Application of caspase-4 gene or encoded protein thereof serving as target spot in screening medicine for preventing or treating neurotoxicity

Abstract

The invention discloses an application of a caspase-4 gene or an encoded protein thereof as a target spot in screening a medicine for preventing or treating neurotoxicity, and belongs to the technical field of gene engineering. The medicine comprises ASO or gRNA for targeted inhibition of expression of the caspase-4 gene or the encoded protein of the caspase-4 gene. According to the invention, the ASO or CRISPR / Cas9 mode is utilized to block the translation of the caspase-4 gene or increase the mRNA degradation of the caspase-4 gene, so that the expression of the caspase-4 protein is inhibited, and finally, the pathological TDP-43 is successfully reversed. The caspase-4 small-molecule inhibitor is a targeting therapy aiming at caspase-4 for the first time, not only can the expression of caspase-4 be specifically reduced from the gene level, but also the expression level of other members of the caspase-4 family is not influenced, and compared with a traditional caspase-4 small-molecule inhibitor, the caspase-4 small-molecule inhibitor has distinct specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the use of a caspase-4 gene or its encoded protein as a target in screening drugs for preventing or treating neurotoxicity. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by degeneration of upper and lower motor neurons, clinically manifested as progressive motor loss, with initial symptoms including muscle twitching and weakness in the arms or legs, difficulty swallowing or slurred speech, and ultimately leading to an inability to control movement, speaking, eating and breathing. Most ALS patients die of respiratory failure or pneumonia within 2-4 years. ALS has a variety of mutated 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 the early development of the body, and its normal expression in the cell nucleus participates in processes such as gene transcription and precursor splicing. However, when the disease occurs, the misfolded protein TDP-43 encoded and translated by it will accumulate in the patient's brain tissue in the patient's cytoplasm, appearing as highly phosphorylated, ubiquitinated and fragmented inclusion bodies. Because it includes a variety of important functional proteins and genes, it exerts downstream neurotoxicity and becomes an important marker for related neurodegenerative diseases including ALS, frontotemporal dementia, Alzheimer's disease and Parkinson's disease.

[0003] According to the "Guidelines for the Diagnosis and Treatment of Amyotrophic Lateral Sclerosis", ALS should be diagnosed and treated early, and the survival period should be prolonged as much as possible. However, there is currently no effective solution to effectively cure ALS, and it is still a no-man's land, and the time and money required for the development of new drugs are huge. There are only three small compound drugs on the market (riluzole, edaravone and AMX0035 compound preparations), but they are very limited in slowing the progression of ALS, because their modes of action include reducing brain glutamate levels, scavenging free radicals, and comprehensively improving the health of intracellular mitochondria and endoplasmic reticulum, etc., and they cannot fundamentally solve the occurrence of the disease at the genetic level. They can only delay the course of the patient's disease for several months and have certain side effects.

[0004] Gene therapy is an emerging clinical medical technology that combines modern medicine and molecular biology. It is a method of introducing target genes into patients through molecular biology techniques to correct erroneous gene expression in patients' cells, thereby treating diseases. It has attracted increasing attention in the field of ALS. Molecular therapy has many forms and can selectively silence, upregulate, edit or introduce 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 the treatment of ALS. In preclinical trials, most approved drugs belong to 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 the expression of FUS; afinersen is an ASO targeting the C9orf72 gene, which is designed to inhibit the expression of C9orf72 transcripts containing repeat expansions; and BIIB105 (ION541) is an ASO that targets ATXN2 mRNA, which can only target ALS patients carrying the pathogenic CAG repeat expansion of this gene.

[0005] However, so far, there is no gene targeting therapy directly targeting TDP-43. The fundamental reason is that endogenous TDP-43 is lost in the nucleus and accumulated in the cytoplasm. This dual toxicity is the key to ALS motor neuron damage and patient death. It is impossible to alleviate the course of the disease by unilaterally correcting the TDP-43 gene level in the nucleus or cytoplasm. For example, many articles at home and abroad have reported that knocking down TDP-43 by shRNA, or overexpressing full-length and truncated wild-type TDP-43, or even establishing the above three stable heritable mouse models, not only has no therapeutic effect, but will cause more severe neurotoxicity due to the imbalance of TDP-43 levels in the body. Therefore, if a suitable upstream target can be found, it will be crucial for the treatment of ALS.

