Exosome for treating pank2 gene defect related disease, preparation method and application thereof

Abstract

The application provides an exosome for treating a PANK2 gene defect related disease, a preparation method and application thereof. The exosome loaded with PANK2 is used for treating the PANK2 gene defect related disease, can effectively restore the PANK2 protein expression in the brain and improve the neurological dysfunction, has good safety and transformation potential, and provides a treatment strategy with innovation and clinical application prospect for the PANK2 gene defect related disease.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy, specifically relating to exosomes for treating diseases related to PANK2 gene defects, their preparation methods, and their applications. Background Technology

[0002] PANK2 gene deficiency-related diseases (such as pantothenic acid kinase-associated neurodegenerative diseases, PKAN / NBIA lineage) are progressive neurodegenerative rare diseases. Patients often present with progressive dystonia, Parkinson's-like symptoms, dysarthria, and dysphagia. The disease burden is heavy and the course of the disease is long.

[0003] Current clinical interventions are primarily symptomatic and supportive, lacking safe, sustainable, repeatable treatment options with clear disease-modifying potential. Strategies focusing on metabolic supplementation or small molecule interventions still face significant uncertainties regarding efficacy consistency, long-term maintenance, and effective central nervous system exposure. Meanwhile, while cell therapy can provide paracrine and immunomodulatory effects, it carries potential risks at the cellular level and issues with quality consistency. Exosomes, as important effector carriers for paracrine signals, possess advantages such as low immunogenicity, the ability to be engineered to load functional molecules, and potential for cross-blood-brain barrier or intranasal delivery to the brain. Theoretically, they can mitigate the risks of cell therapy to some extent and improve the accessibility of central nervous system administration.

[0004] However, for the specific indication of PANK2 gene deficiency, there is still a lack of an implementable, quantifiable, and standardized exosome therapy technology route. In particular, there are key technical gaps in areas such as effective coverage of the etiology and pathology chain, central targeting and payload delivery, preparation process and quality control system, and safety and controllability in long-term medication scenarios, which makes it difficult to achieve stable and reproducible therapeutic effects and meet the needs of clinical translation. Summary of the Invention

[0005] Based on the above-mentioned shortcomings of the existing technology, the present invention mainly provides a mesenchymal stem cell exosome therapy for treating PANK2 gene deficiency disease, which enables effective delivery and exposure to the central nervous system under non-cellular, non-viral or low-invasive drug administration conditions, and produces a disease-modifying effect on the core pathological links related to PANK2 deficiency. Specifically, this invention aims to solve at least the following technical problems: (1) how to construct an exosome preparation and administration regimen that is low in immunogenicity, reusable, dose-adjustable, and suitable for long-term management, so as to reduce the risk of immune response and systemic toxicity and improve the controllability of long-term treatment of rare diseases; (2) how to enable exosomes to effectively reach and act on key brain regions / key cell types after entering the body, thereby overcoming the bottleneck of efficacy caused by the blood-brain barrier limitation and insufficient distribution in the brain; (3) how to effectively intervene in key disease pathways and improve neurofunctional phenotypes by carrying or delivering functional payloads related to PANK2 deficiency pathology through exosomes; (4) how to establish an exosome preparation, purification, preservation and quality control system for translational applications, solve problems such as differences in exosome sources, insufficient batch-to-batch consistency, difficulty in quantifying potency, and unclear stability and transportation and storage conditions, and form production and quality standards that are releaseable, traceable and reproducible, so as to ensure the stability of efficacy and the feasibility of clinical application.

[0006] By solving the above-mentioned technical problems, the present invention aims to develop a treatment strategy that is more controllable, has repeatable dosing capabilities and central delivery advantages compared with existing symptomatic treatments, small molecule / metabolic supplementation regimens and viral vector gene therapy, thereby providing a new, safe and effective disease-modifying intervention for PANK2 gene deficiency diseases.

[0007] The first aspect of this invention provides the use of PANK2-loaded exosomes in the preparation of medicaments for treating diseases related to PANK2 gene defects.

[0008] In this invention, PANK2 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed PANK2, any form of PANK2 derived from cell processing, and naturally occurring variants of PANK2 (e.g., splice variants or allelic variants). The term encompasses, for example, human PANK2, as well as PANK2 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats).

[0009] In this invention, exosomes (Exo) are cell-derived vesicles present in the body fluids of almost all eukaryotes, and are defined as having a diameter of approximately 30 nm. 100nm vesicles, exosomes can be released from cells or directly from the cell membrane, and perform important and special functions such as coagulation and intercellular signal transduction.

[0010] In some implementations, the PANK2 gene deficiency-related diseases include, but are not limited to, pantothenic acid kinase-related neurodegenerative diseases, HARP syndrome, and neuroacanthosis.

[0011] In some implementations, the PANK2 gene deficiency-related diseases are selected from pantothenic acid kinase-related neurodegenerative diseases.

[0012] In some embodiments, the PANK2-loaded exosomes are prepared by the following method: Step 1): Infect mesenchymal stem cells with lentiviral particles containing PANK2.

[0013] Step 2): Isolate and purify exosomes from the mesenchymal stem cells obtained in Step 1).

[0014] In some implementations, mesenchymal stem cells (MSCs) include, but are not limited to, umbilical cord mesenchymal stem cells (including those derived from umbilical cord blood), bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, synovial mesenchymal stem cells, and thymic mesenchymal stem cells. Mesenchymal stem cells are generally available commercially or obtained using methods known in the art.

