Tat-p01 and its use in treating neurodegenerative disease amyotrophic lateral sclerosis

Abstract

The present application relates to TAT-PO1 and its application in treating amyotrophic lateral sclerosis (ALS). The polypeptide connects TAT sequence (SEQ ID NO. 1) and PO1 sequence (SEQ ID NO. 2) through amino hexanoic acid (AHX), and its amino acid sequence is YGRKKRRQRRR{AHX}RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE. TAT-PO1 can competitively block the interaction between PGAM5 and OMA1, thereby interfering with the pathological mechanism of ALS. The polypeptide TAT-PO1 of the present application can be prepared into a drug, and directly targets the central nervous system through intrathecal injection or intravenous administration, thereby providing a new strategy for the treatment of ALS. In addition, the design idea can also be applied to other neurodegenerative diseases caused by abnormal protein interaction, thereby providing a technical paradigm for the development of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to TAT-PO1 and its application in the treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the progressive degeneration of motor neurons. Patients experience muscle atrophy and respiratory failure, resulting in a median survival of only 3-5 years. Currently, only a few drugs are approved globally for the treatment of ALS, including riluzole, edaravone, and AMX0035, but these drugs generally suffer from limited efficacy and unclear target sites.

[0003] Currently, clinical treatments for ALS mainly include small molecule neuroprotective agents, anti-oxidative stress drugs, gene therapy, and supportive therapy. Among FDA-approved drugs, riluzole, by inhibiting glutamate release and blocking sodium channels, can prolong patient survival by approximately 3-6 months, but it does not significantly improve motor function decline. Edaravone, as a free radical scavenger, can reduce oxidative damage, but it is only effective for early-stage ALS patients and requires repeated intravenous infusions. The combination drug of sodium chlorate and taurine glycol (AMX0035), approved in 2022, exerts a neuroprotective effect by regulating the endoplasmic reticulum-mitochondrial stress pathway, delaying functional decline by approximately 6.5 months, but it still cannot reverse neuronal loss. In the field of gene therapy, antisense oligonucleotides can target and silence SOD1 mutant genes, but this is only applicable to specific ALS subtypes with specific gene mutations and requires intrathecal injection. While supportive therapy can improve symptoms and quality of life, it cannot alter the disease course. Overall, existing therapies suffer from limitations such as limited target sites, narrow scope of action, and short-lived efficacy, and have not yet achieved the goal of precision treatment for ALS.

[0004] In particular, the limited efficacy of existing drugs is due to unclear drug targets, and there is a lack of precise intervention tools targeting the core pathological signaling pathways. While currently used drugs (such as riluzole and edaravone) can slow disease progression, their mechanisms of action do not focus on the core pathological processes of the disease. For example, riluzole only exerts limited neuroprotective effects by inhibiting glutamate release and cannot reverse the degeneration of motor neurons driven by mitochondrial dysfunction, resulting in weak clinical efficacy. Furthermore, existing treatment strategies are mostly based on broad antioxidant or anti-inflammatory mechanisms, lacking targeted design against ALS-specific molecular pathways, leading to unstable drug efficacy and significant side effects. These technical problems stem from the complexity of the ALS pathological mechanism and the limitations of existing research tools. The pathogenesis of ALS exhibits a multi-factor synergistic effect, with its core pathological processes involving dynamic cross-linking at multiple levels, including protein homeostasis imbalance, mitochondrial dysfunction, oxidative stress, and axonal transport abnormalities. However, current drug development is mostly based on single-target or linear signaling pathway assumptions, making it difficult to cover the complex cascade effects in disease progression. Therefore, it is urgent to clarify new mechanisms of ALS disease progression, provide new targets for precision intervention and treatment of ALS patients, and develop reliable ALS treatment methods.

[0005] Integrated stress response (ISR) is an adaptive mechanism by which cells cope with various stressors. However, recent studies have found that when it specifically occurs in mitochondria (i.e., mitochondrial ISR, mtISR) and is chronically activated, it can lead to maladaptive responses, mediating neuronal dysfunction and degeneration. In ALS, mitochondrial dysfunction plays a central pathological role. Therefore, targeting this pathological chronic activation of mtISR may be a promising new strategy for ALS intervention. mtISR is mainly mediated by the OMA1-DELE1-HRI signaling axis, which mediates cellular transcriptional and translational reprogramming by phosphorylating the downstream protein eIF2α and inducing the expression of the transcription factor ATF4. Current research has confirmed the presence of aberrant mtISR activation in various ALS subtypes, but intervention strategies targeting this site still require further breakthroughs. Summary of the Invention

[0006] To address the aforementioned technical problems in existing technologies, this invention discovers an interaction between PGAM5 and the upstream core protease OMA1 of mtISR, and designs an interaction-blocking peptide, TAT-PO1, fused with a transmembrane peptide sequence, based on the interaction between PGAM5 and OMA1. This peptide competitively blocks the interaction between PGAM5 and OMA1, effectively inhibiting mtISR activation in ALS models, thereby reducing the loss of neuromuscular junctions in organoid models derived from ALS patients. This invention may provide a new strategy for clinical intervention and treatment of ALS.

[0007] The technical solution of the present invention is as follows:

[0008] The first objective of this invention is to provide a polypeptide TAT-PO1 that competitively blocks the interaction between PGAM5 and OMA1, the polypeptide consisting of aminocaproic acid linked to the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, wherein the polypeptide TAT-PO1 consists of the TAT sequence shown in SEQ ID NO.1 - aminocaproic acid - PO1 sequence shown in SEQ ID NO.2 from the N-terminus to the C-terminus.

[0009] The amino acid sequence shown in SEQ ID NO.1 is YGRKKRRQRRR.

[0010] The amino acid sequence shown in SEQ ID NO.2 is RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE.

[0011] Aminocaproic acid (AHX) is an organic compound with the chemical formula C6H. 13 NO2 is an aliphatic amino acid containing six carbon atoms. Its molecular structure includes an amino group (-NH2) and a carboxyl group (-COOH), and it serves as a linker for polypeptide modification.

[0012] That is, the amino acid sequence of the polypeptide TAT-PO1 is YGRKKRRQRRR{AHX}RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE.

[0013] A second object of the present invention is to provide a medicament for treating amyotrophic lateral sclerosis (ALS) comprising the aforementioned polypeptide TAT-PO1, or a nucleotide sequence encoding the aforementioned polypeptide TAT-PO1.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients for preparing a dosage form suitable for intrathecal or intravenous administration.

[0015] Furthermore, the drug is administered via intrathecal injection or intravenous injection.

[0016] A third object of the present invention is to provide the use of peptide TAT-PO1 or a dosage form thereof suitable for intrathecal injection in the preparation of a medicament for the treatment of amyotrophic lateral sclerosis (ALS).

[0017] The beneficial effects of this invention are as follows:

[0018] The peptide TAT-PO1 provided by this invention can be formulated into a drug that can be administered via intrathecal injection or intravenous administration to directly target the central nervous system, making it suitable for ALS patients with abnormal activation of the mitochondrial stress pathway. TAT-PO1 can be used alone or in combination with existing neuroprotective drugs to delay motor neuron degeneration and neuromuscular junction loss. Furthermore, its design concept can be extended to other neurodegenerative diseases caused by abnormal protein interactions, providing a technological paradigm for related drug development. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the design screening and results of the present invention, wherein:

[0020] A represents the PAGM5 protein sequence and its domain regions. In this invention, the PAGM5 protein sequence is divided into three segments: ΔP, P1, and P2.

[0021] B indicates that the GST pull-down experiment suggests that the P1 region of PGAM5 interacts with the OMA1 protein;

[0022] C is a structural diagram that further divides the P1 region of PGAM5 into three segments, named PO1, PO2, and PO3 in sequence;

[0023] D represents the GST pull-down experiment, which verified that PO1 is the main region in P1 that interacts with the OMA1 protein.

[0024] Figure 2 This is a schematic diagram illustrating the interference effect of the small molecule blocking peptide TAT-PO1 of the present invention on the interaction between PGAM5 and OMA1.

[0025] Figure 3 The diagram illustrates the results of various methods used to verify the binding ability of the small molecule peptide of the present invention to human OMA1 protein, wherein:

[0026] A is a surface plasmon resonance experiment to verify the binding ability of the small molecule polypeptide TAT-PO1 of the present invention to human OMA1 protein;

[0027] B represents the structure of the polypeptide sequence simulated by AlphaFold;

[0028] C represents the binding mode of a small molecule polypeptide to the human OMA1 protein, simulated by molecular docking.

