WO2025044677A1 - Use of prrt2 acting on forebrain excitatory neuron and up-regulator thereof - Google Patents

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 application of its upregulators Technical Field

The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to the application of PRRT2 and its up-regulator acting on forebrain excitatory neurons.

Background Art

Epilepsy is a typical central nervous system disease caused by excitation-inhibition imbalance. The main clinical manifestations include convulsions, muscle spasms, tonic convulsions, atonia and absence, accompanied by a brief loss of consciousness. During the attack, the patient's EEG shows epileptic discharge characteristics. Epileptic seizures are characterized by suddenness and recurrence, which has a serious impact on the patient's physical and mental health and normal social activities. The causes of epilepsy are relatively diverse. Currently known pathogenic factors include developmental abnormalities, genetic mutations, craniocerebral injury, cerebrovascular disease and inflammation. Except for a small number of self-limited epilepsy, most epilepsy patients need to achieve control of epilepsy through treatment. The treatment methods currently used in clinical practice mainly include drug therapy, surgical resection of local lesions, ketogenic diet, vagus nerve stimulation and deep brain stimulation. Among them, more than two-thirds of epilepsy patients mainly use anti-epileptic drugs for epilepsy control. Although more than 30 anti-epileptic drugs targeting different targets have been launched since the 1950s, forming a relatively effective clinical treatment plan, about 30% of patients with refractory epilepsy still cannot benefit from these drugs or drug combinations. In addition, some epilepsy patients who respond well to existing drugs still face the problem of adverse drug reactions during long-term medication and the inability to fully control epilepsy. The above unmet medical needs are an important driving force for the research of new mechanisms and the development of new therapies.

The homeostatic balance of neural networks depends on the cooperation between excitatory and inhibitory neurons, and epilepsy is mainly manifested as abnormal synchronous excitement of the brain caused by imbalance of neural network homeostasis. Therefore, the ideal epilepsy intervention strategy is to selectively reduce the activity of excitatory neurons or increase the activity of inhibitory neurons through drugs or other treatments, so as to correct the abnormal excitement of the neural network and return it to normal homeostasis; at the same time, the intervention plan should try to avoid or reduce interference with non-epilepsy-related neural networks, such as movement, arousal, cognition and emotional circuits, so as to reduce adverse reactions during long-term medication.

Voltage-gated sodium channels (hereinafter referred to as sodium channels or Nav) are important molecules that the generation of action potentials and impulse conduction of neurons in the brain rely on, and are the basis of the excitability of neural tissue. In the field of epilepsy treatment, sodium channels are important targets for anti-epileptic drugs. More than a quarter of the anti-epileptic drugs currently approved for clinical use target such channels, including sodium phenytoin, carbamazepine, oxcarbazepine, and lacosamide. The mechanism of action of these drugs is to reduce the excitability of neural networks by regulating the activity of sodium channels, thereby reducing or preventing related types of epileptic seizures. Although anti-epileptic drugs targeting sodium channels have been widely used in the treatment of epilepsy, their central adverse reactions (such as dizziness, drowsiness, ataxia, and irritability) have not been effectively resolved.

Lack of cell type selectivity and poor cell state selectivity are the main reasons for the adverse reactions of small molecule anti-epileptic 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 types of neurons at different stages of development. For example, in the forebrain directly related to epilepsy, Nav1.2 and Nav1.6 are mainly distributed in excitatory neurons with glutamate as the main neurotransmitter, while Nav1.1 is mainly distributed in inhibitory interneurons with γ-aminobutyric acid (GABA) as the main neurotransmitter; in the cerebellar cortex related to motor regulation, Nav1.1, Nav1.2 and Nav1.6 are expressed separately or in combination in GABAergic interneurons, granule cells and Purkinje neurons. Antiepileptic drugs that target sodium channels generally lack selectivity among sodium channel members. After entering the central nervous system, they will act on non-epilepsy-related neural cell groups and cause adverse central reactions. It is particularly noteworthy 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.

In addition to the adverse reactions caused by the lack of cell type selectivity, the lack of effective cell state selectivity of the drug is also an important reason for its 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 use of drugs, drugs with poor selectivity for cell 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 recurrence of epilepsy.

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

Summary of the invention

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

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.

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

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

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

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

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.

In one or more embodiments, daily behavior is assessed by a mouse behavioral experiment, wherein the behavioral experiment includes but Not limited to: open field test, wheel test, social behavior test.

