Antisense oligonucleotides targeting the Cav3.1 gene and their use

Abstract

To provide drugs that more efficiently suppress the expression of α1G T-type calcium channels for diseases treatable by inhibiting α1G T-type calcium channels, such as Parkinson's disease. [Solution] To treat various α1G subunit-related diseases of T-type calcium channels, more specifically, to provide antisense oligonucleotides for suppressing the expression of genes encoding Cav3.1, targeting specific sequences, and for use in the treatment of various neuropsychiatric disorders, including Parkinson's disease, epilepsy, essential tremor, depression, anxiety disorders, and unconsciousness.

Description

Technical Field

[0001] The present invention relates to novel antisense oligonucleotides and their use, and more specifically, to antisense oligonucleotides targeting the Cav3.1 gene that are effective in the treatment of diseases such as Parkinson's disease, essential tremor, epilepsy, depression, unconsciousness, and their use.

Background Art

[0002] Parkinson's disease means a disease in which the dopamine neurons in the substantia nigra, which is located in the center of the brainstem, are damaged, resulting in movement disorders. Dopamine is an important neurotransmitter that acts on the basal ganglia of the brain to enable us to move our bodies precisely as desired. The symptoms of Parkinson's disease become clear after about 60 - 80% of the dopamine neurons in the substantia nigra compacta are lost. If a pathological examination is performed, Lewy bodies formed by the deposition of pathogenic α-synuclein protein can be confirmed in various parts of the brain and peripheral nerves. Parkinson's disease is a common neurodegenerative disease after Alzheimer's disease, showing a morbidity rate of 1% in people over 60 years old, and the morbidity rate increases with age.

[0003] The core or classical motor characteristics of Parkinson's disease include bradykinesia, tremor, and rigidity, but there are many other movement-related symptoms such as changes in walking and balance, eye movement regulation, speech and swallowing, bladder regulation, etc. Bradykinesia in Parkinson's disease patients is manifested as a decrease in movement amplitude, movement speed, and difficulty in starting movement. It can affect the spontaneous control of many muscle groups, including the eye muscles (resulting in a delay in starting intermittent movements), speech muscles (becoming softer and sometimes leading to unclear speech), and limb muscles (leading to a decrease in dexterity of the fingertips). Patients complain of difficulty in fine motor tasks such as button pressing, writing, and using tools, and the tremor is variable. Most, but not all, patients have the symptom of tremor (McGregor1,2 and Alexandra B.Nelson1,Neuron 101:1042 - 1056,2019).

[0004] Regarding the etiology, only 5-10% of Parkinson's disease cases are genetically inherited, with the vast majority being idiopathic. Studies on environmental factors in Parkinson's disease have identified exposure to toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), insecticides (rotenone, paraquat), heavy metals (manganese, lead, copper), carbon monoxide, organic solvents, and trace metal elements, as well as head injury, as contributing factors to the onset of Parkinson's disease.

[0005] The exact cause of the destruction of dopamine neurons in the substantia nigra is still unknown.

[0006] Levodopa has been used to treat Parkinson's disease since the late 1960s, a groundbreaking development in treatment, and it remains the most effective anti-Parkinsonian drug. Currently, with appropriate drug therapy, Parkinson's disease patients can maintain a good quality of life for a considerable period, and mortality rates have decreased. However, long-term levodopa treatment presents many problems as the disease progresses. Specifically, after more than six years of levodopa use, approximately 75% of patients experience motor fluctuations, abnormal movements, and other complications (Im et al., J. Korean Neurol. Assoc. 19(4):315-336, 2001).

[0007] Recently, the inventors of this invention investigated the correlation between T-type calcium channels and pathological phenomena in Parkinson's disease by confirming that when inhibitory basal ganglia input from the globus pallidus of the cerebrum is stimulated with optogenetic light, muscle contraction is induced in ventrolateral thalamic neurons between the activation potential and the post-inhibition period, and that such activation potentials and muscle contractions are reduced by a decrease in the number of neurons exhibiting rebound firing after inhibition due to local knockout of T-type calcium channels in the ventrolateral thalamus (Kim et al., Neuron 95:1181-1196, 2017).