[0015] In some embodiments, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood.

[0016] In some embodiments, the lentiviral particle containing the PANK2 gene is assembled from a pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, a packaging plasmid, and an envelope plasmid.

[0017] In one specific implementation, the PANK2 gene coding sequence is selected from human wild-type PANK2, and the corresponding transcript reference number is NM_153638.4.

[0018] In some implementations, the packaging plasmid is responsible for the synthesis of structural proteins in viral particles and the production of enzymes such as reverse transcription and integration.

[0019] In some implementations, envelope plasmids can replace lentiviral pro-enveloping proteins, expanding the host range and enhancing viral stability.

[0020] In some implementations, the packaging plasmid includes, but is not limited to, psPAX2.

[0021] In some embodiments, the envelope plasmid includes, but is not limited to, pMD2.G.

[0022] In some implementations, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2).

[0023] In some embodiments, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:1.

[0024] In some implementations, the MOI of lentiviral particles infecting mesenchymal stem cells is 100-200.

[0025] In some implementations, the MOI for lentiviral particle infection of mesenchymal stem cells is 200.

[0026] In some implementations, step 2) further includes screening cells that stably express exosomes loaded with PANK2A.

[0027] A second aspect of the present invention provides a method for preparing exosomes loaded with PANK2, the method comprising the following steps: Step 1): Infect mesenchymal stem cells with lentiviral particles containing PANK2.

[0028] Step 2): Isolate and purify exosomes from the mesenchymal stem cells obtained in Step 1).

[0029] In some embodiments, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood.

[0030] In some embodiments, the lentiviral particle containing the PANK2 gene is assembled from a pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, a packaging plasmid, and an envelope plasmid.

[0031] In some embodiments, the packaging plasmid is selected from psPAX2.

[0032] In some embodiments, the envelope plasmid is selected from pMD2.G.

[0033] In some implementations, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2).

[0034] In some embodiments, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:1.

[0035] In some implementations, step 2) further includes screening cells that stably express exosomes loaded with PANK2A.

[0036] A third aspect of the present invention provides an exosome prepared by the method described in the second aspect of the present invention.

[0037] A fourth aspect of the present invention provides a pharmaceutical composition for treating diseases related to PANK2 gene deficiency, the pharmaceutical composition comprising the exosomes described in the third aspect of the present invention.

[0038] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0039] In some embodiments, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with other components comprising the formulation and / or the mammals treated with it. Pharmaceutically acceptable carriers and / or excipients may include any solvent suitable for the specific target dosage form.

[0040] Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, excipients, buffers, surfactants, and / or preservatives.

[0041] Examples of excipients include one or more viscosity-improving agents. Viscosity-improving agents are one or more relatively non-toxic chemical compounds or agents that alter the viscosity of a pharmaceutical ingredient and / or preparation. Representative examples of viscosity-improving agents include petrolatum, liquid paraffin, light liquid paraffin, castor oil, mineral oil, cottonseed oil, soybean oil, sesame oil, corn oil, petroleum resins, polyethylene glycol, glycerin, polybutene, rosin, polyvinyl alcohol, polystyrene, polyacrylic acid, propylene glycol, piperine butyl ether, hydroxypropyl methylcellulose, talc, gelatin, hydrogenated rosin glyceryl esters, aliphatic hydrocarbon resins, benzyl acetate, copal resin, silica, polysiloxane, dimethyl polysiloxane, magnesium aluminum silicate, xanthan gum, sodium chondroitin sulfate, cyclodextrin, and carboxyethyl acetate. Alkenyl polymers, sodium alginate, propylene glycol alginate, carrageenan, sodium carboxymethyl cellulose, gluconolactone, squalene, stearyl alcohol, aluminum stearate, lanolin, cetyl alcohol, gelatin, sorbitol, dextran, dextrin, tragacanth gum, palmitic acid, hyaluronic acid, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, butanediol, polyoxyethylene polyoxypropylene glycol, polysorbate, sodium metaphosphate, methyl cellulose, methyl vinyl ester maleic anhydride copolymer, locust bean gum, or cellulose polymers, etc.

[0042] Other representative examples of excipients include one or more antioxidants (thiosulfates, sodium thiosulfate, sodium formaldehyde sulfoxylate, sodium formaldehyde sulfoxylate dihydrate, etc.) and tension modifiers (sodium chloride, etc.).

[0043] Examples of buffers include, but are not limited to, acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, hydrochloric acid-potassium chloride, glycine, aconitic acid, citric acid-phosphoric acid, succinic acid, phthalic acid, maleic acid, carcoic acid, tris(tris(hydroxymethyl)aminomethane), barbituric acid, borax, 2-amino-2-methyl-1,3-propanediol (Ammediol), sodium carbonate-sodium bicarbonate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), ADA (N-(2-acetamido)iminodiacetic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine (N... N-bis(2-hydroxyethyl)glycine), Bis-tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid), CAPSO (N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid), CHES (N-cyclohexyl-2-aminoethanesulfonic acid), DIPSO (3-[N- [N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), EPPS (3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid), HEPES-Na (sodium 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), HEPPSO (2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid monohydrate), MES (2-morpholinylethanesulfonic acid monohydrate), MOPS (3-morpholinylpropanesulfonic acid), MOPSO (2-hydroxy-3-morpholinylpropanesulfonic acid), PIPES (piperazin-1-propanesulfonic acid). ,4-bis(2-ethanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate), TAPSO (2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), Tricine (N-[tris(hydroxymethyl)methyl]glycine), hydrochloric acid; bases, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, and sodium lactate; and buffers, such as citrate / glucose, sodium bicarbonate, and ammonium chloride, and citrate, phosphate, borate, bicarbonate, sodium salt, or potassium salt, including combinations thereof.