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

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 (infection) system); the construct includes: an expression drive system, and a PRRT2 encoding gene whose expression is driven by the drive system.

In one or more embodiments, the expression driving system comprises a single or combined forebrain excitatory neuron-specific expression driving system.

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.

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.

In one or more embodiments, the construct includes an operational gene expression regulatory element that depends on Cre recombinase, such as (but not limited to) Double-floxed inverse orientation, DIO); preferably, the element for DIO is LoxP/Lox2272, and the Cre recombinase-dependent expression of PRRT2 is performed using specific recognition sequences based on LoxP and Lox2272.

In one or more embodiments, the construct includes: construct 1, including sequentially connected: a promoter (such as but not limited to beta-actin promoter), LoxP/Lox2272, a PRRT2 encoding gene, LoxP/Lox2272; the PRRT2 encoding gene is reversely connected between two pairs of LoxP/Lox2272 sequences; construct 2: including sequentially connected: 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).

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

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

In one or more embodiments, 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 (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.

In one or more embodiments, the PRRT2 is PRRT2 from zebrafish, PRRT2 from human or PRRT2 from mouse; preferably, PRRT2 from zebrafish.

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 the protein (a), and has the function of the 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 the protein (a) and has the function of the protein (a); or (d) a protein formed by adding a tag sequence to the N or C terminus of any of the polypeptides of (a) to (c), or adding a signal peptide sequence or a secretion signal sequence to its N terminus.

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.

In one or more embodiments, the expression driving system comprises a single or combined forebrain excitatory neuron-specific expression driving system.

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.

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.

In one or more embodiments, the construct includes: an operably linked 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 Cre recombinase-dependent PRRT2 expression is performed using specific recognition sequences based on LoxP and Lox2272.

In one or more embodiments, the construct is contained in an expression vector or is directly inserted into the genome of the intervention object 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.

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

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.

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.

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; 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.

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

Other aspects of the present invention will be apparent to those skilled in the art from the disclosure of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 2. Amino acid sequence of mouse PRRT2 protein.

Figure 3. Schematic diagram of EGFP control plasmid pCAG-empty-IRES-EGFP.

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

Fig. 5. The sequence of the protein coding region (CDS) of the human PRRT2 gene.

Fig. 6. Amino acid sequence of human PRRT2 protein.

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

Fig. 8. The protein coding region (CDS) sequence of the zebrafish Prrt2 gene.

Fig. 9. Amino acid sequence of zebrafish PRRT2 protein.

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

Fig. 11. Effect of PRRT2 overexpression on the fast inactivation process of Nav1.2 sodium channel.

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

(B) The upper part is a schematic diagram of the voltage clamp scheme for activating sodium ion channels. The lower part is a representative sodium channel current curve. When the voltage-gated sodium channel Nav1.2 is clamped to 0 mV, the channel opens and sodium ions flow in to form an inward sodium current. After a short opening, the sodium channel quickly inactivates, the channel closes, and the sodium current decays rapidly, as shown by the arrow.

(C) Rapid inactivation of sodium channels leads to sodium current decay, and the decay rate is characterized by the time constant tau. There was no significant difference in the time constant of sodium current decay between the PRRT2 overexpression group and the EGFP overexpression control group (nsP＞0.05). mPRRT2: mouse PRRT2.

Fig. 12. Effect of PRRT2 overexpression on the recovery of Nav1.2 sodium channel from fast inactivation.

(A) Schematic diagram of the voltage clamp protocol for detecting the recovery of sodium channels from fast inactivation. Below are 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.

(B) The process of sodium channel recovery from the fast inactivation state. There was no significant difference between the mPRRT2 overexpression group and the EGFP overexpression control group (n.s.P＞0.05). mPRRT2: mouse PRRT2.

Fig. 13. Effect of PRRT2 overexpression on the slow inactivation process of Nav1.2 sodium channel.

(A) The top is a schematic diagram of the voltage clamp protocol for detecting the slow inactivation of sodium channels. The bottom is an example of the sodium current measured during the first depolarization of control cells and the sodium current measured after the sodium channels experienced depolarization of different lengths followed by a brief hyperpolarization.

