention can be a combination of two or more antisense oligomers capable of binding to a selected target to induce exon exclusion in a CNOT3 gene transcript. The combination may be a mixture of two or more antisense oligomers and/or a construct containing two or more antisense oligomers linked together.

Table 1. List of antisense oligonucleotide sequences used in this study and efficacy assessment for AO-induced CNOT3 exon skipping. +1: >50% exon skipping, 0: <50% exon skipping, -1: No exon skipping

SEQ ID JSR# Coordinates Sequence 5'-3' 2-OMePS 1 4112 CNOT3_H2A(-6+19) ACUCUCUUGGGAGACGGACGCUGCUA -1 2 4113 CNOT3_H2A(+28+52) AUCUUCCCUGCCCUACAGACGCACU -1 3 4114 CNOT3_H2D(+55-4) GUACCUUGGAGUUUGCGCUUGUCCG -1 4 3697 CNOT3_H3A(+9+33) CGGACACCUCUUGAGGCAGCGAUC 1 5 3698 CNOT3_H3A(+39+63) GCCAAAUAUCUUCAAACUGCUCCAC 0 6 4579 CNOT3_H4A (-8+17) UUGGCUGCAUUGUGGAGCUGAGGGA -1 7 4580 CNOT3_H4A (+12+36) CUUUUCUUUCUGGUUCGCGUUGGCU 1 8 4581 CNOT3_H4A (+38+62) AUCUCCUUCUUUAGGUCAGCCUCAU 0 9 4582 CNOT3_H4D (+13-12) CAGCCCCCUCACUUGUAGCUUCUUA 1 10 4583 CNOT3_H5A (-9+16) UUUGGUCCCUCAGCCGCUGCAGAUG -1 eleven 4584 CNOT3_H5A (+36+60) CUGCCUCUUGUCCUUGAUCUCGUUG 1 12 4585 CNOT3_H5A (+53+77) UUGCGGUUGUCUAUAAGCUGCCUCU -1 13 4586 CNOT3_H5D (+13-12) CUGGGCUCCUACCGGUCUCAAUGAGC -1 14 4587 CNOT3_H6A (-5+20) ACUUUGAACCGUUCCAUUUGCUGUA 1 15 4588 CNOT3_H6A (+12+36) GGUCUCUCGUUCCACAACUUUGAAC 0 16 4589 CNOT3_H6A (+37+61) CCUCUUUGCUGUAAGCUUUGGUUUU 1 17 4590 CNOT3_H6A (+89+113) ACCUCUUCCUUCUCCUUCUGGGCAG 1 18 4591 CNOT3_H6D (+15-10) CCCAACUCACCGUGAGCCACUGGCC 1 19 4592 CNOT3_H7A(-6+19) UGAGCGUGUCGAUGGUAUUCUAGGG -1 20 4593 CNOT3_H7A(+16+40) CAAACUGGUCCACCUGCAUGUUGAG -1 21 4594 CNOT3_H7A(+62+86) CCCUUCUUCUUGCGUGUCUGCACUG -1 22 4595 CNOT3_H7D(+15-10) CCUCACUCACAUCCUUGUCGCCCUU -1 23 3699 CNOT3_H8A(-10+15) CCGCTTCAAGCCCTCGCCCAGGGCC -1 24 3700 CNOT3_H8A(+18+41) GUGGUAGCGGUGCUUCUCGAUGUG -1 25 3701 CNOT3_H8A(+34+58) UGGUCUCUAGCAUGCGCACGUGGUA -1 26 3702 CNOT3_H8A(+75+99) GGCGUCAACGAGGAUGGAGUCAUUG 0 27 3703 CNOT3_H8A(+141+165) CUCCUCGAAGUCGGGGUCCUGGGAU 1 28 3704 CNOT3_H9A(-6+19) GUGGCGACCAGCGCCUGUGCUGUGG -1 29 3705 CNOT3_H9A(+29+52) CUCAUCCUCCAUGUGGCUGUGGCUG -1 thirty 3706 CNOT3_H9A(+56+80) GGGCGUGCUGCUGGACUGGUUGAAG 1 31 3707 CNOT3_H9A(+98+122) GGCUGGGCUGGGCGGGAAUGGGAGAG 0 32 5485 CNOT3_H9A(+52+76) GUGCUGCUGGACUGGUUGAAGAUCU 0 SEQ ID JSR# Coordinates Sequence 5'-3' 2-OMePS 33 4596 CNOT3_H10A(-10+15) AUCUUCAGAGUUUUCCUGAGGUAGG 1 34 4597 CNOT3_H10A(+16+40) CUGUGGAACGUCCCCUCUUCUUAUC 1 35 4598 CNOT3_H10D(+12-13) UCACACCACCUGGCUGACUUCACU 1 36 4892 CNOT3_H11A(+49+73) AGGUGGGCGGCACAGCUGGGGACUG -1 37 4893 CNOT3_H11A(+56+80) GAGGGGUAGGUGGGCGGCACAGCUG -1 38 4894 CNOT3_H11A(+74+98) GCAGCAGGCGGGGGGCCGGAGGGGU -1 39 4115 CNOT3_H11A(-8+17) CCGUUUUUGGCUGGAGACUGCGGGU 0 40 4116 CNOT3_H11A(+171+195) GUGGCUGGGAGCUGGACUGGCCUUG 1 41 3708 CNOT3_H12A(+1+25) CUGUCUGCCACAACUGAGCUGUAAC NT 42 3709 CNOT3_H12A(+85+109) GGGUUGUGGGGGCCGGAAGGGGGGC NT 43 3710 CNOT3_H12D(+19-6) ACUCACGAGGUGCUGGGAGGUGGGU NT 44 4117 CNOT3_H13A(-6+19) UGCCGCACUGGGUUCCUUCCUGGAG 1 45 4118 CNOT3_H13A(+38+62) UGUUCCCUGAGCCUGGGGCCACGCC 0 46 4119 CNOT3_H13A(+83+107) GAGGAUUCACAGGCAGUGGCACCAG 1 47 4120 CNOT3_H14A(-8+17) CUCAGAGGCUCAGGGGCCUGGGGAG 0 48 4121 CNOT3_H14A(+48+72) AGGGUCCUCAAUGCCAGAGCUGAUG 0 49 4895 CNOT3_H15A(+147+171) GCAUGUGGUGCCAGGCGGCCUCUUC -1 50 4896 CNOT3_H15A(+157+181) GAGGGGUGAGGCAUGUGGUGCCAGG -1 51 4897 CNOT3_H15A(+175+199) CGAAUACGCUCAGAGUCAGAGGGGU -1 52 4122 CNOT3_H15A(-11+14) GCUCAGGAUGAUGUCUGUGGGGGAGG -1 53 4123 CNOT3_H15A(+5+29) AGGUGCUGAUGUACUGCUCAGGAUG 1 54 4124 CNOT3_H15A(+42+66) CCUCUGACAGCUGCAGGGGCGGCUG 0 55 4125 CNOT3_H15A(+78+102) CCAUGGACAGACACCCAGCGACAGC 1 56 4126 CNOT3_H15A(+108+132) AGAGCUGCUCCUUGGUGAGGGGCAC 1 57 4127 CNOT3_H15A(+162+186) AGUCAGAGGGGUGAGGCAUGUGGUG 1 58 4128 CNOT3_H16A(-11+14) GGGGGAGGUACUGCCUGUGAGAGCA -1 59 4129 CNOT3_H16A(+36+58) GGUGGCAUCUGGUGGUGGUAGG 1 60 4130 CNOT3_H16A(+109+133) CUCCAGAUAGUAGAAGAUGAAGAAG 0 61 4365 CNOT3_H17A(+28+52) GCCAUGACUGCUUCUUUAGGGCCUU -1 63 4366 CNOT3_H17A(+57+81) GAACCACAUCAUGUACUUGGUGUGG -1 64 4367 CNOT3_H17A(+83+107) AUGGUCUUGGGCUCCUCGUGCCUCU 1 65 --- CNOT3 H17A(+78+102) CUUGGGCUCCUCGUGCCUCUGGAAC NT 66 --- CNOT3 H17A(+88+112) CAGUGAUGGUCUUGGGCUCCUCGUG NT 67 5486 CNOT3 H17A(+102+126) CUGCUCAAACUCGUCAGUGAUGGUC 0 68 3886 CNOT3_H18A(-10+15) GUAGAUGUAGGUGCCCUGGCCGGGG -1 69 3887 CNOT3_H18A(+16+40) GCUGGCCCCCACUUCUCGUAGUCAAA 1 70 3888 CNOT3_H18A(+41+65) UCAAAGGUGAAGCCUUCCUUCUUCC 1 71 3889 CNOT3_H18A(+66+90) GUCCCGGUCCUCCAGGUAGCGGUAC -1 72 5551 CNOT3_H9A(+1+25) GGGGAGGUGGCGACCAGCGCCUGUG 0 73 5555 CNOT3_H17A(+71+95) UCCUCGUGCCUCUGGAACCACAUCA 1 74 4368 CNOT3 H17D(+15-10) GGGCCCUCACCUGCUCAAACUCGUC 0

[0059] A method for controlling splicing in a CNOT3 gene transcript is also provided, comprising the step of:

a) providing one or more antisense oligomers as described herein and causing the oligomer(s) to bind to the target nucleic acid site.