[0008] Voltage-gated calcium channels play a role in increasing intracellular calcium concentration through the activation of nerve cells (Tsien, RW, Annu. Rev. Physiol. 45:341-358, 1983), and are classified into high-voltage-dependent and low-voltage-dependent channels depending on their voltage dependence (Tsien, RW et al., Trends Neurosci. 18, 52-54, 1995). T-type calcium channels are representative low-voltage-gated calcium channels, and in mammals, there are three types based on the genotype of the α1 subunit: Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I) (Perez-Reyes, E., Physiol. Rev. 83:117-161, 2003). Among these, the α1G calcium channel is involved in the generation of burst firings in neurons in the thalamic nucleus, and its important pathological function has recently been revealed (Kim, D. et al., Science 302, 117-119, 2003; Kim, D. et al., Neuron 31: 35-45, 2001).Pathological phenomena associated with α1G calcium channels include abdominal pain (Korean Patent No. 868735), epilepsy (Korean Patent No. 534556), anxiety disorders (Korean Patent No. 958291), Dravet syndrome (Korean Published Patent Publication No. 10-2019-0139302), frontal lobe dysfunction due to irreversible brain injury (Korean Published Patent Publication No. 10-2013-0030011), neuropathic pain (Choi et al., Proc. Natl. Acad. Sci. USA, 113(8):2270-2275, 2016; WO2006064981A1), Angelman syndrome and / or Prader-Willi syndrome. Examples include Cav3.1 syndrome (WO2017070680A1), depression (CN108853510A), rheumatoid arthritis (WO2020203610A1), concentration disorder (Korean Patent No. 1625575), diabetes (CN10264172A), and essential tremor (Korean Patent No. 958286). An interesting point is the transformed mouse in which the Cav3.1 T-type calcium channel is knocked out (Cav3.1). - / - In this case, it is not lethal, and outwardly, it develops normally.

[0009] To improve these pathological symptoms, drugs that block the function of T-type calcium channels are necessary, but there are the following problems. Firstly, various T-type calcium channel blockers, including ethosuximide, do not selectively inhibit only Cav3.1 due to the high homology among the three T-type calcium channels mentioned above. Secondly, in the case of ethosuximide, which is currently used clinically under the brand name Zrotin, it generates nerve signals, but important voltage-dependent Na + Channel and K +They also inhibit the channels (Leresche et al., J. Neurosci. 18(13):4842-4853, 1998), and side effects such as headache, dizziness, and vomiting are known. Thirdly, due to the structural similarity of calcium channels, calcium channel inhibitors cross-act with other types of calcium channels such as N-type and P / Q-type channels. Since N-type and P / Q-type channels are important for the secretion of neurotransmitters in all nerves, blocking them can cause serious impairment to brain function and life. Fourthly, even if an inhibitor selectively acts on the Cav3.1 channel, it may interfere with the function of Cav3.1 expressed in cardiac nerves, potentially leading to abnormalities in cardiac function (Mangoni et al., Circ. Res. 98:1422-1430, 2006).

[0010] For these reasons, nucleic acid molecular-based drugs such as siRNA and shRNA that target only the Cav3.1 gene have been developed. However, these require introduction into a separate vector such as a lentivirus and must be injected locally and precisely into the required brain region using highly invasive devices such as stereotaxic injectors, which presents significant problems for actual clinical application.