(B) The process of sodium channels entering the slow inactivation state. Compared with the control group overexpressing EGFP, the sodium channels in the mPRRT2 overexpression group entered the slow inactivation state faster during sustained depolarization. There was a significant difference between the two groups (P < 0.0001). mPRRT2: mouse PRRT2.

Fig. 14. Effect of PRRT2 overexpression on the recovery of Nav1.2 sodium channel from slow inactivation.

(A) Schematic diagram of the voltage clamp protocol for detecting the recovery of sodium channels from slow inactivation. Below are examples of sodium currents measured during the first depolarization and after the slowly inactivating sodium channels have undergone hyperpolarization recovery periods of varying lengths.

(B) The process of sodium channel recovery from slow inactivation. Compared with the control group overexpressing EGFP, the sodium channel in the mPRRT2 overexpression group recovered more slowly from the slow inactivation state. There was a significant difference between the two groups (P < 0.001).

Fig. 15. Effect of zebrafish PRRT2 overexpression on slow inactivation of Nav1.2 sodium channel.

(A) Comparison of the amino acid sequences of mouse, human and zebrafish PRRT2 proteins. The amino acid sequence identity of mouse and human PRRT2 proteins is 77.46%, and the amino acid sequence identity of zebrafish and mouse PRRT2 proteins is 43.6%. The bottom horizontal line in the sequence marks the C-terminus of PRRT2 (including the transmembrane segment).

(B) The process of sodium channels entering the slow inactivation state. Compared with the control group overexpressing EGFP, the sodium channels in the mPRRT2, hPRRT2, and zPRRT2 overexpression groups entered the slow inactivation state faster during sustained depolarization, and there was a significant difference between the groups (P < 0.0001).

(C) The process of sodium channel recovery from slow inactivation. Compared with the control group overexpressing EGFP, the sodium channels in the mPRRT2, hPRRT2, and zPRRT2 overexpression groups recovered more slowly from the slow inactivation state, with significant differences between the groups (P < 0.0001). In addition, the sodium channels in the zPRRT2 overexpression group recovered more slowly from the slow inactivation state than those in the mPRRT2 and hPRRT2 groups, with significant differences between the groups (P < 0.0001). mPRRT2: mouse PRRT2, hPRRT2: human PRRT2, zPRRT2: zebrafish PRRT2.

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

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

Figure 18. Forebrain excitatory neurons overexpress PRRT2.

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

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

(C) Fluorescent immunohistochemistry verified the expression of PRRT2 in forebrain excitatory neurons.

FIG. 19 . Overexpression of PRRT2 in forebrain excitatory neurons inhibits convulsant-induced epileptic seizures.

(A) Representative examples of epileptic behaviors induced by convulsants (pentylenetetrazole, PTZ, 45 mg/kg, intraperitoneal injection) in mice.

(B) Compared with the control group, overexpression of PRRT2 in forebrain excitatory neurons significantly reduced the severity of epileptic seizures induced by pentylenetetrazol in mice, and there was a significant difference in the severity of seizures between the two groups (P < 0.0001). PTZ: pentylenetetrazol; PRRT2-OE: PRRT2 over-expression.

Figure 20. Overexpression of PRRT2 in forebrain excitatory neurons inhibits epileptic EEG induced by convulsants.

(A) Schematic diagram of EEG recording in mice.

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

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

(A) In the open field test, the total distance covered by the two groups of mice was similar, and no significant difference was detected (n.s.P＞0.05).

(B) In the wheel test, the time that the two groups of mice could maintain on the fixed-speed wheel was similar, and no significant difference was detected (n.s.P＞0.05). The upper limit of the time for this test was 60 seconds.

(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 (n.s.P＞0.05). Empty represents the empty box group, and Novel represents the unfamiliar mouse group.

FIG. 22 . Overexpression of PRRT2 in forebrain inhibitory neurons facilitates convulsant-induced epileptic seizures.

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

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

(C) Fluorescence immunohistochemistry verified the expression of PRRT2 in forebrain inhibitory neurons. The scale bar is 1 mm.

(D) Representative examples of epileptic behaviors induced by subthreshold doses of convulsants (pentylenetetrazol, PTZ, 35 mg/kg, intraperitoneal injection) in Nkx2.1-cre mice; PTZ: pentylenetetrazol.

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

(F) Schematic diagram of mouse EEG recording.