[0060] According to yet another aspect of the present invention, a splicing control target nucleic acid sequence for CNOT3 is provided, comprising DNA equivalents of nucleic acid sequences selected from Table 1 or the group consisting 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 NO: 4, 7, 27, 30, 34 and 64, and sequences complementary thereto .

[0061] Designing antisense oligomers to completely mask consensus splice sites may not necessarily result in altered splicing of the target exon. In addition, the present inventors have discovered that the size or length of the antisense oligomer itself is not always a major factor in the design of antisense oligomers. With some targets, such as exon 3 of IGTA4, antisense oligomers as short as 20 bases are capable of causing some exon skipping, in some cases more efficiently than other longer (e.g., 25 bases) oligomers targeting the same exon.

[0062] The present inventors have also discovered that there is no standard motif that can be blocked or masked by antisense oligomers to redirect splicing. It was found that there is a need to develop antisense oligomers and empirically evaluate their individual effectiveness.

[0063] More specifically, the antisense oligomer may be selected from the antisense oligomers presented in Table 1. The sequences are preferably selected from the group consisting of any one or more of any one or more 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 NO: 4, 7, 27, 30, 34 and 64, and combinations thereof or cocktails. It includes sequences that can hybridize to such sequences under stringent hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that have or modulate pre-mRNA processing activity in the CNOT3 gene transcript.

[0064] An oligomer and DNA, cDNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can bind to each other through hydrogen bonding. Thus, “specifically hybridizable” and “complementary” are terms that are used to denote a sufficient degree of complementarity or pairing such that stable and specific binding occurs between the oligomer and the DNA, cDNA or target RNA. It is understood in the art that the sequence of an antisense oligomer does not need to be 100% complementary to the sequence of its target sequence for specific hybridization. An antisense oligomer specifically hybridizes when binding of a compound to a target DNA or RNA molecule interferes with the normal function of the target DNA or RNA product, and there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense oligomer to a non-target sequence under conditions in which specific binding is desired, i.e. physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays, the conditions under which the assays are performed.

[0065] Selective hybridization can occur under low, medium, or high stringency conditions, but preferably under high stringency conditions. Those skilled in the art will appreciate that hybridization stringency will be influenced by conditions such as salt concentration, temperature or organic solvents, in addition to the host composition, the length of complementary strands, and the number of nucleotide base pairing errors between the hybridizing nucleic acids. Severe temperature conditions typically include temperatures greater than 30°C, typically greater than 37°C, and preferably greater than 45°C, preferably at least 50°C, and typically 60°C-80°C or higher. The hard salt regime will typically be 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 measuring any single parameter. An example of stringent hybridization conditions is 65 ºC and 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Thus, antisense oligomers of this invention may include oligomers that selectively hybridize to the sequences presented in Table 1, or SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16- 18, 27, 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64.

[0066] It should be understood that exon-terminal codon arrangements in structural proteins may not always be disrupted at the codon end, hence it may be necessary to remove more than one exon from the pre-mRNA to allow in-frame reading of the mRNA. In such circumstances, it may be necessary to select multiple antisense oligomers using the method of this invention, each directed to a different region responsible for inducing inclusion of the desired exon and/or intron. At a given ionic strength and pH, Tmelt. represents the temperature at which 50% of the target sequence hybridizes to the complementary polynucleotide. Such hybridization may occur with an “almost” or “substantially” complementary sequence of the antisense oligomer to the target sequence, as well as with an exactly corresponding complementary sequence.

[0067] Typically, selective hybridization occurs when there is at least about 55% identity over a region of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95% 98% or 99% identity with the nucleotides of the antisense oligomer. The homology comparison length, as described, can span longer regions and in some embodiments will often span a region of at least about nine nucleotides, typically at least about 12 nucleotides, more commonly at least about 20, often more 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 sequences of the antisense oligomers of this invention preferably have at least 75%, more preferably at least 85%, more preferably at least 86, 87, 88, 89 or 90% homology with the sequences shown in the listings sequences in this document. 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 greater the homology required to obtain selective hybridization. Therefore, if the antisense oligomer of the present invention consists of less than about 30 nucleotides, it is preferable that the percentage 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 specified in the sequence listings herein. Nucleotide homology comparisons can be made using sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). Thus, sequences having a length similar to or significantly different from those cited herein can be compared by inserting gaps into the alignment, and such gaps can be determined, for example, using the comparison algorithm used by GAP.

[0069] The antisense oligomer of this invention may have regions of reduced homology and regions of exact homology to the target sequence. It is not necessary for an oligomer to have exact homology along its entire length. For example, the oligomer may have contiguous stretches of at least 4 or 5 bases that are identical to the target sequence, preferably contiguous stretches of at least 6 or 7 bases that are identical to the target sequence, more preferably contiguous stretches of at least 8 or 9 bases , identical to the target sequence. The oligomer may have regions of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases that are identical to the target sequence. The remaining regions of the oligomeric sequence may periodically coincide with the target sequence; for example, the remaining sequence may have an identical base followed by a non-identical base followed by an identical base. Alternatively (or also) the oligomer sequence may have multiple regions of identical sequence (eg, 3, 4, 5, or 6 bases) interspersed with regions of less than perfect homology. Such sequence mismatches will preferably have no or very little loss of splicing switch activity.

[0070] The term “modulate” or “modulates” includes “increasing” or “decreasing” one or more quantifiable parameters, optionally by a specific and/or statistically significant amount. The terms "increase" or "enhancing", "enhance" or "enhance" or "stimulate" or "stimulant" generally refer to the ability of one or antisense oligomers or compositions to produce or cause a greater physiological response (i.e. downstream effects) in a cell or subject regarding the response caused by the absence of the antisense oligomer or a control compound. The terms “reducing” or “reducing” generally refer to the ability of one or antisense oligomers or compositions to produce or cause a reduced physiological response (ie, downstream effects) in a cell or subject compared to the response caused by the absence of the antisense oligomer or a control compound.

[0071] Appropriate physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include increased exclusion of specific exons in the CNOT3 pre-mRNA, a decrease in the amount of CNOT3 pre-mRNA, or decreased expression of a functional protein. CNOT3 in the cell, tissue or entity in need. A "reduced" or "reduced" amount is usually a statistically significant value and can include a decrease of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g. 500, 1000 times) (including all whole numbers and decimal numbers between and more than 1, e.g. 1.5, 1.6, 1.7, 1.8) less than the amount received in the absence of antisense oligomer (no agent) or control compound.

[0072] The term “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, when measured in accordance with routine diagnostic methods. Appropriate physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the symptoms or pathology of a disease such as retinitis pigmentosa.

[0073] The "reduction" in response may be statistically significant compared to the response caused by the absence of the antisense oligomer or control composition and may include 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% reduction, including all integers in between.

[0074] The length of the antisense oligomer can vary as long as it is able to selectively bind to the intended site in the pre-mRNA molecule. The length of such sequences can be determined in accordance with the selection techniques described herein. Typically, an antisense oligomer will be from about 10 nucleotides in length to about 50 nucleotides in length. However, it will be understood that any nucleotide length within this range can be used in the method. Preferably, the length of the antisense oligomer is from 10 to 40, from 10 to 35, from 15 to 30 nucleotides in length, or from 20 to 30 nucleotides in length, most preferably from about 25 to 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, the term “antisense oligomer” refers to a linear sequence of nucleotides or nucleotide analogues that allows a nucleotide base to hybridize to a target sequence in RNA through Watson-Crick base pairing to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms "antisense oligomer", "antisense oligonucleotide", "oligomer" and "antisense compound" can be used interchangeably to refer to an oligonucleotide. The cyclic subunits may be based on ribose or other pentose sugar or, in some embodiments, a morpholine group (see description of morpholino oligonucleotides below). Also contemplated are peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl oligonucleotides, among other antisense agents known in the art.