[0044] Examples of surfactants include, but are not limited to, sorbitan ether esters of oleic acid (e.g., polysorbate 80 or Tween 20 and 80), polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, cremophor, sodium alkylbenzene sulfonate, glycerin, lecithin, sucrose esters, polyoxyethylene alkyl ethers, polyoxyethylene stearate, polyethylene glycol 40 stearate, ethylene glycol monostearate, polyethylene glycol monostearate, and polymers of oxyethylated octylphenol (tyloxapine). The ophthalmic compositions of the present invention comprise, are substantially composed of, or further comprise composed of, the following: polysorbate 80, polyoxyethylene hydrogenated castor oil, lecithin, or combinations thereof. In some specific embodiments, the ophthalmic compositions of the present invention comprise, are substantially composed of, or are further composed of: polysorbate 80, polyoxyethylene hydrogenated castor oil, lecithin, or combinations thereof.

[0045] Examples of preservatives include, but are not limited to, imidazolidinyl urea, methylparaben, propylparaben, phenoxyethanol, disodium EDTA, benzalkonium chloride, thimerosal, chlorobutanol, sorbic acid, and combinations thereof.

[0046] In some embodiments, the pharmaceutical composition is administered in a therapeutically effective amount.

[0047] As used herein, the term “effective dose” is equivalent to “therapeutic effective dose” and means that exosomes effectively treat or improve diseases related to PANK2 gene deficiency as described herein and thus produce the desired therapeutic, ameliorative, inhibitory, or preventative effects in subjects in need.

[0048] As used herein, the term "subject" refers to an animal or any living organism that has sensory and motor abilities and requires oxygen and organic food. Non-limiting examples include rodents (e.g., guinea pigs, hamsters, rats, mice), canines (e.g., dogs), cats (e.g., cats), pigs (e.g., pigs), equines (e.g., horses), non-human primates (e.g., monkeys, apes, baboons, gorillas, chimpanzees, orangutans), or humans. In a preferred embodiment, the subject is selected from humans.

[0049] In some implementations, the effective dose for a given subject can be determined through routine testing, which can be performed by a clinician or practitioner in the field based on factors relevant to the subject. Dosage and administration can be adjusted to provide adequate levels of the drug or maintain the desired effect. Factors considered may include, but are not limited to, genetic screening, severity of the disease state, disease progression, the subject's overall health status, race, age, weight, sex, diet, timing and frequency of administration, drug combination, response sensitivity, experience with other therapies, and tolerance / responsiveness to the therapy.

[0050] In some implementations, the exosomes are administered for one, two, three, or even more days.

[0051] In some implementations, the exosomes are administered for 30 to 60 days.

[0052] In some implementations, the exosomes are administered once daily.

[0053] For any exosome, the effective dose can initially be estimated in cell culture assays or in relevant animal models (e.g., mice, guinea pigs, chimpanzees, marmosets, or tamarins). Relevant animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine the effective dose and route of administration to humans. Therapeutic efficacy and toxicity can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as EDTA. 50 (Dose effective for 50% of the population) and LD 50 (The dose that would be lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 In some respects, the effective dose reaches a high therapeutic index. In a more specific sense, the dose includes doses with little or no toxicity in the ED. 50 Within the circulating concentration range. The dosage can vary within this range, depending on the dosage form used, patient sensitivity, and route of administration.

[0054] The exosomes described herein can be administered to subjects using any pharmaceutical dosage form known in the art. Non-limiting examples include non-gastrointestinal dosage forms and gastrointestinal dosage forms.

[0055] In some preferred embodiments, the pharmaceutical composition is selected from non-gastrointestinal dosage forms.

[0056] In some implementations, the non-gastrointestinal dosage form includes, but is not limited to, injectable dosage forms.

[0057] If administered parenterally, examples of such administration include, but are not limited to, one or more of the following: intranasal, intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracardiac, intramuscular, or subcutaneous administration, and / or administration via infusion techniques.

[0058] In some preferred embodiments, the application is selected from the nasal cavity.

[0059] In some implementations, the administration is performed via nasal instillation.

[0060] In some embodiments, the pharmaceutical composition also includes other medicines for treating diseases related to PANK2 gene defects.

[0061] In some embodiments, the pharmaceutical composition also includes other medications for treating pantothenic acid kinase-related neurodegenerative diseases.

[0062] In some implementations, the other drugs for treating pantothenic acid kinase-related neurodegenerative diseases include, but are not limited to, trihexyphenidyl, baclofen, clonazepam, levodopa, deferiphenone, and phosphonocarboxylate.

[0063] The fifth aspect of the present invention provides a lentiviral particle, which is assembled from a pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, a packaging plasmid, and an envelope plasmid.

[0064] In some embodiments, the packaging plasmid is selected from psPAX2.