(G) Representative EEG examples. Subthreshold doses of PTZ could not induce epileptic EEG signals in the cerebral cortex of control mice; however, subthreshold doses of PTZ could induce epileptic discharge events in the cerebral cortex of mice overexpressing PRRT2 in forebrain inhibitory neurons.

DETAILED DESCRIPTION

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 channels in a slow inactivation state, and reduce the effective supply of activatable sodium channels by enhancing the slow inactivation of sodium channels. Furthermore, the inventors selectively overexpressed the PRRT2 protein in forebrain excitatory neurons to feedback reduce the excitability of neurons in abnormally excited states, 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 has both cell type selectivity and cell state selectivity, and has good application prospects in the field of epilepsy treatment.

PRRT2 and sodium channels

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 sequences having the same function as PRRT2.

These variant forms include but are not limited to: deletion, insertion and/or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of PRRT2.

The above term "PRRT2" also includes proteins having a homology of 80% or higher with the polypeptide sequence defined by the above SEQ ID NO: 2, 4 or 6; preferably a homology of 85% or higher, such as 90%, 95%, 98% or 99% homology) and having the same function as the PRRT2 involved in the embodiments of the present invention are also included in the present invention. Methods and tools for aligning sequence identity are also 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 based on the percentage of identical positions.

A polynucleotide sequence encoding PRRT2 or its variant protein (coding sequence) can also be applied to the present invention. The term "coding gene" can be a polynucleotide that includes the protein, or a polynucleotide that also includes additional coding and/or non-coding sequences. In some embodiments, the polynucleotide sequence encoding the PRRT2 protein (coding sequence) is a polynucleotide sequence shown in SEQ ID NO: 1, 3 or 5.

In the research work of the inventors, it was found that the protein encoded by the PRRT2 (proline-rich transmembrane protein 2) gene can selectively act on the sodium ion channels in the slow inactivation state, that is, PRRT2 overexpression can promote the slow inactivation of sodium ion channels, but has no significant effect on the fast inactivation of sodium ion channels. PRRT2 has the characteristics of sodium ion channel state-dependent regulation. By enhancing the slow inactivation of sodium ion channels, it reduces the effective supply of cellular sodium ion channels under the state of continuous depolarization. This feature of PRRT2 is conducive to reducing the abnormal excitability of cells without affecting the normal excitatory activity of cells, so that it has good cell state selectivity.

Based on this discovery, the inventors used adeno-associated virus as a gene delivery vector, selectively overexpressed PRRT2 protein in the excitatory neurons of the forebrain, and feedback-reduced the excitability of abnormally excited neurons. The trial found that selectively targeting PRRT2 therapy can effectively suppress epileptic seizures without interfering with daily behavior.

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

PRRT2 acting on forebrain excitatory neurons and application of its upregulators

Based on the above new discovery of the inventors, the present invention provides a use of an up-regulator of PRRT2 that selectively acts on forebrain excitatory neurons, for preparing a drug for treating or preventing epilepsy.

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

Those skilled in the art know that "epileptic" seizures can be divided into different levels, including but not limited to: tonic-clonic, tonic, clonic, myoclonic, absence or atonic seizures. In the mouse model, epileptic seizures can be divided into six levels, namely: Level 1: The mouse lies motionless on the ground, crawling on the belly; Level 2: sudden convulsions, sudden cessation of behavior, and the tail is raised; Level 3: muscle clonus, head twisting, hand convulsions; Level 4: tonic-clonic, falling to the ground and convulsions, jumping and running wildly; Level 5: falling to the ground and developing to rigid extension of limbs; Level 6: death.

In one or more embodiments, upregulators of PRRT2, acting selectively on forebrain excitatory neurons, can reduce the number, frequency, level and/or duration of epileptic seizures experienced by a subject 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.

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.

The "excitatory neuron" is a neuron that can release neurotransmitters such as glutamate, which can cause the connected postsynaptic neuron to become more likely to be excited. The "inhibitory neuron" is a neuron that can release neurotransmitters such as gamma-aminobutyric acid, which can cause the connected postsynaptic neuron to become less likely to be excited.

As used in the present invention, the PRRT2 upregulator includes agonists, promoters, stimulants, etc., and these terms can be 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.

In one or more embodiments, the PRRT2 up-regulator includes (but is not limited to): a construct, or an expression system formed by the construct (e.g., a viral (infection) system); the construct includes: a forebrain excitatory neuron-specific expression drive system, and a PRRT2 encoding gene driven by the drive system. Typically, the forebrain excitatory neuron-specific expression drive 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.

