An antisense oligonucleotide for inducing skipping of exon 11 of NOTCH3 gene and pharmaceutical composition and application thereof

Abstract

The application discloses an antisense oligonucleotide for inducing skipping of the 11th exon of a NOTCH3 gene, a pharmaceutical composition thereof and application thereof. The application provides six candidate sequences of ASO12, ASO15, ASO16, ASO17, ASO19 and ASO20 for efficiently inducing exon skipping, wherein the half effective concentration (EC50) of ASO17 is as low as 36.53 nM. The application also provides a pharmaceutical composition containing the antisense oligonucleotide and application thereof in preparation of a medicine for treating CADASIL, and is especially suitable for a Chinese CADASIL patient population carrying a mutation of the 11th exon of a NOTCH3 gene. It is verified through experiments that the truncated NOTCH3 protein produced by skipping the 11th exon retains normal expression level, subcellular localization and signal transduction function, and meanwhile, pathogenic protein aggregation is reduced. The application fills the blank of specific treatment of CADASIL for Chinese population, and has clear clinical conversion value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antisense oligonucleotide for inducing exon 11 skipping of the NOTCH3 gene, its pharmaceutical composition, and its application. Background Technology

[0002] Autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL) is the most common inherited small vessel disease of the brain in adults. Typical clinical manifestations include dizziness, headache (including migraine with aura), recurrent transient ischemic attacks or ischemic strokes, progressive cognitive impairment, and mental disorders. This disease is caused by mutations in the NOTCH3 gene. Currently, there are no specific treatments for this disease; patients can only receive symptomatic treatment and preventative interventions, resulting in a severely impaired quality of life.

[0003] The NOTCH3 gene contains 33 exons and encodes a single-pass transmembrane receptor protein. Its extracellular domain contains 34 epidermal growth factor-like repeats (EGFRs), each containing 6 cysteine ​​residues. Pathogenic mutations in CADASIL typically alter the number of cysteine ​​residues within the EGFR domain (changing from an even number to an odd number), disrupting normal disulfide bond formation. This leads to misfolding of the NOTCH3 receptor's extracellular domain, which accumulates around vascular smooth muscle cells (VSMCs) and pericytes, forming characteristic osmophilic granular material (GOM) deposits, ultimately causing vascular lesions.

[0005] Existing technologies have reported antisense oligonucleotide exon skipping strategies targeting exons 2-4 of the NOTCH3 gene, but these strategies mainly target hotspot mutations in Caucasian populations and cannot effectively cover exon 11 mutations that are prevalent in Chinese patients. Furthermore, existing technologies lack systematic studies on the retention of protein function after skipping exon 11 of NOTCH3.

[0006] Therefore, developing a treatment that targets the most common mutations in the Chinese population and is functionally validated as safe and effective is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0007] In view of this, the present invention aims to provide an antisense oligonucleotide for inducing exon 11 skipping of the NOTCH3 gene, a pharmaceutical composition thereof, and its application. The present invention first discovers through systematic research that when exon 11 of the NOTCH3 gene is specifically skipped, the expressed truncated NOTCH3 protein can maintain a normal expression level, subcellular membrane localization, and downstream signal transduction function similar to the wild-type protein.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides an antisense oligonucleotide for inducing exon 11 skipping of the NOTCH3 gene, the antisense oligonucleotide consisting of 20 nucleotides and complementary to the NOTCH3 gene target sequence shown in SEQ ID NO: 1.

[0009] Furthermore, the antisense oligonucleotide includes one or more of the following: ASO 12, the sequence of which is shown in SEQ ID NO: 2; ASO 15, the sequence of which is shown in SEQ ID NO: 3; ASO 16, the sequence of which is shown in SEQ ID NO: 4; ASO 17, the sequence of which is shown in SEQ ID NO: 5; ASO 19, the sequence of which is shown in SEQ ID NO: 6; ASO 20, the sequence of which is shown in SEQ ID NO: 7.

[0010] Furthermore, the nucleoside internucleotide bond of the modified antisense oligonucleotide is a phosphate thioester bond.

[0011] Furthermore, the antisense oligonucleotide is an RNA with a full-chain phosphate thioester backbone and all sugar groups modified with 2'-O-methoxyethyl.

[0012] The present invention also provides a pharmaceutical composition comprising the above-described antisense oligonucleotide or a pharmaceutically acceptable salt thereof.

