Small nucleic acid targeting PIKfyve gene as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a small nucleic acid targeting a PIKfyve gene as well as a preparation method, a pharmaceutical composition and application of the small nucleic acid. According to the small nucleic acid targeting the PIKfyve gene disclosed by the invention, the expression level of PIKfyve mRNA (messenger Ribonucleic Acid) is obviously reduced at the cellular level, and the axon length of neuronal cells is obviously increased; in-vivo verification shows that the body function and the survival condition of mice with amyotrophic lateral sclerosis can be remarkably improved. The small nucleic acid targeting the PIKfyve gene disclosed by the invention provides a new thought for treating neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to small nucleic acids targeting the PIKfyve gene, pharmaceutical compositions thereof, and uses thereof. Background Art

[0002] Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a neurodegenerative disease characterized by the progressive loss of motor neurons and the progressive muscle weakness and atrophy of the muscles of the limbs and trunk, thereby affecting the patient's motor, swallowing, and respiratory functions, and ultimately leading to the death of the patient.

[0003] ALS can be classified into familial (familial ALS, FALS) and sporadic (sporadic ALS, SALS) according to genetics. Familial ALS accounts for about 10%, and the remaining 90% is sporadic ALS. The genetic pattern of FALS is mainly autosomal dominant inheritance and is associated with various gene abnormalities. More than 30 gene mutations related to ALS have been identified so far. Common abnormal genes include C9orf72 (accounting for 39.3%), SOD1 (12%-23.5%), TARDBP (5%), and FUS (4.1%), etc. SALS usually has no family history of the disease, but most SALS patients still carry gene mutations related to the onset of ALS.

[0004] Currently, the pathogenesis of ALS is still unclear. Under the current medical technology conditions, ALS is still an incurable disease, and only through various methods can the development of the disease be delayed as much as possible, thereby prolonging the patient's survival period.

[0005] The drugs approved for the treatment of ALS in China are riluzole, edaravone, and Tofersen. The mechanism of action of riluzole is that on the one hand, it inhibits the release of glutamate and reduces the damage of motor neurons caused by excessive glutamate; on the other hand, riluzole can stabilize the inactivated state of voltage-dependent sodium channels and reduce the overexcitation of neurons. From the perspective of clinical use effects, riluzole can prolong the survival period of patients by 2-3 months.

[0006] Different from riluzole, the medicinal mechanism of edaravone is to inhibit the generation of free radicals, reduce the damage of nerve cells caused by excessive oxidation, and thus achieve the purpose of delaying the progression of ALS. However, edaravone is only effective for a small part of early-onset ALS. Long-term clinical practice has proved that the curative effects of riluzole and edaravone are obviously not so satisfactory in actual clinical practice, and people have begun to explore new directions from the perspective of gene therapy.

[0007] Tofersen is an ASO drug targeting the SOD1 gene and was approved for marketing in China on October 8, 2024. Its mechanism of action is to reduce the synthesis of SOD1 protein and the accumulation of SOD1 toxic protein. In a phase III trial of amyotrophic lateral sclerosis, intrathecal injection of Tofersen did not reach the primary endpoint (total ALSFRS-R score), and about 6.7% of patients reported serious neurological-related adverse events, including myelitis, aseptic meningitis, optic disc edema, and lumbosacral neuritis. However, Tofersen reduced the level of plasma neurofilament light chain (a neurodegenerative biomarker) in patients. Therefore, although Tofersen did not achieve a statistically significant improvement compared with placebo, the FDA finally approved the marketing application of Tofersen. However, this gene-targeted drug is only applicable to ALS patients with the SOD1 genotype, and this part of patients only accounts for 2% of the ALS patient population. The majority of the remaining patients are still in urgent need of broad-spectrum, safe, and effective drugs for treatment.

[0008] ALS is a neurodegenerative disease caused by many different reasons. One of the common characteristics of neurodegenerative diseases is the accumulation of misfolded proteins. Therefore, clearing misfolded proteins has become a research direction for the development of ALS treatment drugs. Research has shown that pharmacological inhibition of the PIKfyve kinase activates an unconventional protein clearance mechanism involving the exocytosis of aggregation-prone proteins. This unconventional protein clearance mechanism can enhance the level of exocytosis in vivo and thus effectively clear the misfolded proteins accumulated in cells. The study also found that PIKfyve inhibitors can extend the survival cycle of motor neurons in various forms of ALS and are expected to become an effective treatment for various forms of ALS.