[0006] Domestic and foreign studies have shown that a variety of caspase hydrolases, including caspase-4, play a role in promoting 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. At present, the specific functions of all family members are not well understood. This is because when some traditional small molecule compounds are used to inhibit a certain caspase, the normal activity of other members in the family will also be affected, leading to a series of unexpected effects. For example, small compounds including VX-765, Terfenadine, Fenbufen, Ac-DEVD, Ac-LEVD and Ac-YVAD, while chemically inhibiting caspase-4, will also cause the inactivation of multiple enzymes such as caspase-1, 3, 5, 7, 9, 11, 13, etc., and the activation of some caspase members is often necessary for the normal physiological function of the body. In addition, long-term stimulation of multiple inhibitors will also lead to serious side effects such as the death of replaced normal cells. Therefore, when using traditional small molecule inhibitors for basic research, there are many disadvantages such as insufficient efficacy, poor target specificity and adverse side effects, which greatly limit their potential for application in the treatment of various diseases. Although a very small number of commercial inhibitors have been able to enter clinical trials, they have also been terminated prematurely due to their unpredictability and adverse side effects. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of a caspase-4 gene or its encoded protein as a target in screening drugs for preventing or treating neurotoxicity. The present invention is the first to perform gene therapy targeting caspase-4 in mice, using ASO or CRISPR / Cas9 to block the translation of the caspase-4 gene or increase its mRNA degradation, thereby inhibiting the expression of caspase-4 protein, and ultimately achieving successful reversal of pathological TDP-43, which is specifically manifested in the reduction of TDP-43 fragmentation products in the cytoplasm, the recovery and increase of full-length TDP-43 in the nucleus, and the pathological toxicity such as the loss of neurons in the mouse brain is significantly alleviated.

[0008] In addition, the present invention is the first targeted therapy for caspase-4, which not only specifically reduces the expression of caspase-4 at the gene level, but also does not affect the expression levels of other members of the caspase family, and has distinct specificity compared with traditional caspase-4 small molecule inhibitors. The targeted therapy for caspase-4 developed by the present invention can alleviate TDP-43 pathology and its neurotoxicity, and provides a new strategy for specific treatment of related diseases including ALS in the future, so it has great potential for drug market development.

[0009] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

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

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

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

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

[0014] Furthermore, the gRNA includes two sequences that serve as targets at the same time, and the specific sequences are 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 specifically targeting 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 specifically targeting the caspase-4 gene or its encoded protein, wherein the gRNA comprises two sequences serving as targets at the same time, and the specific sequences 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 a method for preparing a drug for preventing or treating neurotoxicity by using an agent for inhibiting / interfering with the expression of caspase-4 gene or its encoded protein.

[0023] Further, neurotoxic conditions include ALS.

[0024] Beneficial effects of the present invention:

[0025] The present invention utilizes ASO or CRISPR / Cas9 to block the translation of the caspase-4 gene or increase its mRNA degradation, thereby inhibiting the expression of caspase-4 protein, and ultimately achieving successful reversal of pathological TDP-43. In addition, the present invention is the first to develop a targeted therapy for caspase-4, which not only specifically reduces the expression of caspase-4 at the gene level, but also does not affect the expression levels of other members of the caspase family. Compared with traditional caspase-4 small molecule inhibitors, it has distinct specificity. The effective alleviation of TDP-43 pathology and its neurotoxicity also provides a new strategy for the specific treatment of related diseases including ALS in the future, and therefore has great potential for drug market development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a picture of immunoblotting validation of the targeting effect of caspase-4-ASO using the HeLa cell line;

[0027] Figure 2 This is the immunoblot image after caspase-4-ASO was injected by sustained-release pump for 4 weeks;

