Methods for the treatment of Leber congenital amaurosis

Abstract

The present invention relates to a method for treating a Leber congenital amaurosis in a patient harbouring the mutation c.2991+1655 A>G in the CEP290 gene, comprising the step of administering to said patient at least one antisense oligonucleotide complementary to nucleic acid sequence that is necessary for preventing splicing of the cryptic exon inserted into the mutant c. 2991+1655 A>G CEP290 mRNA.

Claims

1. A method for restoring the function of CEP290 in a cell of a subject having a c.2991+1655A>G mutation present in the CEP290 gene, wherein said method comprises the step of intravitreal injection of a composition comprising a plasmid or viral vector encoding an antisense oligonucleotide that inhibits splicing of the cryptic exon inserted into the mutant c.2991+1655A>G CEP290 mRNA in a cone or rod cell, wherein the antisense oligonucleotide is complementary to a sequence within the mutant c.2991+1655A>G CEP290 pre-mRNA that is required for correct splicing of said targeted cryptic exon in said cone or rod cell, and wherein said sequence is selected from the group consisting of exon splicing enhancer (ESE) sequences and a sequence comprising the donor splice site created by the c.2991+1655A>G mutation.

2. The method of claim 1, wherein the vector is a plasmid vector.

3. The method of claim 1, wherein the vector is a viral vector that is an RNA virus, a DNA virus, an SV-40 type virus, a polyoma virus, an Epstein-Barr virus, a papilloma virus, a herpes virus, a vaccinia virus or a polio virus.

4. The method of claim 3, wherein the vector is a DNA virus selected from the group consisting of an adenovirus and an adeno-associated virus (AAV).

5. A method for treating Leber congenital amaurosis in a patient harboring the mutation c.2991+1655A>G in the CEP290 gene, wherein said method comprises the step of intravitreal injection the subject a composition comprising a plasmid or viral vector encoding an antisense oligonucleotide that inhibits splicing of the cryptic exon inserted into the mutant c.2991+1655A>G CEP290 mRNA in a cone or rod cell of said subject, wherein the antisense oligonucleotide is complementary to a sequence within the mutant c.2991+1655A>G CEP290 pre-mRNA that is required for correct splicing of said targeted cryptic exon in said cone or rod cell, wherein said sequence is selected from the group consisting of exon splicing enhancer (ESE) sequences and a sequence comprising the donor splice site created by the c.2991+1655A>G mutation.

6. The method of claim 5, wherein the vector is a plasmid vector.

7. The method of claim 5, wherein the vector is a viral vector that is an RNA virus, a DNA virus, an SV-40 type virus, a polyoma virus, an Epstein-Barr virus, a papilloma virus, a herpes virus, a vaccinia virus or a polio virus.

8. The method of claim 7, wherein the vector is a DNA virus selected from the group consisting of an adenovirus and an adeno-associated virus (AAV).

Description

FIGURES

(1) FIG. 1 Schematic representation of wild-type and mutant CEP290 transcripts and sequences of AONs. Shown are: ESE (+90+120), (SEQ ID NO:1):; ESE(+50+70), (SEQ ID NO:2); ESEsense (+50+70), (SEQ ID NO:3); H26D (+7−18), (SEQ ID NO: 4); HD26D (+10−11), (SEQ ID NO: 5); and HD26 (+19−11), SEQ ID NO: 6). Four AONs were designed to target ESE sequences in the cryptic exon using the ESEfinder 3.0 program available at //rulai.cshl.edu/cgi-bin/tools/ESE3/.