[0009] So far, no effective PIKfyve inhibitor drug has been marketed for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis. There is an urgent need to develop a safe and effective PIKfyve inhibitor drug to relieve the pathological symptoms of patients with neurodegenerative diseases such as amyotrophic lateral sclerosis, restore their motor function, and thus improve the quality of life of patients or extend the survival time of patients. Summary of the Invention

[0010] Aiming at the defects of the prior art, the present invention rationally designs and synthesizes small nucleic acid molecules targeting the PIKfyve gene and verifies them in vitro and in vivo, aiming to develop a safe and effective PIKfyve small nucleic acid inhibitor.

[0011] In one aspect, the present invention provides a small nucleic acid targeting the PIKfyve gene, which comprises any one or more nucleotide sequences selected from those shown in SEQ ID NO: 1 to 128, SEQ ID NO: 130, SEQ ID NO: 132 to 135, and SEQ ID NO: 136 to 142.

[0012] In one or more embodiments, the small nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 132 or SEQ ID NO: 135.

[0013] Preferably, the small nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 135.

[0014] The nucleotide sequence shown in SEQ ID NO: 135 is , where T represents thymidine deoxynucleotide; G represents guanine deoxynucleotide; C represents cytosine deoxynucleotide; A represents adenine deoxynucleotide; g represents guanine ribonucleotide; c represents cytosine ribonucleotide; a represents adenine ribonucleotide; m5C represents 5-methylcytidine deoxyribonucleotide; m 5 cm represents 2'-O-(2-methoxyethyl)-5-methylcytidine ribonucleotide; x m (italic) represents a 2'-O-(2-methoxyethyl)-modified ribonucleotide (x = a, g, c, or u); * represents that the nucleotide replaces one non-bridging oxygen atom in the phosphodiester bond with a sulfur atom.

[0015] In one or more embodiments, the small nucleic acid comprises a sense strand and an antisense strand. The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 139, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 140; or, the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 141, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 142.

[0016] In one aspect, the present invention provides a small nucleic acid targeting the PIKfyve gene, which comprises the nucleotide sequence shown in SEQ ID NO: 132.

[0017] In one aspect, the present invention provides a small nucleic acid targeting the PIKfyve gene, which comprises the nucleotide sequence shown in SEQ ID NO: 135.

[0018] In one or more embodiments, the small nucleic acid comprises a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 139, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 140.

[0019] The nucleotide sequence shown in SEQ ID NO: 139 is , and the nucleotide sequence shown in SEQ ID NO: 140 is ; wherein, T represents thymidine deoxynucleotide; G represents guanosine deoxynucleotide; A represents adenosine deoxynucleotide; u represents uridine ribonucleotide; g represents guanosine ribonucleotide; c represents cytidine ribonucleotide; a represents adenosine ribonucleotide; x m represents a 2'-O-methyl modified ribonucleotide (x = a, g, c or u); represents a 2'-fluoro modified deoxyribonucleotide (X = A, G, C or U).

[0020] In one or more embodiments, the small nucleic acid comprises a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 141, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 142.

[0021] The nucleotide sequence shown in SEQ ID NO: 141 is ) , and the nucleotide sequence shown in SEQ ID NO: 142 is ) ; wherein, T represents thymidine deoxynucleotide; G represents guanosine deoxynucleotide; C represents cytidine deoxynucleotide; A represents adenosine deoxynucleotide; g represents guanosine ribonucleotide; c represents cytidine ribonucleotide; a represents adenosine ribonucleotide; x m represents a 2'-O-methyl modified ribonucleotide (x = a, g, c or u); * represents that the nucleotide replaces a non-bridging oxygen atom in the phosphodiester bond with a sulfur atom; X f represents a 2'-fluoro modified deoxyribonucleotide (X = A, G, C or U); (C n x) represents a 2'-O-n-carbon atom linear alkyl modified ribonucleotide (x = a, g, c or u), n = 8 - 25; (VPx) represents a 5'-E-vinyl phosphate modified ribonucleotide (x = a, g, c or u).