[0028] Figure 3 This is the immunofluorescence image after caspase-4-ASO was injected with a sustained-release pump for 4 weeks;

[0029] Figure 4 This is the immunoblot image after caspase-4-ASO was injected by sustained-release pump for 4 weeks;

[0030] Figure 5 This is the immunofluorescence image after caspase-4-ASO was injected with a sustained-release pump for 4 weeks;

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

[0032] Figure 7 This is an immunofluorescence image after 4 weeks of targeting of caspase-4 mediated by CRISPR / Cas9 expressed by adeno-associated virus;

[0033] Figure 8 This is an immunoblot image of CRISPR / Cas9-mediated caspase-4 targeting 4 weeks after expression of adeno-associated virus;

[0034] Fig. 9 This is an immunofluorescence image 4 weeks after CRISPR / Cas9-mediated caspase-4 targeting was expressed using adeno-associated virus. DETAILED DESCRIPTION

[0035] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0036] Example 1 Design of ASO targets

[0037] 1. Retrieve the human caspase-4 mRNA sequence from the NCBI database, identify the key functional domains of caspase-4, and preferentially target its coding region or splicing site to disrupt function. Use professional software (such as RNAstructure, mFOLD) to predict the secondary structure of the target mRNA, select the single-stranded exposed region to improve the binding efficiency, design two sequences and use 2'-O-methyl (2'-OMe) for modification to increase binding affinity and reduce immunogenicity. 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 Biotechnology Co., Ltd., and the target sequences were used for the first time.

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

[0039] Example 2 ASO target efficacy detection

[0040] Caspase-4-ASO and Non-targeted-ASO were injected into mice to verify their targeting effect and therapeutic effect. The specific process is as follows:

[0041] (1) Use a 1.0 mL sterile syringe to slowly inject 2 OD / 100 μL of caspase-4-ASO and control non-targeted-ASO into the sustained-release pump. At the same time, inject the drugs into the connecting tube of the flow regulator to avoid bubbles, and connect it to the flow regulator. Place the drug-filled sustained-release pump in 37°C saline for soaking and set aside. The connecting tube should not be immersed in saline.

[0042] (2) Turn on the small animal inhalation gas anesthesia machine and connect it to the brain stereotaxic device. Place the mouse in the gas anesthesia machine and anesthetize it until it is painless and unresponsive. Fix the mouse's head. Transfer the mouse to the stereotaxic instrument and fix its head, and keep the mouse under continuous breathing anesthesia. Transfer the mouse and fix its head to the stereotaxic instrument to prevent it from moving. Use sterile gloves and disinfectant to clean the mouse's head, remove the hair, cut the skin on the head, and use 3% H 2 O 2 Wipe and dissolve the meninges to fully expose the Bregma cross suture. Take Bregma as the origin, move 2mm forward and 1.65mm to the right to mark the drilling coordinates. Then turn on the handheld skull drill and use it to carefully drill at the marked position.

[0043] (3) Use surgical scissors to make a small incision in the neck and then cut it open. Use hemostats to separate the subcutaneous connective tissue on the back. Implant the sustained-release pump into the subcutaneous space. Gently insert the flow regulator needle vertically into the drill hole. Fix it with dental cement. Use surgical sutures to suture the head and neck incisions. The flow rate of the sustained-release pump is 0.13±0.02μL / h.

[0044] (4) During the implantation of the sustained-release pump, the mice were closely observed. After about 4 weeks, the mice were euthanized and their brain tissues were taken for pathological analysis. The results are shown in the table below. Figure 2 to Figure 5 .