[0029] Figure 4 This diagram illustrates the regulatory effects of the small molecule blocking peptide TAT-PO1 of the present invention on mitochondrial integration stress response (mtISR) in different SH-SY5Y cell models, wherein:

[0030] A shows the results and statistical graph of TAT-PO1 downregulating mtISR levels in a VCP R159H point mutation cell model;

[0031] B shows the results and statistical graph of TAT-PO1 downregulating mtISR levels in the SH-SY5Y cell model overexpressing PGAM5.

[0032] Figure 5 This is a schematic diagram illustrating the regulation of mitochondrial integration stress response (mtISR) by the small molecule blocking peptide TAT-PO1 of the present invention, which depends on the OMA1 protein, wherein:

[0033] A is a schematic diagram illustrating the successful construction of the OMA1 gene knockout SH-SY5Y cell line as verified by Western blotting experiment.

[0034] B is a schematic diagram and statistical graph showing the results of the immunoblotting experiment verifying that the regulation of mtISR by TAT-PO1 depends on the OMA1 protein.

[0035] Figure 6 This is a schematic diagram illustrating the effect of the small molecule blocking peptide TAT-PO1 of the present invention in slowing down the degeneration of motor neurons and the loss of neuromuscular junctions in ALS iPSCs, wherein:

[0036] A shows the immunohistochemical staining results of NeuN and ChAT in hSCOs from the control group on Day 28.

[0037] B represents the injection of an acetylcholine probe into hSCOs and the detection of acetylcholine levels.

[0038] C represents the immunohistochemical staining results of NeuN and ChAT in hSCOs of the control group and the sporadic ALS group (ALS401);

[0039] D is a schematic diagram of the co-culture of human spinal cord organoids (hSCOs) and differentiated C2C12 cells (as mouse skeletal muscle cells mSkM) to obtain hSCOs-mSkM co-cultures;

[0040] E is a schematic diagram and statistical graph illustrating the salvage effect of TAT-PO1 on ALS-induced motor neuron degeneration and neuromuscular junction loss, verified by immunofluorescence.

[0041] Figure 7 This diagram illustrates the results of improving neuromuscular junction and other pathological phenotypes, as well as motor phenotypes, in ALS model mice (TDP-43 A315T transgenic mice) after delivery of the small molecule blocking peptide TAT-PO1 of the present invention via an adeno-associated virus (AAV) vector and intrathecal injection.

[0042] A shows a schematic diagram and fluorescence verification image of intrathecal injection of TAT-PO1;

[0043] B shows the results of immunofluorescence verification of TAT-PO1 inhibition of mtISR activation in the TDP-43 (A315T) mouse model and its statistical graph;

[0044] C is a schematic diagram and statistical analysis of the results of immunofluorescence verification of TAT-PO1 in improving neuromuscular junction and motor neuron degeneration;

[0045] D is a schematic diagram and statistical graph showing the results of the foot imprint experiment verifying the improvement of gait of TAT-PO1 on TDP-43 (A315T) model mice;

[0046] E is a schematic diagram illustrating the results of the claw gripping force test verifying the improvement of claw gripping force of TAT-PO1 on TDP-43 (A315T) model mice. Detailed Implementation

[0047] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0048] Example 1: Design and Screening of Small Molecule Peptides

[0049] like Figure 1 A in the diagram shows the PAGM5 protein sequence and its domain regions. This invention first divides the PGAM5 (Phosphoglycerate mutase family member 5) protein sequence into three segments, named ΔP, P1, and P2. The full-length PGAM5 protein (FL) and fragments retaining some domains (ΔP, P1, P2) are constructed into the vector pGEX-6P-1 (GST vector), forming four plasmid vectors: GST-PGAM5-FL, GST-PGAM5-ΔP, GST-PGAM5-P1, and GST-PGAM5-P2. The full-length OMA1 sequence is constructed into the pET-28a(+) (HIS) vector, forming His-OMA1, for subsequent GST pull-down experiments (GST fusion protein sedimentation technology). Figure 1 (B in the middle).

[0050] 1.1 The plasmid construction steps are as follows:

[0051] (1) Extraction of total RNA from cells: Collect the cell pellet, add 1 mL TRizol (Sigma, Cat# T9424), mix well, and lyse on ice for 6-10 minutes. After complete lysis, add 200 μL chloroform, mix by inverting, incubate at 4°C for 10-15 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol to precipitate the RNA, mix well, incubate on ice for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes at 4°C. Discard the supernatant, wash the pellet with 75% ethanol, gently tap the pellet to resuspend it, incubate for 1-2 minutes, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the supernatant and repeat the centrifugation once. Air dry the pellet in a clean bench for 5-10 minutes, add 30 μL DEPC water to dissolve the RNA, measure the concentration with a micro spectrophotometer, and store at -80°C for later use.

[0052] (2) cDNA library construction: Take an appropriate amount of the RNA obtained above and reverse transcribe it according to the following two-step method (Vazyme, Cat# R323-01) to obtain cDNA. Store at -20°C for a long time. The specific steps are as follows:

[0053] Step 1: Preparation of the reaction system: Add 500 ng template RNA; add 4 μL of 4x gDNA wiper mix; finally, add nuclease-free water to the reaction system to bring the total volume to 16 μL. Heat the mixture at 42°C for 2 minutes to complete the removal of genomic DNA.

[0054] Second step reaction system preparation: Take 16 μL of the total system from the previous step, add 4 μL of 5x HiScript IIIqRT SuperMix, mix well and set aside.

[0055] Reverse transcription procedure: First, react at 37°C for 15 minutes to complete reverse transcription; then heat at 85°C for 5 seconds to inactivate the enzyme.

[0056] Storage instructions: After the reaction is complete, store the product at 4°C to maintain the enzyme inactive state.

[0057] (3) Vector construction: Collect an appropriate amount of SH-SY5Y cells, extract RNA and reverse transcribe it to obtain a cDNA library according to the above steps, find the CDS sequence fragment of the target gene to be extracted on the NCBI website, design primers, specifically copy and paste the gene CDS sequence into DNAMAN to form a new sequence file; select about 20 bp each upstream and downstream as initial primers, click the Analyse button on the top toolbar, click the PCR button, simulate whether the upstream and downstream primer sequences match and whether primer dimers will form, etc.; control the melting temperature (TM) value between 58-62℃. Construct the GST vector by digesting it with restriction endonucleases BamHI and EcoI, and homologously recombine it into the basic plasmid pGEX-6P-1; construct the His vector by digesting it with restriction endonucleases BamH1 and SalI, and ligating it into the basic plasmid pET-28a(+) with T4 ligase. The specific experimental steps are as follows:

[0058] The primers used are as follows:

[0059] The vector GST-PGAM5-FL was constructed. The forward sequence of the primers was GST-PGAM5-F: AGGGGCCCCTGGGATCCATGGCGTTCCGGCAGG (SEQ ID NO.4), and the reverse sequence was GST-PGAM5-R: TCGTCAGTCAGTCACGATTCAGGATCGAGTGATCTTG (SEQ ID NO.5).

[0060] The vector GST-PGAM5-ΔP was constructed. The forward sequence of the primers was GST-PGAM5-ΔP-F:AGGGGCCCCTGGGATCCATGGCGTTCCGGCAGG (SEQ ID NO.6), and the reverse sequence was GST-PGAM5-ΔP-R:TCGTCAGTCAGTCACGATTCACGTGGCCTTGGCTTTGTAG (SEQ ID NO.7).

[0061] The vector GST-PGAM5-P1 was constructed. The forward sequence of the primers was GST-PGAM5-P1-F:AGGGGCCCCTGGGATCCATGCGGCACATCTTCCTCATC (SEQ ID NO.8), and the reverse sequence was GST-PGAM5-P1-R:TCGTCAGTCAGTCACGATTCACTGCACAGCTTCCGGCT (SEQ ID NO.9).

[0062] The vector GST-PGAM5-P2 was constructed. The forward sequence of the primers was GST-PGAM5-P2-F:AGGGGCCCCTGGGATCCATGTATTACGAAGACGGAGCCC (SEQ ID NO.10), and the reverse sequence was GST-PGAM5-P2-R:TCGTCAGTCAGTCACGATTCAGGATCGAGTGATCTTG (SEQ ID NO.11).

[0063] The vector GST-PGAM5-PO1 was constructed. The forward sequence of the primers was GST-PGAM5-PO1-F:CAGGCTGAATGAATCGTGACTGACTGACG (SEQ ID NO.12), and the reverse sequence was GST-PGAM5-PO1-R:CACGATTCATTCAGCCTGCTCCCGACC (SEQ ID NO.13).