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

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

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

Construct 2: a promoter specifically expressing forebrain excitatory neurons and a gene encoding cre recombinase (eg, Emx1 promoter drives the expression of the gene encoding cre recombinase).

In the present invention, the PRRT2 polynucleotide sequence can be inserted into a recombinant expression vector, so that it can be transferred into cells to overexpress PRRT2. As long as it can replicate and be stable in the host, any plasmid and vector can be used in the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence of PRRT2 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 the expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element. For example, the expression vector includes: a viral vector, a non-viral vector; preferably, the viral vector includes (but is not limited to): an adeno-associated virus (AAV) vector, a lentiviral vector, an adenoviral vector, a retroviral vector, etc.

In the present invention, since the vector selectively acts on the excitatory neurons of the forebrain, the vector has a 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 vector. 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.

As an 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 a cell that can express the PRRT2, so that the cell expresses 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 (such as 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 cistern) 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.

After research and analysis, the inventors selected PHP.eB serotype AAV virus as the delivery vector of 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 PRRT2 gene to neurons in the brain (such as Cap-B10 serotype AAV virus, etc.) and vectors that help to efficiently deliver PRRT2 gene to excitatory neurons in the forebrain can be applied to this experiment.

In the present invention, after overexpression to produce 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, 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.

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

The terms "treat" and "prevent" generally refer to the use of drugs or methods 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 the progression, reversing the progression, or eradicating or ameliorating the 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.

After knowing the use of the PRRT2, the PRRT2 or its up-regulator can be administered to the subject by various methods well known in the art. Preferably, gene therapy can be used, such as delivering an expression unit (such as an expression vector or virus, etc.) carrying the PRRT2 gene to the target through a certain route, and causing it to express (preferably overexpress) active PRRT2; or, 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.

The term "effective amount" refers to an amount that can achieve a desired result (e.g., a preventive or therapeutic result). The effective amount of PRRT2 or its up-regulator described in the present invention may vary depending on 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 (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the PRRT2 or its up-regulator such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by 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 given per day, or the dose may be reduced proportionally.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

Materials and methods

1. Experimental Materials

Mouse PRRT2 plasmid (Origene, #MR214094).

Human PRRT2 plasmid (Origene, #RC202304).

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

pCAG2IG plasmid (Addgene, #122292).

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

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

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

Patch clamp recording system (HEKA, #EPCIO).

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

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

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

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

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

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

The AAV virus used in the present 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:

AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA;

AAV-PHP.eB-CAG-DIO-mCherry;

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

C57BL/6J mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

Emx1-cre mice, name: B6.129S2-Emx1tm1(cre)Krj/J, from Jackson Lab, catalog number: JAX catalog number #005628.

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

2. Animal husbandry

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

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

3. Orbital vein injection of AAV virus

Take out the AAV virus stored at -80 degrees Celsius and thaw it on ice. Dilute the AAV virus to the injection concentration (2×10 ¹² vg/mL, vg: AAV vector genomes) with pre-cooled sterile PBS. Equilibrate the AAV virus solution to room temperature before injection and add 0.3 Aspirate 50 μL of AAV virus solution into a mL insulin needle for injection.

Mice were anesthetized by injecting sodium pentobarbital solution (90 mg/kg, intraperitoneal injection). After the mouse was anesthetized, the mouse head was fixed with fingers, and the mouse artery was avoided to be pressed. The eye skin was stretched to fully expose the eyeball. The insulin needle was inserted from the corner of the eye at a 20° angle, and the eyeball was avoided when entering the orbit. The needle tip entered the orbit to a depth of 2 mm to reach the orbital venous plexus, and 50 μL was slowly injected. After the injection, the needle was slowly withdrawn and excess fluid and blood in the corner of the eye were wiped off. The same injection was performed in the orbital vein on the other side, and a total titer of 2×10 ¹¹ vg of AAV virus was injected on both sides.

After the injection, the mice were placed back in the cages and sent back to the feeding room after they woke up.