[0011] This highlights the urgent need to develop a new type of drug that selectively inhibits only the α1G T-type calcium channel (Cav3.1) while avoiding other side effects. [Overview of the project] [Problems that the invention aims to solve]

[0012] The present invention aims to solve the various problems described above, and its objective is to provide a drug that can be treated by inhibiting α1G T-type calcium channels, such as in Parkinson's disease, by more efficiently suppressing the expression of α1G T-type calcium channels. However, these problems are illustrative and do not limit the scope of the present invention. [Means for solving the problem]

[0013] According to one aspect of the present invention, an antisense oligonucleotide for suppressing Cav3.1 gene expression is provided, targeting the nucleic acid sequence shown in Sequence ID No. 1.

[0014] According to one aspect of the present invention, a pharmaceutical composition for the treatment of Parkinson's disease is provided, comprising the antisense oligonucleotide as an active ingredient.

[0015] According to another aspect of the present invention, a pharmaceutical composition for the treatment of depression is provided, comprising the antisense oligonucleotide as an active ingredient.

[0016] According to one aspect of the present invention, a pharmaceutical composition for the treatment of essential tremor is provided, comprising the antisense oligonucleotide as an active ingredient.

[0017] According to one aspect of the present invention, a pharmaceutical composition for the treatment of epilepsy is provided, comprising the antisense oligonucleotide as an active ingredient.

[0018] According to one aspect of the present invention, a pharmaceutical composition for the treatment of anxiety disorders is provided, comprising the antisense oligonucleotide as an active ingredient.

[0019] According to another aspect of the present invention, a pharmaceutical composition for restoring unconsciousness is provided, comprising the antisense oligonucleotide as an active ingredient.

[0020] According to another aspect of the present invention, a method for treating Parkinson's disease in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from Parkinson's disease.

[0021] According to another aspect of the present invention, a method for treating depression in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from depression.

[0022] According to another aspect of the present invention, there is provided a method for treating essential tremor in an individual, comprising administering a therapeutically effective amount of the antisense oligonucleotide to an individual suffering from essential tremor.

[0023] According to another aspect of the present invention, there is provided a method for treating epilepsy in an individual, comprising administering a therapeutically effective amount of the antisense oligonucleotide to an individual having epilepsy.

[0024] According to another aspect of the present invention, there is provided a method for treating anxiety disorder in an individual, comprising administering a therapeutically effective amount of the antisense oligonucleotide to an individual having anxiety disorder.

[0025] According to another aspect of the present invention, there is provided a method for restoring consciousness in an individual, comprising administering a therapeutically effective amount of the antisense oligonucleotide to an individual in an unconscious state.

Advantages of the Invention

[0026] The antisense oligonucleotide according to an embodiment of the present invention, unlike conventional T-type channel blockers, not only specifically suppresses only the expression of the Cav3.1 gene, but also has almost no side effects when administered in vivo, and is very efficiently used for the treatment of various diseases related to Cav3.1 activity, such as Parkinson's disease, neuropathic pain, attention deficit disorder, anxiety disorder, etc. However, the effects of the present invention are not limited to the above-described content.