[0064] The vector GST-PGAM5-PO2 was constructed. The forward sequence of the primers was GST-PGAM5-PO2-F:GGATCCATGCTCACTGGGCTCCGCCTG (SEQ ID NO.14), and the reverse sequence was GST-PGAM5-PO2-R:CCCAGTGAGCATGGATCCCAGGGGCCC (SEQ ID NO.15).

[0065] The vector GST-PGAM5-PO3 was constructed. The forward sequence of the primers was GST-PGAM5-PO3-F:GGATCCATGCACCTGCCAGGCGTCTGC (SEQ ID NO.16), and the reverse sequence was GST-PGAM5-PO3-R:TGGCAGGTGCATGGATCCCAGGGGCCCC (SEQ ID NO.17).

[0066] The vector His-OMA1 was constructed, with the forward sequence His-OMA1-F: GGATCCATGAGCTTCATCTGTGGAT (SEQ ID NO.18) and the reverse sequence His-OMA1-R: GTCGACACTGCCCGTTCTTTTCTCAAC (SEQ ID NO.19).

[0067] The vector His-PO1 was constructed. The forward sequence of the primers was His-PO1-F: ACAGCAAATGGGTCGCATGCGGCACATCTTCCTCA (SEQ ID NO.20), and the reverse sequence was His-PO1-R: GTGCGGCCGCAAGCTTTTCAGCCTGCTCCCGACC (SEQ ID NO.21).

[0068] PCR reaction conditions: 94℃ for 2 minutes (initial denaturation); 98℃ for 10 seconds (denaturation); 55-68℃ for 30 seconds (annealing); 68℃ for 10-15 seconds / kb (annealing-extension, 35 cycles); 68℃ for 5 minutes (final extension); 4℃ (Hold).

[0069] PCR product digestion system: PCR product 25 μL; enzyme 1 1 μL; enzyme 2 1 μL; Cutsmart / rCutsmart buffer (10x) 5 μL; double-distilled water 18 μL (total system 50 μL).

[0070] Vector digestion conditions: 2 μg vector; 1 μL enzyme 1; 1 μL enzyme 2; 5 μL rCutSmart buffer (10x); double-distilled water to a final volume of 50 μL. After digestion in a 37°C water bath for 1-2 hours, perform agarose gel electrophoresis and product purification.

[0071] Ligation reaction system: 1 μL vector, 4 μL target fragment; 1 μL T4 DNA ligase, 1 μL T4 DNA ligase buffer (10x) plus 1 μL, 3 μL double-distilled water; ligation at room temperature (recommended optimal temperature 16℃).

[0072] In vitro homologous recombination system: linearized vector X μL (calculated according to the instructions and vector concentration), insert fragment Y μL (calculated according to the instructions), 2x MultiF Seamless Assembly Mix (ABclonal, RK21020) plus 10 μL, double-distilled water to 20 μL; homologous recombination was performed after digesting the GST vector with BamHI and EcoRI.

[0073] The specific steps of the transformation experiment are as follows: First, mix 2 μL of the ligation product or homologous recombination product with 10 μL of DH5α competent cells (1:5 ratio); Step 1, place in a 4℃ ice box and incubate for 30 minutes; Step 2, transfer to a 42℃ water bath for 1 minute; Step 3, immediately return to the 4℃ ice box and incubate for 3 minutes; Step 4, add 600 μL of antibiotic-free LB medium; Step 5, incubate at 37℃ in a shaker for 1-2 hours; Step 6, take 400 μL of bacterial culture and spread it on a plate containing the corresponding antibiotic. Subsequent operations include: using a 20 μL pipette tip to pick single colonies and add them to 600 μL of LB medium containing the antibiotic, incubate at 37℃ with shaking for 6-8 hours, then take 200 μL of bacterial culture for sequencing, and amplify the target plasmid for the successfully sequenced strains.

[0074] 1.2 The experimental steps for GST pull-down are as follows:

[0075] Successfully obtained by following the above steps Figure 1 The GST-PGAM5-FL, GST-PGAM5-ΔP, GST-PGAM5-P1, GST-PGAM5-P2, and His-OMA1 vectors shown in B were used for subsequent GST pull-down experiments:

[0076] (1) Plasmid transformation: 200 ng plasmid was added to 10 μL of BL21 strain, placed on ice for 30 minutes, heat-shocked in a 42℃ water bath for 1 minute, placed on ice for 3 minutes, 600 μL of antibiotic-free LB medium was added, and shaken at 220 rpm for 30 minutes at 37℃. The mixture was then plated onto the corresponding antibiotic-resistant LB plates. GST vector was plated onto ampicillin plates, and His vector was plated onto kanamycin plates. The mixture was then incubated overnight at 37℃ for 16 hours.

[0077] (2) Single clone amplification: Inoculate into 13.5 mL of LB medium with the corresponding resistance, shake at 37°C and 180 rpm for about 10 hours until OD reaches 100%. 600 ≈ 0.6-0.8.

[0078] (3) Induction of protein expression: IPTG was added to the bacterial culture to a final concentration of 0.4 mM, and the fusion protein was induced to express for 6 hours at 28℃ and 180 rpm in a shaker.

[0079] (4) Lysis and sonication: Centrifuge the bacterial culture at 12,000 rpm, discard the supernatant, resuspend in PBS with 1% Triton X-100 and 1% protease inhibitor, and sonicate on ice for 2 seconds on, 9 seconds off, at 40% power until clear. Then centrifuge at 12,000 rpm for 10 min and collect the supernatant, which is the protein solution.

[0080] (5) Verification of expression: The concentration was measured using BCA, and an equal amount of protein supernatant was prepared for Western blot verification of expression.

[0081] (6) His-OMA1 pre-cleaning: Take 1,000 μg HIS protein and add 40 μL GST agarose beads, suspend at 4℃ for 3 hours, centrifuge at 3,000 rpm at 4℃ for 3 minutes, and take the supernatant.

[0082] (7) GST-pull down: Add an equal volume of His-OMA1 protein solution (approximately 150 μL) to each tube of GST protein. Then, add PBS and 1% protease inhibitor to each tube to make up the volume, and incubate overnight at 4°C for approximately 12 hours. The next day, add GST agarose beads and incubate at 4°C for 1 hour; centrifuge at 3,000 rpm for 3 minutes, wash with GST binding buffer, and centrifuge again. Repeat 5 times.

[0083] (8) Sample preparation, loading and gel running: Add 40 μL of GST binding buffer and 10 μL of loading buffer to the precipitate, then boil at 100℃ for 10 minutes; centrifuge at 6,000 rpm for 3 minutes, take the supernatant, divide it in half, prepare duplicate samples and run gels; the rest is the same as the WB experiment procedure.

[0084] 1.3 The specific steps of the WB process are as follows:

[0085] (1) Preparation of separating gel (taking 10% separating gel as an example): Take 4.0 mL of H2O, 3.3 mL of 30% Acr-Bic solution, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 100 μL of 10% ammonium persulfate, 100 μL of 10% SDS, and 4 μL of TEMED and mix them.

[0086] (2) After the separating gel solidifies, prepare the stacking gel (taking single-board stacking gel as an example): Take 2.1 mL of H2O, 0.5 mL of 30% Acr-Bic solution, 0.38 mL of 1.5 M Tris-HCl (pH 8.8), 30 μ L of 10% ammonium persulfate, 30 μ L of 10% SDS, and 3 μ L of TEMED and mix them.

[0087] (3) Mix the sample prepared by the above GST pull-down with the sample loading buffer, heat it in a metal bath at 100°C for 10 minutes, and collect the liquid on the tube wall after a short centrifugation after heating.

[0088] (4) Electrophoresis: After the prepared gel solidifies, fix it in the electrophoresis tank, and add the protein marker and the prepared sample into the gel wells according to the experimental order. Add an appropriate amount of electrophoresis buffer to the electrophoresis tank, adjust the voltage to 100V, and start electrophoresis. Stop electrophoresis when the bromophenol blue in the sample moves to the lower half of the separating gel.

[0089] (5) Transfer: Remove the gel from the glass plate and place it on three layers of filter paper with sponge padding. Cut an NC membrane of appropriate size to cover the gel, then stack three layers of filter paper and sponge. Fix the structure with a clamp and place it in the transfer tank. Add transfer solution and ice box. Adjust the constant current to 320 mA and the time to 70 minutes.

[0090] (6) Closure: After the transfer is completed, take out the NC membrane and immerse it completely in 5% skim milk. Incubate it on a shaker at room temperature for 1 hour.

[0091] (7) Primary antibody incubation: Prepare the corresponding indicator antibody in 5% skim milk at an appropriate ratio, completely immerse the cut NC membrane, and incubate overnight at 4°C.