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

4. EEG/EMG electrode implantation and EEG recording

The experimental mice were first anesthetized with 4% isoflurane, and then the mouse heads were fixed to the stereotaxic adapter. The isoflurane concentration was adjusted to 1.5% during the entire subsequent surgery to keep the mice in a state of continuous anesthesia. In addition, chlortetracycline eye ointment was used to protect the mouse eyes to avoid retinal damage caused by dryness. The hair on the head of the mouse was removed with a depilatory cream, and the head skin was cut along the midline to expose the skull surface. The surgical incision and skull surface were treated with a 2% hydrogen peroxide solution, and the connective tissue on the skull surface was scraped off. The skin around the exposed skull was fixed with tissue glue (Vetbond, 3M Deutschland Gmbh), and the membrane system on the skull surface was cleaned again. The electrode implantation position was marked by stereotaxic method, with coordinates AP-0.8mm, ML±1.1mm. After the skull surface was dry, the skull surface was covered with a light-cured self-etching adhesive (3M ESPE Single Bound universal) to form a solidified layer. A small handheld cranial drill was used to open a hole at the implantation position mark, with a circular hole diameter of approximately 0.6mm. 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, and the recording electrodes and electrode interface components were fixed with dental cement. In order to facilitate electrophysiological recording of mice in the awake state, a lightweight titanium alloy sheet (30 mm × 2 mm × 1 mm) was additionally fixed with dental cement at the position of the mouse head near the electrode interface. The two metal wire electrodes used to record electromyographic signals extended from the bottom of the electrode interface component to the neck of the mouse, and the exposed ends of the 1 mm electrodes were inserted into the muscle layers on both sides of the neck, and the neck skin was sutured and disinfected after fixation. The mice recovered for one week after surgery.

Before ECoG/EMG recording in the awake state, the mouse was fixed to the recording platform using a metal plate on the mouse's head, and the mouse was allowed to adapt to this head-fixed state for 30 minutes. The ECoG and EMG signals were amplified 100 times by a differential amplifier (Model 3000, A-M system), and then band-filtered (ECoG 1-300Hz, EMG 10-1000Hz) and collected (1000Hz) and recorded by a digital-to-analog converter (Digidata 1332A, Molecular Devices).

5. PTZ-induced epilepsy mouse model

Before the test, 3.5 mg/mL (Nkx2.1-cre mouse experiment) or 4.5 mg/mL (Emx1-cre mouse experiment) of pentylenetetrazol solution was prepared with normal saline. According to the weight of mice, pentylenetetrazol was injected intraperitoneally at a dose of 35 mg/kg or 45 mg/kg. Immediately after the injection, the mice were placed in a behavior box for video recording to observe the epileptic seizures of the mice within 30 minutes after administration.

Mouse epilepsy rating criteria:

Level 1: The mouse lies motionless on the ground, crawling on its belly;

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

Grade 3: Myoclonus, head twisting, hand twitching;

Grade 4: Tonic-clonic, falling to the ground and twitching, jumping and running wildly;

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

Level 6: Death.

6. Immunohistochemical detection of PRRT2 expression in the brain

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

7. Sequence information

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

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

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

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

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

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

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

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

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

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

In HEK293 cells stably transfected with Nav1.2, PRRT2 overexpression plasmid or control plasmid was transfected using Lipo3000 reagent. After expression at 37°C for 24 hours, the cells were plated on round glass slides and continued to express for 12 hours.

Sodium channel currents were recorded by 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). Electrode. Under an inverted microscope (MF53, Micro-shot), the microelectrode manipulator (MP225, Butter Instrument) was manipulated to contact the recording electrode to the cell surface, and negative pressure was applied to form a GΩ seal. After the GΩ seal was formed, fast 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. Then slow capacitance compensation was performed and 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.

The entry and recovery of the fast inactivation state of the sodium ion channel were tested using Procedure 1: fast inactivation (Figure 11B) and Procedure 2: Recover from fast inactivation (Figure 12A). The entry and recovery of the slow inactivation state of the sodium ion channel were recorded using Procedure 3: Entry in slow inactivation (Figure 13A) and Procedure 4: Recover from slow inactivation (Figure 14A), respectively.

The testing scheme for the effect of human PRRT2 and zebrafish PRRT2 on the slow inactivation of sodium channels is similar to the testing scheme for mouse PRRT2 mentioned above, but in the process of detecting the recovery of sodium channels from slow inactivation, the inventors used a continuous testing scheme instead of the repeated scanning test method to improve the success rate of cell electrophysiological recording.

