Use of PRRT2 acting on forebrain excitatory neuron and up-regulator thereof

Abstract

Provided in the present invention is the use of an up-regulator of a proline-rich transmembrane protein 2 (PRRT2) selectively acting on a forebrain excitatory neuron in the preparation of a drug for treating or preventing epilepsy.

Description

PRRT2 acting on forebrain excitatory neurons and its upregulatory agents Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to PRRT2 acting on forebrain excitatory neurons and the application of an upregulator thereof. Background Art

[0002] Epilepsy is a typical central nervous system disorder caused by an imbalance between excitation and inhibition. Clinical manifestations include convulsions, muscle spasms, tonic convulsions, atonia, and absences, accompanied by brief loss of consciousness. During an episode, the patient's electroencephalogram (EEG) displays characteristic epileptic-like discharges. Epileptic seizures are characterized by sudden and recurrent onset, severely impacting patients' physical and mental health, as well as their normal social activities. The etiology of epilepsy is diverse, with known contributing factors including developmental abnormalities, genetic mutations, head injury, cerebrovascular disease, and inflammation. With the exception of a small number of self-limited epilepsy cases, most patients with epilepsy require treatment to control their seizures. Current treatments used in clinical practice primarily include medication, surgical resection of localized lesions, the ketogenic diet, vagus nerve stimulation, and deep brain stimulation. Antiepileptic drugs are the primary treatment for over two-thirds of epilepsy patients. Although over 30 antiepileptic drugs targeting various targets have been introduced since the 1950s, establishing a relatively effective clinical treatment regimen, approximately 30% of patients with refractory epilepsy still do not benefit from these drugs or drug combinations. Furthermore, some epilepsy patients who respond well to existing medications still face the challenges of long-term adverse drug reactions and inability to fully control their epilepsy. These unmet medical needs are a key driver for the research and development of novel mechanisms and therapies.

[0003] The homeostatic balance of neural networks depends on the collaboration between excitatory and inhibitory neurons. Epilepsy is primarily manifested by abnormal synchronous brain excitation caused by an imbalance in neural network homeostasis. Therefore, the ideal epilepsy intervention strategy is to selectively reduce excitatory neuronal activity or increase inhibitory neuronal activity through medication or other therapeutic means, thereby correcting the abnormal excitability of the neural network and restoring it to normal homeostasis. At the same time, intervention plans should minimize or avoid interference with non-epilepsy-related neural networks, such as motor, arousal, cognitive, and emotional circuits, to reduce adverse reactions associated with long-term medication use.

[0004] Voltage-gated sodium channels (hereafter referred to as sodium channels or Nav) are important molecules that brain neurons rely on for action potential generation and impulse conduction, and are the basis for the excitability of neural tissue. In the field of epilepsy treatment, sodium channels are important targets for antiepileptic drugs. Over a quarter of the antiepileptic drugs currently approved for clinical use target these channels, including sodium phenytoin, carbamazepine, oxcarbazepine, and lacosamide. The mechanism of action of these drugs is to regulate sodium channel activity, reduce the excitability of neural networks, and thereby reduce or prevent related types of epileptic seizures. Although antiepileptic drugs targeting sodium channels have been widely used in epilepsy treatment, their central nervous system adverse reactions (such as dizziness, drowsiness, ataxia, and irritability) remain unresolved.

[0005] Lack of cell type selectivity and poor cell state selectivity are the main causes of central nervous system adverse reactions (CNS) with current small-molecule antiepileptic drugs. In the central nervous system, there are four main subtypes of voltage-gated sodium channels (Nav1.1, Nav1.2, Nav1.3, and Nav1.6), which are distributed in different neuronal types at different stages of development. For example, in the forebrain, which is directly related to epilepsy, Nav1.2 and Nav1.6 are primarily distributed in excitatory neurons that use glutamate as the primary neurotransmitter, while Nav1.1 is primarily distributed in inhibitory interneurons that use γ-aminobutyric acid (GABA) as the primary neurotransmitter. In the cerebellar cortex, which is associated with motor regulation, Nav1.1, Nav1.2, and Nav1.6 are expressed individually or in combination in GABAergic interneurons, granule cells, and Purkinje neurons. Antiepileptic drugs that target sodium channels, due to their general lack of selectivity among sodium channel members, can act on non-epileptic-related neuronal cell populations after entering the central nervous system, causing CNS adverse reactions. Of particular concern is that the inhibition of forebrain inhibitory interneurons by antiepileptic drugs targeting sodium ion channels will, to a certain extent, reduce the function of the brain's inhibitory neural network, partially offset the inhibitory effect of antiepileptic drugs on excitatory neural networks, weaken their antiepileptic efficacy, and in some cases (such as Dravet syndrome) may even worsen epilepsy.

[0006] In addition to the adverse reactions caused by the lack of cell type selectivity, the lack of effective cell state selectivity of drugs is also an important reason for their adverse reactions. Cell state selectivity refers to the ability of anti-epileptic drugs or treatments to act on cells that are abnormally excited during epileptic seizures without interfering with the normal functions of similar cells in the non-epileptic state. Considering that epilepsy treatment often requires long-term drug use, drugs that have poor selectivity for cellular epileptic states often cause patients to feel uncomfortable during daily medication (non-epileptic state), affecting their daily life and work, and even causing patients to interrupt medication and cause epilepsy to relapse.

[0007] Therefore, there is an urgent need in this field to develop drugs with cell type selectivity and cell state selectivity targeting sodium ion channels, so as to enhance the intervention effect of epilepsy and reduce side effects, and promote the precise treatment of epilepsy.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a use of a proline-rich transmembrane protein 2 (PRRT2) upregulator that selectively acts on forebrain excitatory neurons, for preparing a drug for treating or preventing epilepsy.

[0010] In a first aspect of the present invention, there is provided a use of an upregulator of PRRT2 that selectively acts on forebrain excitatory neurons for preparing a drug for treating or preventing epilepsy.

[0011] In one or more embodiments, the upregulation is a significant upregulation, promotion, increase or improvement; for example, an upregulation, promotion, increase or improvement of 10%, 20%, 30% or more.

[0012] In one or more embodiments, the selective action on forebrain excitatory neurons includes: selectively acting on slow inactivation of sodium ion channels (preferably Nav1.2 and Nav1.6), and / or selectively acting on abnormal cell excitability.

[0013] In one or more embodiments, the treating or preventing epilepsy comprises reducing the number, frequency, level, and / or duration of epileptic seizures.

[0014] In one or more embodiments, the treating or preventing epilepsy does not affect the subject's daily activities.

[0015] In one or more embodiments, the number, frequency, level and / or duration of epileptic seizures are assessed by behavioral experiments in mice. Preferably, the behavioral experiments include EEG and behavioral observations.

[0016] In one or more embodiments, daily behavior is assessed by behavioral experiments on mice, including but not limited to open field test, running wheel test, and social behavior test.

[0017] In one or more embodiments, the subjects of the use include humans, non-human primates, and rodents.

[0018] In one or more embodiments, the upregulator of PRRT2 that selectively acts on forebrain excitatory neurons is a construct, or an expression system formed by the construct (e.g., a viral (infectious) system); the construct includes: an expression drive system, and a PRRT2 encoding gene whose expression is driven by the drive system.

[0019] In one or more embodiments, the expression driving system includes a single or combined forebrain excitatory neuron-specific expression driving system.

[0020] In one or more embodiments, the forebrain excitatory neuron-specific expression driving system includes a single form of forebrain glutamatergic neuron-specific promoter, including (but not limited to): CaMKIIa promoter; a single form of promoter is used to directly drive the expression of PRRT2.

