Use of caspase-4 gene or its encoded protein as a target in screening for drugs for preventing or treating neurotoxicity

Abstract

The application discloses a use of a caspase-4 gene or a coded protein thereof as a target point in screening of a drug for preventing or treating neurotoxicity, and belongs to the technical field of genetic engineering. The drug comprises ASO or gRNA for targeted inhibition of expression of the caspase-4 gene or the coded protein thereof. The application can block translation of the caspase-4 gene or increase degradation of mRNA of the caspase-4 gene by using the ASO or CRISPR / Cas9 mode, so as to inhibit expression of the caspase-4 protein, and finally realize successful reversal of pathological TDP-43. The application is a first targeting therapy for the caspase-4, can specifically reduce expression of the caspase-4 at a gene level, and will not affect expression levels of other members of the caspase family, and has distinct specificity compared with a traditional caspase-4 small molecule inhibitor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a use of a caspase-4 gene or a protein encoded by the caspase-4 gene as a target in screening of a drug for preventing or treating neurotoxicity. BACKGROUND

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is characterized by the degeneration of upper and lower motor neurons, and is clinically manifested as progressive loss of motor function, with initial symptoms including muscle cramps and weakness in arms or legs, difficulty in swallowing or slurred speech, and eventually leading to uncontrolled movement, speech, eating and breathing. Most ALS patients die of respiratory failure or pneumonia within 2-4 years. ALS has multiple mutation gene types, such as TDP-43, SOD1, FUS and C9orf72, among which TDP-43 plays an important role in the pathogenesis of more than 95% of ALS patients. This is because TDP-43 is essential for early development of the body, and normal expression of TDP-43 in the nucleus is involved in processes such as gene transcription and precursor splicing. However, when the disease occurs, the translated misfolded protein TDP-43 accumulates in the cytoplasm of the patient, and is in the form of highly phosphorylated, ubiquitinated and fragmented inclusion bodies, which includes a variety of important functional proteins and genes, and thus exerts downstream neurotoxicity, becoming an important marker of related neurodegenerative diseases including ALS, frontotemporal lobar degeneration, Alzheimer's disease and Parkinson's disease.

[0003] According to the Guidelines for Diagnosis and Treatment of Amyotrophic Lateral Sclerosis, ALS should be diagnosed and treated as early as possible, and the survival period should be as long as possible. However, there is currently no effective solution to effectively cure ALS, and it still belongs to the uncharted territory. The time and money required for the development of new drugs are huge. There are only three small compound drugs (riluzole, edaravone and AMX0035 compound preparation) on the market, but they are very limited in slowing down the progression of ALS. Because their modes of action include reducing brain glutamate levels, removing free radicals, and improving the overall health status of intracellular mitochondria and endoplasmic reticulum, they cannot fundamentally solve the occurrence of the disease from the genetic level, and often can only delay the patient's course of disease for several months, and have certain side effects.

[0004] Gene therapy, a novel clinical medical technology combining modern medicine and molecular biology, uses molecular biology techniques to introduce target genes into a patient's body, correcting erroneous gene expression in the patient's cells and thus treating the disease. It has attracted increasing attention in the field of ALS. Molecular therapies take many forms, selectively silencing, upregulating, editing, or introducing genes or transcripts to temporarily or permanently correct the root cause of the disease. In particular, gene targeting methods, including antisense oligonucleotides (ASO) and CRISPR / Cas9, are becoming a key research area for ALS treatment, and most drugs approved for preclinical trials fall into this category. For example, BIIB067 (tofersen) is an ASO that inhibits the production of SOD1 protein; ION363 (jacifusen) is an ASO that targets FUS mRNA and can reduce FUS expression; afinersen is an ASO that targets the C9orf72 gene and aims to inhibit the expression of transcripts containing duplicate C9orf72; and BIIB105 (ION541) is an ASO that targets ATXN2 mRNA and can only be used for ALS patients carrying pathogenic CAG duplicate amplifications of this gene.