Brief Description of the Drawings

[0027] [Figure 1] It is a genomic map showing the genomic DNA structure of isoform 13 of the cacna1g gene, which is the target of the antisense oligonucleotide of the present invention, and the target position of the antisense oligonucleotide candidate designed to target it. [Figure 2]This is a schematic diagram illustrating the structure of a gapmer, an antisense oligonucleotide produced according to one embodiment of the present invention, and the type of modification used. [Figure 3] This graph shows the knockdown efficiency of the cacna1g gene when cells are transfected with an antisense oligonucleotide candidate according to one embodiment of the present invention. [Figure 4] This graph shows the results of comparing the degree of suppression of cacna1g gene expression depending on the treatment concentration when cells are treated with an antisense oligonucleotide candidate according to one embodiment of the present invention. [Figure 5A] This is a schematic diagram illustrating an experimental schedule for analyzing the toxicity of an antisense oligonucleotide and its ability to regulate the expression of the in vivo cacna1g gene according to one embodiment of the present invention. [Figure 5B] This graph shows the survival rate of experimental animals up to 7 days after administration of eight antisense oligonucleotide candidates according to one embodiment of the present invention. [Figure 5C] This graph shows the results of measuring the expression level of the cacna1g gene in the brains extracted from the aforementioned experimental animals using qRT-PCR. [Figure 6A] This graph shows the results of qRT-PCR analysis performed to measure the presence or absence of cross-reactivity of antisense oligonucleotides to Cav3.2 according to one embodiment of the present invention. [Figure 6B] This graph shows the results of qRT-PCR analysis performed to measure the presence or absence of cross-reactivity of antisense oligonucleotides to Cav3.3 according to one embodiment of the present invention. [Figure 7A] This is a schematic diagram showing the schedule of an animal experiment to verify the therapeutic effect of an antisense oligonucleotide on Parkinson's disease according to one embodiment of the present invention. [Figure 7B] This graph shows the results of measuring the lateral rotational displacement distance in an open-field test conducted one week after administration of an antisense oligonucleotide according to one embodiment of the present invention. [Figure 7C]This graph shows the results of measuring the distance traveled during the open field inspection mentioned above. [Figure 7D] This graph shows the results of measuring the movement speed during the open field inspection mentioned above. [Figure 7E] This graph shows the results of measuring the elapsed time until crashing, which was measured by performing accelerated rotor rod tests at one-day intervals for five days, one week after administration of an antisense oligonucleotide according to one embodiment of the present invention in the aforementioned animal experiment. [Figure 7F] These are a series of drawings showing the results of histochemical analysis performed to confirm the mechanism of action of an antisense oligonucleotide according to one embodiment of the present invention. [Figure 8A] This is a schematic diagram showing the schedule of an animal experiment to verify the therapeutic effect of an antisense oligonucleotide on essential tremor according to one embodiment of the present invention. [Figure 8B] This is a schematic diagram illustrating a device for measuring tremor symptoms caused by essential tremor. [Figure 8C] This figure shows the results of measuring the degree of tremor in the form of a spectrogram when an antisense oligonucleotide is administered according to one embodiment of the present invention. [Figure 8D] The graphs shown above quantify the results of Figure 8C, with the left side showing the results of measuring the duration of tremors, and the right side showing the quantitative intensity of tremors over time. [Figure 9A] This is a schematic diagram showing the schedule of an animal experiment to verify the therapeutic effect of an antisense oligonucleotide on depression caused by chronic and persistent stress, according to one embodiment of the present invention. [Figure 9B] This is a schematic diagram illustrating the process of inducing social stress from day 5 to