[0076] Included are non-naturally occurring antisense oligomers or "oligonucleotide analogues", including antisense oligomers or oligonucleotides having (i) a modified backbone structure, e.g., a backbone different from the standard phosphodiester linkage found in naturally occurring oligo- and polynucleotides, and/or (ii) modified sugar moieties, such as morpholine moieties rather than ribose or deoxyribose moieties. Oligonucleotide analogues contain bases capable of forming hydrogen bonds by Watson-Crick base pairing with standard polynucleotide bases, the backbone of the analogues representing the bases in such a way as to allow such hydrogen bonding in a sequence-specific manner between the oligonucleotide analogue molecule and bases in the standard polynucleotide ( e.g. single-stranded RNA or single-stranded DNA). Preferred analogues are those that contain a substantially uncharged phosphorus-containing backbone.

[0077] One method of producing antisense oligomers is to methylate the 2' position of the hydroxyribose, and inclusion of a phosphorothioate backbone produces molecules that superficially resemble RNA but are much more resistant to nuclease digestion, although those skilled in the art will know other forms of suitable backbones that can be used for the purposes of this invention.

[0078] To avoid degradation of pre-mRNA during duplex formation with antisense oligomers, the antisense oligomers used in this method can be tailored to minimize or prevent degradation by endogenous RNase H. This property is highly advantageous because treatment of RNA with unmethylated oligomers, intracellular or in crude extracts that contain RNase H, lead to the degradation of pre-mRNA:antisense oligomers duplexes. Any form of modified antisense oligomers that can bypass or avoid such degradation can be used in this method. Nuclease resistance can be achieved by modifying the antisense oligomers of this invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, including carboxyl groups, ester groups and alcohol groups.

[0079] Antisense oligomers that do not activate RNase H can be prepared according to known methods (see, for example, US Pat. No. 5,149,797). Such antisense oligomers, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification that sterically interferes with or prevents RNase H from binding to a duplex molecule containing the oligomer as one of its members, where the structural modification does not significantly interfere with or impair duplex formation . Because the portions of an oligomer involved in duplex formation are substantially different from those involved in RNase H binding to it, numerous antisense oligomers are available that do not activate RNase H. For example, such antisense oligomers may be oligomers in which at least one or all The bridging phosphate residues between nucleotides are modified phosphates such as methylphosphonates, methylphosphothioates, phosphoromorpholidates, phosphoropiperazidates, boranophosphates, amide linkages and phosphoramidates. For example, any other of the internucleotide bridging phosphate residues may be modified as described. In another non-limiting example, such antisense oligomers are molecules in which at least one or all of the nucleotides contain a 2'-lower alkyl group (such as, for example, C ₁ -C ₄ , linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl and isopropyl). For example, any other of the nucleotides may be modified as described.

[0080] An example of antisense oligomers that, when duplexed with RNA, are not degraded by cellular RNase H is the 2'-O-methyl derivative. Such 2'-O-methyl oligoribonucleotides are stable in cells and animal tissues, and their RNA duplexes have higher Tm values than their ribo- or deoxyribo-analogues. Alternatively, the nuclease-resistant antisense oligomers of this invention may have at least one of the last 3' terminal nucleotides fluorinated. Alternatively, the nuclease-resistant antisense oligomers of the present invention have phosphorothioate bonds linking at least two of the last 3-terminal nucleotide bases, preferably containing phosphorothioate bonds between the last four 3'-terminal nucleotide bases.

[0081] Enhanced splicing switching can also be achieved using alternative oligonucleotide chemistry. For example, the antisense oligomer can be selected from the list including: phosphoramidate or phosphorodiamidate morpholino oligomer (PMO); PMO-X; PPMO; peptide nucleic acid (PNA); locked nucleic acid (LNA) and derivatives, including alpha-L-LNA, 2'-amino LNA, 4'-methyl LNA and 4'-O-methyl LNA; ethylene bridged nucleic acids (ENAs) and their derivatives; phosphorothioate oligomer; tricyclo-DNA (tcDNA) oligomer; tricyclophosphorothioate oligomer; 2'O-methyl-modified oligomer (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabino (FANA); unblocked nucleic acid (UNA); hexitol nucleic acid (HNA); cyclohexenyl nucleic acid (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine or any combination of the above in the form of mixers or gapmers. To further improve delivery efficiency, the above-mentioned modified nucleotides are often conjugated to fatty acids/lipids/cholesterol/amino acids/carbohydrates/polysaccharides/nanoparticles, etc. with sugar or fragments of nitrogenous bases. These conjugated nucleotide derivatives can also be used to construct antisense exon skipping oligomers. Antisense oligomer-induced splicing modification of human CNOT3 gene transcripts typically uses oligoribonucleotides, PNA, 2OMe, or MOE-modified bases on a phosphorothioate backbone. Although 2OMeAOs are used to construct oligos, due to their efficient in vitro uptake when delivered as cationic lipoplexes, these compounds are sensitive to nuclease degradation and are not considered ideal for in vivo or clinical applications. When alternative chemical methods are used to create antisense oligomers of this invention, uracil (U) of the sequences presented herein can be replaced by thymine (T).

[0082] Although the antisense oligomers described above are the preferred form of antisense oligomers of this invention, the present 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 this invention include oligomers containing modified backbones or non-natural internucleoside linkages. As defined herein, oligomers having modified backbones include those that contain a phosphorus atom in the backbone and those that do not contain a phosphorus atom in the backbone. For the purposes of this description and as is sometimes referred to in the art, modified oligomers that do not have a phosphorus atom in their internucleoside backbone may also be considered antisense oligomers.

[0084] In other preferred oligomeric mimetics, both the sugar and the internucleoside linkage, i.e. backbone of nucleotide units are replaced by new groups. The core units are retained for hybridization with the appropriate target nucleic acid compound. One such oligomeric compound, an oligomer mimetic, which has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PKN compounds, the sugar backbone of the oligomer is replaced by an amide-containing backbone, in particular, an aminoethylglycine backbone. The nucleobases are retained and bound directly or indirectly to the aza nitrogen atoms of the amide backbone.

[0085] Another preferred chemistry is phosphorodiamidate morpholino oligomer (PMO) oligomeric compounds, which are not degraded by any known nuclease or protease. These compounds are uncharged, do not activate RNase H activity upon binding to an RNA strand, and have been shown to exhibit robust modulation of splicing following administration in vivo (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0086] Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also contain modifications or substitutions of nitrogenous bases (often referred to in the art simply as "base"). Certain nitrogenous bases are particularly useful for increasing the binding affinity of the oligomeric compounds of this 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. Substitutions of 5-methylcytosine have been shown to increase nucleic acid duplex stability by 0.6–1.2 °C, especially when combined with 2′-O-methoxyethyl sugar modifications.

[0087] Another modification of the oligomers of this invention involves chemically linking to the oligomer one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligomer. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioester such as hexyl-S-tritylthiol, thiocholesterol, an aliphatic chain such as dodecanediol or undecyl units, a phospholipid such as di-hexadecyl- rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, or adamantaneacetic acid, palmityl moiety, myristyl or octadecylamine or hexylaminocarbonyloxycholesterol moiety.

[0088] Cell-penetrating peptides have been added to morpholino phosphorodiamidate oligomers to enhance cellular uptake and nuclear localization. Various peptide tags have been shown to influence capture efficiency and target tissue specificity, as shown in Jearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624-1629.

[0089] It is not necessary that all positions in a given compound be uniformly modified, and in fact more than one of the above modifications may be included in a single compound or even a single nucleoside in an oligomer. The present invention also includes antisense oligomers, which are chimeric compounds. "Chimeric" antisense oligomers or "chimeras" in the context of this invention are antisense oligomers, in particular oligomers that contain two or more chemically different regions, each of which consists of at least one monomer unit, i.e. nucleotide in the case of an oligomeric compound. These oligomers typically contain at least one region in which the oligomer is modified to provide the oligomer or antisense oligomer with increased resistance to nuclease digestion, increased cellular uptake, and an additional region to increase binding affinity to the target nucleic acid.

[0090] The activity of antisense oligomers and variants thereof can be analyzed using conventional methods in the art. For example, splice patterns and expression levels of the RNAs and proteins of interest can be assessed by any of a variety of well-known methods for detecting splice patterns and/or expression of a 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 techniques such as Northern blotting and/or the use of nucleic acid arrays; nucleic acid amplification methods; immunological methods for detecting proteins; protein purification methods; and protein function or activity assays.

[0091] RNA expression levels can be assessed by obtaining mRNA/cDNA (ie, a transcribed polynucleotide) from a cell, tissue, or organism and by hybridizing the mRNA/cDNA with a reference polynucleotide that is the complement of the nucleic acid of interest or a fragment thereof. The cDNA may optionally be amplified using any of a variety of polymerase chain reaction or in vitro transcription methods prior to hybridization with a complementary polynucleotide; preferably without amplification. Expression of one or more transcripts can also be detected using quantitative PCR to assess the level of expression of the transcript(s).