[0005] To date, there is no gene-targeting therapy that directly targets TDP-43. The root cause lies in the dual toxicity of endogenous TDP-43, which is lost in the cell nucleus and accumulates in the cytoplasm. This dual toxicity is key to motor neuron damage and patient death in ALS. Simply correcting the TDP-43 gene levels in either the nucleus or cytoplasm alone cannot alleviate the disease progression. For example, numerous articles both domestically and internationally have reported that knocking down TDP-43 with shRNA, overexpressing full-length or truncated wild-type TDP-43, or even establishing the three stable heritable mouse models mentioned above, not only fail to provide any therapeutic effect but also cause more severe neurotoxicity due to the imbalance of TDP-43 levels in the body. Therefore, finding a suitable upstream target will be crucial for the treatment of ALS.

[0006] Domestic and international studies have shown that various caspase hydrolases, including caspase-4, play a promoting role in the cleavage and mislocalization of TDP-43. However, this caspase family has as many as 12 member proteins, which are highly homologous to each other. Currently, the specific functions of all family members are poorly understood because inhibiting a certain caspase with some traditional small molecule compounds can also affect the normal activity of other members within the family, leading to a series of unintended consequences. For example, small compounds including VX-765, Terfenadine, Fenbufen, Ac-DEVD, Ac-LEVD, and Ac-YVAD, while chemically inhibiting caspase-4, can also inactivate multiple enzymes such as caspase-1, 3, 5, 7, 9, 11, and 13. Often, the activation of some caspase members is essential for normal physiological functions. Furthermore, long-term stimulation by multiple inhibitors can lead to serious side effects such as the death of the replaced normal cells. Therefore, the use of traditional small molecule inhibitors in basic research suffers from numerous disadvantages, including insufficient efficacy, poor target specificity, and adverse side effects, significantly limiting their potential application in the treatment of various diseases. Although a very small number of commercially available inhibitors have managed to enter clinical trials, these trials have often been terminated prematurely due to their unpredictability and adverse side effects. Summary of the Invention

[0007] To address the aforementioned shortcomings in existing technologies, this invention provides the use of the caspase-4 gene or its encoded protein as a target in screening drugs for the prevention or treatment of neurotoxicity. This invention is the first to demonstrate gene therapy targeting caspase-4 in mice. Utilizing ASO or CRISPR / Cas9, the translation of the caspase-4 gene can be blocked or its mRNA degradation increased, thereby inhibiting caspase-4 protein expression. Ultimately, pathological TDP-43 is successfully reversed, specifically manifested as a reduction in TDP-43 fragmented products in the cytoplasm, a recovery and increase in full-length TDP-43 in the nucleus, and a significant alleviation of pathological toxicity, including the loss of nerve cells in the mouse brain.

[0008] Furthermore, this invention represents the first caspase-4-targeted therapy that not only specifically reduces caspase-4 expression at the gene level but also does not affect the expression levels of other members of the caspase family, exhibiting distinct specificity compared to traditional caspase-4 small molecule inhibitors. The caspase-4-targeted therapy developed in this invention can alleviate TDP-43 pathology and its neurotoxicity, providing a novel strategy for future specific treatments of related diseases, including ALS, and therefore possesses significant potential for drug market development.

[0009] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0010] The purpose of this invention is to provide the use of the caspase-4 gene or its encoded protein as a target in screening drugs for the prevention or treatment of neurotoxicity.

[0011] Furthermore, the drug includes an active ingredient that inhibits the expression of the caspase-4 gene or its encoded protein.

[0012] Furthermore, the active ingredient is ASO or gRNA that targets caspase-4.

[0013] Furthermore, the ASO target sequence is: 5'-UGAUGTCAAAGTCAGCUCCA-3' (SEQ ID NO.1) (when creating the sequence listing, the U base in the sequence listing is replaced with T).