[0065] In some embodiments, the envelope plasmid is selected from pMD2.G.

[0066] In some implementations, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2).

[0067] In some embodiments, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:1.

[0068] A sixth aspect of the present invention provides a cell infected with the lentiviral particles described in the fifth aspect.

[0069] In some implementations, the cells include mesenchymal stem cells.

[0070] In some embodiments, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood.

[0071] A seventh aspect of the present invention provides a nasal drug delivery device, the nasal drug delivery device comprising a drug delivery unit containing exosomes as described in the third aspect of the present invention.

[0072] The eighth aspect of the present invention provides a method for constructing an animal model of pantothenic acid kinase-related neurodegenerative diseases, the method comprising causing non-human animals to express or lose Pank2 activity.

[0073] In some embodiments, causing the non-human animal Pank2 expression or activity to be lost includes knocking out and / or knocking down Pank2 in the non-human animal.

[0074] In some embodiments, causing the non-human animal to lose Pank2 expression or activity includes knocking out and / or knocking down exon 2 region of the Pank2 gene in the non-human animal.

[0075] In some implementations, the knockout and / or knockdown may use one or more of the following technologies, including but not limited to: Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, phiC31, CRISPR-Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, interfering RNA, and homologous recombination.

[0076] In some implementations, the technique used for knockout and / or knockdown is selected from CRISPR-Cas9.

[0077] In some implementations, the CRISPR-Cas9 uses sgRNA and the Cas9 protein.

[0078] In some implementations, the sgRNA sequence used by the CRISPR-Cas9 is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0079] In some implementations, the method further includes genotyping non-human animal individuals to obtain Pank2 homozygous knockout animals.

[0080] In some implementations, the genotyping is performed using a PCR method.

[0081] In some embodiments, the genotyping primers include: F1: 5′-GATTTAGGGTTGGGAAGGGACATA-3′; R1: 5′-CTTTCTCTCGATTCTTCACTAGCC-3′; F2: 5′-AGGGGATAAGAGCCTACAAGGAAT-3′.

[0082] In some implementations, the genotyping results show that homozygous Pank2 knockout individuals are represented by a 679 bp band.

[0083] In some implementations, the method further includes phenotypic assessment of non-human animal individuals.

[0084] In some implementations, the phenotypic assessment includes neurobehavioral testing.

[0085] In some implementations, the neurobehavioral testing includes motor coordination ability testing and / or assessment of behavioral abnormalities.

[0086] In some implementations, the animal model exhibits motor impairment, decreased coordination, and / or neurological dysfunction.

[0087] In some implementations, the animal model is used for pathogenesis studies of pantothenic acid kinase-related neurodegenerative diseases and / or for drug efficacy evaluation / therapeutic drug screening.

[0088] In some implementations, the non-human animal is a mammal.

[0089] In some implementations, the mammals include, but are not limited to, rodents, carnivores, chiropterans, hedgehogs, and insectivores.

[0090] In some embodiments, the mammal is selected from rodents.

[0091] In some embodiments, the rodents include, but are not limited to, hamsters, rats, dwarf rats, spiny rats, moles, thorny rats, and rock rats.

[0092] In some embodiments, the rodent is selected from the Muridae family.

[0093] In this invention, the rat family includes, but is not limited to, black rats, brown rats, gerbils, New World rats, Old World rats, SD rats, Polynesian rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, climbing rats, shrews, Sri Lankan mice, Sikkimese mice, Javan mice, Indian mice, field mice, brown mice, Kushner's mice, Cyprus mice, South Indian mice, Thai mice, Yugoslavian mice, house mice, Burmese mice, Hungarian mice, Mediterranean mice, earth-colored mice, Ivorian mice, toad mice, Angola mice, hill mice, Botswana mice, crested mice, Somali mice, Ghanaian mice, South African mice, shrew-like mice, valley mice, Central African mice, bristle mice, Zambian mice, Ugandan mice, delicate mice, Neptune mice, Freund's mice, Felix's mice, flat-haired mice, cave mice, and Shore's mice.

[0094] In some implementations, the murine mice are selected from house mice.

[0095] In some implementations, the mice include the C57BL / 6 subspecies.

[0096] In some embodiments, the C57BL / 6 subspecies mouse is selected from the C57BL / 6JCya subspecies mouse.

[0097] The ninth aspect of the present invention provides a method for treating pantothenic acid kinase-related neurodegenerative diseases, characterized in that the method comprises administering exosomes as described in the third aspect of the present invention or pharmaceutical compositions as described in the fourth aspect of the present invention.

[0098] The advantages and beneficial effects of this invention are as follows: 1. Strong brain-targeting ability and high delivery efficiency This invention uses MSC-derived exosomes as a delivery carrier to achieve targeted delivery to the central nervous system via nasal administration. Exosomes can cross the blood-brain barrier and are enriched in brain regions closely related to motor regulation, such as the hippocampus, prefrontal cortex, and basal ganglia.

[0099] 2. Significantly restores PANK2 protein expression and metabolic function. In a PANK2 gene knockout mouse model, the exosomes of this invention can significantly increase the expression level of PANK2 protein in brain tissue.

[0100] 3. Good long-term safety. After 30 and 60 days of continuous intranasal administration, the serum ALT, TBIL, UREA and CK levels in the treatment group mice remained within the normal range, and no obvious histological damage was observed in the major organs. This indicates that the present invention has good performance in terms of system safety and nervous system compatibility, and is suitable for long-term intervention.