[0092] (8) Secondary antibody incubation: Prepare the corresponding indicator antibody in 5% skim milk at an appropriate ratio. After the primary antibody incubation is completed, recover the antibody, rinse the NC membrane with TBST solution for 10 minutes, repeat three times, add the secondary antibody, incubate at room temperature for 1 hour, rinse with TBST three times again after the incubation is completed, and it can be used for subsequent development.

[0093] 1.4 The development steps using ECL chemiluminescence are as follows:

[0094] According to experimental requirements, equal volumes of solutions A and B of the ECL luminescent solution were mixed and reacted at room temperature in the dark for 1 minute. Then, the NC membrane was immersed in the mixture, gently agitated to ensure complete coverage, and reacted at room temperature in the dark for 1 minute. Finally, the NC membrane was placed in an imager for image acquisition.

[0095] GST pull-down experiments suggest that the P1 region of PGAM5 interacts with the OMA1 protein. Figure 1 (B in the diagram); Subsequently, we further divide the P1 region of PGAM5 into three structural segments, named PO1, PO2, and PO3 respectively. Figure 1 (C in the text); GST-PGAM5-PO1, GST-PGAM5-PO2, and GST-PGAM5-PO3 vectors were constructed using vector truncation. The primers for vector construction are described in section 1.1 of Example 1. Further GST pull-down experiments revealed that PO1 is the main region in P1 that interacts with the OMA1 protein (C in the text). Figure 1The sequence P1 is located at amino acids 98-196 of PGAM5, and the sequence PO1 is located at amino acids 98-129 of the PGAM5 protein. The base sequence corresponding to PO1 is: CGGCACATCTTCCTCATCAGGCATTCCCAGTACCACGTGGATGGCTCCCTGGAGAAGGACCGCACTCTGACCCCGCTGGGTCGGGAGCAGGCTGAA (SEQ ID NO.3); the encoded amino acid sequence is: RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE (SEQ ID NO.2).

[0096] Example 2: Verification of blocking peptide interference with PGAM5-OMA1 interaction

[0097] In Example 1, through GST pull-down experiments and structural partitioning, we have preliminarily determined that the PO1 region of PGAM5 is the key region for its interaction with the OMA1 protein. Figure 1 This discovery lays the foundation for further exploration of the functional properties of PO1. To further verify whether PO1 can competitively antagonize the interaction between PGAM5 and OMA1, we designed the experiment in Example 2 (…). Figure 2 In this experiment, the PO1 fragment was amplified by PCR, and then the His vector was constructed by digesting the His vector with BamHI / SalI and ligating it with T4 enzyme. GST pull-down experiments were performed simultaneously on PGAM5, PO1, and OMA1, following the same vector construction and experimental procedures. The results showed that the interaction between PGAM5 and OMA1 was significantly weakened after the addition of His-PO1, and this weakening effect was positively correlated with the dose of His-PO1. Figure 2 As shown, the binding degree of PGAM5 and OMA1 gradually decreased with the increase of His-PO1 concentration, further confirming the antagonistic effect of PO1 on their interaction.

[0098] Example 3: Verification of the binding ability of PO1 to human OMA1 protein using multiple methods

[0099] The GST pull-down experiment results in Example 2 showed that PO1 significantly attenuated the interaction between PGAM5 and OMA1 in a dose-dependent manner, confirming its competitive antagonistic effect. However, to gain a more comprehensive understanding of the interaction mechanism between PO1 and OMA1, we further employed multiple methods ( Figure 3 To verify the binding ability of PO1 to the human OMA1 protein, these methods include surface plasmon resonance assays (SPR). Figure 3In A), AlphaFold structure prediction ( Figure 3 (B) and molecular docking experiments ( Figure 3 (C in the text) to explore the functional characteristics of PO1 from different perspectives.

[0100] 3.1 The procedure for surface plasmon resonance (SPR) experiments is as follows:

[0101] His-OMA1 protein was obtained through transformation with BL21 strain and IPTG-induced expression, followed by purification to obtain His-OMA1 protein solution. The relevant vector construction and protein purification process are described in Example 1. Simultaneously, a TAT-PO1 peptide with the amino acid sequence YGRKKRRQRRR{AHX}RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE was synthesized by a company. This peptide was used for subsequent surface plasmon resonance (SPR) experiments, the specific steps of which are as follows:

[0102] (1) Instrument preparation and initialization: Turn on the Biacore X100 instrument, preheat to 25°C, rinse the system with deionized water and HBS-EP buffer to confirm that there are no air bubbles, insert the CM5 chip and initialize the instrument.

[0103] (2) Chip activation: Mix EDC and NHS solutions in a 1:1 ratio to prepare 100 μL of the solution at a flow rate of 10 μL / min to activate the carboxyl groups on the chip surface for 8 minutes.

[0104] (3) Ligand coupling: Sodium acetate buffer solution with pH 4.0 / 4.5 / 5.0 was injected sequentially. pH 4.0 is the best coupling condition for the curve. The protein sample and sodium acetate solution with pH 4.0 were injected again at a ratio of 1:20, at a flow rate of 10 μL / min, and the mixture was treated for 8 minutes.

[0105] (4) Add ethanolamine (1 M, pH 8.5) for blocking at a flow rate of 10 μL / min for 8 minutes.

[0106] (5) Inject HBS-EP buffer at a flow rate of 30 μL / min and process until the baseline is stable.

[0107] (6) Set up ligand samples with gradient concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.782, and 0 μM. Inject the ligand samples sequentially at a flow rate of 30 μL / min, with a binding time of 100 s and a dissociation time of 120 s.

[0108] (7) After all cycles are completed, the results are fitted with the Langmuir model or the Kinetic Analysis model to obtain the SPR sensing curve, which reflects the change of reflection intensity (RU) value under different ligand concentrations during binding and dissociation.

[0109] SPR results are as follows Figure 3 As shown in Figure A, this polypeptide has a good binding affinity to the OMA1 protein (Kd = 1.47M).

[0110] 3.2 The AlphaFold structure prediction and molecular docking steps are as follows:

[0111] The structures of PO1 and human OMA1 were simulated using AlphaFold3 software, and molecular docking was performed using Meastro software. Specifically:

[0112] (1) Simulate receptor ligand structure: Obtain the amino acid sequence of human OMA1 protein from NCBI, and use AlphaFold3 to simulate the structure of OMA1 protein and PO1.

[0113] (2) Receptor structure pretreatment: Water molecules and other ligands of OMA1 were removed by using the Protein Preparation Wizard tool in Schrödinger software, while peptide chain breaks and incorrect secondary structures were corrected and their energy was minimized.

[0114] (3) Ligand structure preprocessing: The 3D structure of PO1 is processed using the Ligprep tool to generate possible protonated states and conformations, while ensuring that the energy of each conformation is minimized.

[0115] (4) Generate docking box: Use the Receptor Grid Generation tool to select the protein active site to generate docking box. The docking range includes the groove structure where it exerts phosphatase activity and the adjacent region. Since the OMA1 protein has no clear active site, we choose to include most of its protein structure within the docking box range to avoid interference from the disordered surrounding structure.

[0116] (5) Molecular docking: Molecular docking is performed using the Ligand Docking tool. Each conformation of the ligand will produce a docking result. The protein-ligand 2D interaction mode is used as a reference, and the structure with the highest docking score is selected for subsequent optimization.

[0117] (6) Image optimization: The color of the molecular docking structure was adjusted using Pymol software, the hydrogen bonds between amino acids and ligands were drawn using Wizard-Measurement tool, the background surface structure was blurred, and the structure was optimized by ray rendering.

[0118] The results of molecular docking are as follows Figure 3 B in Figure 3 As shown in Figure C, the PO1 structure predicted by AlphaFold3 consists of multiple α-helices connected by a flexible region. The variable conformation of the flexible region allows it to integrate into the ring structure of the OMA1 protein, revealing their interaction.

[0119] Example 4: Cell model validation of the inhibitory peptide TAT-PO1 inhibiting mitochondrial integration stress response (mtISR).

[0120] Example 3 above comprehensively verified the binding ability of PO1 to human OMA1 protein through various in vitro experimental methods (such as surface plasmon resonance experiments, AlphaFold structure prediction, and molecular docking experiments). Figure 3 This provides strong theoretical support for the functional mechanism of PO1 at the molecular level. However, in order to further apply these in vitro research results to the regulation of biological functions, we need to verify the actual biological effects of the blocking peptide TAT-PO1 at the cellular level. Therefore, Example 4 ( Figure 4 By constructing different SH-SY5Y cell models (including VCP R159H mutant cell lines), Figure 4 The A in the text, and the cell model overexpressing PGAM5, Figure 4 (B) In-depth investigation into the regulatory role of TAT-PO1 in mitochondrial integrated stress response (mtISR) (ATF4 and CHOP are key indicators and upstream regulators of mtISR activation) to assess its functional performance in the cellular environment.