[0021] In one or more embodiments, the forebrain excitatory neuron-specific expression driving system includes a combination of forebrain glutamatergic neuron-specific promoters, including (but not limited to): a forebrain excitatory neuron-specific promoter for driving Cre recombinase expression (such as Emx1 promoter or CaMKIIa promoter) and a strong promoter for driving PRRT2 expression (such as but not limited to beta-actin promoter (CAG) promoter); the combined expression driving system is used to take into account both forebrain excitatory neuron specificity and high-efficiency expression characteristics.

[0022] In one or more embodiments, the construct includes an operational gene expression regulatory element that is dependent on Cre recombinase, such as (but not limited to) double-floxed inverse orientation (DIO); preferably, the element for DIO is LoxP / Lox2272, and the specific recognition sequences based on LoxP and Lox2272 are used to express PRRT2 in a Cre recombinase-dependent manner.

[0023] In one or more embodiments, the construct includes: Construct 1, comprising sequentially linked: a promoter (such as but not limited to a beta-actin promoter), LoxP / Lox2272, a PRRT2 encoding gene, LoxP / Lox2272; the PRRT2 encoding gene is reversely linked between two pairs of LoxP / Lox2272 sequences; Construct 2: comprising sequentially linked: a forebrain excitatory neuron-specific expression promoter, a Cre recombinase encoding gene (such as an Emx1 promoter driving the expression of the Cre recombinase encoding gene).

[0024] In one or more embodiments, the expression of the gene encoding Cre recombinase is driven by a forebrain excitatory neuron-specific expression promoter; Cre recombinase acts on LoxP / Lox2272, converting the reverse PRRT2 encoding gene into a forward connection, and its promoter drives expression.

[0025] In one or more embodiments, an inducible expression system (such as but not limited to Tet-on / off) may be provided to achieve regulatable on / off of PRRT2 expression.

[0026] In one or more embodiments, the construct is contained in an expression vector or is directly inserted into the genome of the intervention subject through gene editing; the expression vector includes: viral vector, non-viral vector; preferably, the viral vector includes: adeno-associated virus (AAV) vector, lentiviral vector, adenoviral vector, retroviral vector; more preferably, the adeno-associated virus vector includes: PHP.eB serotype AAV vector, Cap-B10 serotype AAV vector; or the gene editing includes (but is not limited to) gene editing based on CRISPR-Cas (such as Cas9) technology.

[0027] In one or more embodiments, the PRRT2 is zebrafish-derived PRRT2, human-derived PRRT2, or mouse-derived PRRT2; preferably, zebrafish-derived PRRT2.

[0028] In one or more embodiments, the PRRT2 is: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 2, 4 or 6; (b) a protein derived from (a) which is obtained by replacing, deleting or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues in the amino acid sequence of protein (a), and has the function of protein (a); (c) a protein derived from (a) which has more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the amino acid sequence of protein (a) and has the function of protein (a); or (d) a protein formed by adding a tag sequence to the N- or C-terminus of any of the polypeptides (a) to (c), or adding a signal peptide sequence or a secretion signal sequence to its N-terminus.

[0029] In another aspect of the present invention, a construct for treating or preventing epilepsy is provided, comprising: an expression drive system, and a PRRT2 encoding gene whose expression is driven by the drive system.

[0030] In one or more embodiments, the expression driving system includes a single or combined forebrain excitatory neuron-specific expression driving system.

[0031] In one or more embodiments, the forebrain excitatory neuron-specific expression driving system includes a single form of forebrain glutamatergic neuron-specific promoter, including (but not limited to): CaMKIIa promoter; a single form of promoter is used to directly drive the expression of PRRT2.

[0032] In one or more embodiments, the forebrain excitatory neuron-specific expression driving system includes a combination of forebrain glutamatergic neuron-specific promoters, including (but not limited to): a forebrain excitatory neuron-specific promoter for driving Cre recombinase expression (such as Emx1 promoter or CaMKIIa promoter) and a strong promoter for driving PRRT2 expression (such as but not limited to beta-actin promoter (CAG) promoter); the combined expression driving system is used to take into account both forebrain excitatory neuron specificity and high-efficiency expression characteristics.

[0033] In one or more embodiments, the construct includes an operably linked: an operative gene expression regulatory element dependent on Cre recombinase, such as (but not limited to) double-floxed inverse orientation (DIO); preferably, the element for DIO is LoxP / Lox2272, and the specific recognition sequences based on LoxP and Lox2272 are used to express PRRT2 in a Cre recombinase-dependent manner.

[0034] In one or more embodiments, the construct is contained in an expression vector or is directly inserted into the genome of the intervention subject through gene editing, and the expression vector includes: a viral vector, a non-viral vector; preferably, the viral vector includes: an adeno-associated virus (AAV) vector, a lentiviral vector, an adenoviral vector, a retroviral vector; more preferably, the adeno-associated virus vector includes: a PHP.eB serotype AAV vector, a Cap-B10 serotype AAV vector.

[0035] In one or more embodiments, the gene editing includes (but is not limited to) gene editing based on CRISPR-Cas (such as Cas9) technology.

[0036] In another aspect of the present invention, an expression system for treating or preventing epilepsy is provided, which is a viral system obtained by packaging the viral vector.

[0037] In another aspect of the present invention, a drug for treating or preventing epilepsy or a drug kit containing the drug is provided, wherein the drug contains the expression system.

[0038] In another aspect of the present invention, a method for selectively delivering an effective amount of PRRT2, or the PRRT2 upregulator that selectively acts on forebrain excitatory neurons, to forebrain excitatory neurons is provided, comprising contacting the forebrain excitatory neurons with an expression vector comprising an effective amount of PRRT2, or its upregulator; preferably, the expression vector comprises: a viral vector, a non-viral vector; more preferably, the viral vector comprises: an adeno-associated virus (AAV) vector, a lentiviral vector, an adenoviral vector, a retroviral vector; even more preferably, the adeno-associated virus vector comprises: a PHP.eB serotype AAV vector, a Cap-B10 serotype AAV vector; preferably, the expression vector is delivered by intraorbital injection (preferably orbital vein injection), intracranial injection, intrathecal (spinal cord) injection, intrathecal (cerebral cisterna magna) injection, intracerebral injection, intraventricular injection, or direct injection into the epileptic focus in the brain.

[0039] In another aspect of the present invention, a method is provided for selectively acting on the slow inactivation of sodium ion channels, selectively acting on abnormal cell excitability, and / or selectively acting on forebrain excitatory neurons, comprising administering to a subject an effective amount of PRRT2, or an upregulator thereof (preferably an expression vector that overexpresses the PRRT2).

[0040] Other aspects of the present invention will be apparent to those skilled in the art in view of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1. The protein coding region (CDS) sequence of the mouse Prrt2 gene.

[0042] Figure 2. Amino acid sequence of mouse PRRT2 protein.

[0043] Figure 3. Schematic diagram of the EGFP control plasmid pCAG-empty-IRES-EGFP.

[0044] Figure 4. Schematic diagram of the mouse PRRT2 overexpression plasmid pCAG-mouse PRRT2(HA)-IRES-EGFP.

[0045] Figure 5. Sequence of the protein coding region (CDS) of the human PRRT2 gene.

[0046] Figure 6. Amino acid sequence of human PRRT2 protein.

[0047] Figure 7. Schematic diagram of the human PRRT2 overexpression plasmid pCAG-human PRRT2(HA)-IRES-EGFP.

[0048] Figure 8. The protein coding region (CDS) sequence of the zebrafish Prrt2 gene.

[0049] Figure 9. Amino acid sequence of zebrafish PRRT2 protein.

[0050] Figure 10. Schematic diagram of the zebrafish PRRT2 overexpression plasmid pCAG-zebra PRRT2(HA)-IRES-EGFP.

[0051] Figure 11. Effect of PRRT2 overexpression on the fast inactivation process of Nav1.2 sodium channel.