[0092] The present invention provides an antisense oligomer-induced switch in CNOT3 gene transcript splicing, a clinically relevant oligomer chemistry, and a delivery system for manipulating CNOT3 splicing to therapeutic levels. A significant reduction in the amount of full-length CNOT3 mRNA and, consequently, CNOT3 protein from transcription of the CNOT3 gene is achieved by:

1) in vitro oligomer refinement using fibroblast cell lines through experimental evaluation of (i) intronic enhancer target motifs, (ii) antisense oligomer length and development of oligomer cocktails, (iii) chemistry selection, and (iv) addition of cell penetrating peptides (CPPs) ), to enhance the delivery of oligomers; And

2) detailed evaluation of a new approach to generating CNOT3 transcripts with one or more missing exons.

[0093] Thus, this document demonstrates that CNOT3 pre-mRNA processing can be manipulated using specific antisense oligomers. In this way, a functionally significant reduction in the amount of CNOT3 protein can be achieved, thereby reducing the severe pathology associated with retinitis pigmentosa.

[0094] Antisense oligomers used in accordance with this invention can be conveniently prepared using well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, California). One method for synthesizing oligomers on a modified solid support is described in US Pat. No. 4,458,066.

[0095] Any other means for such synthesis known in the art may be used additionally or alternatively. The use of similar methods for the preparation of oligomers such as phosphorothioates and alkylated derivatives is well known. In one such automated embodiment, diethyl phosphoramidites are used as starting materials and can be synthesized as described by Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

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

[0097] Antisense oligomers can be formulated for oral, topical, parenteral or other delivery, particularly as ocular topical delivery and ocular injection formulations. The compositions can be formulated to aid in capture, distribution and/or absorption at the site of delivery or activity. Preferably, the antisense oligomers of this invention are formulated for local delivery to the eye, either by intraocular injection or intraocular implant, such that the effects on CNOT3 production are limited spatially and are not systemic.

Method of treatment

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

[0099] More specifically, the antisense oligomer may be selected from Table 1 or the group consisting of any one or more of SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18 , 27, 30, 34, 35, 64 and 67, even more preferably, SEQ ID NO: 4, 7, 27, 30, 34 and 64 and combinations or cocktails thereof. These include sequences that can hybridize to such sequences under stringent hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that have or modulate pre-mRNA processing activity in the CNOT3 gene transcript.

[00100] The invention also extends to a combination of two or more antisense oligomers capable of binding to a selected target to induce exon exclusion in a CNOT3 gene transcript. The combination may be a cocktail of two or more antisense oligomers, a construct containing two or more, or two or more antisense oligomers combined together for use in antisense oligomer-based therapy.

[00101] Thus, there is provided a method of treating, preventing or reducing the effects of a disease associated with the expression of CNOT3, comprising the step of:

a) administering to the patient an effective amount of one or more antisense oligomers or a pharmaceutical composition containing one or more antisense oligomers, as described herein.

[00102] A preferred disease associated with CNOT3 expression in a patient is retinitis pigmentosa.

[00103] Thus, the invention provides a method of treating, preventing or reducing the effects of retinitis pigmentosa, comprising the step of:

a) administering to the patient an effective amount of one or more antisense oligomers or a pharmaceutical composition containing one or more antisense oligomers, as described herein.

[00104] Preferably, therapy is used to reduce functional CNOT4 protein levels using an exon skipping strategy. Reduction of CNOT3 levels is preferably achieved by reducing transcript levels by modifying pre-mRNA splicing in the CNOT3 gene transcript or part thereof.

[00105] Reducing CNOT3 levels will preferably result in a reduction in the number, duration, or severity of symptoms of a condition or pathology associated with CNOT3, such as retinitis pigmentosa.

[00106] As used herein, the term “treating” a subject (eg, a mammal, such as a human) or cell is any type of intervention used in an attempt to alter the natural history of a disease or condition in an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and can be carried out either prophylactically or after the onset of a pathological event or exposure to an etiological agent. Also included are “preventative” treatments, which may be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily indicate complete elimination, cure, or prevention of a disease or condition or associated symptoms.

[00107] The subject with the disease associated with the expression of CNOT3 may be a mammal, including a human.

[00108] The antisense oligomers of this invention can also be used in combination with alternative treatments, such as drug therapy.

[00109] Thus, the present invention provides a method of treating, preventing, or ameliorating the effects of a disease or condition associated with the expression of CNOT3, wherein the antisense oligomers of the present invention are administered sequentially or concurrently with another alternative therapy associated with the treatment, prevention, or amelioration of the disease condition. or a condition associated with CNOT3 expression. Preferably, the disease or condition is retinitis pigmentosa.

Delivery

[00110] Antisense oligomers of this invention can also be used as prophylactic or therapeutic agents that can be used to treat a disease. Accordingly, in one embodiment, the present invention provides antisense oligomers that bind to a selected target in CNOT3 pre-mRNA to induce efficient and sequential exon skipping, as described herein, in a therapeutically effective amount, in admixture with a pharmaceutically acceptable carrier, diluent, or excipient.

[00111] Also provided is a pharmaceutical, prophylactic or therapeutic composition for treating, preventing or reducing the effects of a disease associated with the expression of CNOT3 in a patient, wherein the composition contains:

a) one or more antisense oligomers as described herein; And

b) one or more pharmaceutically acceptable carriers and/or diluents.

[00112] It is preferred that the antisense oligomer of this invention be delivered by a localized ocular route to avoid systemic effect. Routes of administration include, but are not limited to, intravitreal, intracameral, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, or topical (drops, eye washes, creams, etc.). Delivery methods include, for example, injection using a syringe and a drug delivery device, such as an implanted intravitreal delivery device (eg, VITRASERT®).

[00113] In one embodiment, the antisense oligomer is administered intravenously at a dose of 20 mg/kg. For example, the antisense oligomer can be administered intravenously to mice at a dose of 20 mg/kg.

[00114] Preferably, the antisense oligomer is administered by intravitreal injection at a dose of 0.01-1.5 mg/kg body weight, 0.1-0.1 mg/kg body weight, 0.2-0.8 mg/kg body weight body, 0.4-0.7 mg/kg body weight, or more preferably 0.4-0.6 mg/kg body weight. The antisense oligomer can be administered by intravitreal injection, for example, at a dose of 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, 1, 5 mg/kg body weight. Preferably, the antisense oligomer is administered by intravitreal injection at a dose of about 0.5 mg/kg body weight. For example, the antisense oligomer can be administered to mice via intravitreal injection at a dose of about 0.5 mg/kg body weight.

[00115] More preferably, the antisense oligomer is administered by intravitreal injection in doses of 0.5-50 mg per eye, 0.5-40 mg per eye, 0.5-30 mg per eye, 2-30 mg per eye, 2- 20 mg per eye, 0.5 to 20 mg per eye, or more preferably 5 to 20 mg per eye. The antisense oligomer can be administered by intravitreal injection, for example, at a dose of about 0.5 mg per eye, 1.0 mg per eye, 2.0 mg per eye, 3.0 mg per eye, 4.0 mg per eye, 5. 0 mg per eye, 6.0 mg per eye, 7.0 mg per eye, 8.0 mg per eye, 9.0 mg per eye, 10.0 mg per eye, 11.0 mg per eye, 12.0 mg per eye, 13.0 mg per eye, 14.0 mg per eye, 15.0 mg per eye, 16.0 mg per eye, 17.0 mg per eye, 18.0 mg per eye, 19.0 mg per eye, 20.0 mg per eye, 21 mg per eye, 22 mg per eye, 23 mg per eye, 24 mg per eye, 25 mg per eye, 30 mg per eye, 35 mg per eye, 40 mg per eye, 45 mg per eye or 50 mg per eye. Preferably, the antisense oligomer is administered by intravitreal injection at a dose of about 5-20 mg per eye.

[00116] The antisense oligomer can be administered at regular intervals for a short period of time, for example, daily for two weeks or less. However, in many cases the oligomer is administered intermittently over a longer period of time. The administration may be accompanied by or simultaneously with the administration of an antibiotic or other therapeutic treatment. The treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated based on the results of immunoassays, other biochemical tests, and physiological examination of the subject being treated.

[00117] Dosage may depend on the severity and sensitivity of the disease state being treated, with the course of treatment lasting from several days to several months, or until a cure or improvement in the disease state is achieved. Alternatively, the dosage can be adjusted based on the rate of disease progression. First, a baseline progression level is established. The rate of progression after a single initial dose is then monitored to ensure that it is decreasing. Preferably, no progression is observed after administration. Preferably, repeat administration is only necessary if the rate of progression has not changed. Successful treatment preferably results in cessation of further disease progression or even some restoration of vision. Optimal dosing regimens can be calculated based on measurements of drug accumulation in the patient's body. Those skilled in the art can readily determine optimal dosages, dosing techniques, and repetition rates.