[0022] In another aspect, the present invention provides the use of the small nucleic acid targeting the PIKfyve gene as described in any one of the embodiments herein in the preparation of a medicament for treating neurodegenerative diseases.

[0023] In one or more embodiments, the neurodegenerative disease is selected from one or more of amyotrophic lateral sclerosis, Parkinson's syndrome, and Alzheimer's disease.

[0024] In another aspect, the present invention provides a method for preparing a small nucleic acid targeting the PIKfyve gene as described in any one of the embodiments herein, the method comprising synthesizing nucleotide monomers into the small nucleic acid.

[0025] Preferably, the method is the phosphoramidite triester method.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a small nucleic acid targeting the PIKfyve gene as described in any one of the embodiments herein, and a pharmaceutically acceptable carrier.

[0027] In another aspect, the present invention provides the use of the pharmaceutical composition as described in any one of the embodiments herein in the preparation of a medicament for treating neurodegenerative diseases.

[0028] In one or more embodiments, the neurodegenerative disease is selected from one or more of amyotrophic lateral sclerosis, Parkinson's syndrome, and Alzheimer's disease.

[0029] Compared with the prior art, the small nucleic acid targeting the PIKfyve gene provided by the present invention has the following beneficial effects: 1. Different types of modifications contained in the small nucleic acid molecule significantly enhance its stability, and a very low degradation rate can still be achieved after treatment with in vitro serum for 24 h.

[0030] 2. In vitro, it can achieve a significant decrease in the relative expression level of PIKfyve mRNA at the cellular level at a transfection concentration of nM, and significantly increase the axon length of neuronal cells.

[0031] 3. In vivo, it can significantly improve the physical function and survival status of mice with amyotrophic lateral sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 are the results of serum stability determination of ASO molecules and siRNA molecules. Among them, Figure 1 A in is the result of serum stability determination of ASO molecules; Figure 1 B in is the result of serum stability determination of siRNA molecules.

[0033] Figure 2 is the relative mRNA expression level of the PIKfyve gene in DRG cells after knockdown with ASO-7 molecules or siRNA-60' molecules. Figure 2A in it is the relative mRNA expression level of PIKfyve gene in DRG cells after knockdown with ASO-7 molecule; Figure 2 B in it is the relative mRNA expression level of PIKfyve gene in DRG cells after knockdown with siRNA-60’ molecule.

[0034] Figure 3 is the immunofluorescence result of the effect of ASO-7 molecule transfection on axon growth in DRG cells. Among them, Figure 3 A in it is the observation result of the blank cell group; Figure 3 B in it is the observation result of the positive control group; Figure 3 C in it is the observation result of the ASO-7 group.

[0035] Figure 4 is the proportion of axons of DRG cells in each group in different length intervals.

[0036] Figure 5 is the average axon length of DRG cells in each group. Specific implementation manners

[0037] Example 1: Sequence design and synthesis of small nucleic acids (1) Sequence design of small nucleic acids Obtain the sequence of PIKfyve gene (Gene ID: 200576) from the NCBI database, avoid the 5'untranslated region (5'UTR) and 3'untranslated region (3'UTR) and the sequences near its start codon, design siRNAs and ASOs targeting the PIKfyve gene, and introduce nucleotide modifications. The siRNA sequences targeting the PIKfyve gene, the ASO sequences containing modifications, and the siRNA sequences containing modifications are shown in Table 1, Table 2, and Table 3 respectively.