day 10 after administration of an antisense oligonucleotide according to one embodiment of the present invention, and the method of testing for chronic social defeat stress-induced depression performed 17 days later. [Figure 9C]The following is a series of graphs showing the quantitative measure of chronic depression upon administration of antisense oligonucleotides according to one embodiment of the present invention, the left side showing the level of Cav3.1 mRNA expression quantified by qRT-PCR, the center showing the measured social interaction time, and the right side showing the measured sociality index. [Figure 10A] This is a schematic diagram of a forced swimming animal experiment schedule to verify the therapeutic effect of an antisense oligonucleotide on depression induced by short-term acute stress according to one embodiment of the present invention. [Figure 10B] This graph shows a quantitative measure of acute depression induced by forced swimming when an antisense oligonucleotide is administered according to one embodiment of the present invention. The left side shows the level of Cav3.1 mRNA expression quantified by qRT-PCR, and the right side shows the results of measuring immobility time, which is a measure of the degree of stress. [Figure 11A] This is a schematic diagram showing an animal experiment schedule for verifying the therapeutic effect of antisense oligonucleotides on epilepsy according to one embodiment of the present invention, which shows an experimental process in which electroencephalography (EEG) probe insertion is performed 12 days after administration of antisense oligonucleotides, and EEG is measured 30 minutes before and after treatment with GBL (70 mg / kg) to induce absence seizures on the 14th day. [Figure 11B] The following is a series of graphs showing the degree of reduction in Spike and wave discharge (SWDs) in the absence seizure-specific EEG wavelength band, based on EEG measurement results when an antisense oligonucleotide according to one embodiment of the present invention is administered to the experimental animals described above, wherein the left side shows the duration of SWDs and the total SWD time, and the right side shows the number of SWDs. [Figure 12A] This is a schematic diagram showing the schedule of an animal experiment to verify the therapeutic effect of avatin, an antisense oligonucleotide anesthetic, on loss of consciousness according to one embodiment of the present invention. [Figure 12B]This graph shows the results of verifying the therapeutic effect of administering an antisense oligonucleotide according to one embodiment of the present invention, by measuring the time from loss of consciousness to recovery (LORR: Loss of Righting Reflex) after loss of consciousness induced by avatin. [Figure 12C] This is a schematic diagram showing a schedule of further animal experiments to verify the stimulating effect after ethanol treatment of an antisense oligonucleotide according to one embodiment of the present invention. [Figure 12D] The following is a series of graphs showing the results of verifying the therapeutic effect of loss of consciousness by measuring the degree of awakening with low-concentration ethanol when administering antisense oligonucleotides according to one embodiment of the present invention. The left side shows the results of measuring the speed of the experimental animals, the center shows the results of measuring the distance traveled by the experimental animals, and the right side shows the results of measuring the rotation frequency of the experimental animals. [Figure 12E] This graph shows the time taken to lose consciousness (LORR latency) after treatment with a high concentration (3.5 g / kg) of ethanol. [Figure 13A] This is a schematic diagram illustrating a general schedule for an animal experiment using an antisense oligonucleotide (a91) that targets the vicinity of the target sequence according to one embodiment of the present invention. [Figure 13B] This graph shows the survival rate of experimental animals up to 7 days after administration of various antisense oligonucleotides according to one embodiment of the present invention. [Figure 13C] This graph shows the results of measuring the expression level of Cav3.1 in brains extracted from experimental animals 7 days after administration of various antisense oligonucleotides according to one embodiment of the present invention. [Modes for carrying out the invention]