[00118] Optimal dosages may vary depending on the relative activity of individual oligomers and can generally be estimated based on EC50s that have been shown to be effective in in vitro and in vivo animal models.

[00119] Typically, the dose is 0.01 to 1.5 mg/kg body weight or intravitreal injection ranging from 0.5 to 50 mg per eye and may be administered one or more times per day, week, month, or year, or even once every 2-20 years. The frequency of repeat dosing depends on the rate of disease progression. Those skilled in the art can readily estimate the frequency of dosage repetition based on the measured residence time and concentration of the drug in body fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, with the oligomer administered in maintenance doses, the dose may be 0.01-1.5 mg/kg body weight or administered by intravitreal injection at a dose of 0. 5-50 mg per eye one or more times per day, week, month or year, or even once every 2-20 years.

[00120] An effective in vivo treatment regimen using the antisense oligomers of this invention may vary depending on the duration, dose, frequency and route of administration, as well as the condition of the subject being treated (i.e., prophylactic administration versus responsive administration for localized or systemic infection). Accordingly, such in vivo therapy often requires monitoring with tests appropriate for the specific type of disease being treated and appropriate adjustments in dosage or treatment regimen to achieve optimal therapeutic outcome.

[00121] Treatment can be monitored, for example, by general disease indicators known in the art. The effectiveness of the in vivo administered antisense oligomers of the present invention can be determined from biological samples (tissue, blood, urine, etc.) collected from a subject before, during and after administration of the antisense oligomer. Analyzes of such samples include (1) monitoring the presence or absence of heteroduplex formation with target sequences and non-target sequences using procedures known to those skilled in the art, such as gel electrophoretic mobility assays; (2) monitoring the amount of mutant mRNA relative to a reference normal mRNA or protein, as determined by standard methods such as RT-PCR, Northern blotting, ELISA, or Western blotting.

[00122] Intranuclear oligomer delivery is a major problem for antisense oligomers. Various cell penetrating peptides (CPPs) localize PMO to varying degrees in different conditions and in different cell lines, and the inventors have evaluated novel CPPs for their ability to deliver PMO to target cells. The terms CPP or "cellular uptake enhancing peptide fragment" are used interchangeably and refer to cationic cell-penetrating peptides, also called "transport peptides", "carrier peptides" or "peptide transduction domains". The peptides, as shown herein, have the ability to induce cell entry in about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells in a given culture population and lead to macromolecular translocation in several tissues in vivo when administered systemically. CPPs are well known in the art and are disclosed, for example, in US Patent Application No. 2010/0016215, which is incorporated herein by reference in its entirety.

[00123] Thus, the present invention provides antisense oligomers of the present invention that benefit from combination with cell penetrating peptides for the production of therapeutic pharmaceutical compositions.

Excipients

[00124] The antisense oligomers of the present invention are preferably delivered in a pharmaceutically acceptable composition. The composition may contain from about 1 nM to 1000 nM of each of the desired antisense oligomers of this invention. Preferably, the composition may contain from about 1 to 500 nM, from 10 to 500 nM, from 50 to 750 nM, from 10 to 500 nM, from 1 to 100 nM, from 1 to 50 nM, from 1 to 40 nM, 1 nM up to 30 nM, from 1 to 20 nM, most preferably from 1 to 10 nM of each antisense oligomer(s) of this invention.

[00125] The composition 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 or 1000 nm of each desired antisense oligomer(s) of this invention.

[00126] The present invention further provides one or more antisense oligomers adapted to assist in the prophylactic or therapeutic treatment, prevention or alleviation of symptoms of a disease, such as a disease or pathology associated with CNOT3 expression, in a form suitable for delivery to a patient.

[00127] The phrase “pharmaceutically acceptable” refers to molecular compounds and compositions that are physiologically tolerable and do not typically cause an allergic or similar adverse reaction such as stomach upset or the like when administered to a patient. The term "carrier" refers to the diluent, adjuvant, excipient or carrier with which the compound is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The preferred carriers are water or saline solutions and aqueous solutions of dextrose and glycerol, especially for injection solutions. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, PA (2013).

[00128] In a more specific form of the invention, pharmaceutical compositions are provided containing therapeutically effective amounts of one or more antisense oligomers of this invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents with varying buffer levels (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, as well as additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, acid, sodium metabisulfite), preservatives (eg thimerosol, benzyl alcohol) and excipients (eg lactose, mannitol). The material may be incorporated into particulate polymer compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. You can also use hylauronic acid. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the present proteins and derivatives. 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 in dry powder form, for example, in lyophilized form.

[00129] It should be understood that the pharmaceutical compositions provided in accordance with this invention can be administered by any means known in the art. Pharmaceutical compositions for administration are administered by injection, orally, topically, or by the pulmonary or nasal route. For example, antisense oligomers can be delivered by intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous routes of administration. The appropriate route can be determined by one skilled in the art depending on the condition of the patient being treated. Preferably, the antisense oligomers are delivered to the eye locally, by intraocular injection or intraocular implant, so that the effects on CNOT3 production are localized and not systemic.

[00130] Formulations for topical administration include those in which the oligomers of this invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Lipids and liposomes can be neutral (eg, dioleoylphosphatidyl DOPE, ethanolamine, dimyristoylphosphatidylcholine, DMPC, distearolyphosphatidylcholine), negative (eg, dimyristoylphosphatidylglycerol, DMPG) and cationic (eg, dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other administration, the oligomers herein may be encapsulated in or complexed with liposomes, in particular cationic liposomes. Alternatively, the oligomers can form complexes with lipids, in particular cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts and their uses are further described in US Patent No. 6,287,860 and/or US Patent Application No. 09/315,298 filed May 20, 1999.

[00131] In some embodiments, the antisense oligomers herein may be delivered by topical or transdermal routes (e.g., by incorporating the antisense oligomers, e.g., in emulsions, with such antisense oligomers optionally packaged in liposomes), including delivery to ocular surfaces. Such topical or transdermal and emulsion/liposome-mediated delivery methods are described for the delivery of antisense oligomers in the art, for example, in US Pat. No. 6,965,025. Preferably, local delivery is to the eye.

[00132] Antisense oligomers described herein can also be delivered via an implantable device. The creation of such a device is a recognized method in the art, for example, the design of a synthetic implant described, for example, in US Pat. No. 6,969,400. Preferably, the implant can be implanted into the eye for long-term delivery of antisense oligomers.

[00133] Compositions and formulations for ocular administration, including ocular injection, ocular topical delivery, and ocular implant, may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, compounds- carriers and other pharmaceutically acceptable carriers or excipients.

[00134] Delivery of a therapeutically useful amount of antisense oligomers can be achieved using previously published methods. For example, delivery of an antisense oligomer can be accomplished by a composition comprising a mixture of an antisense oligomer and an effective amount of a block copolymer. An example of this method is described in US patent application 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 in Gebski et al. (2003) Human Molecular Genetics, 12(15): 1801-1811. A method for introducing a nucleic acid molecule into a cell via an expression vector, either in the form of naked DNA or in complex with lipid carriers, is described in US 6806084.

[00135] Antisense oligomers can be introduced into cells using methods recognized in the art (for example, transfection, electroporation, fusion, liposomes, colloidal polymer particles and viral and non-viral vectors, as well as other methods known in the art) . The delivery method chosen will depend at least on the cells being treated and the location of the cells and will be apparent to one skilled in the art. For example, localization can be achieved using liposomes with specific markers on the surface to guide the liposome, direct injection into tissue containing the target cells, specific receptor-mediated uptake, and the like.

[00136] It may be desirable to deliver the antisense oligomer in a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposomal compositions. These colloidal dispersion systems can be used in the production of therapeutic pharmaceutical compositions.

[00137] Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. These formulations may have pure cationic, anionic or neutral characteristics and are useful for in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles can encapsulate a significant percentage of an aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated within an aqueous cavity and delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).

[00138] For liposomes to be an effective gene transfer vehicle, the following characteristics must be present: (1) encapsulate the antisense oligomer of interest with high efficiency without compromising its biological activity; (2) preferential and significant binding to the target cell compared to non-target cells; (3) delivery of the aqueous contents of the vesicle to the cytoplasm of the target cell with high efficiency; and (4) accurate and efficient expression of genetic information (Mannino, et al., Biotechniques, 6: 682, 1988). The liposome composition is usually a combination of phospholipids, especially high phase transition temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can 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 a stable complex. pH-sensitive or negatively charged liposomes are thought to capture DNA rather than form complexes with it. Both cationic and non-cationic liposomes are used to deliver DNA into cells.

