Small nucleic acid targeting PIKfyve gene and pharmaceutical composition and use thereof

Abstract

The present invention discloses a small nucleic acid targeting the PIKfyve gene, as well as its preparation method, pharmaceutical composition, and uses. The small nucleic acid targeting the PIKfyve gene disclosed herein significantly reduces PIKfyve mRNA expression levels at the cellular level and significantly increases axon length in neuronal cells. In vivo validation demonstrates significant improvements in the physical function and survival of mice with amyotrophic lateral sclerosis (ALS). The small nucleic acid targeting the PIKfyve gene disclosed herein provides a novel approach for treating neurodegenerative diseases such as ALS, Parkinson's disease, and Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small nucleic acid targeting the PIKfyve gene, a pharmaceutical composition 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 gradual loss of motor neurons, progressive muscle weakness and atrophy of the limbs and trunk muscles, thereby affecting the patient's movement, swallowing and breathing functions, and ultimately leading to death.

[0003] Based on genetics, ALS can be divided into familial ALS (FALS) and sporadic ALS (SALS). Familial ALS accounts for approximately 10%, while sporadic ALS accounts for the remaining 90%. FALS is inherited primarily in an autosomal dominant manner and is associated with multiple genetic abnormalities. Currently, over 30 ALS-related gene mutations have been identified, including C9orf72 (39.3%), SOD1 (12%-23.5%), TARDBP (5%), and FUS (4.1%). SALS typically has no family history, but most SALS patients still carry gene mutations associated with the onset of ALS.

[0004] The pathogenesis of ALS is still unclear. Under current medical technology conditions, ALS is still an incurable disease. We can only use various methods to delay the progression of the disease as much as possible and thus prolong the patient's survival.

[0005] Approved drugs for the treatment of ALS in my country include riluzole, edaravone, and tofersen. Riluzole's mechanism of action is to inhibit glutamate release, reducing motor neuron damage caused by excessive glutamate. It also stabilizes the inactivated state of voltage-dependent sodium channels, reducing neuronal hyperexcitability. Clinically, riluzole has been shown to prolong patient survival by 2–3 months.

[0006] Unlike riluzole, edaravone works by inhibiting free radical production, reducing neuronal damage caused by excessive oxidation, and thereby slowing the progression of ALS. However, edaravone is only effective for a small subset of early-onset ALS cases. Long-term clinical practice has proven that the efficacy of riluzole and edaravone is clearly unsatisfactory in actual clinical practice, prompting researchers to explore new avenues through the lens of gene therapy.

[0007] Tofersen, an ASO drug targeting the SOD1 gene, was approved for marketing in my country on October 8, 2024. Its mechanism of action is to reduce SOD1 protein synthesis and the accumulation of toxic SOD1 proteins. In a Phase III trial for amyotrophic lateral sclerosis (ALSFSRS-R) treatment, intrathecal tofersen did not meet the primary endpoint (ALSFSRS-R total score). Approximately 6.7% of patients experienced serious neurological adverse events, including myelitis, aseptic meningitis, optic disc edema, and lumbar neuritis. However, tofersen reduced plasma neurofilament light chain (a biomarker of neurodegeneration) levels in patients. Consequently, the FDA ultimately approved tofersen despite not achieving a statistically significant improvement compared to placebo. However, this gene-targeted drug is only suitable for ALS patients with a specific SOD1 genotype, which accounts for only 2% of the ALS patient population. The majority of patients remain in urgent need of broad-spectrum, safe, and effective treatments.

[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 therapeutic drugs. Studies have shown that pharmacological inhibition of 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 the body, thereby effectively clearing misfolded proteins accumulated in cells. The study also found that PIKfyve inhibitors can prolong the survival period of various forms of ALS motor neurons, and are expected to become an effective treatment for various forms of ALS.