[0052] (A) Schematic diagram of sodium channel current recording in HEK293 cells stably expressing Nav1.2.

[0053] (B) Schematic diagram of the voltage-clamp protocol for activating sodium channels (top). Representative sodium channel current curves are shown below. When the voltage-gated sodium channel Nav1.2 is clamped to 0 mV, the channel opens, allowing sodium ions to flow inward, forming an inward sodium current. After a brief opening, the sodium channel rapidly inactivates, closing, and the sodium current decays rapidly, as indicated by the arrow.

[0054] (C) Rapid inactivation of sodium channels leads to sodium current decay, and the decay rate is characterized by the time constant tau. The time constant of sodium current decay in the PRRT2 overexpression group was not significantly different from that in the EGFP overexpression control group (nsP>0.05). mPRRT2: mouse PRRT2.

[0055] Figure 12. Effect of PRRT2 overexpression on the recovery of Nav1.2 sodium channel from fast inactivation.

[0056] (A) Schematic diagram of the voltage-clamp protocol for measuring sodium channel recovery from fast inactivation. Examples of sodium currents measured in control cells during the first depolarization and in fast-inactivating sodium channels after hyperpolarization recovery periods of varying lengths are shown below.

[0057] (B) The process of sodium channel recovery from the fast inactivation state. No significant difference was observed between the mPRRT2 overexpression group and the EGFP overexpression control group (nsP>0.05). mPRRT2: mouse PRRT2.

[0058] Figure 13. Effect of PRRT2 overexpression on the slow inactivation process of Nav1.2 sodium channel.

[0059] (A) Schematic diagram of the voltage-clamp protocol for detecting the slow inactivation of sodium channels. Examples of sodium currents measured in control cells during the first depolarization and after depolarization of sodium channels for varying durations followed by a brief hyperpolarization are shown below.

[0060] (B) The process of sodium channels entering the slow inactivation state. Compared with the EGFP-overexpressing control group, the sodium channels in the mPRRT2-overexpressing group entered the slow inactivation state more rapidly during sustained depolarization. The difference between the two groups was significant (P < 0.0001). mPRRT2: mouse PRRT2.

[0061] Figure 14. Effect of PRRT2 overexpression on the recovery of Nav1.2 sodium channels from slow inactivation.

[0062] (A) Schematic diagram of the voltage-clamp protocol for monitoring sodium channel recovery from slow inactivation. Examples of sodium currents measured during the first depolarization and after hyperpolarization recovery periods of varying lengths are shown below.

[0063] (B) The process of sodium channel recovery from slow inactivation. Compared with the EGFP-overexpressing control group, the mPRRT2-overexpressing group showed a slower recovery of sodium channel from slow inactivation. There was a significant difference between the two groups (P < 0.001).

[0064] Figure 15. Effect of zebrafish PRRT2 overexpression on slow inactivation of Nav1.2 sodium channel.

[0065] (A) Amino acid sequence alignment of mouse, human, and zebrafish PRRT2 proteins. The amino acid sequence identity between mouse and human PRRT2 proteins is 77.46%, while the amino acid sequence identity between zebrafish and mouse PRRT2 proteins is 43.6%. The bottom horizontal line in the sequence indicates the C-terminus of PRRT2 (including the transmembrane region).

[0066] (B) The process of sodium channels entering the slow inactivation state. Compared with the EGFP-overexpressing control group, the sodium channels in the mPRRT2, hPRRT2, and zPRRT2-overexpressing groups entered the slow inactivation state faster during sustained depolarization, with significant differences between the groups (P < 0.0001).

[0067] (C) Recovery of sodium channels from slow inactivation. Compared with the EGFP-overexpressing control group, the mPRRT2, hPRRT2, and zPRRT2 overexpressing groups showed a slower recovery rate from slow inactivation, with significant differences between groups (P < 0.0001). Furthermore, the zPRRT2 overexpressing group showed a significantly slower recovery rate from slow inactivation than the mPRRT2 and hPRRT2 groups, with significant differences between groups (P < 0.0001). mPRRT2: mouse PRRT2, hPRRT2: human PRRT2, zPRRT2: zebrafish PRRT2.

[0068] Figure 16. Schematic diagram of adeno-associated virus overexpressing mCherry plasmid pAAV-CAG-DIO-mCherry.

[0069] Figure 17. Schematic diagram of adeno-associated virus overexpressing mouse PRRT2 plasmid pAAV-CAG-DIO-mouse PRRT2-HA.

[0070] Figure 18. Overexpression of PRRT2 in forebrain excitatory neurons.

[0071] (A) Schematic diagram of orbital vein injection of AAV-PHP.eB virus into Emx1-cre mice.

[0072] (B) Schematic diagram of selective overexpression of PRRT2 in forebrain excitatory neurons. PRRT2-OE: PRRT2 over-expression.

[0073] (C) Fluorescence immunohistochemistry verified the expression of PRRT2 in forebrain excitatory neurons.

[0074] FIG19 . Overexpression of PRRT2 in forebrain excitatory neurons inhibits convulsant-induced epileptic seizures.

[0075] (A) Representative examples of epileptic behaviors in mice induced by a convulsant (pentylenetetrazole, PTZ, 45 mg / kg, intraperitoneal injection).

[0076] (B) Compared with the control group, overexpression of PRRT2 in forebrain excitatory neurons significantly reduced the severity of pentylenetetrazol-induced epileptic seizures in mice (P < 0.0001). PTZ: pentylenetetrazol; PRRT2-OE: PRRT2 over-expression.

[0077] Figure 20. Overexpression of PRRT2 in forebrain excitatory neurons inhibits convulsant-induced epileptiform electroencephalograms.

[0078] (A) Schematic diagram of EEG recording in mice.

[0079] (B) Representative EEG examples. Pentylenetetrazol (PTZ) can induce epileptiform EEG signals in the mouse cerebral cortex; overexpression of PRRT2 in excitatory neurons in the forebrain can effectively inhibit PTZ-induced epileptiform discharge events in the mouse cerebral cortex.

[0080] Figure 21. Overexpression of PRRT2 in forebrain excitatory neurons does not affect the normal behavioral activities of mice.

[0081] (A) In the open field test, the total distance covered by the two groups of mice was similar, and no significant difference was detected (nsP>0.05).

[0082] (B) In the rotarod test, the time the two groups of mice spent on the fixed-speed rotarod was similar, with no significant difference detected (nsP>0.05). The upper limit of the test time was 60 seconds.

[0083] (C) In the social behavior test, both groups of animals showed a tendency to communicate with their companion mice, and no significant difference was detected between the two groups (nsP>0.05). Empty represents the empty box group, and Novel represents the unfamiliar mouse group.

[0084] FIG22 . Overexpression of PRRT2 in forebrain inhibitory neurons facilitates convulsant-induced epileptic seizures.

[0085] (A) Schematic diagram of orbital vein injection of AAV-PHP.eB virus into Nkx2.1-cre mice.

[0086] (B) Schematic diagram of selective overexpression of PRRT2 in forebrain inhibitory neurons.

[0087] (C) Fluorescent immunohistochemistry validates the expression of PRRT2 in forebrain inhibitory neurons. Scale bar is 1 mm.

[0088] (D) Representative examples of epileptic behaviors induced by a subthreshold dose of a convulsant (pentylenetetrazolyl, PTZ, 35 mg / kg, intraperitoneal injection) in Nkx2.1-cre mice; PTZ: pentylenetetrazol.

[0089] (E) Compared with the control group, overexpression of PRRT2 in forebrain inhibitory neurons significantly enhanced the severity of epileptic seizures induced by subthreshold doses of pentylenetetrazol in Nkx2.1-cre mice, and the difference in seizure severity between the two groups was significant (P < 0.01). PRRT2-OE: PRRT2 over-expression.

[0090] (F) Schematic diagram of mouse EEG recording.