[00139] Liposomes also include “sterically stabilized” liposomes, a term which as used herein refers to liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in increased circulation time compared to liposomes not containing such specialized lipids . Examples of sterically stabilized liposomes are those in which the vesicle-forming portion of the lipid portion of the liposome includes one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their use are further described in US6287860.

[00140] As is known in the art, antisense oligomers can be delivered using, for example, methods including liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake and receptor-mediated endocytosis, as well as using additional non-endocytic delivery methods , such as microinjection, permeabilization (eg, streptolysin-O permeabilization, anionic peptide permeabilization), electroporation, and various non-invasive, non-endocytic delivery methods that are known in the art (see Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35 -49, incorporated by reference in its entirety).

[00141] The antisense oligomer can also be combined with other pharmaceutically acceptable carriers or diluents to form a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example, phosphate buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.

[00142] The routes of administration described are intended as a guide only, as one skilled in the art will readily be able to determine the optimal route of administration and any dosage for any particular animal and condition.

[00143] Numerous attempts have been made to introduce functional new genetic material into cells both in vitro and in vivo (Friedmann (1989) Science, 244: 1275-1280). These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S); integration into non-retroviral vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of a transgene linked to a heterologous promoter-enhancer element by liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288; Am. J. Resp. Biol., 4:206-209; Sci. (USA), 84:7851-7855); in combination with ligand-specific cation-based 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 tissue causes 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 of only mouse lungs following intravenous or intratracheal administration of a liposome DNA complex. . An example of a review article on human gene therapy procedures is: 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.

[00144] Antisense oligomers of this invention include any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound that, when administered to an animal, including a human, is capable of delivering (directly or indirectly) a biologically active metabolite or residue. Accordingly, by way of example, the disclosure also relates to prodrugs and pharmaceutically acceptable salts of the compounds of this invention, pharmaceutically acceptable salts of such prodrugs and other bioequivalents.

[00145] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of this invention: that is, salts that retain the desired biological activity of the parent compound and do not cause undesirable toxicological effects on it. For oligomers, preferred examples of pharmaceutically 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, etc.; (b) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids, such as, for example, 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, naphthalene sulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid and the like; and (d) salts formed with elemental anions such as chlorine, bromine and iodine. The pharmaceutical compositions of this invention can be administered in several ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be by topical (including ophthalmic, mucosal, and rectal delivery), pulmonary (e.g., inhalation or insufflation of powders or aerosols, including nebulizer, intratracheal, intranasal, epidermal, and transdermal), oral, or parenteral routes. . Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intraocular, or intramuscular injection or infusion; or intracranial, for example, intrathecal or intraventricular administration. It is believed that oligomers with at least one 2'-O-methoxyethyl modification are particularly useful for intraocular administration. Preferably, the antisense oligomer is delivered intraocularly.

[00146] The pharmaceutical compositions of this invention, which can be conveniently presented in unit dosage form, can be prepared in accordance with conventional methods well known in the pharmaceutical industry. Such methods include the step of combining the active ingredients with pharmaceutical carrier(s) or excipient(s). Typically, formulations are prepared by uniformly and thoroughly combining the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Swiss style implementation options

[00147] In another aspect, the present invention provides the use of one or more antisense oligomers as described herein in the manufacture of a medicament for modulating or controlling a disease associated with CNOT3 expression.

[00148] This invention also provides the use of purified and isolated antisense oligomers, as described herein, for the production of a drug for the treatment of a disease associated with the expression of CNOT3.

[00149] The use of purified and isolated antisense oligomers as described herein is also proposed for the production of a medicament for treating, preventing, or reducing the effects of a disease associated with CNOT3 expression.

[00150] Preferably, the pathology or disease associated with CNOT3 is retinitis pigmentosa.

[00151] The invention extends, according to yet another aspect, to cDNAs or cloned copies of antisense oligomeric sequences of the invention, as well as vectors containing antisense oligomeric sequences of the invention. The invention also extends to cells containing such sequences and/or vectors.

Sets

[00152] Also provided is a kit for treating, preventing, or mitigating the effects of a disease associated with CNOT3 expression in a patient, which includes at least an antisense oligomer as described herein, and combinations or cocktails thereof, packaged in a suitable container along with instructions on its application.

[00153] In a preferred embodiment, the kits will contain at least one antisense oligomer, as described herein or as shown in Table 1, or SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67, even more preferably, SEQ ID NO: 4, 7, 27, 30, 34 and 64 or a mixture of antisense oligomers as described herein. Kits may also contain peripheral reagents such as buffers, stabilizers, etc.

[00154] Therefore, a kit is provided for treating, preventing, or alleviating a disease or condition associated with CNOT3 expression in a patient, which includes at least an antisense oligomer described herein or as shown in Table 1, and combinations or cocktails thereof, packaged in a suitable container, along with instructions for its use.

[00155] Also provided is a kit for treating, preventing or alleviating a disease or condition associated with CNOT3 expression in a patient, which contains at least an antisense oligomer selected from the group consisting of any one or more of SEQ ID NO: 1-74, more preferably SEQ ID NO: 4, 7, 9, 11, 14, 16-18, 27, 30, 34, 35, 64 and 67, even more preferably SEQ ID NO: 4, 7, 27, 30, 34 and 64 , as well as combinations or cocktails thereof, packaged in a suitable container, together with instructions for its use.

[00156] Preferably, the disease or condition is retinitis pigmentosa.

[00157] The contents of the kit may be lyophilized, and the kit may further contain a suitable solvent for reconstituting the lyophilized components. The individual components of the kit will be packaged in separate containers and, associated with such containers, there may be notices in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice reflects the agency's approval of the manufacture, use, or sale for administration to humans.

[00158] When the components of the kit are provided in one or more liquid solutions, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. For in vivo use, the expression construct can be formulated into a pharmaceutically acceptable composition for administration by syringe. In this case, the container may be an inhaler, syringe, dropper, eye dropper, or other similar device from which the composition may be applied to an affected area of the animal, such as the lungs, administered to the animal, or even applied and mixed with other components of the kit.

[00159] In one embodiment, the kit of this invention contains a composition containing a therapeutically effective amount of an antisense oligomer capable of binding to a selected target on a CNOT3 gene transcript to modify pre-mRNA splicing in the CNOT3 gene transcript or a portion thereof. In an alternative embodiment, the composition is in pre-measured, pre-mixed and/or pre-packaged form. It is desirable that the intraocular solution be sterile.

[00160] The kit of this invention may also include instructions designed to facilitate compliance with the administration schedule by the user. The instructions used in this document apply to any label, insert, etc. and may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet or any combination thereof. For example, in one embodiment, a kit of this invention contains instructions for administering the compositions of this invention. In one embodiment, the instructions indicate that the composition of this invention is suitable for the treatment of retinitis pigmentosa. Such instructions may also include dosage instructions, as well as instructions for use by topical delivery to the eye or by intraocular injection.

[00161] Antisense oligomers and suitable excipients can be individually packaged to allow the practitioner or user to formulate the components into a pharmaceutically acceptable composition as desired. Alternatively, the antisense oligomers and suitable excipients can be packaged together, requiring a minimal formulation step by the practitioner or user. In any case, packaging must maintain the chemical, physical and aesthetic integrity of the active ingredients.

General points

[00162] Those skilled in the art will understand that the invention described herein is subject to changes and modifications other than those specifically described. The invention is intended to include all such variations and modifications. The invention also includes all steps, features, compositions and compounds mentioned or indicated in the specification, individually or together, and any and all combinations or any two or more steps or features.

[00163] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, which means that it should be read and considered by the reader as part of this text. That a document, reference, patent application or patent cited in this text is not repeated in this text is for the sake of brevity.

[00164] Any manufacturer's instructions, descriptions, product specifications and product sheets for any products mentioned herein or in any document incorporated by reference herein are hereby incorporated herein by reference and may be used in the practice of the invention.

[00165] The scope of this invention is not limited to any of the specific embodiments described herein. These embodiments are for illustration purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

[00166] The invention described herein may include one or more ranges of values (eg, size, offset, field strength, etc.). A range of values will mean all values within the range, including values that define the range and values adjacent to the range that produce the same or substantially the same result as values immediately adjacent to the value that defines the boundary range. Accordingly, unless otherwise indicated, figures set forth in the specification and claims are approximate and may vary depending on the desired properties sought to be achieved by the invention. Therefore, "about 80%" means "about 80%" as well as "80%". At a minimum, each numeric parameter should be considered in light of the number of significant figures and conventional rounding approaches.