[0009] To date, there is no effective PIKfyve inhibitor available for the treatment of neurodegenerative diseases such as ALS. There is an urgent need to develop a safe and effective PIKfyve inhibitor to alleviate the symptoms of patients with neurodegenerative diseases such as ALS, restore motor function, and thereby improve their quality of life or prolong their survival. Summary of the Invention

[0010] In response to the shortcomings of the existing technology, the present invention aims to develop a safe and effective PIKfyve small nucleic acid inhibitor by rationally designing and synthesizing small nucleic acid molecules targeting the PIKfyve gene and using them for in vitro and in vivo verification.

[0011] In one aspect, the present invention provides a small nucleic acid targeting the PIKfyve gene, comprising any one or more nucleotide sequences selected from SEQ ID NOs: 1 to 128, SEQ ID NO: 130, SEQ ID NOs: 132 to 135, and SEQ ID NOs: 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-methylcytosine deoxyribonucleotide; m 5 cm stands for 2'-O-(2-methoxyethyl)-5-methylcytosine ribonucleotide; x m (italics) denote 2'-O-(2-methoxyethyl)-modified ribonucleotides (x = a, g, c, or u); * denotes a nucleotide in which a sulfur atom replaces one of the non-bridging oxygen atoms in the phosphodiester bond.

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

[0019] The nucleotide sequence shown in SEQ ID NO: 139 is , the nucleotide sequence shown in SEQ ID NO: 140 is ; Wherein, T represents thymidine deoxynucleotide; G represents guanine deoxynucleotide; A represents adenine deoxynucleotide; u represents uracil ribonucleotide; g represents guanine ribonucleotide; c represents cytosine ribonucleotide; a represents adenine 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 comprising the nucleotide sequence shown in SEQ ID NO: 141, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 142.

[0021] The nucleotide sequence shown in SEQ ID NO: 141 is) , the nucleotide sequence shown in SEQ ID NO: 142 is) ; Wherein, 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; x m represents a 2'-O-methyl modified ribonucleotide (x = a, g, c, or u); * indicates that the nucleotide has a sulfur atom replacing one of the non-bridging oxygen atoms in the phosphodiester bond; X f represents a 2'-fluoro modified deoxyribonucleotide (X = A, G, C or U); (C n x) represents a 2'-n carbon atom n-alkyl-modified ribonucleotide (x = a, g, c or u), with n = 8 to 25; (VPx) represents a 5'-E-vinyl phosphate-modified ribonucleotide (x = a, g, c or u).

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

[0023] In one or more embodiments, the neurodegenerative disease is selected from one or more of amyotrophic lateral sclerosis, Parkinson's disease, 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 embodiment herein, the method comprising synthesizing nucleotide monomers into a 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 embodiment herein, and a pharmaceutically acceptable carrier.

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

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

[0030] 1. The different types of modifications contained in the small nucleic acid molecules significantly enhance their stability, and they can still achieve an extremely low degradation rate after 24 hours of in vitro serum treatment.

[0031] 2. In vitro, the relative expression level of PIKfyve mRNA at the cellular level can be significantly reduced at a transfection concentration of nM, and the axon length of neuronal cells can be significantly increased.

[0032] 3. It can significantly improve the physical functions and survival conditions of mice with amyotrophic lateral sclerosis in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of serum stability tests of ASO and siRNA molecules are shown in Table 1. Figure 1 A in the table is the serum stability test result of the ASO molecule; Figure 1 B in the figure is the result of serum stability test of siRNA molecules.

[0034] Figure 2 It is the relative mRNA expression level of PIKfyve gene in DRG cells after knockdown using ASO-7 or siRNA-60' molecules. Figure 2A in the figure represents the relative mRNA expression level of PIKfyve gene in DRG cells after knockdown by ASO-7; Figure 2 Panel B shows the relative mRNA expression level of the PIKfyve gene in DRG cells after knockdown with siRNA-60' molecules.

[0035] Figure 3 This is the immunofluorescence result of the effect of ASO-7 on axon growth after transfection of DRG cells. Figure 3 A in the figure is the observation result of the blank cell group; Figure 3 B in the figure is the observation result of the positive control group; Figure 3 Panel C shows the observation results of the ASO-7 group.