[0038] Table 1: siRNA sequences targeting the PIKfyve gene

[0039] Table 2: ASO sequences containing modifications targeting the PIKfyve gene

[0040] Table 3: siRNA sequences containing modifications targeting the PIKfyve gene

[0042] Among them, T represents thymidine monophosphate; G represents guanosine monophosphate; C represents cytidine monophosphate; A represents adenosine monophosphate; u represents uridine monophosphate; g represents guanosine monophosphate; c represents cytidine monophosphate; a represents adenosine monophosphate; x m represents a 2'-O-methyl modified ribonucleotide (x = a, g, c or u); m5C represents 5-methylcytidine monophosphate; m5cm represents 2'-O-(2-methoxyethyl)-5-methylcytidine monophosphate; x m (italic) represents a 2'-O-(2-methoxyethyl) modified ribonucleotide (x = a, g, c or u); * represents that the nucleotide replaces a non-bridging oxygen atom in the phosphodiester bond with a sulfur atom (i.e., P=S replaces P=O); X f represents a 2'-fluoro modified deoxyribonucleotide (X = A, G, C or U); (C n x) represents a 2'-O-n-carbon normal alkyl modified ribonucleotide (x = a, g, c or u), n = 8 - 25; (VPx) represents a 5'-E-vinyl phosphate modified ribonucleotide (x = a, g, c or u). Some of the ASOs in Table 2 contain 5-methylcytidine monophosphate (C), 2'-O-(2-methoxyethyl)-5-methylcytidine monophosphate, 2'-O-methyl modified ribonucleotides (a, g, c or u) and / or 2'-O-(2-methoxyethyl) modified ribonucleotides (a, g, c or u); some of the siRNAs in Table 3 contain thymidine monophosphate monomers (T) and / or 2'-fluoro modified deoxyribonucleotides (A, G, C or U).

[0043] (2) Synthesis of small nucleic acids The ASO and siRNA molecules are synthesized by the phosphoramidite triester method, that is, the 5 procedures of "deprotection - activation - coupling - capping - oxidation" are repeated. After each round of repetition, the oligonucleotide chain is extended by one nucleotide, and finally the crude synthesis products of ASO and siRNA with the target length are obtained. The deprotection reagent is TCA Deblock, the activator is 5-benzylthiotetrazole, the capping reagents are CAPA and CAPB, and the oxidant is iodine solution or (E)-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine.

[0044] The crude synthesis product of ASO is deprotected and purified to obtain the crude ASO molecule; the crude synthesis product of siRNA is deprotected, annealed and purified to obtain the crude siRNA molecule. Among them, the HPLC method is used to purify the crude product.

[0045] Purification materials: Anion exchange column Diamond Q Mustang (purchased from Bogolong Company) and eluent (a mixture of different ratios of Solution A and Solution B, Solution A: 100 mM Tris, 10 mM EDTA, 300 mM NaCl, pH 9.0; Solution B: 100 mM Tris, 10 mM EDTA, 1000 mM NaCl, pH 9.0).

[0046] Purification procedure: Elute with 5 CV (column volume) of Solution A; elute with 50 CV of eluent (containing 0%-100% B); elute with 5 CV of Solution B. Desalt the target elution peak product using a G25M column (purchased from Bogolong Company). After desalting, concentrate the product by ultrafiltration to obtain the purified product. After freeze-drying, perform vacuum packaging and store it at -20°C for future use.

[0047] Example 2: Verification of the effect of small nucleic acids at the cellular level In this example, the siRNAs and ASOs constructed in Tables 1 and 2 of Example 1 were used to verify the effect at the cellular level.

[0048] Culture HaCat cells, and the culture medium composition is 90% DMEM + 10% FBS + 1% PS. When the cell growth reaches 90% confluence, inoculate them into a six-well plate and culture for 24 h. Dilute ASO and siRNA with DMEM medium to a concentration of 100 μM in advance for standby. After the culture, add different concentrations of ASO (final concentration 50 nM, 1000 nM or 5000 nM) or siRNA (final concentration 30 nM or 100 nM) to the cells in each well for transfection, and continue to culture the cells; use the cells without adding ASO or siRNA as the control group (control). 48 h after transfection, extract the total cellular RNA by the Trizol method, and detect the expression level of PIKfyve mRNA in the cells of each well relative to the control group by RT-qPCR method.