[0013] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating CADASIL.

[0014] Furthermore, the CADASIL patient carries a mutation in exon 11 of the NOTCH3 gene.

[0015] It contains at least the following beneficial technical effects: Existing antisense oligonucleotide exon-skipping strategies targeting the NOTCH3 gene primarily focus on exons 2-4, corresponding to hotspot mutations in Caucasian populations. However, nearly 50% of pathogenic mutations in Chinese CADASIL patients are located in exon 11. This invention is the first to systematically design and screen antisense oligonucleotides targeting exon 11, providing a targeted, genetically based treatment for the large proportion of Chinese patients worldwide, thus resolving the prominent clinical problem of population mismatch in existing technologies.

[0016] This invention is the first to experimentally demonstrate that the truncated NOTCH3 protein (ΔExon11 NOTCH3) expressed after skipping exon 11 of the NOTCH3 gene can be normally expressed, correctly located in the cell membrane, and maintain downstream signal transduction functions. This key discovery provides a solid theoretical basis and safety evidence for ASO therapy targeting exon 11, eliminating the technical prejudice of those skilled in the art that skipping this exon may lead to loss of protein function.

[0017] This invention employs an ASO walking strategy to systematically cover the splice site of exon 11 and the internal splice enhancer (ESE) motif, screening six ASO molecules with highly efficient exon jumping activity from numerous candidate sequences. In particular, ASO17 exhibits an in vitro EC50 value as low as 36.53 nM. This extremely low EC50 value means that a lower dose can be expected to achieve therapeutic effects when administered in vivo, effectively reducing the potential risks of drug toxicity and manufacturing costs, demonstrating excellent drug development potential.

[0018] The antisense oligonucleotides of this invention employ a full-thiophosphate (PS) backbone and 2'-MOE glycosyl modification. This modification combination has been validated by several FDA-approved ASO drugs (such as Nusinersen for the treatment of spinal muscular atrophy), exhibiting advantages such as strong resistance to nuclease degradation, long plasma half-life, high tissue uptake efficiency, and low immunogenicity. Compared to emerging technologies such as gene editing, the ASO technology platform used in this invention is highly mature, with reversible action and controllable dosage, making it more suitable for chronic neurological genetic diseases requiring long-term medication control. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the ASO design strategy of the present invention, wherein (A) is a schematic diagram of the ASO skipping strategy; (B) is a prediction map of ASO targeted splicing sites and exon splicing enhancement sites; and (C) is a schematic diagram of the ASO walking strategy.

[0020] Figure 2 The results of ASO screening in patient-derived fibroblasts are shown in (A), where (A) is an agarose gel electrophoresis image of RT-PCR products; and (B) is a quantitative statistical graph of exon jumping efficiency based on band grayscale analysis.

[0021] Figure 3 Concentration gradient experiments and EC50 fitting curves for six highly active ASO candidate sequences.

[0022] Figure 4 To validate the function of the Exon11 NOTCH3 protein. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0025] Example 1: Design and Synthesis of ASO Sequences The sequences of exon 11 and its flanking introns were obtained from the NOTCH3 gene sequence (reference sequence NM_000435.3) in the GenBank database. The 5' splice site, 3' splice site, and internal exon splice enhancer (ESE) motif of exon 11 were predicted using the Human Splicing Finder (HSF) bioinformatics tool.

[0026] Based on the predicted results, a series of antisense oligonucleotide sequences of 20 nucleotides in length were designed using the ASO walking strategy. All ASOs were synthesized by a commercial synthetic company and modified with a full-thiophosphate (PS) backbone and a full-chain 2'-O-methoxyethyl (2'-MOE) modification. The synthesized products were purified by high-performance liquid chromatography (HPLC) to a purity of not less than 95%.

[0027] The specific sequences are shown in Table 1: Table 1

[0028] Example 2: Cell Culture and ASO Transfection Screening Patient-derived fibroblasts carrying a mutation in exon 11 of NOTCH3 were used. Cells were seeded in 24-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. When cell confluence reached 60-70%, ASO transfection was performed using the liposome transfection reagent Lipofectamine™ RNAiMAX at a final concentration of 100 nM. A negative control group (transfected with nonsense ASO sequences) was included. After transfection, cells were cultured for another 72 hours. Total RNA was extracted from the cells using the TRIzol method and reverse transcribed into cDNA. PCR amplification was performed using primers flanking exon 11 (forward primer located in exon 10, reverse primer located in exon 12).