[0036] Figure 4 is the proportion of axons of DRG cells in each group in different length ranges.

[0037] Figure 5 is the mean axon length of DRG cells in each group. DETAILED DESCRIPTION

[0038] Example 1: Sequence design and synthesis of small nucleic acids

[0039] (1) Sequence design of small nucleic acids

[0040] The PIKfyve gene sequence (Gene ID: 200576) was obtained from the NCBI database. siRNAs and ASOs targeting the PIKfyve gene were designed, avoiding the 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), and sequences near the start codon. These siRNAs and ASOs were then modified. The sequences of the siRNAs targeting the PIKfyve gene, the ASOs containing these modifications, and the siRNAs containing these modifications are shown in Tables 1, 2, and 3, respectively.

[0041] Table 1: siRNA sequences targeting the PIKfyve gene

[0042]

[0043] Table 2: ASO sequences containing modifications targeting the PIKfyve gene

[0044]

[0045] Table 3: siRNA sequences containing modifications targeting the PIKfyve gene

[0046]

[0047] Wherein, T represents thymidine deoxynucleotide; G represents guanine deoxynucleotide; C represents cytosine deoxynucleotide; A represents adenine deoxynucleotide; u represents uracil ribonucleotide; g represents guanine ribonucleotide; c represents cytosine ribonucleotide; a represents adenine ribonucleotide; x represents thymidine deoxynucleotide; m represents a 2'-O-methyl modified ribonucleotide (x = a, g, c, or u); m5C represents a 5-methylcytosine deoxyribonucleotide; m5cm stands for 2'-O-(2-methoxyethyl)-5-methylcytosine ribonucleotide; x m (italics) represent 2'-O-(2-methoxyethyl) modified ribonucleotides (x = a, g, c, or u); * represents a nucleotide with a sulfur atom replacing a non-bridging oxygen atom in the phosphodiester bond (i.e., P=S instead of P=O); X f represents a 2'-fluoro modified deoxyribonucleotide (X = A, G, C or U); (C n (x) represents a 2'-n carbon atom n-alkyl-modified ribonucleotide (x = a, g, c, or u), with n = 8-25; (VPx) represents a 5'-E-vinyl phosphate-modified ribonucleotide (x = a, g, c, or u). Some ASOs in Table 2 contain 5-methylcytosine deoxyribonucleotides (C), 2'-O-(2-methoxyethyl)-5-methylcytosine ribonucleotides, 2'-O-methyl-modified ribonucleotides (a, g, c, or u), and / or 2'-O-(2-methoxyethyl)-modified ribonucleotides (a, g, c, or u); some siRNAs in Table 3 contain thymidine deoxyribonucleotide monomers (T) and / or 2'-fluoro-modified deoxyribonucleotides (A, G, C, or U).

[0048] (2) Synthesis of small nucleic acids

[0049] ASO and siRNA molecules are synthesized using the phosphoramidite triester method, which involves repeating a five-step process of "deprotection - activation - coupling - capping - oxidation." After each cycle, the oligonucleotide chain is extended by one nucleotide, ultimately yielding crude ASO and siRNA products of the target length. The deprotection reagent is TCA Deblock, the activation agent 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.

[0050] The crude ASO product is deprotected and purified to obtain a crude ASO molecule; the crude siRNA product is deprotected, annealed, and purified to obtain a crude siRNA molecule. The crude product is purified using HPLC.

[0051] Purification materials: anion exchange column Diamond Q Mustang (purchased from Boglon) and eluent (a mixture of solution A and solution B in different proportions, 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).

[0052] Purification procedure: elution with 5 column volumes (CV) of solution A; elution with 50 CV of eluent (containing 0%-100% solution B); and elution with 5 CV of solution B. The target elution peak was desalted using a G25M column (purchased from Boglund). The desalted product was concentrated by ultrafiltration to obtain the purified product, which was lyophilized, vacuum-packed, and stored at -20°C until use.