(2) FIG. 2A-C Effect of AON-mediated exon skipping of the mutant cryptic CEP290 exon on messenger RNAs, protein and primary cilia expression. All measures were recorded in treated (+) or untreated (−) fibroblasts derived from skin biopsies of control individuals (C.sub.1-C.sub.4), heterozygous unaffected carriers (S.sub.1-S.sub.3) and/or patients (P.sub.1-P.sub.4). The error bars represent the standard deviation of the mean from three independent experiments. a) Relative expression levels of wild-type (plain bars) and c.2991+1655 A>G mutant (hatched bars) messengers were determined by RT-qPCR. Results were normalized using the software geNorm taking as reference the RPLP0 and GUSB genes or TBP, RPLP0 and GUSB. Basal expression levels of wild-type CEP290 messengers were strikingly reduced in patients' cell lines compared to controls. Transfections with the antisense but not the sense oligonucleotide ESE(+50+70) (upper and lower panels, respectively) resulted in a statistically significant increase in the expression of the CEP290 wild-type allele in patients and heterozygous carriers (p<0.0001). b) Correlation between the mutant messenger knock-down and the synthesis of the wild-type CEP290 protein. The expression of the CEP290 proteins in cell lines before (−) and after treatment (+) with the AON ESE (+50+70) was determined by Western blot. The relative variations in CEP290 concentrations (central panel) were determined by computed-densitometry analysis of CEP290 and α-tubulin expression in each sample. RT-qPCR analysis of patient samples used for Western blot analysis confirmed efficient exon-skipping in all treated cell lines (lower panel). The results of RT-qPCR were normalized by the software geNorm taking as reference three genes TBP, RPLP0 and GUSB. c) Effect of exon-skipping on the ciliogenesis. Nuclei, cilia and basal bodies of untreated cell lines and fibroblasts transfected with the ESE(+50+70) and ESEsense (+50+70) oligonucleotides were stained using DAPI (blue), anti-acetylated-tubulin (green) and anti-γ-tubulin (red) antibodies, respectively (right panel). The proportions of fibroblasts presenting a primary cilium among cells were calculated by numbering at least 200 cells (individual numbers are given under each bars; left panel). The error bars represent the Standard deviation from counts of at least 4 fields (mean n=9) recorded from two independent experiments. AON-mediated exon skipping of the mutant cryptic CEP290 exon on messenger RNAs resulted in increased proportions of cells harbouring a primary cilium for the four patients (statistically significant in ¾ patients' cell lines P1, P2, P3).

(3) FIG. 3A-B. (a) Expression of wild type transcripts and mutant (c.2991 1655 A>G) of the CEP290 gene in cells of patient (P2) and control (C4) after transfection or not (=condition “untreated”) of five different antisense oligonucleotides. The results of RT-qPCR were normalized using geNorm software and using as reference the two following genes: GUSB and RPLP0. The graph shows the amounts of transcribed wild-type (WT CEP290, black bars) and mutant (Mutant CEP290, hatched bars). (b) Effectiveness of skipping of exon cryptic on the CEP290 messenger RNA mutants. All measurements were performed on untransfected cells (−) or transfected (+) by antisense oligonucleotides HD26 (7-18) and ESE (90 120). Cells used are fibroblasts derived from healthy individuals (C1-C4), heterozygous carriers of non-patients (S1-S3) and individuals with LCA (P1-P4). The error bars represent the standard deviation of the average derived from three independent experiments. The expression levels of mRNA wild-type (WT CEP290, black bars) and mutant c.2991+1655 A>G (Mutant CEP290; hatch bars) were determined by RT-qPCR. The results were normalized using geNorm logicile and using as reference genes RPLP0 and GUSB.

EXAMPLE 1: ANTISENSE OLIGONUCLEOTIDE-MEDIATED EXON SKIPPING ALLOWS EFFICIENT CORRECTION OF ABNORMAL CEP290 SPLICING IN LEBER CONGENITAL AMAUROSIS DUE TO THE FREQUENT CEP290 C.2991+1655A>G MUTATION

(4) Materials and Methods:

(5) Transfection Agent and AONs:

(6) The 26 residues long cationic transfecting peptide LAH4-L1.sup.1 was prepared by automated solid-phase synthesis on Millipore 9050 or ABI 431 synthesizers using fmoc chemistry (a kind gift by A J Mason and B. Bechinger). The transfection agents Lipofectamine2000 and DOTAP were obtained from (Invitrogen) and (Sigma-Aldrich), respectively. The 2′-O-methylphosphorothioate oligonucleotides were obtained from Sigma.