[0091] (G) Representative EEG examples. Subthreshold doses of PTZ failed to induce epileptiform EEG signals in the cerebral cortex of control mice; however, subthreshold doses of PTZ induced epileptiform discharge events in the cerebral cortex of mice overexpressing PRRT2 in forebrain inhibitory neurons. DETAILED DESCRIPTION

[0092] After in-depth research, the inventors discovered for the first time that the protein encoded by the PRRT2 (proline-rich transmembrane protein 2) gene can selectively act on sodium ion channels in the slow inactivation state, and reduce the effective supply of activatable sodium ion channels by enhancing the slow inactivation of sodium ion channels. Furthermore, the inventors selectively overexpressed the PRRT2 protein in the excitatory neurons of the forebrain to feedback-reduce the excitability of neurons in the abnormally excited state, and achieved the goal of effectively suppressing epileptic seizures without interfering with daily behavior in a mouse epilepsy model. The specific expression of PRRT2 in forebrain excitatory neurons is the key to successfully intervening in epilepsy and reducing side effects. The expression of PRRT2 in forebrain excitatory neurons, while having both cell type selectivity and cell state selectivity, has good application prospects in the field of epilepsy treatment.

[0093] PRRT2 and sodium channels

[0094] In the present invention, the term "PRRT2" includes proteins having SEQ ID NO: 2 (mouse), SEQ ID NO: 4 (human), or SEQ ID NO: 6 (zebrafish), and also includes variant forms of the sequence having the same function as PRRT2.

[0095] These variations include, but are not limited to, deletions, insertions, and / or substitutions of several amino acids (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), as well as additions or deletions of one or several amino acids (usually within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, in the art, substitutions with amino acids having similar or similar properties generally do not alter the function of the protein. For another example, additions or deletions of one or several amino acids at the C-terminus and / or N-terminus generally do not alter the function of the protein. The term also includes active fragments and active derivatives of PRRT2.

[0096] The term "PRRT2" also encompasses proteins having 80% or greater homology to the polypeptide sequences defined in SEQ ID NOs: 2, 4, or 6; preferably, 85% or greater homology, such as 90%, 95%, 98%, or 99% homology, and having the same functions as the PRRT2 described in the embodiments of the present invention. Methods and tools for comparing sequence identity are well known in the art, such as BLAST. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides, as measured by the percentage of identical positions.

[0097] Polynucleotide sequences encoding PRRT2 or variant proteins thereof (coding sequences) may also be used in the present invention. The term "coding gene" may include a polynucleotide encoding the protein or a polynucleotide further comprising additional coding and / or non-coding sequences. In some embodiments, the polynucleotide sequence encoding the PRRT2 protein (coding sequence) is the polynucleotide sequence set forth in SEQ ID NO: 1, 3, or 5.

[0098] The inventors' research has revealed that the protein encoded by the PRRT2 (proline-rich transmembrane protein 2) gene can selectively act on sodium channels in the slow-inactivation state. That is, overexpression of PRRT2 can promote the slow inactivation of sodium channels, but has no significant effect on the fast inactivation of sodium channels. PRRT2 has a state-dependent regulation characteristic for sodium channels. By enhancing the slow inactivation of sodium channels, it reduces the effective supply of sodium channels to cells under sustained depolarization. This characteristic of PRRT2 helps it reduce abnormal cell excitability without affecting the normal excitatory activity of cells, giving it good cell state selectivity.

[0099] Based on this discovery, the inventors used adeno-associated virus as a gene delivery vector to selectively overexpress PRRT2 protein in excitatory neurons in the forebrain, thereby feedback-reducing the excitability of neurons in abnormally excited states. Through animal experiments on mouse epilepsy models, they found that selectively targeting PRRT2 for treatment can effectively inhibit epileptic seizures without interfering with daily behavior.

[0100] In the present invention, the terms "voltage-gated sodium ion channel", "sodium ion channel" and "Nav" are used interchangeably.

[0101] PRRT2 acting on forebrain excitatory neurons and its upregulatory agents

[0102] Based on the above new findings of the inventors, the present invention provides a use of an upregulator of PRRT2 that selectively acts on forebrain excitatory neurons, for preparing a drug for treating or preventing epilepsy.

[0103] The "subject" described in the present invention includes but is not limited to: humans, non-human primates, and rodents (such as mice).

[0104] Those skilled in the art will recognize that epileptic seizures can be classified into different levels, including but not limited to tonic-clonic, tonic, clonic, myoclonic, absence, or atonic seizures. In mouse models, epileptic seizures can be categorized into six levels: Level 1: The mouse lies motionless, crawling on its belly; Level 2: Sudden convulsions, with sudden cessation of movement and raised tail; Level 3: Muscle jerks, head twisting, hand twitching; Level 4: Tonic-clonic seizures, with the mouse falling to the ground, twitching, and jumping and running wildly; Level 5: The mouse falls to the ground and develops tonic limb extension; Level 6: Death.

[0105] In one or more embodiments, selectively acting on forebrain excitatory neurons through an upregulator of PRRT2, the number, frequency, level and / or duration of seizures experienced by a subject can be reduced by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100%, including all ranges and subranges therebetween.

[0106] The "selective effect" is relative, for example, the effect on forebrain excitatory neurons is increased by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100%, relative to non-forebrain excitatory neurons, including all ranges and subranges therebetween.

[0107] The term "excitatory neuron" refers to a neuron that releases a neurotransmitter, such as glutamate, which causes the connected postsynaptic neuron to become more likely to fire. The term "inhibitory neuron" refers to a neuron that releases a neurotransmitter, such as gamma-aminobutyric acid, which causes the connected postsynaptic neuron to become less likely to fire.

[0108] As used in the present invention, the PRRT2 upregulator includes agonists, promoters, and stimulants, and these terms are used interchangeably. The PRRT2 upregulator refers to any substance that can increase the activity of PRRT2, enhance the stability of PRRT2, upregulate the expression of PRRT2, increase the effective action time of PRRT2, or promote the transcription and translation of the PRRT2 gene. These substances can be used in the present invention as substances useful for upregulating PRRT2, and thus can be used to treat or prevent epilepsy.

[0109] In one or more embodiments, the PRRT2 upregulator includes (but is not limited to): a construct, or an expression system formed by the construct (e.g., a viral (infectious) system); the construct includes: a forebrain excitatory neuron-specific expression driver system, and a PRRT2 encoding gene driven by the driver system. Typically, the forebrain excitatory neuron-specific expression driver system includes a forebrain excitatory neuron-specific expression promoter. As a preferred embodiment of the present invention, the forebrain excitatory neuron-specific expression promoter includes: Emx1 promoter and CaMKIIa promoter.

[0110] In one or more embodiments, the construct comprises: a promoter, an element for DIO, a PRRT2 encoding gene expression cassette (preferably, the PRRT2 encoding gene is reversely linked), and a Cre recombinase encoding gene driven by a forebrain excitatory neuron-specific expression promoter; preferably, the element for DIO is a LoxP / Lox2272 element, and a DIO regulation method based on LoxP and Lox2272 is used to achieve Cre recombinase-dependent PRRT2 expression.

[0111] As a preferred embodiment of the present invention, the construct comprises:

[0112] Construct 1, comprising: a promoter (such as but not limited to beta-actin promoter), LoxP / Lox2272, and a PRRT2 encoding gene, LoxP / Lox2272, connected in sequence; the PRRT2 encoding gene is connected in reverse order;

[0113] Construct 2: a forebrain excitatory neuron-specific expression promoter and a cre recombinase encoding gene (eg, Emx1 promoter drives the expression of the cre recombinase encoding gene).