[0049] The results of detecting the relative expression levels of PIKfyve mRNA in HaCat cells are shown in Tables 4 and 5. Among them, at the transfection concentrations of 30 nM and 100 nM of siRNA, except for siRNA-6, siRNA-31, and siRNA-33, the expression of PIKfyve mRNA can be significantly knocked down. Among them, siRNA-11, siRNA-25, siRNA-40, and siRNA-60 have better knockdown effects; at the transfection concentrations of 50 nM, 1000 nM, and 5000 nM of ASO, the expression of PIKfyve mRNA can be significantly knocked down. Among them, ASO-1, ASO-3, ASO-4, and ASO-7 have better effects. It can be seen that at the cellular level, the siRNA and ASO targeting the PIKfyve gene in Tables 1 and 2 can basically significantly knock down the expression of the PIKfyve gene.

[0050] Table 4: Knockdown effect of siRNA on the expression level of PIKfyve mRNA

[0051] Table 5: Knockdown effect of ASO on the expression level of PIKfyve mRNA

[0052] Example 3: Serum stability detection of small nucleic acids Under normal circumstances, the degradation rate of siRNA and ASO in the blood is relatively fast. According to the references (doi:10.1038 / mt.2009.91, doi: 10.4062 / biomolther.2023.001), in the blood, the half-lives of unmodified siRNA and ASO are only a few minutes, and their half-lives can be effectively extended after reasonable modification.

[0053] In this example, the serum enzymatic hydrolysis experiment was used to detect the effects of different modification schemes on the stability of siRNA and ASO. ASO-1, ASO-3, ASO-4, and ASO-7 in Table 2 of Example 1 and siRNA-11’, siRNA-25’, siRNA-40’, and siRNA-60’ in Table 3 (corresponding to siRNA-11, siRNA-25, siRNA-40, and siRNA-60 without modification in Table 1 respectively) were selected.

[0054] 800 ng of each purified ASO was mixed with 10% fetal bovine serum and incubated in a 37 °C incubator for 24 h. The incubated samples were used for 2% agarose gel electrophoresis. The electrophoresis conditions were 140 V, 8 min; after electrophoresis, the gel was placed in a UV gel imager for imaging and observation. The gel imaging results are as Figure 1As shown in A of [Figure ID], after 24 h of serum co-incubation, no visible bands were observed in the samples of ASO-1 and ASO-3, while obvious bands were visible in the samples of ASO-4 and ASO-7 at 0 h or 24 h, indicating that ASO-4 and ASO-7 have high stability.

[0055] Mix 800 ng of each of the aforementioned purified siRNAs with 10% fetal bovine serum, and co-incubate in a 37 °C incubator for 24 h. The samples obtained from the incubation are used for 2% agarose gel electrophoresis, and the electrophoresis conditions are 140 V for 8 min; after electrophoresis, the gel is placed in a UV gel imager for development and observation. The gel imaging results are as Figure 1 shown in B of [Figure ID]. After 24 h of serum co-incubation, no visible bands were observed in the samples of siRNA-11’ and siRNA-25’, while obvious bands were observed in the samples of siRNA-40’ and siRNA-60’ at 0 h or 24 h, indicating that siRNA-40’ and siRNA-60’ have high stability.

[0056] Example 4: Promoting effect of small nucleic acids on dorsal root ganglion neurons In this example, ASO-7 and siRNA-60’ constructed in Example 1 were used to study the effect on dorsal root ganglion (DRG) cells.

[0057] (1) Obtaining and culturing of DRG primary cells Healthy C56BL / 6 mice were sacrificed by cervical dislocation, and the epidermis was disinfected with 75% ethanol. The DRG cell bodies at the spinal cord were dissected and placed in a pre-cooled 60 mm dish containing HANKS solution. The HANKS solution was aspirated, and the DRG cell bodies were washed 3 times with pre-cooled HANKS solution. Add 2 mg / mL collagenase I and digest at 37 °C for 1 - 2 h, then add 2 mL of 0.25% trypsin and continue to digest at 37 °C until there are no obvious tissue blocks. Finally, add DMEM medium containing FBS to dilute to terminate the digestion, and obtain the digestion solution. The digestion solution was filtered through a 200-mesh cell sieve, and the filtrate was centrifuged at 900 rpm for 5 min. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended with 5% - 15% BSA and centrifuged again at 900 rpm for 5 min. The supernatant was discarded, and the cells were resuspended with neurobasal-A complete medium and inoculated into a 12-well plate for culture to obtain DRG primary cells. The medium was changed with neurobasal-A complete medium the next day and reserved for use.