(7) Cell Culture and AON Transfection:

(8) Skin biopsies were obtained from 4 LCA patients harbouring the c.2991+1655 A>G mutation (¾ homozygous, P1, P2, P4; ¼ compound heterozygous with the c.5850delT, p.Phe1950LeuFsX14 mutationP3) 3 heterozygous unaffected carriers (S.sub.1 to S.sub.3) and control individuals (C.sub.1 to C.sub.4). Written consent was obtained for each individual and research was approved by Institutional review board.

(9) Primary fibroblasts were isolated by selective trypsinisation and proliferated at 37° C., 5% CO.sub.2, in Opti-MEM Glutamax I medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1% ultroser G substitute serum (Pall France) and 1% streptomycin/penicillin (Invitrogen). Fibroblasts between passage 7 and 9 were plated at 4×10.sup.5 cells/well in 6-well plates 24 hours before transfection. Cells at a confluence of 80% were transfected with 2′-OMePS AONs (150 nM) in Opti-MEM using LAH4-L1 at a 1:10 (w:w) AON:peptide ratio. After 3 hours of incubation at 37° C., the transfection medium was replaced by fresh culture medium.

(10) For the inhibition of nonsense mediated decay, 25 μg/ml of emetine dihydrochloride hydrate (Sigma-Aldrich) was added to the medium for 12 hours.

(11) RNA Extraction and cDNA Synthesis:

(12) Twenty four hours after transfection, the transfected and untreated cells were recuperated. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer's protocol. All samples were DNase treated by the RNase-free DNase set (Qiagen). Concentration and purity of total RNA was assessed using the Nanodrop-8000 spectrophotometer (Nanodrop Technologies) before storage at −80° C. Qualitative analysis of total RNA was performed using the Bioanalyzer 2220 (RNA 6000 Nano kit; Agilent) to verify that the RIN was between 8 and 10. First-stranded cDNA synthesis was performed from 500 ng of total RNA extracted using Verso cDNA kit (Thermo Fisher Scientific) with Random Hexamer:Anchored Oligo(dT) primers at a 3:1 (v:v) ratio according to the manufacturer's instructions. A non-RT reaction (without enzyme) for one sample was used as control and also analyzed by qPCR.

(13) Reverse Transcription Quantitative PCR (RT-qPCR):

(14) To measure the level of expression of CEP290 messengers, the wild-type and mutant alleles were amplified as 93 pb and 117 bp fragments, respectively. Regions of 132 bp, 80 bp, 84 bp, 101 bp and 95 bp within the human TATA box-binding protein mRNA (TBP, NM_003194), the human beta-2-microglobulin mRNA (B2M, NM_004048.2), the human beta glucuronidase mRNA (GUSB, NM_000181.3), the human hypoxanthine phosphoribosyltransferase 1 mRNA (HPRT1, NM_000194) and the human P0 large ribosomal protein mRNA (RPLP0, NM_001002.3) were used for normalization, respectively. A 99 pb fragment of the human albumin gene (ALB, NM_000477) was used to control the non-contamination of cDNAs by genomic DNA. Primers were designed using the Oligo Primer Analysis Software v.7 available at http://oligo.net. The specificity of primer pairs to PCR template sequences was checked against the NCBI database using the Primer-BLAST software available at www.ncbi.nlm.nih.gov/tools/primer-blast.

(15) cDNAs (5 μl of a 1:25 dilution in nuclease-free water) were subjected to real-time PCR amplification in a buffer (20 μl) containing MESA BLUE qPCR Master Mix Plus for Sybr Assay (Eurogentec) and 300 nM of forward and reverse primers, on a Taqman 7900 HT Fast Real-Time PCR System (Applied Biosystems) under the following conditions: initial denaturation at 95° C. for 5 min, followed by 50 cycles of 15 sec at 95° C. and 1 min at 65° C. The specificity of amplification products was determined from melting curve analysis performed at the end of each run using a cycle at 95° C. for 15 sec, 65° C. for 15 sec and 95° C. for 15 sec. Data were analyzed using the SDS 2.3 software (Applied Biosystems).