[0053] Example 2: Verification of the effect of small nucleic acids at the cellular level

[0054] This example uses the siRNA and ASO constructed in Table 1 and Table 2 in Example 1 to verify their effects at the cellular level.

[0055] HaCat cells were cultured in a medium consisting of 90% DMEM + 10% FBS + 1% PS. When cells reached 90% confluence, they were seeded into six-well plates and cultured for 24 hours. ASOs and siRNAs were pre-diluted in DMEM to a concentration of 100 μM. Following incubation, cells were transfected with various concentrations of ASO (50 nM, 1000 nM, or 5000 nM) or siRNA (30 nM or 100 nM) and cultured further. Cells without ASO or siRNA served as controls. 48 hours after transfection, total RNA was extracted using Trizol, and PIKfyve mRNA expression in each well was measured by RT-qPCR relative to the control.

[0056] The relative expression levels of PIKfyve mRNA in HaCat cells are shown in Tables 4 and 5. At transfection concentrations of 30 nM and 100 nM, all siRNAs, except siRNA-6, siRNA-31, and siRNA-33, significantly knocked down PIKfyve mRNA expression. Among them, siRNA-11, siRNA-25, siRNA-40, and siRNA-60 showed better knockdown effects. ASOs at transfection concentrations of 50 nM, 1000 nM, and 5000 nM significantly knocked down PIKfyve mRNA expression. Among them, ASO-1, ASO-3, ASO-4, and ASO-7 showed better effects. Therefore, at the cellular level, the siRNAs and ASOs targeting the PIKfyve gene listed in Tables 1 and 2 are generally able to significantly knock down PIKfyve gene expression.

[0057] Table 4: Knockdown effect of siRNA on PIKfyve mRNA expression level

[0058]

[0059] Table 5: ASO knockdown effect on PIKfyve mRNA expression level

[0060]

[0061] Example 3: Serum stability test of small nucleic acids

[0062] Generally, siRNA and ASO degrade rapidly in the blood. According to references (doi:10.1038 / mt.2009.91, doi: 10.4062 / biomolther.2023.001), the half-life of unmodified siRNA and ASO in the blood is only a few minutes. After reasonable modification, their half-life can be effectively extended.

[0063] In this example, the effects of different modification schemes on the stability of siRNA and ASO were tested by serum enzymatic hydrolysis experiments. 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 unmodified siRNA-11, siRNA-25, siRNA-40, and siRNA-60 in Table 1, respectively) were selected.

[0064] 800 ng of each purified ASO was mixed with 10% fetal bovine serum and incubated in a 37°C incubator for 24 hours. The resulting samples were subjected to 2% agarose gel electrophoresis at 140V for 8 minutes. After the electrophoresis, the gel was visualized using a UV gel imager. Figure 1 As shown in A, after 24 h of serum co-incubation, no bands were visible 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 higher stability.

[0065] 800 ng of each purified siRNA was mixed with 10% fetal bovine serum and incubated in a 37°C incubator for 24 hours. The resulting samples were subjected to 2% agarose gel electrophoresis at 140V for 8 minutes. After electrophoresis, the gel was visualized using a UV gel imager. Figure 1 As shown in Figure B, after 24 hours of serum co-incubation, no bands were visible in the samples of siRNA-11' and siRNA-25', while obvious bands were seen in the samples of siRNA-40' and siRNA-60' at 0 hours or 24 hours, indicating that siRNA-40' and siRNA-60' were more stable.

[0066] Example 4: Growth-promoting effect of small nucleic acids on dorsal root ganglion neurons

[0067] This example uses ASO-7 and siRNA-60' prepared in Example 1 to study their effects on dorsal root ganglion (DRG) neurons.