[0121] 4.1 Construction of the VCP R159H mutant SH-SY5Y cell line:

[0122] A cell line with the VCP R159H point mutation was constructed using CRISPR-Cas9 technology, and the activation of mtISR induced by the VCP mutation was verified, as follows:

[0123] 4.1.1 Design and construction of sgRNA:

[0124] Oligonucleotide sequences for sgRNA were selected and designed based on the VCP gene. An ACCG sequence was added to the 5' end of the upstream primer and an AAAC sequence was added to the 5' end of the downstream primer of the designed sgRNA-targeting DNA sequence (without the PAM sequence). Oligo was synthesized and annealed. The system and procedure are as follows:

[0125] The sgRNA annealing system was prepared as follows: After isolating the obtained primer powder, it was prepared into a 100 μM stock solution (50 μL of ddH2O was added to the powder before subsequent use); 4.5 μL of primer 1, 4.5 μL of primer 2, and 1 μL of NEB buffer were added (total volume 10 μL). The annealing program was set as follows: react at 95℃ for 5 minutes; cool from 95-85℃ at a rate of -2℃ / s; cool from 85-25℃ at a rate of -0.1℃ / s; and finally store at 4℃.

[0126] Take 3 μg of pGL3-U6-sgRNA vector plasmid, add 5 μL of CutSmart Buffer and 1.5 μL of rapid restriction enzyme BsaI for digestion (steps as above).

[0127] After obtaining the products from the previous two steps, the sgRNA is ligated to the vector using T4 ligase, followed by transformation, single clone selection, and sequencing screening (the steps are the same as above) to obtain the correct sgRNA plasmid.

[0128] VCP-sgRNA sequence: CCGTGGTGGGGATGSCGTGCTG (SEQ ID NO. 22);

[0129] R159H-single-stranded DNA (ssDNA):

[0130] 5'-TTGACACCTCTAACTGTGCTTGTACTGTTTGCTCTCGCAGGAGACATTTTTCTTGTCCGTGGTGGGATGCACGCTGTGGAGTTCAAAGTGGTGGAAACAGATCCTAGCCCTTATTGCATTGTTGCTCCAGACACAGTGAT-3' (SEQ ID NO. 23).

[0131] 4.1.2 Electroporation of SH-SY5Y cells:

[0132] (1) Change the medium 1 hour before electroporation to ensure that the cell density reaches 70%-80%;

[0133] (2) After 1 h, remove the cell culture flask from the cell culture incubator, aspirate the culture medium, rinse with 1 mL of PBS, add 1 mL of trypsin digestion solution, observe under a microscope, and after seeing that the cells have been digested into single cells, add 1 mL of culture medium to stop the digestion, use 1 mL of pipette to collect the cells into a 15 mL centrifuge tube, centrifuge at 3,000 rpm at room temperature for 3 min, and remove the supernatant;

[0134] (3) Resuspend the precipitate in 1 mL of culture medium, then take 100 μL of culture medium for cell counting, each 1 x 10 6 Each cell was grouped together for subsequent operations;

[0135] (4) Prepare 50 μL of electroporation solution B and add plasmid, etc. (per 1 x 10 μL) to 50 μL of electroporation solution A. 6 For each cell, add 62.5 pM ssDNA, 1.25 μg sgRNA, and 2.5 μg Cas9 (the total amount of plasmid should not exceed 15% of the total volume).

[0136] (5) Centrifuge the cells, discard the supernatant, and resuspend the precipitate in PBS; group the cells, centrifuge the cells, and discard the supernatant;

[0137] (6) After mixing electroporation solution B and solution A, resuspend the cell pellet;

[0138] (7) Add the mixed cell suspension to the electroporation cup (be careful not to generate bubbles, and do not add too much solution).

[0139] (8) Turn on the electro-rotator and select the program to perform the electro-rotation;

[0140] (9) After electroporation, let stand for 1 min, add an equal volume of culture medium, and then add it to a six-well plate. Observe the cell density after 12-24 h (if the cell density is high, it needs to be passaged in time). If the density is appropriate, add Puromycin for screening (the time for adding the screening drug should not exceed 48 h, and a control well can be set up) to amplify and culture the single clone cells.

[0141] 4.1.3 Screening of VCP-R159H mutant cell lines using the infinite dilution method:

[0142] (1) Remove the culture medium, rinse with 1 mL PBS, digest with 1 mL trypsin, centrifuge at 6,000 rpm at room temperature for 3 min, collect the precipitate, and resuspend the precipitate with 1 mL culture medium;

[0143] (2) Take 10 μL of 1 mL of cell suspension and place it in a clean 1.5 mL centrifuge tube. Dilute it with 100 μL of culture medium and add it to one side of a hemocytometer. Begin counting under a microscope, pressing the counter once for each cell seen (note that the counting should follow the principle of counting from top to bottom and from left to right; count the top cells but not the bottom cells, and count the left cells but not the right cells). Finally, calculate: Cell concentration in the cell culture flask (cells / mL) = (Total number of cells in four large squares / 4) × 10 4 ×Dilution factor;

[0144] (3) After calculating the number of cells, gradually dilute to one cell per 100 μL. For example, if the cell concentration is 500 cells / mL, take 200 μL and dilute to 10 mL. The concentration is 100 cells / 10 mL, which is 10 cells / mL = 1 cell / 100 μL.

[0145] (4) Add 100 μL of cell suspension (2 drops are about 100 μL) to each well of the 96-well plate.

[0146] (5) Change the medium after about 7-8 days and gradually expand the culture to 24-well plates for subsequent experimental operations.

[0147] (6) The genome was extracted using the TIANamp Genomic DNA Kit and sent to the company for sequencing and comparison to obtain the correct VCP-R159H mutant cell line. The cell line was then expanded and cryopreserved for subsequent experiments.

[0148] 4.2 Validation of peptide TAT-PO1 in VCP R159H mutant cell model:

[0149] Control group 1: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator.

[0150] Control group 2: Human neuroblastoma cell line with VCP R159H point mutation (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator.

[0151] Experimental group 1: The VCP R159H point mutant was cultured in a 37℃, 5% CO2 incubator. The cells were treated with 1 μM peptide TAT-PO1 for 12 hours.

[0152] Experimental Group 2: The VCP R159H point mutant cell line was cultured in a 37℃, 5% CO2 incubator. Cells were treated with 1 μM peptide + 2GG for 12 hours.

[0153] Experimental group 3: The VCP R159H point mutant was cultured in a 37℃, 5% CO2 incubator. The cells were treated with 1 μM peptide PO1 for 12 hours.

[0154] To enhance the localization of PO1 in mitochondria, a TAT-PO1 fusion transmembrane peptide sequence was designed and synthesized. Simultaneously, a TAT-2GG-PO1 peptide with two glycine linkers was constructed to avoid the influence of TAT on the peptide conformation.

[0155] After cell treatment, total protein was extracted as follows: First, the cell culture medium was discarded, and the cells were washed with an appropriate amount of PBS. Next, an appropriate amount of total protein extraction lysis buffer was added according to the specific experimental requirements, and the cells were placed on ice and incubated for 10 minutes, with continuous shaking to ensure complete lysis. Then, the cells were scraped off with a cell scraper and transferred to a 1.5 mL EP tube, centrifuged at 12,000 rpm and 4°C for 10 minutes, and the supernatant was collected for subsequent protein concentration determination, Western blotting, development, etc., following the same experimental procedures as in Example 1.

[0156] The protein levels of key mtISR transcription factors ATF4 and CHOP were detected by Western blotting. The results are as follows: Figure 4 As shown in Figure A, the VCP R159H mutant cell line model, by mutating arginine at position 155 of the VCP gene to histidine, showed a significant increase in mtISR levels. TAT-PO1 significantly inhibited mtISR activation, while the addition of the linker weakened this inhibitory effect. Furthermore, the PO1 peptide without TAT fusion showed no significant inhibitory effect in experimental group 3. In summary, this indicates that TAT-PO1 can effectively inhibit mtISR activation in this mutant model. Figure 4 (A in the middle).

[0157] 4.3 Validation of peptide TAT-PO1 in a PGAM5 overexpression cell model:

[0158] Control group 1: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator. The Flag plasmid was transfected for 48 hours.

[0159] Experimental Group 1: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator. Forty-eight hours after transfection with the Flag plasmid, the cells were treated with 1 μM peptide TAT-PO1 for 12 hours.

[0160] Control group 2: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator. Transfected with Flag-PGAM5 plasmid for 48 hours.