[0114] In the present invention, the PRRT2 polynucleotide sequence can be inserted into a recombinant expression vector, which can then be transferred into cells to overexpress PRRT2. As long as it can replicate and be stable in the host, any plasmid or vector can be used in the present invention. Methods familiar to those skilled in the art can be used to construct expression vectors containing the PRRT2 DNA sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and translation control elements. For example, the expression vector includes: viral vectors, non-viral vectors; preferably, the viral vector includes (but is not limited to): adeno-associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, retroviral vectors, etc.

[0115] In the present invention, since the vector selectively acts on the excitatory neurons of the forebrain, the vector has good blood-brain barrier penetration ability, for example, the vector that can penetrate the blood-brain barrier accounts for more than 2%, more than 5%, more than 10%, more than 20%, more than 30% or more of the total vectors. In one or more embodiments of the present invention, the viral vector is an AAV vector. "AAV virion" or "AAV virus" or "AAV virus particle" or "AAV vector particle" or "AAV vector" refers to a viral particle comprising at least one AAV capsid polypeptide and a polynucleotide (such as a PRRT2 polynucleotide sequence). AAV vectors are usually named according to the name (serotype) of the capsid polypeptide. The AAV vectors include (but are not limited to): PHP.eB serotype AAV vector, Cap-B10 serotype AAV vector.

[0116] As one embodiment of the present invention, the gene encoding PRRT2 can be cloned into an appropriate vector (such as a conventional prokaryotic or eukaryotic expression vector, or a viral vector such as a herpes virus vector or an adeno-associated virus vector) by conventional methods, and the vector is introduced into cells that can express the PRRT2, so that the cells express PRRT2. The expression of PRRT2 can be achieved by introducing an appropriate amount of the cells into an appropriate part of the subject's body. As a preferred embodiment of the present invention, the vector is administered by intraorbital injection (e.g., orbital vein injection) so that it selectively acts on excitatory neurons in the forebrain and reduces the influence of the blood-brain barrier. It is known to those skilled in the art that other modes of administration can also be used in the present invention, such as but not limited to: intracranial injection, intrathecal (spinal cord) injection, intrathecal (cerebral cisterna magna injection), intracerebral injection, intraventricular injection, direct injection into the epileptic lesion in the hippocampus, and direct injection into the epileptic lesion in the temporal lobe.

[0117] After research and analysis, the inventors selected the PHP.eB serotype AAV virus as the delivery vector for the PRRT2 gene, which has good blood-brain barrier penetration ability. Considering that gene delivery vectors are in a period of rapid iteration and development, vectors that help to efficiently deliver the PRRT2 gene to neurons in the brain (such as Cap-B10 serotype AAV virus, etc.) and vectors that help to efficiently deliver the PRRT2 gene to excitatory neurons in the forebrain can be applied to this experiment.

[0118] In the present invention, after overexpression of PRRT2, methods well known to those skilled in the art can be used to detect whether the overexpression of PRRT2 is successful. Preferably, when performing gene-level detection, primers that specifically amplify PRRT2 can be used; or probes that specifically recognize PRRT2 can be used to determine the presence or absence of the PRRT2 gene; when performing protein-level detection, antibodies or ligands that specifically bind to the protein encoded by PRRT2 can be used to determine the expression of the PRRT2 protein, for example, by using an anti-PRRT2 antibody to bind to the PRRT2 protein, and then using a secondary antibody with a detection signal (preferably a fluorescent label) to bind to the anti-PRRT2 antibody, and judging by the presence or absence of the detection signal, or the strength of the detection signal.

[0119] The present invention also provides a method for treating or preventing epilepsy, comprising selectively administering an effective amount of PRRT2 or an upregulator thereof (such as an expression vector overexpressing the PRRT2) to excitatory neurons in the forebrain of a subject.

[0120] The terms "treat" and "prevent" generally refer to the use of an agent or method to reduce, eliminate, or prevent the symptoms of a disease, and include achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to slowing the progression of the condition or symptoms of the condition being treated, halting progression, reversing progression, or eradicating or ameliorating symptoms. A prophylactic benefit includes reducing the risk of a condition, delaying the progression of a condition, or reducing the likelihood of a condition occurring.

[0121] After knowing the use of the PRRT2, the PRRT2 or its upregulator can be administered to the subject using a variety of methods well known in the art. Preferably, gene therapy can be used. For example, an expression unit (such as an expression vector or virus, etc.) carrying the PRRT2 gene can be delivered to the target site through a certain route, and the expression (preferably overexpression) of active PRRT2 can be achieved. Alternatively, the expression of endogenous PRRT2 in forebrain excitatory neurons can be enhanced by in vivo gene editing (such as CRISPR-Cas9 gene editing method), thereby achieving the purpose of treating or preventing epilepsy.

[0122] The term "effective amount" refers to an amount that can achieve a desired result (such as a preventive or therapeutic result). The effective amount of PRRT2 or its upregulator according to the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the PRRT2 or its upregulator, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated for the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0123] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Molecular Cloning Laboratory Manual (ed. by J. Sambrook et al., Science Press), or according to the conditions recommended by the manufacturer.

[0124] Materials and methods

[0125] 1. Experimental Materials

[0126] Mouse PRRT2 plasmid (Origene, #MR214094).

[0127] Human PRRT2 plasmid (Origene, #RC202304).

[0128] Zebrafish PRRT2 plasmid (laboratory constructed, cloned from adult zebrafish brain tissue cDNA library).

[0129] pCAG2IG plasmid (Addgene, #122292).

[0130] Lipofectamine 3000 transfection kit (ThermoFisher Scientific, #L3000001).

[0131] Nav1.2 stably transfected cell line (Beijing Ice-Eye Biotechnology Co., Ltd., #ICE-Nav1.2-HEK).

[0132] CO2 constant temperature incubator (ThermoFisher Scientific, #3111).

[0133] Patch clamp recording system (HEKA, #EPCIO).

[0134] Extracellular solution: 140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM D-Glucose, 1.25 mM NaH2PO4, 10 mM HEPES (pH = 7.4 adjusted with NaOH).

[0135] Electrode internal solution: 50 mM CsCl, 10 mM NaCl, 10 mM HEPES, 20 mM EGTA, 60 mM CsF, pH = 7.2 (CsOH).

[0136] AAV-CAG-DIO-mCherry plasmid (Figure 16) (provided by the Gene Editing Platform of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences).

[0137] pAAV-CAG-DIO-mouse PRRT2-HA plasmid (Figure 17) (provided by the Gene Editing Platform of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences).

[0138] Anti-PRRT2 antibody (Wiiget Biotech, #Rp3246-a).

[0139] Anti-HA antibody (ROAHAHA Roche, #11867423).

[0140] The AAV virus used in this invention is produced and provided by the Gene Editing Platform of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences:

[0141] AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA;

[0142] AAV-PHP.eB-CAG-DIO-mCherry;

[0143] Pentylenetetrazol (PTZ) (Sigma-Aldrich, #P6500-25G).

[0144] C57BL / 6J mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0145] Emx1-cre mice, named: B6.129S2-Emx1tm1(cre)Krj / J, were obtained from Jackson Lab, catalog number: JAX catalog number #005628.

[0146] Nkx2.1-cre mice, named: C57BL / 6J-Tg(Nkx2-1-cre)2Sand / J, from Jackson Lab, catalog number: JAX catalog number #008661.

[0147] 2. Animal husbandry

[0148] Three to six mice were housed in each cage with free access to food and water in the cage under a 12-h light / dark cycle, 22-23°C, and 50-60% humidity.

[0149] All experimental animal operations and use complied with the requirements of the Animal Care Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences for experimental animal operations and animal welfare.

[0150] 3. Orbital vein injection of AAV virus

[0151] Take out the AAV virus stored at -80 degrees Celsius and thaw it on ice. Dilute the AAV virus with pre-cooled sterile PBS to the injection concentration (2×10 12 vg / mL, vg: AAV vector genomes). Before injection, the AAV virus solution was equilibrated to room temperature, and 50 μL of the AAV virus solution was drawn up using a 0.3 mL insulin needle for injection.