[0029] PCR products were separated by 2% agarose gel electrophoresis. After taking pictures with a gel imaging system, the band grayscale was analyzed using ImageJ software.

[0030] The formula for calculating exon skipping efficiency is: Skip efficiency (%) = [skipped strip gray value / (skipped strip gray value + unskipped strip gray value)] × 100%.

[0031] The results are as follows Figure 2 As shown, at a concentration of 100 nM, ASO12, ASO15, ASO16, ASO17, ASO19, and ASO20 all exhibited significantly higher jumping efficiencies than the control group.

[0032] Example 3: EC50 Measurement Six ASOs with high hopping efficiency from Example 2 were selected, and gradient transfection experiments were performed in the concentration range of 25-200 nM. The method was the same as in Example 2, and the concentration gradient was verified (25 nM, 50 nM, 100 nM, 200 nM). The dose-response curve was fitted using GraphPadPrism software, and the half-maximal effective concentration (EC50) was calculated.

[0033] The results are as follows Figure 3 As shown, ASO17 exhibits the lowest EC50 value (approximately 36.53 nM), indicating that it possesses optimal in vitro pharmacological activity.

[0034] Example 4: Functional verification of Exon11 NOTCH3 protein To assess whether the NOTCH3 protein that skips exon 11 (NOTCH3 △Exon11) can be normally expressed and localized, NOTCH3 expression vectors with Flag tags (WT, p.R544C, p.R607C, and △Exon11) were constructed and transiently transfected into HEK293T cells. Figure 4 Western blot results showed that the expression levels of NOTCH3 p.R544C and p.R607C mutant proteins were increased compared to the WT NOTCH3, suggesting an increased tendency for mutant protein aggregation. In contrast, the NOTCH3 ΔExon11 protein showed the same expression level as the WT, indicating that protein stability was preserved. Previous studies have shown that NOTCH3 mutant proteins form aggregates in vascular wall cells, therefore, the soluble and insoluble components of total cellular protein were detected separately. Compared with the WT and WT NOTCH3 ΔExon11 proteins, the mutant proteins NOTCH3 p.R544C and p.R607C showed significant accumulation in the insoluble components, while the soluble protein levels remained consistent. These results suggest that exon 11 skipping can reduce NOTCH3 protein aggregation.

[0035] Further transfection of HeLa cells with the NOTCH3 expression vector was performed to analyze whether the cellular localization of WT and mutant proteins was altered. Immunofluorescence results showed that all proteins exhibited perinuclear localization. Previous studies have shown that both WT and mutant NOTCH3 proteins, when overexpressed, remain in the endoplasmic reticulum and Golgi apparatus, which is consistent with the findings in this study. These results indicate that NOTCH3 ΔExon11 maintains its normal expression pattern and subcellular localization.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antisense oligonucleotide for inducing jumping of exon 11 of the NOTCH3 gene, characterized in that, The antisense oligonucleotide consists of 20 nucleotides and is complementary to the NOTCH3 gene target sequence shown in SEQ ID NO:

1.

2. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotides include one or more of the following: ASO 12, the sequence of which is shown in SEQ ID NO: 2; ASO 15, the sequence of which is shown in SEQ ID NO: 3; ASO 16, the sequence of which is shown in SEQ ID NO: 4; ASO 17, the sequence of which is shown in SEQ ID NO: 5; ASO 19, the sequence of which is shown in SEQ ID NO: 6; ASO 20, the sequence of which is shown in SEQ ID NO:

7.

3. The antisense oligonucleotide according to claim 1 or 2, characterized in that, The nucleoside internucleotide bond of the modified antisense oligonucleotide is a thiophosphate bond.

4. The antisense oligonucleotide according to claim 1 or 2, characterized in that, The antisense oligonucleotide is an RNA with a full-chain phosphate thioester backbone and all sugar groups modified with 2'-O-methoxyethyl.

5. A pharmaceutical composition, characterized in that, It comprises the antisense oligonucleotide of any one of claims 1-4 or a pharmaceutically acceptable salt thereof.

6. Use of the pharmaceutical composition of claim 5 in the preparation of a medicament for treating CADASIL.

7. The application according to claim 5, characterized in that, The CADASIL patient carried a mutation in exon 11 of the NOTCH3 gene.

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