[0161] Experimental Group 2: Human neuroblastoma cell line (SH-SY5Y) was cultured in a 37℃, 5% CO2 incubator, transfected with Flag-PGAM5 plasmid for 48 hours, and then treated with 1 μM peptide TAT for 12 hours.

[0162] The protein levels of key mtISR transcription factors ATF4 and CHOP were detected by Western blotting. The results are as follows: Figure 4 As shown in B, overexpression of PGAM5 protein significantly upregulated mtISR levels. However, after the addition of the peptide TAT-PO1, overexpression of PGAM5 could no longer upregulate mtISR levels, indicating that TAT-PO1 can block PGAM5-mediated mtISR activation. Figure 4 (B in the middle).

[0163] Example 5: Verification that TAT-PO1 regulates mitochondrial integration stress response (mtISR) in a manner dependent on OMA1 protein.

[0164] In Example 4, we constructed different SH-SY5Y cell models to investigate the regulatory effect of the small molecule blocking peptide TAT-PO1 on mitochondrial integration stress response (mtISR). To gain a more comprehensive understanding of the mechanism of action of TAT-PO1, we further explored whether its regulation of mtISR depends on the OMA1 protein (…). Figure 5 In this embodiment, we first constructed the OMA1 gene knockout SH-SY5Y cell line ( Figure 5 (A in the text), and then the peptide TAT-PO1 was added to the cell line to detect whether it could still regulate mtISR levels ( Figure 5 (B in the middle).

[0165] 5.1 Construction of the OMA1 gene knockout SH-SY5Y cell line:

[0166] First, sgRNA was designed targeting the OMA1 genome, and corresponding primers were synthesized. The sequences are as follows:

[0167] sgOMA1-F-1:ACCGCACGGGGCTGTCATCAAGTA (SEQ ID NO. 24);

[0168] sgOMA1-R-1: AAACTACTTGATGACAGCCCCGTG (SEQ ID NO. 25);

[0169] Using the primers described above, gene editing was performed on the SH-SY5Y cell line via CRISPR / Cas9 technology. The specific steps included cell electroporation and cell line screening, with the same procedure as in Example 4. After screening and verification, the OMA1 gene knockout SHSY-5Y cell line was successfully obtained. Subsequently, the cell line was cultured, expanded, and cryopreserved for subsequent experiments.

[0170] To verify the successful construction of the OMA1 gene knockout cell line, we collected cell pellets, extracted total protein (using the same steps as above), and detected OMA1 protein expression using Western blotting. The experimental results are as follows: Figure 5 As shown in A, the OMA1 protein is completely absent in the knockout cell line, indicating that the OMA1 gene knockout cell line was successfully constructed. Figure 5 (A in the middle).

[0171] 5.2 Regulation of mitochondrial integration stress response (mtISR) by peptide TAT-PO1 depends on OMA1 protein.

[0172] Control group 1: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator.

[0173] Experimental Group 1: Human neuroblastoma cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator. Cells were treated with 1 μM peptide TAT-PO1 for 12 hours.

[0174] Control group 2: OMA1 gene knockout cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator.

[0175] Experimental group 2: The OMA1 gene knockout cell line (SH-SY5Y) was routinely cultured in a 37℃, 5% CO2 incubator. The cells were treated with 1 μM peptide TAT-PO1 for 12 hours.

[0176] After the cells in each group were treated as described above, we collected the cell pellet according to the previously described steps and extracted total protein. The protein levels of the key mtISR transcription factors ATF4 and CHOP were detected by Western blotting, and the results are as follows: Figure 5 As shown in Figure B, the addition of peptide TAT-PO1 significantly reduced the basal levels of ATF4 and CHOP proteins in SHSY-5Y cells, indicating that it can effectively inhibit the activation of intracellular mtISR. However, in OMA1 gene knockout cells, the inhibitory effect of TAT-PO1 was not significantly different from that of the untreated group. This suggests that the inhibitory effect of TAT-PO1 on mtISR depends on the presence of its target protein OMA1. Figure 5 (B in the middle).

[0177] Example 6: Verification of the effect of peptide TAT-PO1 on motor neuron degeneration and neuromuscular junction loss in ALS iPSCs

[0178] Through the five examples described above, we systematically verified that the small-molecule blocking peptide TAT-PO1 can regulate the mitochondrial integrated stress response (mtISR), and that its regulatory effect depends on the OMA1 protein. These findings not only reveal the molecular mechanism of TAT-PO1 but also provide a theoretical basis for its potential application in disease treatment.

[0179] Based on these findings, we further explored the application potential of the peptide TAT-PO1 in more complex physiological and pathological models. In Example 6, we differentiated induced pluripotent stem cells (iPSCs) derived from patients with amyotrophic lateral sclerosis (ALS) into spinal cord organoids (… Figure 6 The study investigated the effects of TAT-PO1 on slowing motor neuron degeneration and neuromuscular junction loss in this model (AC), and further examined its effectiveness. Figure 6 DE in the middle.

[0180] 6.1 The revival and cultivation of hiPSCs:

[0181] (1) Resuscitation of hiPSCs: Vitronectin was diluted 1 / 100 with DMEM / F12 medium and mixed well. One day before revival, 1 mL of the diluent was added to each well of a six-well plate to cover the bottom of the culture dish. On the day of revival, 5 mL of preheated DMEM / F12 medium was added to a 15 mL centrifuge tube in advance in a biosafety cabinet. The water bath was preheated to 37°C, and then the corresponding hiPSC cryovials were quickly removed from the liquid nitrogen tank and rapidly shaken in the water bath for one minute to thaw. The thawed cell suspension was added to 5 mL of the prepared DMEM / F12 medium, and then centrifuged at 1,000 rpm for three minutes at room temperature to pellet the cells. After discarding the supernatant, the precipitate was resuspended in 1 mL of preheated heterologous-free Essential 8 (E8) medium. The precipitate was then transferred to a Vitronectin-coated culture dish, mixed well, and incubated in a 37°C constant temperature incubator with 5% CO2.

[0182] (2) Culture of hiPSCs: E8 medium was used to maintain cell growth, and half of the medium was changed daily. The cell status of hiPSCs was recorded at all times, with high nucleocytoplasmic ratio and regular cell shape as the standard. When the hiPSC clones formed large sheets and the outlines and gaps between cells could not be clearly distinguished under a light microscope, the cells were passaged. The day before passage, the bottom of the wells of a six-well plate was coated with 1 mL of Vitronectin diluted in DMEM / F12. On the day of passage, the E8 medium in the dish was first discarded and 1 mL of preheated EDTA was added, and then the plate was placed in an incubator for dissociation for 1-2 minutes. The edges of the hiPSC clones were observed under a light microscope. When the edges became shiny and the internal structure of the clones became loose, the EDTA was removed in a biosafety cabinet to terminate the dissociation process, and the cells were gently rinsed once with 1 mL of preheated DMEM / F12 medium. After discarding the washing solution, 1 mL of preheated E8 medium was added and the cells were pipetted to allow the clones to detach in sheets. Cell suspensions were aspirated and seeded into Vitronectin-coated dishes at a ratio of 1:10 to 1:20 to complete passage.

[0183] 6.2 Differentiation methods of human spinal cord organoids (hSCOs):

[0184] (1) In order to differentiate hiPSCs into organoids with characteristics of spinal cord neuron cell populations, we modified the relevant methods to obtain highly reproducible hSCOs. When the hiPSC clones proliferated to 30-40 cells, the E8 culture medium was discarded and preheated NIM differentiation medium (containing 3 μM CHIR99021, 2 μM DMH1, 2 μM SB431542 and 5% KOSR) was added, which was marked as day 1 (D1). The cells were cultured continuously for 6 days, with half of the medium being changed every other day, to induce hiPSCs to become neuroepithelial (NEP) cells.

[0185] (2) On day 6, culture dishes were coated with Matrigel diluted 1:50. On day 7, NEP cells were digested with preheated Dispase and seeded in coated culture dishes at a ratio of 1:6. The cells were cultured for 6 days in NIM differentiation medium containing 1 μM CHIR99021, 2 μM DMH1, 2 μMSB431542, 0.1 μM retinoic acid and 0.2 μM SAG to induce NEP cells to become Oligo2-positive motor neuron progenitor cells (MNPs).

[0186] (3) At D13, Oligo2 and MNPs were digested with Dispase for 5-10 minutes. After observing the curling of the clone edges under a light microscope, the digestion solution was removed. The clones were rinsed once with DMEM / F12, and the clones were blown off and transferred to a T25 ultra-low adsorption culture flask. The clones were suspended and cultured in NIM differentiation medium containing 0.5 μM RA and 0.1 μM SAG for 6 days to induce MNPs to differentiate into hSCOs containing HB9-positive motor neurons (MNs).