[00167] In this specification, unless the context otherwise requires, the word “comprise” or variants such as “comprises” or “comprising” will be understood to imply the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or groups of integers. It should also be noted that in this document and, in particular, in the claims and/or paragraphs, terms such as “comprises”, “comprising”, “contain”, etc. may have the meaning ascribed to them in the Patent Law USA; for example, they can mean "includes", "included", "including" and the like; and that terms such as “consisting essentially of” and “consisting essentially of” have the meaning ascribed to them in the United States Patent Law, for example, they admit elements not expressly listed but exclude elements that appear in the prior art or which affect the basic or novel characteristics of the invention.

[00168] Other definitions of selected terms used herein can be found in the detailed description of the invention and apply throughout the document. Unless otherwise specified, all other scientific and technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs. The term "active agent" may refer to one active agent or may include two or more active agents.

[00169] Sequence identification numbers (“SEQ ID NO:”) containing the nucleotide and amino acid sequence information included in this specification are collected at the end of the specification and were obtained using PatentIn Version 3.0. Each nucleotide or amino acid sequence is identified in the sequence list by a numeric indicator <210> followed by a sequence identifier (eg, <210> 1, <210> 2, etc.). The length, sequence type, and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numerical indicator fields <211>, <212>, and <213>, respectively. The nucleotide and amino acid sequences reported in the specification are determined by the information provided in the numeric indicator field <400> followed by the sequence identifier (e.g., <400> 1, <400> 2, etc.).

[00170] An antisense oligomer nomenclature system has been proposed and published to distinguish between various antisense oligomers (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature became especially relevant when testing several slightly different antisense oligomers targeting the same target region, as shown below:

H # A/D (x:y)

the first letter indicates the species (for example, H: human, M: mouse)

"#" denotes the target exon number

“A/D” indicates the splice acceptor or donor site at the beginning and end of the exon, respectively.

(xy) represents annealing coordinates, where “-” or “+” denote intronic or exonic sequences, respectively. For example, A (-6+18) would point to the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The nearest splice site will be the acceptor, so these coordinates will be preceded by the letter "A". The annealing coordinates at the donor splice site can be described as D (+2-18), where the last 2 exonic bases and the first 18 intronic bases correspond to the annealing site of the antisense oligomer. Full exon annealing coordinates, which will be represented as A (+65+85), that is, the location between the 65th and 85th nucleotides inclusive from the start of that exon.

[00171] The following examples serve to more fully describe the method of using the invention described above, as well as to set forth the best methods contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented by way of illustration.

EXAMPLES

Example 1

AO-mediated exon skipping causing a frameshift in CNOT3

[00172] The structure of the CNOT3 exon is shown in FIG. 1. Splice switch AOs were designed to target enhancer sites in frameshift exons in CNOT3 pre-mRNA (Figure 1) to cause exon skipping and consequent open reading frame loss and CNOT3 knockdown.

[00173] Normal human fibroblasts and PR11 patient fibroblasts (PRPF31 c.1205 C>A Ser402*) were obtained from skin biopsies and cultured in DMEM supplemented with 10% FBS. Antisense sequences (Table 1) synthesized in situ as 2′O-methylphosphorothioate 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 altering target exon selection were then synthesized as phosphorodiamidate morpholino oligomers (PMOs) and transfected into fibroblasts as i) uncomplexed ii) annealed to a sense ODN anchor and delivered with Lipofectamine 3000 (as above), or iii) nucleofection using the 4-D Nucleofector X S primary cell kit P2 (Lonza) according to the manufacturer's instructions.

[00174] Total RNA was extracted from transfected and control cells using a MagMax RNA extraction system (Life Technologies). Transcripts of interest were assessed first by semiquantitative RT-PCR and then by RT-qPCR. cDNA (RNA 300 ng) was synthesized according to the manufacturer's instructions using the SuperScript IV reverse transcriptase kit (Life Technologies). RT-PCR was performed using LA Taq DNA polymerase with GC buffer I (TAKARA) according to the manufacturer's instructions.

[00175] Three different primer sets were used to evaluate CNOT3 transcripts after cells were transfected with CNOT3 exon skipping AO:

1. For AOs targeting exon 3, the forward primer annealing to exon 2 was paired with a reverse primer in exon 6.

2. For AOs targeting exons 8 and 9, a forward primer in exon 7 was paired with a reverse primer in exon 11.

3. For AOs targeting exons 16 and 17, a forward primer in exon 15 was paired with a reverse primer in exon 18.

[00176] RT-qPCR was used to quantify PRPF31 transcript levels after transfection with CNOT3 exon skipping AO. The forward primer across the junction of exons 2 and 3 of PRPF31 was paired with a reverse primer across the junction of exons 3 and 4 (Table 2). Results for all cells treated with CNOT3 AO were normalized to the expression of two constitutive genes, TBP and GAPDH, and compared with cells treated with Anti ISS-N1 and placebo AO to calculate extremes in changes in PRPF31 transcript levels.

Table 2: Primers for RT-PCR and RT-qPCR for CNOT3, PRPF31 and constitutive genes, TBP and GAPDH.

SEQ ID NO Gene Primer name Primer sequence 5'-3' Conc. Analysis 75 CNOT3 Exon 2 forward GAAGATGGCGGACAAGCGCAA 50 nM RT-PCR 76 CNOT3 Exon 6 back CTGGCCAACCTCTTCCTTCTC 50 nM RT-PCR 77 CNOT3 Exon 7 forward CTGTCAGTGCAGACACGCAA 50 nM RT-PCR 78 CNOT3 Exon 11 back GGACAGGCTGGAGCCGTTTT 50 nM RT-PCR 79 CNOT3 Exon 15 forward CATCCTGAGCAGTACATCAGC 50 nM RT-PCR 80 CNOT3 Exon 18 back CTCCAGGTAGCGGTACTCAA 50 nM RT-PCR 81 PRPF31 Exon 2/3 forward GGGATAGTAAGATGTTTGCTGAG 250 nM RT-qPCR 82 PRPF31 Exon 3/4 back GTCCCATCACTTCTGAAGCTTTGG 250 nM RT-qPCR 83 TBP F TCAGGCGTTCGGTGGATCGAGT 500 nM RT-qPCR 84 TBP R AGTGATGCTGGGCACTGCGGAGAA 500 nM RT-qPCR 85 GAPDH F ACAGTCAGCCGCATCTTCTT 250 nM RT-qPCR 86 GAPDH R AGGGGTCTACATGGCAACTG 250 nM RT-qPCR 87 CNOT3 Exon 10 forward GACGTTCCACAGACAGTGAAG 88 CNOT3 Exon 8 back GTGGTAGCGGTGCTTCTCGATG 89 CNOT3 3_5`UTR Forward TATATTCGGGACTCGGGGG

[00177] Sequencing of induced (smaller) CNOT3 transcript products, which are predicted to result from induced exon skipping, revealed that AO 3697 and 3698 mediated exon 3 skipping, AO 3702 induced both partial and complete skipping of exon 8, and AO 3703 mediated exon 8 skipping in a dose-dependent manner (data not shown). AOs that mediated efficient skipping of CNOT3 exons 3, 8, or 9 were then transfected into PR patient fibroblasts for 48 hours, and CNOT3 transcripts were analyzed by RT-PCR (Figure 2). CNOT3 exon skipping was evident 48 hours after transfection, with some reduction in full-length transcript product levels. Exon 3 was skipped by AO 3697, exon 8 was skipped by AO 3702 and 3703, and exon 9 was skipped by AO 3706.

[00178] We have previously shown that phosphorodiamidate morpholino (PMO) chemistry mediates splicing modification more efficiently than the same sequences synthesized using 2'O-methyl PS chemistry. Therefore, the most promising AO sequences for CNOT3 transcript knockdown would be synthesized as PMO and transfected into normal fibroblasts without forming complexes, using a leash/lipoplex or via nucleofection (Nucleofector, Lonza). Evaluation and optimization of the AO sequence targeting CNOT3 is ongoing.

[00179] SH-SY5Y cells are often used as in vitro models of neuronal function and differentiation. They are adrenergic in phenotype but also express dopaminergic markers. AOs that modify CNOT3 transcripts in fibroblasts will be assessed in differentiated SH-SY5Y cells for the ability to suppress CNOT3 and increase PRPF31 expression (transcript and protein).

Example 2

AO-mediated terminal intron retention for CNOT3 suppression

[00180] Antisense sequences have been designed to target the terminal exon of CNOT3 to cause terminal intron retention and knockdown of CNOT3.

[00181] 2'O-methyl AO targeting the terminal exon of CNOT3 was transfected into patient adRP11 fibroblasts for 48 hours. RT-PCR analysis showed that CNOT3 AOS 3887, 3888, and 3889 transcripts (Table 1) induced terminal intron retention and a dose-dependent decrease in the level of full-length transcript product (Fig. 3).