[0101] 4. Overcoming the limitations of traditional treatment methods Currently, there is a lack of targeted molecular therapies for pantothenic acid kinase-related neurodegenerative diseases. Traditional treatments mainly focus on symptomatic support and cannot fundamentally correct metabolic defects. This invention, by supplementing functional PANK2 protein, restores the function of the CoA metabolic pathway from its source. This is a gene function compensation-based precision treatment strategy, providing a new intervention approach for PANK2-related neurodegenerative diseases.

[0102] 5. The delivery method aligns with clinical translation trends. Exosomes, as naturally derived nanovesicle carriers, possess advantages such as low immunogenicity, high biocompatibility, and scalability for mass production. Compared to traditional integrative viral vectors, they do not introduce the risk of genome integration, making them more aligned with the development trends of modern precision medicine and personalized treatment. The technical solution of this invention is scalable in its production process, suitable for development towards clinical-grade preparation and industrialization.

[0103] In summary, the present invention provides a technical solution for delivering PANK2 via MSC-derived exosomes, which can not only effectively restore PANK2 protein expression in the brain and improve neurological dysfunction, but also has good safety and translational potential, providing an innovative and clinically promising treatment strategy for PANK2 deficiency-related diseases. Attached Figure Description

[0104] Figure 1 This is a schematic diagram of the carrier structure; Figure 2 This is a diagram of the exosome identification results; Figure 3 It is PANK2 - / - Diagram of mouse model construction and validation strategies; Figure 4 It is PANK2 - / - Mouse validation results (image); Figure 5 This is a graph showing the improvement in motor dysfunction in mice after 30 days of exosome administration; Figure 6 This is a graph showing the improvement in motor dysfunction in mice after 60 days of exosome administration; Figure 7 This is a graph showing the results of exosome administration in restoring PANK2 expression levels in the basal ganglia; Figure 8 This is a safety assessment of intranasal administration of exosomes. Detailed Implementation

[0105] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0106] Example 1: Preparation of exosomes (1) Construction of lentiviral vector plasmids The lentiviral expression vector pLV[Exp]-Kan-EF1A>hHBA1_5′UTR / hPANK2NM_153638.4 / hHBB_3′UTR:WPREmut6 used in this invention is a third-generation self-inactivated (SIN) lentiviral system with a total length of 9181 bp. The vector structure includes key functional elements such as 5′LTR-ΔU3, RRE, cPPT, EF1A promoter, hHBA1 5′UTR, Kozak sequence, hPANK2 coding region, hHBB 3′UTR, WPREmut6, and 3′LTR-ΔU3. Based on theoretical analysis of the restriction endonuclease sites according to the complete vector sequence, it is predicted that NdeI has two cleavage sites on the plasmid (235 bp and 6569 bp), theoretically capable of cutting the 9181 bp plasmid into two fragments of 6334 bp and 2847 bp. This theoretical analysis is used to confirm the logical integrity of the carrier structure.

[0107] Based on structural analysis, Sanger sequencing was performed to validate key regions of the expression cassette. Specific primers covering the EF1A promoter, hHBA1 5′UTR, Kozak sequence, full-length hPANK2 open reading frame, hHBB 3′UTR, and WPREmut6 region were designed, and a segmented sequencing strategy was used to achieve full cassette coverage. Sequencing results were compared base-by-base with the reference sequence hPANK2 (NM_153638.4) and the standard vector sequence. No base mismatches, insertions, or deletions were found; the open reading frame was intact, and there were no frameshifts or premature stop codons.

[0108] In summary, the structure of this lentiviral expression vector ( Figure 1 The design is reasonable and the sequence is accurate, which can be used for subsequent virus packaging and functional experimental research.

[0109] (2) Packaging and concentration of recombinant lentiviruses To obtain high-titer recombinant lentiviral particles, the expression plasmid was first amplified and purified. Glycerol-containing bacteria were removed from an 80°C freezer and streaked onto LB agar plates containing kanamycin, then incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing antibiotics for shake culture amplification. After the bacterial culture reached the logarithmic growth phase, high-purity plasmid DNA was extracted using an endotoxin-free plasmid extraction kit. The DNA concentration and purity were measured using a nanospectrophotometer to ensure that the A260 / 280 ratio was between 1.8 and 2.0 to meet the requirements for subsequent transfection.

[0110] Lentiviral packaging uses a three-plasmid system. One day before transfection, healthy HEK293T cells were seeded into culture dishes and cultured in DMEM medium containing 10% FBS to achieve 70%–80% cell confluence at transfection. On the day of transfection, the target plasmid pLV-PANK2, packaging plasmid psPAX2, and envelope plasmid pMD2.G were mixed in a 4:3:1 ratio in serum-free medium (Opti-MEM (gibco)). Separately, PEI (MedChemExpress Cat.No.: HY-K2014 Product Name: PEI Transfection Reagent HY-K2014 - 10 mL) transfection reagent was diluted in serum-free medium and incubated at room temperature for 5 minutes. Then, the PEI solution was gently mixed with the plasmid mixture at a 1:1 ratio and incubated at room temperature for approximately 20 minutes to form the DNA–PEI complex. The complex was then evenly added dropwise to a 293T cell culture dish and gently mixed. The medium was replaced with fresh complete medium 6–8 hours after transfection.