[0187] (4) At D19, 0.1 μM Compound E, 20 ng / mL BDNF, 10 ng / mL LIGF-1 and 50 nM cAMP were added to the differentiation medium, and the cells were cultured until D28 to differentiate hSCOs containing ChAT and MN. At D19, hSCOs could also be adhered to Matrigel-coated culture dishes for further differentiation. After D28, half of the medium was replaced every two days with NIM differentiation medium containing 0.5 μM RA, 20 ng / mL BDNF, 10 ng / mL LIGF-1 and 50 nM cAMP to maintain culture.

[0188] Using the methods described above, we directed the differentiation of hiPSCs derived from ALS patients. Immunohistochemical analysis of the mature motor neuron marker ChAT and the cholinergic neuron marker VAChT, combined with an acetylcholine probe, revealed that this experiment successfully differentiated hSCOs rich in motor neurons. Figure 6 AC in the middle.

[0189] 6.3 Construction of human spinal cord organoid-mouse skeletal muscle cell (hSCOs-mSkM) co-culture:

[0190] Motor neuron degeneration in ALS manifests initially as neuromuscular junction (NMJ) dysfunction, followed by motor axonal abnormalities, and finally neuronal cell body degeneration. To assess the impact of ALS on NMJ, we co-cultured differentiated C2C12 cells (mouse skeletal muscle cells mSkM) with human spinal cord organoids (hSCOs) differentiated from stem cells derived from ALS patients, constructing an hSCOs-mSkM co-culture system.

[0191] Two peptides, TAT-PO1 (amino acid sequence: YGRKKRRQRRR{AHX}RHIFLIRHSQYHVDGSLEKDRTLTPLGREQAE) and TAT (amino acid sequence: YGRKKRRQRRR), were synthesized by a commissioned company. TAT-PO1 was the experimental group, and TAT was the control group. The specific treatments were as follows:

[0192] Control group 1: Mouse skeleton muscle (mSkM) differentiated from C2C12 cells were routinely cultured.

[0193] Control group 2: Human spinal cord organoids (hSCOs: NC3-1) in the control group were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a conventional manner.

[0194] Control group 3: Human spinal cord organoids (hSCOs: RC01001-A) in the control group were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a conventional manner.

[0195] Control group 4: Human spinal cord organoids (hSCOs: IMR90-4) in the control group were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a conventional manner.

[0196] Control group 5: Human spinal cord organoids (hSCOs: TDP43-A382T) from patients carrying the TDP43-A382T gene mutation were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a routine manner.

[0197] Control group 6: Human spinal cord organoids (hSCOs: SOD1-D90A) derived from patients carrying the SOD1-D90A gene mutation were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a routine manner.

[0198] Control group 7: Human spinal cord organoids (hSCOs: FUS-R521G) from patients carrying the FUS-R521G gene mutation were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a routine manner.

[0199] Control group 8: Human spinal cord organoids (hSCOs: ALS401) in the sporadic ALS group were pretreated with peptide TAT (0.5 μM) for one week and then co-cultured with mSkM in a conventional manner.

[0200] Experimental Group 1: Human spinal cord organoids (hSCOs: TDP43-A382T) from patients carrying the TDP43-A382T gene mutation were pretreated with peptide TAT-PO1 (0.5 μM) for one week and then co-cultured with mSkM as usual.

[0201] Experimental Group 2: Human spinal cord organoids (hSCOs: SOD1-D90A) derived from patients carrying the SOD1-D90A gene mutation were pretreated with peptide TAT-PO1 (0.5 μM) for one week and then co-cultured with mSkM in a routine manner.

[0202] Experimental Group 3: Human spinal cord organoids (hSCOs: FUS-R521G) derived from patients carrying the FUS-R521G gene mutation were pretreated with peptide TAT-PO1 (0.5 μM) for one week and then co-cultured with mSkM as usual.

[0203] Experimental Group 4: Human spinal cord organoids (hSCOs: ALS401) from the sporadic ALS group were pretreated with peptide TAT-PO1 (0.5 μM) for one week and then co-cultured with mSkM.

[0204] 6.3.1 C2C12 cell culture and differentiation of mouse skeleton muscle (mSkM) cells:

[0205] C2C12 cells were cultured in C2C12 medium at 37°C in a 5% CO2 incubator. When the cell confluence reached 80-90%, they were digested with trypsin containing EDTA at 37°C for 2-3 minutes until the cells shrank into spheres. Digestion was then stopped with an equal volume of culture medium, and the cells were centrifuged at 1,000 rpm for 3 minutes. After discarding the supernatant, the cell pellet was resuspended in 1 mL of culture medium and passaged at a 1:4 ratio. When the cell confluence reached 80-90%, differentiation into mSkM cells was induced. The culture medium was then discarded, and the cells were replaced with differentiation medium. Successful differentiation was indicated by the observation of myotube formation under a light microscope after 6-7 days. Only C2C12 cells up to passage 12 were used for differentiation.

[0206] 6.3.2 Construction of hSCOs-mSkM co-culture:

[0207] To construct an in vitro NMJ functional evaluation system, hSCOs at D30 (±2 days) were added to the surface of mSkM at D7 (±1 day) for co-culture. After discarding the C2C12 differentiation medium, the cells were co-cultured in NIM differentiation medium containing 50 nM cAMP, B27 additive, and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. After standing for 48 hours, the medium was partially changed daily.

[0208] Figure 6 The diagram in D shows a schematic of hSCOs-mSkM co-culture obtained by mixing hSCOs and mSkM. Figure 6 (D in the text). Subsequently, we stained the hSCOs-mSkM co-culture with α-BTX, Tuj-1, and α-Actinin using immunofluorescence experiments, and statistically analyzed the ratio of α-BTX-positive area to Tuj-1-positive area to clarify the differences in neuromuscular junction formation. The results showed that the addition of the peptide TAT-PO1 could significantly slow down ALS-induced motor neuron degeneration and neuromuscular junction loss (D in the text). Figure 6 (E in the text).

[0209] Example 7: Verification that peptide TAT-PO1 can improve the pathological phenotype and motor function of ALS model mice.

[0210] In Example 6, we verified through in vitro experiments that the small molecule blocking peptide TAT-PO1 of the present invention can significantly slow down the degeneration of motor neurons and the loss of neuromuscular junctions in ALS iPSCs. Figure 6 ).

[0211] To further investigate the effects of TAT-PO1 in in vivo models, we delivered the peptide TAT-PO1 into the spinal cord of classic ALS model mice (TDP-43 A315T transgenic mice) using an adeno-associated virus (AAV) vector and observed its effects. Figure 7 The specific steps are as follows:

[0212] Control group 1: One-month-old littermate wild-type (WT) mice were injected intrathecally with TAT-P2A-EGFP virus and fed routinely for 2.5 months before the experiment was conducted.

[0213] Experimental group: One-month-old littermate wild-type (WT) mice were injected intrathecally with TAT-PO1-P2A-EGFP virus and fed routinely for 2.5 months before the experiment was carried out.

[0214] Control group 2: One-month-old ALS model mice (TDP-43 A315T transgenic mice) were injected intrathecally with TAT-P2A-EGFP virus and fed routinely for 2.5 months before the experiment was carried out.

[0215] Experimental Group 2: One-month-old ALS model mice (TDP-43 A315T transgenic mice) were injected intrathecally with TAT-PO1-P2A-EGFP virus and fed routinely for 2.5 months before the experiment was carried out.

[0216] 7.1 Intrathecal injection of TAT-PO1 delivered via AAV vector:

[0217] (1) We commissioned a company to construct an adeno-associated virus (AAV) vector expressing the peptide TAT-PO1 and its control virus. The small molecule peptide TAT-PO1 was linked with green fluorescent protein (EGFP) via P2A to construct two viruses: TAT-PO1-P2A-EGFP (experimental group) and TAT-P2A-EGFP (control group).

[0218] (2) We commissioned a company to construct TDP-43 A315T transgenic mice and raised them to about 1 month old to start the experiment.

[0219] (3) For intrathecal injection, mice were first anesthetized with 3-5% isoflurane, and the anesthesia was maintained with 1-2% isoflurane. The mice were placed in a prone position with their limbs fixed and the spine kept naturally curved. The hair on the back was shaved off with a razor and disinfected with povidone-iodine three times. The injection site was selected at the L5-L6 intervertebral space (located slightly above the line connecting the iliac crests, where a feeling of emptiness will be felt when inserting the needle). The needle was inserted vertically, and after breaking through the ligamentum flavum, 10 μL of the drug was slowly injected. After the injection, the needle was held for 3 seconds and then slowly withdrawn to prevent drug reflux. After the injection, the mice were placed on a heating pad to maintain their body temperature and observed for 30 minutes. After confirming that there were no abnormalities, the mice were returned to the animal room for subsequent experiments.