(16) For each cDNA sample, the mean of quantification cycle (Cq) values was calculated from triplicates (standard deviation SD<0.5 Cq). CEP290 Expression levels were normalized to the “normalization factor” obtained from the geNorm software for Microsoft Excel.sup.2 which uses the most stable reference genes and amplification efficiency estimates calculated for each primer-pair using 4-fold serial dilution curves (1:5, 1:25, 1:125, 1:625). No reverse transcriptase (non-RT), no template (NTC) reactions and non-contamination of cDNAs by genomic DNA (ALBh) were used as negative controls in each run (Cq values NTC=Undetermined, non-RT>40 and ALBh>40).

(17) The quantitative data are the means±SEM of three independent experiments and these are presented as ratio among values for individual mRNAs. The significance of variations among samples was estimated using the Protected List Significant Difference (PLSD) of Fisher according to the significance of analysis of variance (ANOVA test).

(18) Western Blot Analysis:

(19) 24 h after transfection cells were harvested and submitted to lysis in a Triton/SDS buffer (25 mM Tris-base pH7.8, 1 mM DTT, 1 mM EDTA, 15% Glycerol, 8 mM MgCl.sub.2, 1% Triton and 1% SDS) containing complete protease inhibitor cocktail (Roche) on ice for 30 min with repeated mixing. Released DNA was fragmented by 20 s of Ultra-turrax homogenizer (Ika-Werke) and the lysates were centrifuged (15000 g at 4° C. for 10 min). Protein concentrations were determined from the detergent-soluble fractions using the DC™ Protein Assay kit according to the manufacturer protocol (Bio-Rad). Proteins (125 μg) were denatured at 90° C. for 10 minutes in 4× premixed protein sample buffer (XT sample Buffer, Biorad) and separated by electrophoresis (50 volts for 30 minutes followed by 140 volts for 90 minutes at room temperature) on NuPAGE 3-8% Tris Acetate gels (Invitrogen). Proteins were transferred (100 volts, 2 h at 4° C.) to Immobilon-P PVDF membranes (Millipore). Membranes were blocked with PBS 0.5% Tween-20/5% dry milk powder and incubated over night at 4° C. under agitation with polyclonal rabbit anti-human CEP290 (Novus Biologicals) or monoclonal mouse anti-α-tubulin (Sigma) primary antibodies in 1:1800 and 1:500000 dilutions, respectively. Membranes were washed three times in PBS 0.5% Tween-20 solution (15 min) and incubated for 1 hour at room temperature with HRP-conjugated donkey anti-rabbit and sheep anti-mouse immunoglobulins secondary antibodies (Amersham GE Healthcare) in 1:10000 dilutions, respectively. ECL Western Blotting Detection Reagents (Amersham GE Healthcare) was applied according to the manufacturer's instructions and the blot was exposed to Amersham Hyperfilm ECL (Amersham GE Healthcare). The relative expression of the CEP290 protein was estimated by densitometry using α-tubulin as reference on a G:Box from Syngene with the GeneSnap and GeneTool Softwares.

(20) Flow Cytometry:

(21) ESEsense(+50+70)) and antisense ESE (+50+70) AON carrying a 3′ end fluorescein group were obtained from Sigma. Fibroblasts were transfected as described previously. After 3 h of incubation, cells were trypsinated, washed twice in PBS, and maintained into PBS containing 3% serum. Cells were analysed on a Facscalibur flow cytometer (Becton Dickinson, Grenoble, France) using the software CELLQuest. Percentages of fluorescent cells were calculated from 20,000 morphologically intact cells.