[0152] Anesthetize the mouse by injecting sodium pentobarbital solution (90 mg / kg, intraperitoneal injection). After the mouse is anesthetized, fix the mouse's head with your fingers, avoid pressing the mouse's artery, and stretch the skin around the eye to fully expose the eyeball. Insert the insulin needle at a 20° angle from the corner of the eye, and avoid injuring the eyeball when entering the eye socket. The tip of the injection needle enters the eye socket to a depth of 2mm to reach the orbital venous plexus, and slowly inject 50μL. After the injection, slowly withdraw the needle and wipe off excess fluid and blood from the corner of the eye. Perform the same injection in the orbital vein on the other side, and the total injection titer on both sides is 2×1011 vg's AAV virus.

[0153] After the injection, the mice were placed back in the cage and returned to the breeding room after they woke up.

[0154] Electrode implantation surgery was performed two weeks after viral expression, and epilepsy evaluation experiments were performed three weeks after viral expression.

[0155] 4. EEG / EMG electrode implantation and EEG recording

[0156] The experimental mice were first anesthetized with 4% isoflurane, and their heads were then fixed to the stereotaxic adapter. The isoflurane concentration was adjusted to 1.5% throughout the surgery to keep the mice under continuous anesthesia. Additionally, chlortetracycline eye ointment was used to protect the mouse's eyes to prevent retinal damage caused by dryness. Hair was removed from the mouse's head using a depilatory cream, and the skin was incised along the midline to expose the skull surface. A 2% hydrogen peroxide solution was used to treat the surgical incision and skull surface, and connective tissue on the skull surface was scraped off. The skin surrounding the exposed skull was secured with tissue glue (Vetbond, 3M Germany GmbH), and the membranes on the skull surface were cleaned again. The electrode implantation locations were marked using stereotaxic methods, with coordinates AP -0.8 mm and ML ±1.1 mm. After the skull surface dried, it was covered with a light-curing self-etching adhesive (3M ESPE Single Bound Universal) to form a solidified layer. A small handheld cranial drill was used to create a hole with a diameter of approximately 0.6 mm at the implantation site mark. Carefully clean the bone debris at the opening and keep the surface of the cerebral cortex moist. Then, two sterile ECoG recording electrodes (0.5 mm in diameter) were vertically inserted into the cerebral cortex at a depth of 0.35 mm (with the surface of the cerebral cortex as a reference) along the skull opening. The recording electrodes and electrode interface components were fixed with dental cement. To facilitate electrophysiological recordings in awake mice, a lightweight titanium alloy sheet (30 mm × 2 mm × 1 mm) was additionally fixed to the mouse head near the electrode interface with dental cement. Two metal wire electrodes for recording electromyographic signals extended from the bottom of the electrode interface component to the mouse neck. The exposed ends of the 1 mm electrodes were inserted into the muscle layer on both sides of the neck. After fixation, the neck skin was sutured and disinfected. The mice recovered for one week after surgery.

[0157] Before awake ECoG / EMG recordings, mice were fixed to the recording platform using a metal plate around their heads and allowed to acclimate to this head-fixed position for 30 minutes. ECoG and EMG signals were amplified 100x using a differential amplifier (Model 3000, AM system) and then band-filtered (ECoG 1-300 Hz, EMG 10-1000 Hz) for acquisition (1000 Hz) and recording using a digital-to-analog converter (Digidata 1332A, Molecular Devices).

[0158] 5. PTZ-induced epilepsy mouse model

[0159] Before testing, a 3.5 mg / mL (Nkx2.1-cre mouse) or 4.5 mg / mL (Emx1-cre mouse) PTZ solution was prepared in normal saline. Depending on mouse weight, PTZ was injected intraperitoneally at a dose of 35 mg / kg or 45 mg / kg. Immediately following injection, mice were placed in a behavioral chamber and video recorded for seizure activity within 30 minutes of administration.

[0160] Mouse epilepsy rating criteria:

[0161] Level 1: The mouse lies motionless, crawling on its belly;

[0162] Level 2: Sudden convulsions, sudden cessation of behavior, and raised tail;

[0163] Grade 3: Myoclonus, head twisting, hand lifting and twitching;

[0164] Grade 4: Tonic-clonic, convulsions on the ground, jumping and running wildly;

[0165] Grade 5: Falling to the ground and developing tonic extension of limbs;

[0166] Level 6: Death.

[0167] 6. Immunohistochemical detection of PRRT2 expression in the brain

[0168] After deep anesthesia with sodium pentobarbital, mice were perfused through the heart with 10 mL of room-temperature phosphate-buffered saline (PBS) to flush the blood, and then perfused with 10 mL of 4% paraformaldehyde (PFA) at 4°C for tissue fixation. The mouse brain tissue was dissected and removed, placed in 4% paraformaldehyde solution and fixed overnight. The brain tissue was then dehydrated in PBS containing 30% sucrose for 2 days, and the sucrose solution was replaced once every 24 hours. After dehydration, the brain tissue was sagittally sectioned at -20°C with a thickness of 25 μm using a freezing microtome (CM1950, Leica). The brain slices were collected in PBS solution and stored at 4°C. Conventional immunohistochemistry was performed using anti-PRRT2 antibodies.

[0169] 7. Sequence information

[0170] (1) Mouse Prrt2 gene (protein coding region, CDS) sequence (SEQ ID NO: 1):

[0171] (2) Mouse PRRT2 protein amino acid sequence (346 amino acids) (SEQ ID NO: 2):

[0172] (3) Human Prrt2 gene (protein coding region, CDS) sequence (SEQ ID NO: 3):

[0173] (4) Human PRRT2 protein amino acid sequence (340 amino acids) (SEQ ID NO: 4):

[0174] (5) Zebrafish Prrt2 gene (protein coding region, CDS) sequence (SEQ ID NO: 5):

[0175] (6) Zebrafish PRRT2 protein amino acid sequence (226 amino acids) (SEQ ID NO: 6):

[0176] Example 1: Verification of the effect of overexpression of PRRT2 on slow inactivation of sodium channels

[0177] The mouse PRRT2 protein coding region cDNA sequence (Figures 1 and 2) was inserted into the multiple cloning site region of the pCAG2IG plasmid (i.e., EGFP control plasmid, Figure 3) to construct the mouse PRRT2 overexpression plasmid pCAG-mouse PRRT2(HA)-IRES-EGFP (Figure 4).

[0178] In the same manner, the human PRRT2 overexpression plasmid pCAG-human PRRT2(HA)-IRES-EGFP was constructed ( Figures 5 , 6 , and 7 ).

[0179] In the same manner, the zebrafish-derived PRRT2 overexpression plasmid pCAG-zebra PRRT2(HA)-IRES-EGFP was constructed ( Figures 8 , 9 , and 10 ).

[0180] HEK293 cells stably expressing Nav1.2 were transfected with PRRT2 overexpression plasmid or control plasmid using Lipo3000 reagent. After expression at 37°C for 24 hours, the cells were plated onto round glass slides and expression continued for 12 hours.

[0181] Sodium channel currents were recorded using whole-cell recording (Figure 11A). First, a capillary glass tube (BF150-86-10, Sutter Instruments) was pulled into a recording electrode using a microelectrode puller (P97, Sutter Instruments). Under an inverted microscope (MF53, Micro-shot), a microelectrode manipulator (MP225, Butter Instrument) was used to contact the recording electrode to the cell surface, and negative pressure was applied to form a gigahertz (GΩ) seal. After the gigahertz seal was formed, rapid capacitance compensation was performed, and then negative pressure was continued to be applied to break the cell membrane and form a whole-cell recording mode. Slow capacitance compensation was then performed and the membrane capacitance and series resistance were recorded. All electrophysiological experiments were performed at room temperature. The experimental data were collected by an EPC-10 amplifier (HEKA) and stored in the PatchMaster (HEKA) software.