Example 3

Recruitment and screening of families with BD

[00182] We studied 3 generations of 2 families in Western Australia (1 Caucasian and 1 Aboriginal) with PRPF31 mutations (Figure 4). We obtained dermal fibroblasts from 7 patients and began monitoring disease progression in 10 of the 24 patients in these families.

Table 3: Identified mutations causing PR11 and the corresponding number of patients with each confirmed mutation (information from the Australian Inherited Retinal Diseases Registry as of 2018).

Mutation Protein replacement Phenotype Number of affected (state) PRPF31.1205C>A Ser402* Dominant PR 9 (WA) PRPF31.267delA Glu89Aspfs Dominant PR 8 (WA, Vic) PRPF31.527+3A>G N/A: SPLICE Dominant PR 3 (NSW) PRPF31.319C>G Leu107Val Dominant PR 1 (SA) PRPF31.527+1G>T N/A: SPLICE Dominant PR 1 (WA) PRPF31.1289_1290insA X Dominant PR 1 (SA)

Example 4

Obtaining induced pluripotent stem cells from a patient with PR11 and control fibroblasts and assessing gene expression as a consequence of CNOT3 knockdown

[00183] Induced pluripotent stem cells will be derived from patient fibroblasts and control fibroblasts. The patient's fibroblasts will be transfected with reprogrammable episomes (ThermoFisher®) using the NEON® electroporation system. In a typical reprogramming experiment, 12-15 iPSC-like colonies are selected 3-4 weeks post-transfection and subcultured to assess pluripotent gene expression by immunostaining and RT-PCR analysis (Figure 5A-C). Three clones will then be selected for additional testing, including gene expression profiling using TaqMan Arrays (Human Stem Cell Pluripotency Arrays, ThermoFisher) and virtual karyotyping using chromosomal G-band analysis with QuantiSNP analysis (AGRF).

[00184] To demonstrate the differentiation potential of the three lineages, iPSCs were cultured as embryoid bodies for 2-4 weeks and examined for expression of ectoderm, mesoderm, and endoderm differentiation markers, as well as suppression of pluripotency markers using RT-PCR (Figure 5C) . iPSCs will differentiate into retinal organoids using the published CIF 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.), (Fig. 5D-H).

[00185] Differentiated cells will be transfected with CNOT3-targeting lead AO synthesized as morpholino compounds in triplicates for each assay.

[00186] CNOT3, PRPF31 and other splicing factor transcripts will be analyzed. CNOT3, PRPF31 and selected parapecle proteins and splicing factors will be assessed by Western blotting and immunofluorescence to reflect the integrity of the splicing machinery and pathways.

Example 5

Dose-dependent testing of AO

[00187] AO sequences were designed to skip selected exons from CNOT3 messenger RNA and transfected as AO 2'O-methylphosphorothioates into fibroblasts after complexing with cationic liposomes for efficient transfection. Skipping the target exon results in a truncated messenger RNA RT-PCR product, identified by separating and staining the products on a 2% agarose gel (Figures 6 and 7).

[00188] Preferred sequences exhibit dose-dependent skipping of the target exon. Lead sequences were then synthesized as phosphorodiamidate-morpholino oligomers (PPMO) for testing by transfection into patient fibroblasts.

[00189] In patients with PR11, PRPF31 levels are approximately 50% of those in the healthy population. Messenger RNA levels for CNOT3 and PRPF31 were quantified by reverse transcription-quantitative PCR (RT-qPCR) in members of PR 11 families and healthy controls. Fig. Figure 8a shows that higher CNOT3 expression correlates with decreased PRPF31 levels and clinical outcome in heterozygous PRPF31 mutation carriers. CNOT3 target exon skipping was assessed by RT-qPCR (Fig. 8b, 8c), and PRPF31 expression after AO treatment is shown in comparison with that in cells treated with the AO control sequence (25 nM AO transfection control value set to 1), which did not target any region of the genome of a healthy person (no effect expected - negative control). Data for untreated cells are included for comparison.

[00190] In patients with PR11, PRPF31 levels are about 50% of those in the healthy population, whereas in asymptomatic carriers of the mutation, PRPF31 levels are at least 70% of those in the healthy population or higher. A 1.5-fold increase in PRPF31 expression (e.g., skipping CNOT3 exon 3 or 10, Figure 8b and skipping CNOT3 exons 9, 16, or 17, Figure 8C) is expected to restore splicing function in the retinal pigment epithelium PR11.

Example 6

Antisense oligomer-mediated CNOT3 exon skipping enhances PRPF31 expression in iPSC retinal pigment epithelium (RPE) derived from patient PR11 and restores primary cilia length and number.

[00191] ASO6 (SEQ ID NO: 64, CNOT3_H17A(+83+107), targeting exon 17 of CNOT3) was synthesized as a phosphorodiamidate morpholino oligomer (PMO) and transfected into RPE derived from PR11 iPSCs by direct transfection using 5 µM ASO in culture medium.

[00192] Immunocytochemistry was used to immunostain cilia and basal bodies in wild-type and PR11 RPE with or without antisense oligomer treatment.

Protocol for immunostaining of cilia

[00193] RPE cells on a chamber slide were fixed using ice-cold acetone-methanol (1:1) for 4 minutes, then air dried. Cells were blocked for 30 minutes in 10% filtered goat serum in PBS. To stain the basal body, cells were incubated with mouse anti-pericentrin antibody (1:100) for 1 hour at room temperature. Primary antibodies were detected using anti-mouse AlexaFluoro488 (1:400). To stain cilia, cells were incubated with rabbit anti-ARL13B antibodies (1:500) and incubated overnight at 4°C. The second primary antibody was detected using anti-rabbit AlexaFluoro568 (1:400) for 1 hour at room temperature and counterstained with Hoechst to detect nuclei (1:125 dilution). The coverslip was mounted onto a glass slide using Prolong Gold anti-reflective agent.

Confocal analysis and quantification

[00194] Primary RPE cilia were assessed to assess PRPF31 function using confocal microscopy. About 300 RPE cells/sample were measured for cilia length using NIS-Elements Imaging software.

Claims (20)

1. An isolated or purified antisense oligomer for modifying pre-mRNA splicing in a transcript of the CNOT3 gene or part thereof, selected from the list consisting of SEQ ID NO: 1-61 or 63-74. 2. The antisense oligomer according to claim 1, selected from the list consisting of: SEQ ID NO: 4, 7, 9, 11, 15, 16-18, 27, 30, 34, 35 and 64. 3. An antisense oligomer according to claim 1 or 2, selected from the list consisting of SEQ ID NO: 4, 7, 27, 30, 34 and 64. 4. Antisense oligomer according to any one of paragraphs. 1-3, and: (a) the antisense oligomer contains one or more nucleotide positions subject to alternative chemical treatment or modification selected from the list including: (i) modification of the backbone structure; (ii) modification of sugar moieties; (iii) conferring RNase H resistance; (iv) use of a chemical approach to create mimetic oligomers; and/or (b) When any uracil (U) is present in the nucleotide sequence, uracil (U) is replaced by thymine (T). 5. Antisense oligomer according to any one of paragraphs. 1-4, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. 6. Antisense oligomer according to any one of paragraphs. 1-5, in which: (i) the antisense oligomer is a phosphorodiamidate morpholino oligomer; (ii) the sequence of the antisense oligomer corresponds to SEQ ID NO: 64; And (iii) any uracil (U) present in the nucleotide sequence is replaced by thymine (T). 7. A pharmaceutical composition for treating the effects of a disease associated with the expression of CNOT3 in a patient, wherein the composition contains: a) one or more antisense oligomers according to any one of paragraphs. 1-6, and b) one or more pharmaceutically acceptable carriers and/or diluents. 8. A method for treating a disease associated with the expression of CNOT3, comprising the step of: a) administering to the patient an effective amount of an antisense oligomer according to any one of claims. 1-6 or the pharmaceutical composition according to claim 7. 9. A kit for treating the effects of a disease associated with the expression of CNOT3 in a patient, wherein the kit contains at least an antisense oligomer according to any one of claims. 1-6 and their combinations or cocktails, packaged in a suitable container together with instructions for their use. 10. The pharmaceutical composition according to claim 7, the method according to claim 8 or the kit according to claim 9, wherein the disease associated with the expression of CNOT3 is retinitis pigmentosa. 11. The pharmaceutical composition according to claim 7 or 10, the method according to claim 8 or 10, or the kit according to claim 9 or 10, wherein the subject suffering from a disease associated with the expression of CNOT3 is a human. 12. Antisense oligomer according to any one of paragraphs. 1-6, composition according to any one of paragraphs. 7, 10 or 11, method according to any one of paragraphs. 8, 10 or 11 or a set according to any one of paragraphs. 9-11, resulting in PRPF31 protein expression levels 1.5-5 times higher than PRPF31 protein expression levels in subjects with symptomatic PRPF31 mutations.