[0220] 7.2 Spinal cord sections and immunofluorescence staining:

[0221] (1) Spinal cord sampling: After anesthetizing the mice, the heart was exposed by opening the chest and PBS was perfused through the left ventricle. The spinal cord was then cut off. The lamina on both sides of the spinal cord were cut along the coronal plane to expose the entire spinal cord, which was then clamped and removed. At the same time, the anterior tibialis muscle of the mouse was taken for later use.

[0222] (2) Fixation and dehydration: The spinal cord tissue was placed in 4% neutral formaldehyde fixative and fixed at room temperature for 1 day. The fixative was then discarded and the tissue was then placed in 20% sucrose solution for 1 day and 30% sucrose solution for 2 days for gradient dehydration.

[0223] (3) Embedding and sectioning: The processed spinal cord tissue was embedded in an OCT chamber and placed in a -20℃ cryostat. After the embedding medium was completely solidified, sections were prepared with a thickness of 30 μm. After sectioning, the sections were mounted on glass slides and stored in a -20℃ freezer for subsequent experiments.

[0224] (4) Immunofluorescence staining and imaging: First, incubate with immunofluorescence blocking solution at room temperature for 1 hour to block non-specific binding sites. Then, add immunofluorescence antibody dilution solution containing the specified primary antibody and incubate overnight at 4°C. The next day, wash three times with PBS, then add immunofluorescence antibody dilution solution containing the corresponding species' fluorescent secondary antibody and DAPI, and incubate at room temperature for 1 hour. After incubation, wash three times with PBS, and then encapsulate. Finally, acquire images using an upright fluorescence microscope.

[0225] Through spinal cord sections and immunofluorescence staining, we found that EGFP was successfully expressed in the anterior horn of the spinal cord, indicating that TAT-PO1 was successfully delivered and expressed in the anterior horn of the mouse spinal cord. Figure 7 In the TDP-43 A315T transgenic mouse model, the phosphorylation level of eIF2α, a key upstream regulator of mtISR (p-eIF2α), was significantly increased, while the phosphorylation level of eIF2α was significantly decreased after intrathecal injection of the peptide TAT-PO1. Figure 7The B in the figure indicates that TAT-PO1 can effectively inhibit the activation of mtISR in this classic ALS mouse model.

[0226] Simultaneously, immunofluorescence staining was performed on the tibialis anterior muscle of mice (steps as above), and the ratio of α-BTX-positive area to Tuj-1-positive area was calculated. The results showed that intrathecal injection of TAT-PO1 significantly slowed motor neuron degeneration and neuromuscular junction loss in TDP-43 (A315T) mice. Figure 7 (C in the middle).

[0227] 7.3 Exercise-related behavioral tests:

[0228] (1) Footprint Experiment: First, prepare a track 100 cm long and 10 cm wide, with 15 cm high walls on both sides, and cover it with white paper of appropriate size for recording mouse footprints. After allowing the mice to adapt to the environment before the experiment, apply non-toxic red pigment evenly to the hind feet of the mice, and then place the mice at the starting point of the track to allow them to walk freely. When the mice walk on the track, they will leave a series of footprints on the white paper. Record the shape and distribution of these footprints and take pictures with a camera. Measure the distance of each step, 5 steps for each foot, for a total of 10 steps, and take the average value as the mouse's stride length. Through quantitative analysis of parameters such as stride length, evaluate the mouse's motor function.

[0229] (2) Claw gripping force test: A claw gripping force measuring instrument (including an adjustable metal gripping bar and a force sensor connected to a display device) was used, calibrated to zero before the experiment. After the mice were acclimatized to the environment, they were gently placed on the platform below the gripping bar, allowing their front paws to grasp the bar. The mice were then gently pulled, and the maximum gripping force value displayed by the force sensor was recorded. Each mouse was measured 3-5 times, and the average value was taken as the final result. The mice were given a short rest between measurements. The gripping force values ​​were recorded and the average value was calculated. Statistical analysis was performed on the claw gripping forces of different groups of mice to compare the differences in order to assess motor function and muscle strength.

[0230] Statistical analysis results showed that in the footprint test, TDP-43 (A315T) mice injected intrathecally with TAT-PO1 had significantly longer strides than mice injected with the control virus, indicating improved gait. Figure 7 In the claw grip test, the claw grip of mice in the TAT-PO1 injection group was significantly higher than that in the control group, indicating enhanced motor function. Figure 7 (E in the text). Therefore, TAT-PO1, delivered via an AAV vector, significantly improved the pathological and motor function phenotypes in ALS model mice.

[0231] Statistical analyses were performed on all the experimental results. GraphPad Prism 8 software was used for statistical processing and analysis of the experimental data, and quantitative results are expressed as mean ± standard error (Mean ± SEM). The results analysis employed t-tests, one-way ANOVA, and Tukey's paired comparison test. In the results, * indicates... P <0.05; ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001.

[0232] The antibody information used in the Western blot test (1.3) of Example 1 and the spinal cord sections and immunofluorescence staining (7.2) of Example 7 is as follows: Anti-His antibody, rabbit antigen, provided by CST Biotechnology, catalog number 12698S, used ratio 1:1000; Anti-GST antibody, rabbit antigen, provided by CST Biotechnology, catalog number 2624S, used ratio 1:1000; Anti-DYKDDDDK (Flag) antibody, rabbit antigen, provided by CST Biotechnology, catalog number 14793S, used ratio 1:1000; Anti-ATF4 antibody, rabbit antigen, provided by Proteintech, catalog number 10835-1-AP, used ratio 1:1000; Anti-CHOP antibody, rabbit antigen, provided by Proteintech, catalog number 15205-1-AP, used ratio 1:1000; Anti-Actin antibody, mouse antigen, provided by Ori The following antibodies were provided by Gene, catalog number TA-09, at a ratio of 1:1000: Anti-OMA1 antibody (rabbit antigen), provided by Proteintech, catalog number 17116-1-AP, at a ratio of 1:1000: Anti-Tuj1 antibody (rabbit antigen), provided by Covance, catalog number PRB-435P, at a ratio of 1:500: Anti-α-BTX antibody, provided by Bosunlife, catalog number (B00018), at a ratio of 1:1000: Anti-NeuN antibody (mouse antigen), provided by ARG, catalog number ARG52283, at a ratio of 1:1000: Anti-ChAT antibody (goat antigen), provided by Millipore, catalog number AB144P, at a ratio of 1:200: Anti-VAChT antibody (rabbit antigen), provided by Synaptic The following secondary antibodies were provided by Systems, catalog number 139103, used at a ratio of 1:1000; Anti-α-Actinin antibody, mouse antigen, was provided by Sigma-Aldrich, catalog number A7811, used at a ratio of 1:1000; Hoechst was provided by Solarbio, catalog number C0030, used at a ratio of 1:1000; the Western blot secondary antibodies were provided by Thermo Fisher Scientific, with anti-rabbit secondary antibody catalog number 31460 and anti-mouse secondary antibody catalog number 31430, both used at a ratio of 1:5000; the fluorescent secondary antibodies were all provided by Thermo Fisher Scientific, including 488-channel Donkey Anti-Mouse, catalog number A21202; 546-channel Donkey Anti-Mouse, catalog number A10036; 546-channel Donkey Anti-Rabbit, catalog number A10040; and 647-channel Donkey Anti-Rabbit, catalog number A31573.647-channel Donkey Anti-Goat, catalog number A21447, with a fluorescent secondary antibody usage ratio of 1:500.

[0233] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The polypeptide TAT-PO1, characterized in that, The polypeptide is an aminocaproic acid linked to the TAT sequence shown in SEQ ID NO.1 and the PO1 sequence shown in SEQ ID NO.

2. The polypeptide TAT-PO1 consists of the TAT sequence shown in SEQ ID NO.1, aminocaproic acid, and the PO1 sequence shown in SEQ ID NO.2, from the N-terminus to the C-terminus.

2. A drug for treating amyotrophic lateral sclerosis (ALS), characterized in that, It comprises the polypeptide TAT-PO1 of claim 1, or the nucleotide sequence encoding the polypeptide TAT-PO1 of claim 1.

3. The drug according to claim 2, characterized in that, It also contains pharmaceutically acceptable excipients.

4. The drug according to claim 2, characterized in that, The drug is administered via intrathecal injection or intravenous injection.

5. The use of the polypeptide TAT-PO1 of claim 1 or the drug of claim 2 in the preparation of a medicament for treating amyotrophic lateral sclerosis (ALS).

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