[0014] Furthermore, the gRNA includes two sequences that simultaneously act as target sites, as follows:

[0015] gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; (SEQ ID NO. 2)

[0016] gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'. (SEQ ID NO.3)

[0017] Another object of the present invention is to provide an ASO that specifically targets the caspase-4 gene or its encoded protein, the sequence of which is as follows:

[0018] 5'-UGAUGTCAAAGTCAGCUCCA-3'. (SEQ ID NO.1)

[0019] Another object of the present invention is to provide a gRNA that specifically targets the caspase-4 gene or its encoded protein, the gRNA comprising two sequences that simultaneously serve as target sites, the specific sequences of which are as follows:

[0020] gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; (SEQ ID NO. 2)

[0021] gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'. (SEQ ID NO.3)

[0022] Another object of the present invention is to provide the use of agents that inhibit / interfere with the expression of the caspase-4 gene or its encoded protein in the preparation of medicaments for the prevention or treatment of neurotoxicity.

[0023] Furthermore, neurotoxicity-related conditions include ALS.

[0024] The beneficial effects of this invention are:

[0025] This invention utilizes ASO or CRISPR / Cas9 to block the translation of the caspase-4 gene or increase its mRNA degradation, thereby inhibiting caspase-4 protein expression and ultimately achieving successful reversal of pathological TDP-43. Furthermore, this invention is the first to develop a caspase-4-targeted therapy that not only specifically reduces caspase-4 expression at the gene level but also does not affect the expression levels of other members of the caspase family, exhibiting distinct specificity compared to traditional small molecule caspase-4 inhibitors. The effective relief of TDP-43 pathology and its neurotoxicity also provides a novel strategy for future specific treatments of related diseases, including ALS, thus possessing significant potential for drug market development. Attached Figure Description

[0026] Figure 1 This is an immunoblot verification image showing the effect of HeLa cell line on caspase-4-ASO targeting;

[0027] Figure 2 This is an immunoblot image obtained 4 weeks after caspase-4-ASO was administered using a sustained-release pump.

[0028] Figure 3 Immunofluorescence image after 4 weeks of infusion of caspase-4-ASO using a sustained-release pump;

[0029] Figure 4 This is an immunoblot image obtained 4 weeks after caspase-4-ASO was administered using a sustained-release pump.

[0030] Figure 5 Immunofluorescence image after 4 weeks of infusion of caspase-4-ASO using a sustained-release pump;

[0031] Figure 6 This is an immunoblot verification image showing the effect of HeLa cell line on caspase-4-Cas9 targeting;

[0032] Figure 7 Immunofluorescence image of adeno-associated virus expressing CRISPR / Cas9-mediated caspase-4 4 weeks after targeting;

[0033] Figure 8 This is an immunoblot image obtained 4 weeks after targeting adeno-associated virus (AAV) with CRISPR / Cas9-mediated caspase-4.

[0034] Figure 9 Immunofluorescence image after 4 weeks of targeting CRISPR / Cas9-mediated caspase-4 with adeno-associated virus. Detailed Implementation

[0035] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0036] Example 1: Design of ASO Targets

[0037] 1. Human caspase-4 mRNA sequences were retrieved from the NCBI database to identify key functional domains of caspase-4, and their coding regions or splicing sites were preferentially targeted to disrupt their function. Specialized software (such as RNAstructure and mFOLD) was used to predict the secondary structure of the target mRNA, and single-stranded naked regions were selected to improve binding efficiency. Two sequences were designed, and 2'-O-methyl (2'-OMe) modification was used to increase binding affinity and reduce immunogenicity. The 2'-OMe-modified caspase-4-specific antisense oligonucleotides caspase-4-ASO target 1 (5'-UGACUGTGAAGAGGCCACUU-3') and caspase-4-ASO target 2 (5'-UGAUGTCAAAGTCAGCUCCA-3') were synthesized and purified by Shanghai Sangon Biotech Co., Ltd., and both target sequences were used for the first time.