[0182] The entry into and recovery from the fast inactivation state of the sodium channel were measured using Procedure 1: Fast Inactivation (Figure 11B) and Procedure 2: Recovery from Fast Inactivation (Figure 12A). The entry into and recovery from the slow inactivation state of the sodium channel were recorded using Procedure 3: Entry into Slow Inactivation (Figure 13A) and Procedure 4: Recovery from Slow Inactivation (Figure 14A), respectively.

[0183] The testing protocol for the effects of human PRRT2 and zebrafish PRRT2 on the slow inactivation of sodium channels is similar to the testing protocol for mouse PRRT2 described above. However, in the process of detecting the recovery of sodium channels from slow inactivation, the inventors used a continuous testing protocol instead of the repeated scanning test method to improve the success rate of cell electrophysiological recordings.

[0184] During the recording process, the initial clamping voltage of the cell was -120mV, at which time the sodium ion channel was in a closed state or resting state. When the clamping voltage was raised from -120mV to 0mV, the cell depolarized, the sodium ion channel opened, and extracellular sodium ions rapidly flowed inward to form a sodium current (Figure 11B). Subsequently (about 0.5-1ms), the sodium ion channel began to enter an inactivated state (fast inactivation), the channel closed, and the sodium current decayed (Figures 11B and 11C). The sodium ion channel enters a fast inactivation state within a short depolarization period and can return to an activatable resting state during a hyperpolarization process of tens to tens of milliseconds (Figures 12A and 12B).

[0185] The results showed that overexpression of mouse PRRT2 had no significant effect on the formation of fast inactivation of sodium channels and the recovery from fast inactivation ( Figures 11-12 ).

[0186] In addition to the fast inactivation mechanism, sodium channels also exhibit slow inactivation. It takes longer for sodium channels to enter or recover from the slow inactivation state than for fast inactivation, typically taking hundreds of milliseconds to tens of seconds. Based on the above rules, a brief 10-millisecond hyperpolarization after the depolarization process can restore the fast-inactivating sodium channel, effectively isolating the slow-inactivating component of the sodium channel (Figure 13A).

[0187] The results showed that overexpression of mouse PRRT2 promoted the speed at which sodium ion channels entered the slow inactivation state during depolarization ( Figure 13B ) and prolonged the time it took for sodium ion channels to recover from the slow inactivation state to the activatable resting state ( Figures 14A and 14B ).

[0188] The regulatory effect of human PRRT2 on slow inactivation of sodium ion channels was similar to that of mouse PRRT2 ( Figure 15A- Figure 15C ).

[0189] PRRT2 is conserved across many species. Surprisingly, in previous work, the inventors investigated the effects of PRRT2 from various species on the slow inactivation of sodium channels and found that zebrafish-derived PRRT2 had a significantly stronger inhibitory effect on the recovery of slow inactivation of sodium channels (compared to mouse and human PRRT2, overexpression of zebrafish PRRT2 resulted in a longer recovery time for slow inactivation of sodium channels, which can be understood as having a stronger effect on slow inactivation of sodium channels) than human and mouse PRRT2. Figure 15C shows the comparison of zebrafish, human, and mouse PRRT2.

[0190] In summary, PRRT2 overexpression promotes slow inactivation of sodium channels but has no significant effect on fast inactivation. Therefore, PRRT2 exhibits state-dependent regulation of sodium channels, favoring a reduction in the effective supply of cellular sodium channels under sustained depolarization. This characteristic of PRRT2 allows it to reduce abnormal cellular excitability without affecting normal cellular excitatory activity, resulting in excellent cell-state selectivity.

[0191] Example 2: Overexpression of PRRT2 in forebrain excitatory neurons intervenes in epileptic seizures

[0192] To detect the expression and distribution of PRRT2 protein, the inventors performed immunofluorescence staining on brain slice samples using anti-PRRT2 antibodies and anti-HA tag antibodies. Anti-PRRT2 antibodies are used to detect both endogenous and overexpressed PRRT2 proteins, while anti-HA antibodies can only detect overexpressed HA-tagged PRRT2 proteins.

[0193] Brain slices were first rinsed three times with PBS for 10 minutes each, then incubated in blocking buffer containing 0.3% Triton X-100 and 5% bovine serum protein (BSA) in PBS for 1 hour at room temperature. Following blocking, primary antibody incubation was performed, with brain slices incubated overnight at 4°C in an anti-PRRT2 antibody (1:500, rabbit). Brain slices were then rinsed three times with PBS and incubated in a secondary antibody solution. Brain slices were incubated in a fluorescent secondary antibody (1:2000, donkey anti-rabbit 647nm, donkey anti-rat 488nm) and Hoechst (1:5000) for 2 hours at room temperature. After rinsing three times with PBS, brain slices were mounted on glass slides, air-dried, and then covered with 80% glycerol in PBS. Images were observed and photographed using a fluorescence microscope (VS-120, Olympus) with a 10x objective.

[0194] Adeno-associated virus (AAV) is the most commonly used vector for in vivo gene delivery. In this study, the PHP.eB serotype AAV was selected as the delivery vector for the PRRT2 gene. Experiments have shown that it has good blood-brain barrier penetration and facilitates PRRT2 gene expression in the brain.

[0195] PRRT2 protein overexpression utilizes a Cre recombinase-dependent approach, achieved by incorporating the DIO system (Loxp / Lox2272) into a viral expression vector (Figure 17, pAAV-CAG-DIO-mouse PRRT2-HA). This indirectly relies on the Emx1 promoter (a forebrain excitatory neuron-specific promoter) to drive Cre recombinase expression, achieving specific expression of PRRT2 protein in forebrain excitatory neurons.

[0196] At the same time, since the experimental animals are mice, the present inventors used a mouse strain (emx1-cre) that specifically expresses cre recombinase. In this strain of mice, the expression of cre recombinase depends on the Emx1 promoter.

[0197] The experimental results showed that in Emx1-cre mice, a total of 2×10 11 The AAV-PHP.eB-CAG-DIO-mCherry virus (vg) can achieve overexpression of the mCherry fluorescent protein in forebrain excitatory neurons. Injection of the same total amount of the AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA virus resulted in overexpression of the PRRT2 protein in forebrain excitatory neurons (Figures 18A-18C).

[0198] Compared with the control group, the mice in the PRRT2 overexpression group showed stronger anti-epileptic ability in the PTZ-induced epilepsy model ( FIG19A ).

[0199] In the evaluation based on the epileptic behavior grade, after intraperitoneal injection of PTZ (45 mg / kg), the average seizure grade of mice in the group overexpressing PRRT2 in forebrain excitatory neurons was 1.4, while the average seizure grade of mice in the control group was 4. There was a very significant statistical difference in the epileptic seizure grade between the two groups (Figure 19B).

[0200] The results of EEG recordings also showed that the epileptiform EEG signals of mice in the group overexpressing PRRT2 in forebrain excitatory neurons after PTZ injection were significantly weaker than those of the control group ( Figure 20A and Figure 20B ).

[0201] Compared with control mice, mice overexpressing PRRT2 in excitatory neurons showed no significant behavioral abnormalities in the open field test, wheel test, and social interaction test ( Figures 21A-21C ).

[0202] In the same manner, a total of 2 × 10 11 vg of AAV-PHP.eB-CAG-DIO-mCherry virus (control) or AAV-PHP.eB-CAG-DIO-zebra PRRT2 virus.