[0111] Cell supernatants were collected at 48 and 72 hours post-transfection. The collected viral supernatants were filtered through a 0.45 μm filter to remove cell debris. The filtrate was concentrated by ultracentrifugation (4°C, 25,000 rpm, 2 hours), and the supernatant was discarded. The viral pellet was gently resuspended in PBS. The virus was then aliquoted and stored... Store at 80℃ for later use.

[0112] (3) Lentiviral infection of MSCs and construction of stable cell lines Before large-scale infection, the optimal infection and screening conditions for MSCs need to be determined through preliminary experiments. First, a puromycin kill curve experiment is performed: MSCs are seeded in 24-well plates, and different concentrations of puromycin (e.g., 0.5, 1.0, 1.5, 2.0 μg / mL) are added, and the plates are cultured continuously for 3–5 days. The lowest concentration that kills all uninfected MSCs within a specified time is taken as the optimal lethal concentration for subsequent stable cell transformation screening. Simultaneously, infection efficiency and cell status can be observed in the preliminary experiments using different virus dosages (or different MOIs) to determine the optimal conditions for subsequent formal infection.

[0113] During formal infection, healthy, low-passage MSCs were seeded into culture plates. When cell confluence reached 30%–50%, an appropriate amount of concentrated lentiviral solution (pLV-PANK2) was added, gently mixed, and incubated in an incubator. Approximately 8 hours after infection, the medium was replaced with fresh complete medium to reduce toxicity and promote cell recovery. Approximately 15 hours after infection, cells were passaged at a ratio of 1:3–1:5 and replaced with selection medium containing a pre-determined lethal concentration of puromycin for drug screening. During screening, the selection medium was changed every 2–3 days for approximately 1–2 weeks until all uninfected control group cells died, while drug-resistant cell clones stably survived and expanded in the infected group. Finally, the surviving clones were collected and expanded to establish a stable MSC cell line overexpressing PANK2. To verify the success of stable transfection, cell proteins were extracted and the expression level of PANK2 protein in MSCs was detected by Western Blot. At the same time, PANK2 mRNA was detected by qPCR. The stable high expression of PANK2 was confirmed by comparison with control MSCs, thus completing the construction and identification of the PANK2-MSC stable transfection cell line.

[0114] (4) Production and purification of PANK2-MSC-derived exosomes To obtain exosomes from stably overexpressing PANK2 MSCs (PANK2-MSCs), the stably transfected PANK2-MSCs, validated by Western blotting, were first expanded to the required number. When cell confluence reached approximately 70%–80%, the cells were gently washed twice with sterile PBS to remove residual serum-derived exosomes. The cells were then cultured for another 48 hours in serum-free medium or exosome-specific de-exosome medium to promote exosome secretion into the culture supernatant. During culture, cell stability was maintained to prevent apoptosis or contamination that could affect exosome quality.

[0115] After culture, the cell supernatant was collected and first centrifuged at low speed to remove cells and cell debris (e.g., 300 × g for 10 min, followed by 2,000 × g for 10 min). If necessary, further centrifugation at 10,000 × g could be performed to remove larger vesicles and apoptotic bodies. The treated supernatant was concentrated and then purified into exosomes using a qEV size exclusion column. Specifically, 500 μL of concentrated cell supernatant was added to a equilibrated qEV column (izon Product code: ICO-70). After the sample had completely entered the column, 3 mL of 1×PBS was added for elution, and the initial eluent was discarded. Then, 2 mL of 1×PBS was added for elution, and the corresponding fractions were collected. The collected eluent was the enriched exosome suspension. After separation, the column was thoroughly rinsed with 1×PBS (5 times in total) to ensure column cleanliness and reusability. The resulting PANK2-MSC-derived exosomes were preserved and stored at 4–8 °C for short-term storage and at [other locations] for long-term storage. Keep in the refrigerator at 80℃.

[0116] (5) Identification of particle size, morphology and marker proteins of exosomes derived from PANK2-MSC To systematically characterize the physicochemical properties and purity of exosomes derived from stably overexpressing PANK2 MSCs (PANK2-MSCs), their particle size distribution, concentration, morphology, and marker protein expression were detected. First, nanoparticle tracking analysis (NTA) was used to determine the exosome particle size and concentration. A Zetaview instrument was used to track the Brownian motion of particles in the sample in real time, and the hydrodynamic diameter and particle concentration were calculated by analyzing the particle trajectory. Before formal detection, the instrument was calibrated using 100 nm standard nanoparticles diluted at a ratio of 1:250,000. The exosome sample was diluted 1:1,000 and slowly injected into the detection cell using a 5 mL syringe, ensuring that air bubbles were expelled to avoid detection errors. The results showed that the exosome particle size was mainly distributed in the 30–150 nm range, consistent with typical exosome particle size characteristics.

[0117] To further observe the morphology of exosomes, ultrastructural analysis was performed using transmission electron microscopy (TEM). A suitable amount of exosome suspension was dropped onto a copper mesh support, negatively stained, and observed, revealing a typical bilayer membrane structure and cup-shaped morphology. To verify the purity and specific marker expression of exosomes, exosome proteins were extracted and analyzed by Western blotting. The results showed that the exosome samples expressed classic positive marker proteins (such as CD9, CD63, CD81, Alix, and HSP70), while the organelle marker protein GM130 was not detected, indicating that the samples were not contaminated by the Golgi apparatus and had good purity. Furthermore, the expression level of PANK2 protein in exosomes was detected by Western blotting, and the results showed that PANK2 was significantly enriched in PANK2-MSC-derived exosomes, confirming that the target protein was successfully loaded and stably present in the exosomes. The exosome identification results are as follows: Figure 2 As shown.