[0038] 2. The 2'OMe-modified caspase-4-specific antisense oligonucleotide caspase-4-ASO and the control non-targeted-ASO were thoroughly dissolved in DEPC water. After verifying the targeting efficacy using HeLa cell lines, it was found that caspase-4-ASO target 2 (5'-UGAUGTCAAAGTCAGCUCCA-3'), abbreviated as caspase-4-ASO (hereinafter referred to as caspase-4-ASO), dissolved in 100 μL of DEPC water, had the best effect on inhibiting endogenous caspase-4 protein. Figure 1 ).

[0039] Example 2: ASO Target Efficacy Detection

[0040] The targeting effect and therapeutic efficacy of caspase-4-ASO and non-targeted-ASO were verified by injecting them into mice, as follows:

[0041] (1) Using a 1.0 mL sterile syringe, slowly infuse 2 OD / 100 μL of caspase-4-ASO and control Non-targeted-ASO into the sustained-release pump. At the same time, inject the drug into the connecting tube of the flow regulator to avoid air bubbles and connect it to the flow regulator. Place the drug-filled sustained-release pump in 37°C physiological saline for later use, without immersing the connecting tube in physiological saline.

[0042] (2) Turn on the small animal inhalation gas anesthesia machine and connect the stereotaxic device. Place the mouse in the gas anesthesia machine until it is anesthetized and unresponsive. Fix the mouse's head. Transfer the mouse to the stereotaxic device and fix its head, maintaining continuous respiratory anesthesia. Transfer the mouse and fix its head to the stereotaxic device to prevent head movement. Clean the mouse's head with sterile gloves and disinfectant, remove hair, and cut open the scalp. Use 3% H2O2 to wipe and dissolve the meninges to fully expose the Bregma suture. Mark the drilling coordinates by moving 2 mm forward and 1.65 mm to the right, using Bregma as the origin. Then, turn on the handheld cranial drill and carefully drill at the marked locations.

[0043] (3) Make a small incision in the neck using surgical scissors and then cut it open. Use hemostatic forceps to separate the subcutaneous connective tissue in the back. Insert the sustained-release pump into the subcutaneous space, gently insert the flow regulator needle vertically into the drill hole, fix it with dental cement, and suture the head and neck incision with surgical sutures. The sustained-release pump flow rate is 0.13±0.02μL / h.

[0044] (4) During the implantation of the sustained-release pump, the mice were closely monitored. Approximately 4 weeks later, the mice were euthanized, and brain tissue was harvested for pathological analysis. Results are shown below. Figures 2 to 5 .

[0045] Western blot analysis showed that, compared with the non-targeted control, caspase-4-ASO effectively inhibited caspase-4 levels, while reducing the fragmentation product of endogenous TDP-43 in mice. Figure 2 Simultaneously, immunofluorescence staining also confirmed that, compared with the non-targeted control, mice treated with caspase-4-ASO showed reduced cytoplasmic accumulation of endogenous TDP-43. Figure 3Furthermore, we verified whether caspase-4-ASO knockdown reduced neurotoxicity in mice. Immunoblotting showed that after 4 weeks of sustained-release caspase-4-ASO administration, neuronal marker proteins MAP2 and NeuN were restored upon caspase-4-ASO inhibition. Figure 4 Immunofluorescence results also showed that the expression levels of MAP2 and NeuN increased after caspase-4-ASO inhibition. Figure 5 )

[0046] Example 3: CRISPR / Cas9 Inhibits Caspase-4 Expression

[0047] 1. Obtain target points

[0048] The targeting sites designed for exon 2 of the full-length transcript of the caspase-4 gene (ENST00000444739.7) were validated using HeLa cell lines. Two effective specific target sites (5'-CACTGGTGTTTTGGATAACT-3' and 5'-CAAGAGAAGCAACGTATGGC-3) were selected. This targeting combination is being used for the first time.

[0049] 2. Transfection of HeLa cells

[0050] The AAV-CASP4-gRNA adeno-associated virus vector was constructed by tandemly constructing it on the ssAAV.U6-gRNA1-U6-gRNA2.EFS.ZsGreen plasmid. Next, human HeLa cells were co-transfected with different ratios of AAV-CASP4-gRNA:Cas9, and it was determined that a 1:1 ratio of AAV-CASP4-gRNA:Cas9 had the best effect on inhibiting endogenous caspase-4 protein. Figure 6 The control group used the empty backbone sequence of ssAAVEFS.ZsGreen. Both viruses were packaged by Paizhen Biotechnology Co., Ltd. to obtain 5e10 sequences. 12 After the infection particles were infecting the mice, they were used for subsequent in vivo experiments.