[0068] (1) Acquisition and culture of primary DRG cells

[0069] Healthy C56BL / 6 mice were sacrificed by cervical dislocation. The epidermis was disinfected with 75% ethanol, and DRG cells were dissected from the spinal cord and placed in a 60 mm dish filled with pre-chilled HANKS solution. The HANKS solution was aspirated, and the DRG cells were washed three times with pre-chilled HANKS solution. Then, 2 mg / mL collagenase I was added and digested at 37°C for 1–2 h. Then, 2 mL of 0.25% trypsin was added and digestion continued at 37°C until no tissue fragments were visible. Finally, the digestion was terminated by dilution with DMEM supplemented with FBS to 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, discard the supernatant, take the cell pellet and resuspend it with 5%-15% BSA, centrifuge it again at 900 rpm for 5 minutes, discard the supernatant, resuspend the cells with complete medium containing neurobasal-A, and inoculate them into 12-well plates for culture to obtain DRG primary cells. Replace the medium with complete medium containing neurobasal-A the next day and set aside.

[0070] (2) Determination of PIKfyve gene expression level

[0071] When the DRG cells reached an appropriate confluence, they were seeded into six-well plates and cultured for 24 hours. ASO-7 and siRNA-60 were pre-diluted in DMEM medium to a concentration of 100 μM. Following incubation, cells in each well were transfected with varying concentrations of ASO (50 nM, 1000 nM, or 5000 nM) or siRNA (30 nM or 100 nM), and the cells were cultured further. Cells without ASO or siRNA served as controls. Forty-eight hours after transfection, total RNA was extracted using Trizol, and the relative expression level of PIKfyve mRNA in each well was determined by RT-qPCR.

[0072] The relative expression level of PIKfyve mRNA in DRG cells was detected as follows Figure 2 As shown in the figure, compared with the control group, ASO-7 can achieve a knockdown effect of about 50% at a transfection concentration of 1000nM, and the knockdown effect is further increased to about 60% at a transfection concentration of 5000nM, while no obvious knockdown effect was observed at a transfection concentration of 50nM ( Figure 2 , A); siRNA-60' can achieve a knockdown effect of about 40% at a transfection concentration of 30nM, and the knockdown effect is further increased to about 60% at a transfection concentration of 100nM ( Figure 2 , B). It can be seen that for DRG cells, both ASO and siRNA targeting the PIKfyve gene can significantly knock down the expression of the PIKfyve gene, and the knockdown effect has a certain degree of dose dependence.

[0073] (3) Determination of neuronal axon length

[0074] For the cells transfected with 1000 nM ASO in Example (2), a blank cell group (no-treatment control) and a positive control group (positive control-tofersen) were also set up; wherein, the blank cell group did not add ASO or siRNA, and the positive control group added 1000 nM Tofersen instead of ASO-7; 48 hours after transfection, the cells were fixed with 4% paraformaldehyde for 10 to 30 minutes, and then immunofluorescence staining was performed to facilitate the statistical analysis of the changes in neuronal axon length after PIKfyve target knockdown, wherein the primary antibody was a β-tubulin antibody and the secondary antibody was a fluorescently labeled antibody.

[0075] Each group of cells was observed under a fluorescence microscope. Figure 3For each cell hole observed, 6 fields of view were randomly photographed, the axon lengths of neurons in the fields of view were counted, and the axons were grouped by length intervals to obtain the following: Figure 4 The analysis results are shown. In the blank cell group (no-treatment control), the axons with a length of 20-40 μm were the most numerous; in the positive control group (positive control-tofersen), the axons with a length of 40-60 μm were the most numerous. Compared with the blank cell group, the proportion of axons with a length of 40-60 μm, 60-80 μm, 80-100 μm and 100-120 μm in the ASO-7 group (ASO-PIKfyve) increased; in addition, the mean value of the axon length of each group was calculated, as shown below. Figure 5 The results shown. Figure 4 and Figure 5 It can be seen that after ASO-7 reduced the expression level of PIKfyve, it significantly promoted the growth of axons of dorsal root ganglion neurons. When the transfection concentration was 1000nM, its effect in promoting neuronal axon growth was no less than that of Tofersen.