[0058] (2) Determination of PIKfyve gene expression level When the above DRG cells grew to an appropriate confluence, they were seeded into six-well plates and cultured for 24 h. ASO-7 and siRNA-60’ were pre-diluted with DMEM medium to a concentration of 100 μM for standby. After the culture, different concentrations of ASO (final concentrations of 50 nM, 1000 nM or 5000 nM) or siRNA (final concentrations of 30 nM or 100 nM) were added to the cells in each well for transfection, and the cells were continuously cultured; the cells without adding ASO or siRNA were used as the control group (control). After 48 h of transfection, the total cellular RNA was extracted by the Trizol method, and the relative expression levels of PIKfyve mRNA in the cells of each well were detected by RT-qPCR method.

[0059] The results of the detection of the relative expression levels of PIKfyve mRNA in DRG cells are as Figure 2 shown. Compared with the control group, ASO-7 could achieve a knockdown effect of about 50% at a transfection concentration of 1000 nM, and the knockdown effect was further improved to about 60% at a transfection concentration of 5000 nM, while no obvious knockdown effect was observed at a transfection concentration of 50 nM ( Figure 2 , A); siRNA-60’ could achieve a knockdown effect of about 40% at a transfection concentration of 30 nM, and the knockdown effect was further improved to about 60% at a transfection concentration of 100 nM ( Figure 2 , B). It can be seen that for DRG cells, both ASO and siRNA targeting the PIKfyve gene can significantly knockdown the expression of the PIKfyve gene, and the knockdown effect has a certain degree of dose-dependence.

[0060] (3)Measurement of neuron axon length For the cells transfected with 1000 nM ASO in Example (2) of this embodiment, a blank cell group (no-treatment control) and a positive control group (positive control - tofersen) were additionally set up; among them, the blank cell group did not add ASO or siRNA, and the positive control group added 1000 nM Tofersen instead of ASO-7; at 48 h after transfection, the cells were fixed with 4% paraformaldehyde for 10 - 30 min, and then immunofluorescence staining was performed to facilitate the statistical analysis of the changes in the neuron axon length after the knockdown of the PIKfyve target. Among them, the primary antibody was the β-tubulin antibody, and the secondary antibody was the antibody with a fluorescent label.

[0061] The cells in each group were observed under a fluorescence microscope, and the observation results are as Figure 3 shown. Six fields of view were randomly photographed for each observed cell well, the axon lengths of the neurons in the fields of view were statistically analyzed, and grouped according to the length intervals, and the results are as Figure 4The analysis results shown. In the no-treatment control group, the axons with a length of 20 - 40 μm were the most numerous; in the positive control-tofersen group, the axons with a length of 40 - 60 μm were the most numerous. Compared with the no-treatment control group, the proportions of axons with lengths of 40 - 60 μm, 60 - 80 μm, 80 - 100 μm, and 100 - 120 μm in the ASO-7 group (ASO-PIKfyve) all increased; in addition, the mean values of the axon lengths in each group were statistically calculated to obtain the results as shown in Figure 5 shown. As can be seen from Figure 4 and Figure 5 , after ASO-7 reduced the expression level of PIKfyve, it significantly promoted the axon growth of dorsal root ganglion neurons. When the transfection concentration was 1000 nM, its effect on promoting neuron axon growth was not inferior to that of the Tofersen drug.

[0062] Example 4: Application of ASO in a mouse model of amyotrophic lateral sclerosis In this example, ASO-7 constructed in Example 1 was used to study its effect on a mouse model of amyotrophic lateral sclerosis at the animal level. The mouse model of amyotrophic lateral sclerosis was a Tg(Prnp-TARDBP*A315T)95Balo / J male mouse. The Tg(Prnp-TARDBP*A315T) 95Balo / J male mice and C57BL / 6 mice in this example were commercially available.