(22) Immunofluorescence Microscopy:

(23) Fibroblasts were seeded at 2.5×10.sup.5 cells/well on glass coverslips in 12-well plates. 24 hours before transfection with the ESEsense (+50+70) and ESE (+50+70) AONs, in the conditions described previously. Ten hours after transfection, cells were washed with PBS and incubated 30 h in serum free medium (37° C., 5% CO2). Untreated fibroblasts were processed in the same conditions. Subsequently, cells were fixed in ice-cold methanol (5 minutes at −20° C.) and washed twice in PBS. Cells were permeabilized in PBS supplemented with 3% BSA and 0.1% Triton for 1 hour at room temperature before being incubated overnight at 4° C. in permeabilization buffer containing (rabbit anti-γ-tubulin (1:1000), mouse monoclonal anti-acetylated tubulin (1:1000); Sigma-Aldrich) primary antibodies. After three washes with PBS, cells were incubated for 1 hour at room temperature in permeabilization buffer containing secondary antibodies (Alexa-Fluor 594- and Alexa-Fluor 488-conjugated goat anti-rabbit IgG (1:1000) and goat anti-mouse IgG (1:1000); Molecular Probes) followed by three washes PBS. A mounting media containing DAPI (DAPI Fluoromount G; SouthernBiotech) was used to label nuclei. Immunofluorescence images were obtained using a Leica DM IRBE microscope and a MicroPublisher 3.3 RTV camera (Q-Imaging). The final images were generated using the Cartograph version 7.2.3 (Microvision Instruments) and ImageJ (National Institutes of Health). The percentage of ciliated cells was calculated from two independent experiments (n>300 cells for each cell line). The significance of variations among samples was estimated using the PLSD of Fisher according to the significance of the ANOVA test.

(24) Results

(25) Leber congenital amaurosis (LCA, MIM204000) is a common cause of blindness in childhood (10%)′. It is the most severe inherited retinal dystrophy, responsible for blindness or profound visual deficiency at birth or in the first months of life. In the following months, the disease will either present as a dramatically severe and stationary cone-rod disease with extremely poor visual acuity (VA≤light perception; type I) or a progressive, yet severe, rod-cone dystrophy with measurable visual acuity over the first decade of life (20/200≤VA≤60/200; type II).sup.2.

(26) Hitherto, alterations of 16 genes with highly variable patterns of tissular distribution and functions have been reported in LCA.sup.1,3. In Western countries, mutations affecting the centrosomal protein 290 (CEP290) are the main cause of the disease (20%).sup.4,5. Among them, the c.2991+1655 A>G mutation accounts for over 10% of all cases, making this change an important target for therapy.

(27) The c.2991+1655 A>G mutation is located deep in intron 26 where it creates a splice-donor site 5 bp downstream of a strong cryptic acceptor splice site (FIG. 1). As a result, a cryptic 128 bp exon which encodes a stop codon is inserted in the CEP290 mRNA, between exons 26 and 27.sup.4. Considering the great potential of exon skipping as a therapy to by-pass protein-truncating gene lesions.sup.6,7, we assessed the possibility to use antisense oligonucleotides (AON) to hide the cryptic donor splice site created by the c.2991+1655 A>G mutation from the splicing machinery, and correct the abnormal splicing in carrier patients' cell lines.

(28) Fibroblasts were derived from skin biopsies of controls (C1-C4), unaffected heterozygous carriers (S1-S3) and homozygous (P1, P2 and P4) or compound heterozygous (P3) patients.

(29) The efficiency of the skipping strategy was assessed after optimizing 2′O-methyl phosphorothioate-modified AON sequences (FIG. 1) and concentrations, transfection conditions and treatment time (not shown). Expression levels of the wild-type and mutant CEP290 mRNAs were measured by quantitative reverse transcription PCR (RT-qPCR) in untreated fibroblasts and cells transfected with an AON designed to target exon splicing enhancer (ESE) sequences (ESE(+50+70)) or a sense version (ESEsense(+50+70)).