[0118] Example 2: Functional Verification of Exosomes To evaluate the therapeutic efficacy and safety of MSC-derived PANK2 exosomes in vivo, a disease model was established using 6–8 week old PANK2 gene knockout (PANK2^- / -) mice. Existing technology reports that PANK2 gene knockout mice do not exhibit the typical neurobehavioral phenotype consistent with human pantothenic kinase-associated neurodegeneration (PKAN). However, this invention, through a series of experiments, found that knocking out exon 2 of the PANK2 gene in mice can exhibit neurological dysfunction and motor abnormalities. The disease model mice were C57BL / 6JCya-Pank2^em1 / Cya, constructed within the C57BL / 6JCya genetic background, belonging to a conventional knockout model. The targeting strategy is as follows: Figure 3 As shown: Deletion or disruption of the key functional region of the mouse Pank2 gene (involving exon 1 to exon 2) results in a coding sequence interruption, thereby achieving Pank2 loss of function. The specific construction method is as follows: (1) Using CRISPR / Cas9 technology, single-stranded RNAs (sgRNAs) targeting key exon regions of the Pank2 gene were designed. The sgRNAs are shown in SEQ ID NO.1 and SEQ ID NO.2. SEQ ID NO.1: gRNA-A1: TAGGCTTTCCTAGCACGTTA-GGG SEQ ID NO.2: gRNA-A2: ACGCCTATTTGCGGTTGTCT-TGG (2) sgRNA and Cas9-mRNA were introduced into the fertilized egg together; (3) The fertilized egg is transferred to the surrogate recipient to obtain an F0 generation through surrogacy; (4) The F0 generation was crossbred to obtain homozygous PANK2^- / - mice.

[0119] Genotyping of the model mice was performed using PCR. The validation strategy was as follows: Figure 3 and Figure 4 As shown, two sets of specific primers were used for detection: the first set of primers was F1: 5′-GATTTAGGGTTGGGAAGGGACATA-3′, R1: 5′-CTTTCTCTCGATTCTTCACTAGCC-3′, amplifying the mutant allele fragment size of 679 bp, corresponding to the wild-type allele size of 2069 bp; the second set of primers was F2: 5′-AGGGGATAAGAGCCTACAAGGAAT-3′, R1: 5′-CTTTCTCTCGATTCTTCACTAGCC-3′, amplifying the wild-type allele fragment size of 569 bp. The total volume of the PCR reaction system was 25 μL, including 9.0 μL ddH2O, 1.0 μL upstream primer, 1.0 μL downstream primer, 12.5 μL Premix Taq, and 1.5 μL DNA template. The PCR reaction conditions were: 94 ℃ pre-denaturation for 3 min, followed by 35 cycles (94 ℃ for 30 s, 60 ℃ for 35 s, 72 ℃ for 35 s), and a final extension at 72 ℃ for 5 min. Genotypes were determined based on electrophoresis results: homozygous knockout mice (Pank2^- / -) showed a single band of 679 bp, heterozygous mice showed two bands of 679 bp and 569 bp, and wild-type mice showed a single band of 569 bp. The validation results are as follows: Figure 4 As shown.

[0120] After obtaining homozygous PANK2^- / - mice through the above screening method, further behavioral screening was conducted before incorporating them into subsequent experiments. Results showed that these model mice exhibited significant neurological dysfunction and abnormal motor coordination. PANK2 deficiency leads to impaired coenzyme A (CoA) metabolic pathways, subsequently causing mitochondrial dysfunction, abnormal lipid metabolism, and neurodegenerative changes. In this embodiment, PANK2-MSC-derived exosomes were delivered intranasally, allowing them to enter the central nervous system via the olfactory nerve-related pathway, thereby compensating for PANK2 function in the brain and being used for subsequent treatment efficacy evaluation.

[0121] Experimental animals were randomly divided into five groups: ① Wild-type control group, receiving an equal volume of physiological saline (20 μL / animal) intranasally; ② PANK2 KO model group, receiving an equal volume of physiological saline (20 μL / animal) intranasally; ③ Low-dose treatment group, receiving 2 × 10⁻⁶ saline intranasally. 6 particles / each; ④ Medium-dose treatment group, 2 × 10 particles / each administered intranasally. 7 particles / each; ⑤ High-dose treatment group, 2 × 10 particles / each administered intranasally. 8 Particles / each. Dosage was administered once daily for 30 and 60 days to assess interim and sustained treatment efficacy, respectively.

[0122] Systematic behavioral assessments were performed after 30 and 60 days of continuous administration, including the open field test (autonomic activity), balance beam test (fine motor coordination), pole climbing test (motor initiation and descent), grip strength test (muscle strength level), rotarod test (motor coordination and endurance), and treadmill test (motor tolerance). All behavioral tests were analyzed in a blinded manner. Results showed that, compared with the KO model group, mice treated with PANK2 exosomes exhibited significant improvements in rotarod dwell time, balance beam passage time, and grip strength, demonstrating a clear trend towards motor function recovery, with dose-dependent changes and more significant effects in the medium- and high-dose groups. Figure 5 , Figure 6 ).