[0063] (1) Preparation of the preparation 100 mg (concentration 6.7 mg / mL) of ASO-7 or 100 mg of Tofersen; 0.21 mg / mL of calcium chloride dihydrate; 0.11 mg / mL of disodium hydrogen phosphate; 0.16 mg / mL of magnesium chloride hexahydrate; 0.03 mg / mL of sodium dihydrogen phosphate; 0.22 mg / mL of potassium chloride; 8.77 mg / mL of sodium chloride; made up to volume with sterile injection water and adjusted the pH value to 7.2 (a pH range of 6.7 - 7.7 is an acceptable range).

[0064] (2) Determination of the effect of ASO-7 on a mouse model of amyotrophic lateral sclerosis The mice were grouped according to Table 6 below. Among them, the mice in the positive control group, low-dose group, and high-dose group were mouse models of amyotrophic lateral sclerosis, and the mice in the negative control group were C57BL / 6 mice; according to the recommended dose in the Tofersen drug instructions, each mouse in the positive control group was injected with 0.033 mg of Tofersen.

[0065] Table 6: Grouping of experimental mice

[0067] All mice were raised in a standardized animal house. When the mice were 41 days old, they were weighed and administered the drug for the first time via intrathecal injection. When the mice were 61 days old, they were administered the drug for the second time, and the survival rate and body weight of the surviving mice were counted, and their performance in the righting test and rotarod test was measured.

[0068] The survival results of the mice are shown in Table 7 below. The survival rates of the mice in the positive control group and the high-dose group were 50%, higher than 12.5% in the low-dose group. In the high-dose group, 25% of the mice had an increase in body weight, while there were no mice with an increase in body weight in the positive control group and the low-dose group. It can be seen that ASO-7 is beneficial to improving the survival status of mice with amyotrophic lateral sclerosis.

[0069] Table 7: Statistical results of mouse survival rate and body weight

[0071] The measurement results of the righting test of the mice are shown in Table 8 below. The average righting time of the mice in the high-dose group and the low-dose group was significantly less than that in the positive control group. Among them, the average righting time in the high-dose group was 0.1 second, which was significantly less than that in the low-dose group and the same as that in the negative control group. It can be seen that ASO-7 shortened the righting time of mice with amyotrophic lateral sclerosis, significantly improved their physical activity function, and enabled them to achieve rapid righting.

[0072] Table 8: Measurement results of mouse righting test

[0074] The measurement results of the rotarod test of the mice are shown in Table 9 below. The dropping time of the mice in the low-dose group was less than that in the positive control group, while the dropping time of the mice in the high-dose group was greater than that in the positive control group. It can be seen that ASO-7 at high dose prolonged the time that mice with amyotrophic lateral sclerosis could stay on the rotarod without falling, significantly improving their physical activity function.

[0075] Table 9: Measurement results of mouse rotarod test

[0077] In summary, the small nucleic acid targeting the PIKfyve gene provided by the present invention can significantly decrease the expression level of PIKfyve mRNA at the cellular level at a transfection concentration of nM in vitro, and significantly increase the axon length of neuronal cells; in vivo, it can significantly improve the physical function and survival status of mice with amyotrophic lateral sclerosis. Therefore, it can be used to treat diseases mediated by PIKfyve or to prepare drugs for treating diseases mediated by PIKfyve, and the diseases include but are not limited to amyotrophic lateral sclerosis, Parkinson's syndrome, and Alzheimer's disease.

Claims

1. Small nucleic acid targeting the PIKfyve gene, characterized in that, The small nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO:

135.

2. Use of the small nucleic acid targeting the PIKfyve gene according to claim 1 in the preparation of a medicament for treating neurodegenerative diseases.

3. Use according to claim 2, wherein the neurodegenerative disease is selected from one or more of amyotrophic lateral sclerosis, Parkinson's syndrome, and Alzheimer's disease.

4. A method for preparing the small nucleic acid targeting the PIKfyve gene according to claim 1, the method comprising synthesizing the small nucleic acid from nucleotide monomers.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the small nucleic acid targeting the PIKfyve gene according to claim 1 and a pharmaceutically acceptable carrier.

6. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating neurodegenerative diseases.

7. Use according to claim 6, wherein the neurodegenerative disease is selected from one or more of amyotrophic lateral sclerosis, Parkinson's syndrome, and Alzheimer's disease.

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