[0051] 3. Animal experiments

[0052] Mice were placed in an induction box and anesthetized with isoflurane as preoperative anesthesia. After complete anesthesia, the mice were placed on a sponge pad, their mouths were placed inside an anesthesia mask, and the skull was fixed to a stereotaxic apparatus, ensuring its stability and bilateral symmetry. The scalp and neck skin on the top of the skull were disinfected with 75% alcohol using a sterile cotton swab. Hair on the top of the skull was removed with scissors, and part of the scalp and subcutaneous fascia were cut. The soft tissue on the surface of the skull was digested with a cotton swab dipped in 3% H2O2 until the anterior and posterior fontanelles were clearly exposed. Using bregma as the origin, the coordinates for drilling were marked 2mm forward and 1.65mm to the right, and holes were drilled at these marked locations.

[0053] The viral fluid was drawn up using a microsyringe, which was then fixed to a stereotaxic instrument. Air was expelled from the needle, and the needle was inserted into the drilled holes on both sides at a speed of 0.1 mm / s to a depth of 2.0 mm. The needle was stopped for 10 minutes. The experimental group and control group were then injected with AAV-CASP4-gRNA and AAV-CTRL-gRNA viruses at a rate of 100 nmol / min, respectively, with a viral dose of 1.25 μL on each side. The needle was then stopped for 10 minutes and withdrawn at a speed of 0.1 mm / s.

[0054] The incision was sutured shut, and the mice were observed until they regained consciousness. After bilateral prefrontal cortex viral injection, the mice were allowed to stand for five minutes, then the microsyringe was slowly removed, the scalp was sutured, and penicillin-streptomycin solution was applied. The mice were kept warm post-surgery, and their condition was closely monitored. Approximately four weeks later, the mice were euthanized, and brain tissue was harvested for a series of pathological examinations, including Western blotting and immunofluorescence staining, as detailed below:

[0055] To investigate whether knocking out caspase-4 with AAV-CASP4-gRNA adeno-associated virus (AAV-CASP4-gRNA) alleviates TDP-43 pathology and neurotoxicity in mice, immunofluorescence assays were performed. The results are shown below. Figure 7 .

[0056] like Figure 7 As shown, compared with the AAV-Ctrl-gRNA non-target control, the mean fluorescence intensity of caspase-4 was significantly reduced in mice treated with caspase-4-Cas9, indicating that it effectively knocked down caspase-4 levels, thereby reducing the cytoplasmic accumulation of endogenous TDP-43 in mice. Our immunoblotting also confirmed this result, showing that compared with the non-target control, caspase-4-Cas9 effectively inhibited caspase-4 levels, reduced the fragmentation products of endogenous TDP-43 in mice, and that the neuronal marker protein MAP2 could be restored after caspase-4-Cas9 inhibition. Figure 8 and Figure 9).

[0057] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of the caspase-4 gene or its encoded protein as a target in screening drugs for the prevention or treatment of ALS, characterized in that, The drug comprises an active ingredient, ASO or gRNA, that inhibits the expression of the caspase-4 gene or its encoded protein; the ASO sequence is: 5'-UGAUGTCAAAGTCAGCUCCA-3'; the gRNA comprises two sequences that simultaneously serve as targets, the specific sequences of which are as follows: gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'.

2. An ASO that specifically targets the caspase-4 gene or its encoded protein, characterized in that, The sequence of the ASO is as follows: 5'-UGAUGTCAAAGTCAGCUCCA-3'.

3. A gRNA that specifically targets the caspase-4 gene or its encoded protein, characterized in that, The gRNA comprises two sequences that simultaneously serve as target sites, as follows: gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'.

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