[0045] Western blot analysis showed that caspase-4-ASO effectively inhibited the level of caspase-4 compared with the non-targeted control, and the fragmentation products of endogenous TDP-43 in mice were reduced ( Figure 2 Meanwhile, immunofluorescence staining also confirmed that the cytoplasmic accumulation of endogenous TDP-43 was reduced in mice treated with caspase-4-ASO compared with non-targeted controls ( Figure 3). We further verified whether targeted knockdown of caspase-4-ASO could reduce neurotoxicity in mice. Immunoblotting showed that after 4 weeks of caspase-4-ASO administration using a sustained-release pump, the neuronal marker proteins MAP2 and NeuN could be restored after caspase-4-ASO inhibition ( Figure 4 ), and immunofluorescence results also showed that the expression levels of MAP2 and NeuN could be increased after caspase-4-ASO inhibition ( Figure 5 )

[0046] Example 3 CRISPR / Cas9 inhibits caspase-4 expression

[0047] 1. Obtaining the target

[0048] The targeting sites designed for the exon 2 sequence of the full-length transcript of the caspase-4 gene (ENST00000444739.7) were verified by the HeLa cell line, and two effective specific target sites (5'-CACTGGTGTTTTGGATAACT-3' and 5'-CAAGAGAAGCAACGTATGGC-3, this targeting combination was used for the first time) were selected.

[0049] 2. Transfection of HeLa cells

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

[0051] 3. Animal experiments

[0052] Place the mouse in an induction box and use isoflurane to induce anesthesia as preoperative anesthesia. After the mouse is fully anesthetized, place the mouse on a sponge pad, put the mouse's mouth into the anesthesia mask, and fix the skull on a stereotaxic apparatus to ensure that the skull is stable and bilaterally symmetrical. Use a sterile cotton swab dipped in 75% alcohol to disinfect the mouse's scalp and shoulder and neck skin; remove the hair on the top of the skull with scissors, cut part of the scalp and the fascia under the scalp, and dip a cotton swab in an appropriate amount of 3% H 2 O2 Digest the soft tissue on the surface of the skull until the anterior and posterior fontanelles are clearly exposed. Take bregma as the coordinate origin, move 2 mm forward and 1.65 mm to the right to mark the drilling coordinates, and drill at the marked locations.

[0053] A microsyringe was used to draw up the virus liquid, and the syringe was fixed on the stereotaxic apparatus. The air in the needle was emptied and the needle was inserted into the holes on the left and right 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 the control group were injected with AAV-CASP4-gRNA and AAV-CTRL-gRNA viruses at a speed of 100 nl / min, respectively, and the virus dose was 1.25 μL on each side. After that, the needle was stopped for 10 minutes and the needle was removed at a speed of 0.1 mm / s.

[0054] The incision was sutured with sutures, and the mice were observed until they woke up; after the virus injection into the bilateral prefrontal motor cortex was completed, the mice were left to stand for five minutes, the microsyringe was slowly removed, the scalp was sutured, and penicillin-streptomycin solution was applied. The mice were kept warm after surgery, and their status was closely observed. About 4 weeks later, the mice were euthanized and brain tissues were taken for a series of pathological tests such as immunoblotting and immunofluorescence staining, as follows:

[0055] To investigate whether knocking out caspase-4 using AAV-CASP4-gRNA adeno-associated virus would reduce TDP-43 pathology and neurotoxicity in mice, immunofluorescence detection was performed. Figure 7 .

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

[0057] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

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

2. The use according to claim 1, characterized in that The medicine comprises an effective component for inhibiting the expression of caspase-4 gene or its coding protein.

3. The use according to claim 2, characterized in that: The active ingredient is ASO or gRNA targeting caspase-4.

4. The use according to claim 3, characterized in that The ASO target sequence is: 5'-UGAUGTCAAAGTCAGCUCCA-3'.

5. The use according to claim 3, characterized in that: gRNA includes two sequences that serve as targets at the same time. The specific sequences are as follows: gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'.

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

7. A gRNA specifically targeting caspase-4 gene or its encoded protein, characterized in that: The gRNA includes two sequences that serve as targets at the same time, and the specific sequences are as follows: gRNA-1: 5'-CACTGGTGTTTTGGATAACT-3'; gRNA-2: 5'-CAAGAGAAGCAACGTATGGC-3'.

8. Use of an agent that inhibits / interferes with the expression of caspase-4 gene or its encoded protein in the preparation of a drug for preventing or treating neurotoxicity.

9. The use according to claim 8, characterized in that Such neurotoxic conditions include ALS.

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