[0203] The results are shown in Table 1. Surprisingly, overexpression of zebrafish PRRT2 in forebrain excitatory neurons significantly inhibited PTZ-induced epileptic seizures. Zebrafish-derived PRRT2 exhibited significantly better effects than PRRT2 from other species.

[0204] Table 1. Overexpression of zebrafish PRRT2 in forebrain excitatory neurons inhibits convulsant-induced epileptic seizures

[0205] Example 3: Effects of Overexpression of PRRT2 in Non-forebrain Excitatory Neurons

[0206] To verify the importance of selective expression of PRRT2, the inventors tested overexpression of PRRT2 in non-forebrain excitatory neurons to analyze whether it induces adverse reactions.

[0207] In Nkx2.1-cre mice, the expression of cre recombinase depends on the Nkx2.1 gene promoter, which drives the selective expression of cre recombinase in forebrain inhibitory neurons (i.e., γ-aminobutyric acid neurons). The inventors used Nkx2.1-cre mice and adopted the strategy of adeno-associated virus delivery to inject a total of 2×10 11 vg AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA virus was used to overexpress PRRT2 in forebrain inhibitory neurons ( Figures 22A and 22B ).

[0208] The inventors found that in NKX2.1-cre mice, compared with the control mice, the mice in the inhibitory neuron overexpression PRRT2 group showed epilepsy susceptibility in the PTZ-induced epilepsy model (Figure 22D and Figure 22E). After intraperitoneal injection of subthreshold doses of PTZ (35 mg / kg), the mice in the inhibitory neuron overexpression PRRT2 group had higher epileptic seizure levels than the control mice (Figure 22D and Figure 22E). The results of EEG recordings also showed that the epileptic-like EEG signals of the mice in the forebrain inhibitory neuron overexpression PRRT2 group were significantly stronger than those in the control mice after injection of subthreshold doses of PTZ (Figure 22F and Figure 22G).

[0209] In addition, the inventors also observed that mice were susceptible to epilepsy by selectively overexpressing PRRT2 in astrocytes. Therefore, overexpression of PRRT2 in non-forebrain excitatory neurons leads to adverse reactions.

[0210] In summary, overexpression of PRRT2 protein in forebrain excitatory neurons can effectively enhance the anti-epileptic ability of mice without affecting normal movement and social behavior, and this selective expression strategy avoids the epilepsy susceptibility caused by overexpression of PRRT2 protein in forebrain inhibitory neurons.

[0211] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. Use of a PRRT2 upregulator selectively acting on forebrain excitatory neurons for preparing a drug for treating or preventing epilepsy.

2. The use according to claim 1, characterized in that The selective action on forebrain excitatory neurons includes: selective action on slow inactivation of sodium ion channels, and / or selective action on abnormal cell excitability.

3. The use according to claim 1, characterized in that The treatment or prevention of epilepsy includes reducing the number, frequency, level and / or duration of epileptic seizures.

4. The use according to claim 1, characterized in that The PRRT2 up-regulator selectively acting on forebrain excitatory neurons is a construct, or an expression system formed by the construct; the construct includes: an expression drive system, and a PRRT2 encoding gene whose expression is driven by the drive system.

5. The use according to claim 4, characterized in that The expression drive system includes a single or combined forebrain excitatory neuron-specific expression drive system; The forebrain excitatory neuron-specific expression driving system includes a single form of forebrain glutamatergic neuron-specific promoter, including: CaMKIIa promoter; the single form of promoter is used to directly drive the expression of PRRT2; The forebrain excitatory neuron-specific expression driving system includes a forebrain glutamatergic neuron-specific promoter in a combined form, including: a forebrain excitatory neuron-specific promoter for driving Cre recombinase expression and a strong promoter for driving PRRT2 expression; the combined expression driving system is used to take into account both forebrain excitatory neuron specificity and high-efficiency expression characteristics.

6. The use according to claim 4, characterized in that The construct includes an operational gene expression regulatory element dependent on Cre recombinase, such as Double-floxed inverse orientation, DIO; preferably, the element for DIO is LoxP / Lox2272, and the Cre recombinase-dependent PRRT2 expression is performed using specific recognition sequences based on LoxP and Lox2272.

7. The use according to claim 6, characterized in that The construct comprises: Construct 1, comprising: a promoter, LoxP / Lox2272, a PRRT2 encoding gene, LoxP / Lox2272 connected in sequence; the PRRT2 encoding gene is reversely connected between two pairs of LoxP / Lox2272 sequences; Construct 2: includes the following: a forebrain excitatory neuron-specific expression promoter and a Cre recombinase encoding gene connected in sequence.

8. The use according to any one of claims 4 to 7, characterized in that: The construct is contained in an expression vector or is directly inserted into the genome of the intervention object through gene editing; the expression vector includes: viral vector, non-viral vector; preferably, the viral vector includes: adeno-associated virus vector, lentivirus vector, adenovirus vector, retrovirus vector; more preferably, the adeno-associated virus vector includes: PHP.eB serotype AAV vector, Cap-B10 serotype AAV vector; or the gene editing includes gene editing based on CRISPR-Cas technology.

9. The use according to claim 1, characterized in that The PRRT2 is PRRT2 from zebrafish, human or mouse; preferably PRRT2 from zebrafish.

10. A construct for treating or preventing epilepsy, comprising: An expression drive system, and a PRRT2 encoding gene whose expression is driven by the drive system; Preferably, the expression drive system comprises a single or combined forebrain excitatory neuron-specific expression drive system; The forebrain excitatory neuron-specific expression driving system includes a single form of forebrain glutamatergic neuron-specific promoter, including: CaMKIIa promoter; the single form of promoter is used to directly drive the expression of PRRT2; The forebrain excitatory neuron-specific expression driving system includes a forebrain glutamatergic neuron-specific promoter in a combined form, including: a forebrain excitatory neuron-specific promoter for driving Cre recombinase expression and a strong promoter for driving PRRT2 expression; the combined expression driving system is used to take into account both forebrain excitatory neuron specificity and high-efficiency expression characteristics.

11. The construct according to claim 10, wherein The construct includes operably linked: an operative gene expression regulatory element dependent on Cre recombinase, such as; preferably, the element for DIO is LoxP / Lox2272, and the expression of PRRT2 dependent on Cre recombinase is performed using specific recognition sequences based on LoxP and Lox2272.

12. The construct according to claim 10 or 11, which is contained in an expression vector or directly inserted into the genome of the intervention subject through gene editing, wherein the expression vector comprises: Viral vectors, non-viral vectors; Preferably, the viral vector includes: an adeno-associated viral vector, a lentiviral vector, an adenoviral vector, a retroviral vector; more preferably, the adeno-associated viral vector includes: a PHP.eB serotype AAV vector, a Cap-B10 serotype AAV vector; The gene editing includes gene editing based on CRISPR-Cas technology.

13. An expression system for treating or preventing epilepsy, which is a viral system obtained by packaging the viral vector according to claim 12.

14. A drug for treating or preventing epilepsy or a drug kit containing the drug, wherein the drug contains the expression system according to claim 13.

15. A method for selectively delivering an effective amount of PRRT2, or a PRRT2 up-regulator selectively acting on forebrain excitatory neurons, to forebrain excitatory neurons, comprising contacting the forebrain excitatory neurons with an expression vector comprising an effective amount of PRRT2, or an up-regulator thereof; Preferably, the expression vector includes: a viral vector, a non-viral vector; more preferably, the viral vector includes: an adeno-associated viral vector, a lentiviral vector, an adenoviral vector, a retroviral vector; more preferably, the adeno-associated viral vector includes: a PHP.eB serotype AAV vector, a Cap-B10 serotype AAV vector; Preferably, the expression vector is delivered by intraorbital injection, intracranial injection, intrathecal injection, intrathecal injection, intracerebral injection, intraventricular injection, or direct injection into the epileptic focus in the brain.

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