[0123] After the behavioral experiments were completed, brain tissue and serum samples were collected for molecular and histological analysis. Western blot results showed that brain tissue and serum samples were collected from the hippocampus, prefrontal cortex, and basal ganglia. Figure 7 In key brain regions such as the brain, the expression level of PANK2 protein was significantly upregulated in the treatment group, partially recovering to near wild-type levels, indicating that exosome-mediated PANK2 supplementation alleviated metabolic defects in KO mice to some extent.

[0124] Regarding safety assessment, serum biochemical tests (ATL, TBTL, CK, and UREA, etc.) remained within the normal range, and no significant abnormalities in liver and kidney function were observed. HE staining of major organs (heart, liver, spleen, lungs, and kidneys) showed no significant histological damage, and inflammatory factor levels did not show abnormal elevations, suggesting that long-term nasal administration has good systemic safety. Figure 8 ).

[0125] In summary, in the PANK2 gene knockout mouse model, intranasal administration of MSC-derived PANK2 exosomes effectively increased the expression level of PANK2 protein in the brain, improved motor behavior disorders, and partially restored neurological function, while no obvious toxic side effects were observed, indicating that it has good therapeutic potential and biosafety.

[0126] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of PANK2-loaded exosomes in the preparation of drugs for treating diseases related to PANK2 gene defects.

2. Use according to claim 1, characterized in that, The PANK2 gene deficiency-related diseases are selected from pantothenic acid kinase-related neurodegenerative diseases.

3. Use according to any one of claims 1-2, characterized in that, The PANK2-loaded exosomes were prepared by the following method: Step 1): Infect mesenchymal stem cells with lentiviral particles containing PANK2; Step 2): Isolate and purify exosomes from the mesenchymal stem cells obtained in Step 1); Preferably, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood; Preferably, the lentiviral particle containing the PANK2 gene is assembled from a pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, a packaging plasmid, and an envelope plasmid. Preferably, the packaging plasmid is selected from psPAX2; Preferably, the envelope plasmid is selected from pMD2.G; Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2). Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:1; Preferably, step 2) further includes screening cells that stably express exosomes loaded with PANK2A.

4. A method of preparing PANK2-loaded exosomes, characterized in that, The method includes the following steps: Step 1): Infect mesenchymal stem cells with lentiviral particles containing PANK2; Step 2): Isolate and purify exosomes from the mesenchymal stem cells obtained in Step 1); Preferably, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood; Preferably, the lentiviral particle containing the PANK2 gene is assembled from a pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, a packaging plasmid, and an envelope plasmid. Preferably, the packaging plasmid is selected from psPAX2; Preferably, the envelope plasmid is selected from pMD2.G; Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2). Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:1; Preferably, step 2) further includes screening cells that stably express exosomes loaded with PANK2.

5. An exosome, characterized in that, The exosomes are prepared by the method of claim 4.

6. A pharmaceutical composition for treating diseases related to PANK2 gene deficiency, characterized in that, The pharmaceutical composition comprises the exosomes as described in claim 5; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients; Preferably, the PANK2 gene deficiency-related diseases are selected from pantothenic acid kinase-related neurodegenerative diseases. Preferably, the pharmaceutical composition is administered via nasal administration.

7. A lentiviral particle, characterized in that, The lentiviral particles are assembled from pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, packaging plasmid, and envelope plasmid. Preferably, the packaging plasmid is selected from psPAX2; Preferably, the envelope plasmid is selected from pMD2.G; Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is (1-5):(1-5):(0.5-2). Preferably, the ratio of the pLV[Exp]-EF1A plasmid containing the PANK2 gene coding sequence, the packaging plasmid, and the envelope plasmid is 4:3:

1.

8. A cell, characterized in that, The cells were infected with the lentiviral particles of claim 7; Preferably, the cells comprise mesenchymal stem cells; Preferably, the mesenchymal stem cells are selected from mesenchymal stem cells derived from umbilical cord blood.

9. A nasal drug delivery device, characterized in that, The nasal delivery device includes a delivery unit containing the exosomes as described in claim 5.

10. A method for constructing a non-human animal model of pantothenic acid kinase-related neurodegenerative diseases, characterized in that, The method includes causing non-human animals to express or lose the activity of Pank2; Preferably, causing the non-human animal Pank2 expression or activity to be lost includes knocking out and / or knocking down Pank2 in non-human animals; Preferably, the loss of expression or activity of the non-human animal Pank2 includes knocking out and / or knocking down the exon 2 region of the Pank2 gene in the non-human animal. Preferably, the technique used for knockout and / or knockdown is selected from CRISPR-Cas9; Preferably, the sgRNA sequence used by the CRISPR-Cas9 is shown in SEQ ID NO.1 and SEQ ID NO.2; Preferably, the method further includes genotyping screening of non-human animal individuals to obtain Pank2 homozygous knockout animals; Preferably, the non-human animal is a mammal; Preferably, the mammal includes rodents; Preferably, the rodent is selected from the Muridae family; Preferably, the murine mouse is selected from the house mouse family; Preferably, the house mouse includes the C57BL / 6 subspecies of mouse; Preferably, the C57BL / 6 subspecies mouse is selected from the C57BL / 6JCya subspecies mouse.

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