[0076] Example 4: Application of ASO in Amyotrophic Lateral Sclerosis Model Mice

[0077] This example uses ASO-7, constructed in Example 1, to study its effects on ALS model mice. The ALS model mice are Tg(Prnp-TARDBP*A315T)95Balo / J male mice. The Tg(Prnp-TARDBP*A315T)95Balo / J male mice and C57BL / 6 mice used in this example are commercially available.

[0078] (1) Preparation of preparations

[0079] 100 mg (6.7 mg / mL) ASO-7 or 100 mg tofersen; 0.21 mg / mL calcium chloride dihydrate; 0.11 mg / mL sodium phosphate dibasic; 0.16 mg / mL magnesium chloride hexahydrate; 0.03 mg / mL sodium phosphate monobasic; 0.22 mg / mL potassium chloride; 8.77 mg / mL sodium chloride; make up to volume with sterile water for injection and adjust the pH to 7.2 (pH 6.7 to 7.7 is acceptable).

[0080] (2) Determination of the effects of ASO-7 on amyotrophic lateral sclerosis model mice

[0081] The mice were grouped according to Table 6 below, wherein the mice in the positive control group, low-dose group, and high-dose group were amyotrophic lateral sclerosis model mice, and the mice in the negative control group were C57BL / 6 mice; according to the recommended dose of Tofersen in the drug instructions, each mouse in the positive control group was injected with 0.033 mg of Tofersen.

[0082] Table 6: Experimental mice grouping

[0083]

[0084] All mice were housed in a standardized animal room. When the mice were 41 days old, they were weighed and given the first dose by intrathecal injection. The second dose was given when the mice were 61 days old. The survival rate and body weight of the surviving mice were counted, and their performance in the righting test and rotarod test was measured.

[0085] The survival results of mice are shown in Table 7 below. The survival rate of mice in the positive control and high-dose groups was 50%, higher than the 12.5% ​​in the low-dose group. 25% of the mice in the high-dose group gained weight, while none of the mice in the positive control and low-dose groups gained weight. This indicates that ASO-7 improves the survival of mice with ALS.

[0086] Table 7: Statistics of mouse survival rate and body weight

[0087]

[0088] The results of the mouse righting test are shown in Table 8 below. The average righting time for mice in the high-dose and low-dose groups was significantly shorter than that of the positive control group. The average righting time for the high-dose group was 0.1 seconds, significantly shorter than that of the low-dose group and the same as that of the negative control group. This indicates that ASO-7 shortened the righting time of mice with ALS, significantly improving their physical function and enabling them to right themselves quickly.

[0089] Table 8: Results of mouse righting test

[0090]

[0091] The results of the rotarod test in mice are shown in Table 9 below. The low-dose group showed a shorter time to fall than the positive control group, while the high-dose group showed a longer time to fall than the positive control group. This indicates that the high-dose ASO-7 prolonged the time that mice with ALS could remain on the rotarod without falling, significantly improving their physical function.

[0092] Table 9: Results of the mouse rotarod test

[0093]

[0094] In summary, the small nucleic acids targeting the PIKfyve gene provided by the present invention can significantly reduce PIKfyve mRNA expression levels at the cellular level at a nanomolar transfection concentration in vitro and significantly increase axon length in neuronal cells. In vivo, they can significantly improve the physical function and survival of mice with amyotrophic lateral sclerosis (ALS). Therefore, they can be used to treat PIKfyve-mediated diseases or to prepare medicaments for treating PIKfyve-mediated diseases, including but not limited to ALS, Parkinson's disease, and Alzheimer's disease.

Claims

1. A small nucleic acid targeting the PIKfyve gene, characterized in that: The nucleotide sequence of the small nucleic acid is shown in SEQ ID NO:

135.

2. Use of the small nucleic acid targeting the PIKfyve gene as claimed in claim 1 in the preparation of a drug for treating amyotrophic lateral sclerosis.

3. A method for preparing the small nucleic acid targeting the PIKfyve gene as claimed in claim 1, wherein the method is a phosphoramidite triester method.

4. 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.

5. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating amyotrophic lateral sclerosis.

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