During the recording process, the initial clamping voltage of the cell is -120mV, at which time the sodium ion channel is in a closed state or a resting state. When the clamping voltage rises from -120mV to 0mV, the cell depolarizes, the sodium ion channel opens, and extracellular sodium ions rapidly flow inward to form a sodium current (Figure 11B). Subsequently (about 0.5-1ms), the sodium ion channel begins to enter an inactivated state (fast inactivation), the channel closes, and the sodium current decays (Figures 11B and 11C). The sodium ion channel enters a fast inactivation state within a short depolarization period, and can be restored to an activatable resting state during a hyperpolarization process of ten to tens of milliseconds (Figures 12A and 12B).

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

In addition to the fast inactivation mechanism, sodium channels also have the characteristics of slow inactivation. It takes longer for sodium channels to enter the slow inactivation state or recover from the slow inactivation state than the fast inactivation state, usually hundreds of milliseconds to tens of seconds. According to the above rules, after the depolarization process is completed, a brief hyperpolarization of 10 milliseconds can restore the fast inactivation state of sodium channels, thereby effectively separating the slow inactivation component of the sodium channel (Figure 13A).

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

The regulatory effect of human PRRT2 on slow inactivation of sodium channels is similar to that of mouse PRRT2 ( Figure 15A-Figure 15C ).

PRRT2 is conserved in many species. Surprisingly, in the previous work, the inventors found that the inhibitory effect of zebrafish PRRT2 on the recovery of sodium channel slow inactivation (compared with mouse and human PRRT2, zebrafish PRRT2 overexpression makes the recovery time of sodium channel slow inactivation longer, which can be understood as having a stronger effect on sodium channel slow inactivation) was significantly stronger than that of human and mouse PRRT2. Figure 15C is the comparison result of zebrafish, human and mouse PRRT2.

In summary, PRRT2 overexpression can promote the slow inactivation of sodium channels, but has no significant effect on the fast inactivation of sodium channels. Therefore, PRRT2 has the characteristics of sodium channel state-dependent regulation, and is more inclined to reduce the effective supply of cell sodium channels under continuous depolarization. This feature of PRRT2 allows it to reduce the effective supply of cell sodium channels under continuous depolarization without affecting the normal excitatory activity of cells. Reduce the abnormal excitability of cells, giving them good cell state selectivity.

Example 2: Overexpression of PRRT2 in forebrain excitatory neurons to intervene in epileptic seizures

In order to detect the expression and distribution of PRRT2 protein, the inventors used anti-PRRT2 antibody and anti-HA tag antibody to perform immunofluorescence staining on brain slice samples. Anti-PRRT2 antibody is used to detect PRRT2 protein including endogenous and overexpressed, while anti-HA antibody can only detect overexpressed PRRT2 protein with HA tag.

First, the brain slices were rinsed three times with PBS solution for 10 minutes each time, and then incubated with blocking solution containing 0.3% Triton X-100 and 5% bovine serum protein (BSA; dissolved in PBS) at room temperature for 1 hour. After the blocking was completed, the primary antibody was incubated, and the brain slice samples were incubated in anti-PRRT2 antibody (1:500, rabbit) solution at 4°C overnight. The brain slices were rinsed three times with PBS solution and then incubated with secondary antibodies. The brain slice samples were incubated in fluorescent secondary antibodies (1:2000, donkey anti-rabbit-647nm, donkey anti-rat 488nm) and Hoechst (1:5000) solutions at room temperature for 2 hours. After rinsing three times with PBS solution, the brain slices were attached to the slides, and after drying, PBS solution containing 80% glycerol was added and the slides were sealed. The images were observed and photographed under a 10x objective lens using a fluorescence microscope (VS-120, Olympus).

Adeno-associated virus (AAV) is the most commonly used vector for gene delivery in vivo. In this experiment, PHP.eB serotype AAV virus was selected as the delivery vector of PRRT2 gene. The experiment showed that it has good blood-brain barrier penetration ability and is conducive to the expression of PRRT2 gene in the brain.

Overexpression of PRRT2 protein was achieved by Cre recombinase-dependent method, by setting up DIO system (Loxp/Lox2272) in viral expression vector to achieve Cre recombinase-dependent expression (Figure 17, pAAV-CAG-DIO-mouse PRRT2-HA). Indirectly relying on Emx1 promoter (forebrain excitatory neuron-specific expression promoter)-driven Cre recombinase expression to achieve specific expression of PRRT2 protein in forebrain excitatory neurons.