(30) Untreated patient's fibroblasts expressed significantly reduced levels of the wild-type CEP290 allele compared to controls and heterozygous carriers (FIG. 2a). When the cells were transfected with the AON ESE(+50+70), patients, unaffected carriers and controls fibroblasts expressed the wild-type messengers to equivalent levels, suggesting highly efficient skipping of the mutant cryptic exon (FIG. 2a). The increase in expression of the wild-type allele was strikingly higher than the decrease in expression of the mutant allele. These data suggested the degradation of mutant messengers through cellular mRNA control quality processes, in particular nonsense mediated decay (NMD). The significant increase of the mutant but not the wild-type mRNA levels in the fibroblasts of a homozygous patient (P1) treated with the NMD inhibitor emetine.sup.8 gave strong support to this hypothesis.

(31) The splicing was unchanged when cell lines were treated with the sense oligonucleotide (FIG. 2a). To ascertain that the absence of effect of the sense version of the AON on CEP290 splicing was not due to a reduced delivery efficiency, the cell line of Patient P1 was transfected using fluorescently labeled sense and antisense ESE(+50+70) AONs, respectively. Evidence for similar transfection efficiencies with both AONs gave strong support to the sequence-dependent skipping of the CEP290 cryptic exon. In this regard, it is worth noting that similar levels of skipping were reached when cell lines were treated with an antisense AONs designed to target an ESE site predicted downstream of the +50+70 sequence (ESE(+90+120)) (FIG. 1). These data indicate that different AONs may allow efficient skipping of the cryptic exon inserted into the mutant c.2991+1655 A>G CEP290 mRNA.

(32) Three other AON were identified (Table 1) which are able to induce exon skipping.

(33) TABLE-US-00001 TABLE 1 Sequence of the 2′OMe-P S Oligonucleotides AON Sequence H26D 5′-gguaugagauacucacaauuac-3 (+10−11) (SEQ ID NO: 5) H26D 5′-gguaugagauacucacaauuacaacuggggc-3′ (+19−11) (SED ID NO: 6) H26D 5′-gggauagguaugagauacucacaau-3 (+7−18) (SEQ ID NO: 4)

(34) To determine whether AON-induced exon skipping in the fibroblasts of patients influenced CEP290 protein levels, we performed Western blot analysis using a polyclonal antibody recognizing the C-terminus of the CEP290 protein. Increased levels of the wild-type CEP290 protein were evidenced when the cell lines of patients and unaffected carriers were treated with the antisense oligonucleotide but not the sense version.

(35) CEP290 is an integral component of the ciliary gate that bridges the transition zone between the cilia and cytoplasm. The protein plays an important role in maintaining the structural integrity of this gate, and thus has a crucial role in maintaining ciliary function.sup.9 We previously reported that nasal epithelial cells of LCA patients harbouring CEP290 mutations, including the c.2991+1655A>G change, present short cilia and heterogeneous axonemal abnormalities suggestive of a defect in cilia assembly or maintenance.sup.10. Here, we show that following serum-starvation, primary cilia expression is significantly reduced in LCA patients' fibroblasts harbouring the c.2991+1655A>G change compared to control cell lines (mean.sub.P1-P4=48.6% t 6.5% versus mean.sub.C1,C3=83.6%±3.2%; p=0.0097; FIG. 2c).

(36) Interestingly, upon transfection with the AON ESE(+50+70) but not the ESEsense(+50+70) oligonucleotide, the proportion of ciliated patients' cells increased significantly, reaching levels similar to controls: mean.sub.P1-P4+ESE (+50+70)=75.3%±3.5% vs mean.sub.P1-P4+ESEsense(+50+70)=58.75%±8.77%, p<0.01; mean.sub.P1-P4+ESE (+50+70) vs mean.sub.C1,C3+ESE(+50+70)=78.3%±3.4%; p=0.624 (FIG. 2c). This suggests that AON-mediated exon skipping resulted in a significant improvement of cilia assembly and/or maintenance.

(37) CEP290 mutations are the most common cause of LCA, yet no curative treatment exists. Our present results show therapeutic potential of exon skipping for the treatment of the mutation c.2991+1655A>G which accounts alone for 10% of all LCA cases.

REFERENCES

(38) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.