At the same time, since the experimental animals are mice, the 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.

The experimental results showed that in Emx1-cre mice, orbital vein injection of a total of 2×10 ¹¹ vg of AAV-PHP.eB-CAG-DIO-mCherry virus could achieve overexpression of mCherry fluorescent protein in forebrain excitatory neurons. Injection of the same total amount of AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA virus could observe overexpression of PRRT2 protein in forebrain excitatory neurons (Figure 18A-Figure 18C).

Compared with the control group mice, the mice in the PRRT2 overexpression group showed stronger anti-epileptic ability in the PTZ-induced epilepsy model ( FIG. 19A ).

In the evaluation based on epileptic behavior grade, after intraperitoneal injection of PTZ (45 mg/kg), the average seizure grade of mice in the forebrain excitatory neuron overexpression group 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).

The results of EEG recording also showed that the epileptic-like EEG signals of mice in the group overexpressing PRRT2 in forebrain excitatory neurons after PTZ injection were significantly weaker than those of mice in the control group ( FIGS. 20A and 20B ).

Compared with control mice, mice whose excitatory neurons overexpressed PRRT2 showed significantly higher performance in the open field test, wheel running test, and social interaction test. No significant behavioral abnormalities were observed in the experiments (Figure 21A-21C).

In the same manner, Emx1-cre mice were injected intraorbitally with 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.

The results are shown in Table 1. Surprisingly, overexpression of zebrafish PRRT2 in forebrain excitatory neurons can significantly inhibit PTZ-induced epileptic seizures. Zebrafish-derived PRRT2 exhibits significantly better effects than PRRT2 from other species.

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

Example 3: Effects of overexpressing PRRT2 in non-forebrain excitatory neurons

To verify the importance of selective expression of PRRT2, the inventors tested whether overexpression of PRRT2 in non-forebrain excitatory neurons would induce adverse reactions.

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 a strategy of adeno-associated virus delivery to inject a total of 2×10 ¹¹ vg of AAV-PHP.eB-CAG-DIO-mouse PRRT2-HA virus into the orbital vein to overexpress PRRT2 in forebrain inhibitory neurons (Figures 22A and 22B).

The inventors found that in NKX2.1-cre mice, compared with control mice, 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), mice in the inhibitory neuron overexpression PRRT2 group had higher epileptic seizure levels than mice in the control group (Figure 22D and Figure 22E). The results of EEG recording also showed that the epileptic EEG signals of mice in the forebrain inhibitory neuron overexpression PRRT2 group were significantly stronger than those of mice in the control group after injection of subthreshold doses of PTZ (Figure 22F and Figure 22G).

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.

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.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims (15)

A use of a PRRT2 upregulator selectively acting on forebrain excitatory neurons for preparing a drug for treating or preventing epilepsy. The use according to claim 1, characterized in that the selective action on forebrain excitatory neurons includes: selectively acting on slow inactivation of sodium ion channels, and/or selectively acting on abnormal cell excitability. The use according to claim 1, characterized in that the treatment or prevention of epilepsy comprises: reducing the number, frequency, level and/or duration of epileptic seizures. The use according to claim 1, characterized in that the upregulator of PRRT2 that selectively acts on forebrain excitatory neurons is a construct, or an expression system formed by the construct; the construct comprises: an expression drive system, and a PRRT2 encoding gene driven to be expressed by the drive system. The use according to claim 4, characterized in that 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 combined forebrain glutamatergic neuron-specific promoter, 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. The use as described in claim 4 is 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. 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. 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: a viral vector, a non-viral vector; preferably, the viral vector includes: an adeno-associated virus 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; or the gene editing includes gene editing based on CRISPR-Cas technology. The use according to claim 1, characterized in that the PRRT2 is PRRT2 from zebrafish, PRRT2 from human or PRRT2 from mouse; preferably PRRT2 from zebrafish. 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 combined forebrain glutamatergic neuron-specific promoter, 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. The construct as described in claim 10 is characterized in that the construct includes: 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. The construct according to claim 10 or 11, which is contained in an expression vector or directly inserted into the genome of the intervention object through gene editing, wherein the expression vector includes: a viral vector, a non-viral vector; 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. An expression system for treating or preventing epilepsy, which is a viral system obtained by packaging the viral vector according to claim 12. 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. 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.