[0028] Definitions of terms: As used herein, the term "oligonucleotide" refers to a polynucleotide formed from a natural nucleobase and a pentafuranosyl group (sugar)-forming nucleoside, which are linked together by natural phosphodiester linkages. Therefore, the term "oligonucleotide" refers to a natural species or natural subunit or a synthetic species formed from closely related homologs thereof.

[0029] As used herein, the term “oligonucleotide” encompasses nucleotides, preferably deoxyribonucleotides (DNA), ribonucleotides (RNA), or DNA / RNA hybrid nucleic acid molecules containing both DNA and RNA. This term refers solely to the primary structure of the molecule. Therefore, this term includes double-stranded or single-stranded DNA and double-stranded or single-stranded RNA.

[0030] Furthermore, the term "oligonucleotide" refers to an artificial form that is not of the natural form but includes a portion that performs a function similar to that of a natural oligonucleotide. Oligonucleotides may have a sugar moiety, a nucleic acid base moiety, or links between modified nucleotides. Of the possible modifications, preferred modifications are 2'-O-alkyl derivatives of the sugar moiety, particularly 2'-O-ethyloxymethyl or 2'-O-methyl, and / or phosphorothioates, phosphorodithioates, or methylphosphonic acids to the nucleotide backbone.

[0031] As used herein, the term "nucleoside-bonding group" refers to a group that can covalently bond two nucleosides, such as between DNA units, between a DNA unit and a nucleotide analog, between two non-LNA units, between a non-LNA unit and an LNA unit, or between two LNA units. In native nucleic acid molecules, phosphodiesters are used as such nucleoside-bonding groups, but as mentioned above, such phosphodiesters can be substituted with similar bonds such as phosphorothioates. Such a bonding group is one or more selected from the following group of configurations: -OP(O)2-O-, -OP(O,S)-O-, -OP(S)2-O-, -SP(O)2-O-, -SP(O,S)-O-, -SP(S)2-O-, -OP(O)2-S-, -OP(O,S)-S-, -SP(O)2-S-, -O-PO(R)-O-, O-PO(OCH3)-O-, -O-PO(NR)-O-, -O-PO(OCH2CH2S-R)-O-, -O-PO(BH3)-O-, -O-PO(NR)-O-, -OP(O)2-NR-, -N R-P(O)2-O-, -NR-CO-O-, -NR-CO-NR-, -O-CO-O-, -O-CO-NR-, -NR-CO-CH2-, -O-CH2-CO-NR-, -O-CH2-CH2-NR-, -CO-NR-CH2-, -CH2-NR-CO-, -OCH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NR-, -O-CH2-CH2-NR-CO-, -CH2-NCH3-O-CH2- (where R is hydrogen or an alkyl group having 1 to 4 carbon atoms).

[0032] As used herein, the term "targeted" means that a nucleic acid sequence can hybridize complementaryally with a specific nucleic acid sequence.

[0033] As used herein, the terms “can hybridize,” “can be hybridized,” “hybridize,” or “hybridize” are typically used to mean the formation of hydrogen bonds between two strands of nucleic acid, also known as Watson-Crick bonds between complementary bases, to form a double helix duplex or triplex (when oligonucleotides consist of two strands).

[0034] As used herein, the term "gene encoding Cav3.1" refers to the genomic sequence of cacna1g, which includes the introns and exons of the gene encoding the α1G subtype of the T-type calcium channel. In humans, the cacna1g gene is located on chromosome 17 and is known to have a size of 66,760 bp according to the NCBI Reference Sequence:NC_000017.11.

[0035] As used herein, the term "product of the gene encoding Cav3.1" means the mRNA sequence produced by transcription of the cacna1g genome sequence. This mRNA may include both the pre-splicing and post-splicing stages.

[0036] An antisense oligonucleotide according to one embodiment of the present invention preferably specifically hybridizes with the gene encoding Cav3.1 or its products. The antisense oligonucleotide according to the present invention can hybridize with the DNA of the gene encoding Cav3.1 and / or mRNA derived from these genes. In particular, the antisense oligonucleotide according to one embodiment of the present invention targets the nucleic acid sequence shown in SEQ ID NO: 1, which includes intron 37 and exon 38 in the mRNA encoding Cav3.1. That is, the antisense oligonucleotide according to one embodiment of the present invention can suppress the expression of Cav3.1 by hybridizing with either pre-splicing or post-splicing mRNA of Cav3.1, provided that the position is appropriate. The antisense oligonucleotide according to one embodiment of the present invention contains enough identical nucleotides to specifically hybridize.

[0037] As used herein, the term "specific hybridization" means, in particular, that the oligonucleotide has a sufficient degree of complementarity to avoid nonspecific fixation of the oligonucleotide to the untargeted sequence under conditions where specific fixation is required. The oligonucleotide does not need to be 100% complementary to the target nucleic acid sequence to which it specifically hybridizes. In particular, oligonucleotides with at least approximately 80% complementarity can specifically hybridize to the nucleic acid selected as the target.

[0038] As used herein, the terms “LNA unit,” “LNA monomer,” “LNA residue,” “Lock nucleic acid unit,” “Lock nucleic acid monomer,” or “Lock nucleic acid residue” mean a biring nucleoside analog. LNA units are described in detail in WO1999014226A, WO2000056746A, WO2000056748A, WO200125248A, WO2002028875A, WO2003006475A, and WO2003095467A. An LNA unit may also be defined by its chemical formula. Therefore, as used in this invention, an “LNA unit” means a compound whose chemical formula is represented by the following structural formula: [ka] (Structural formula 1a) or [ka] (Structural formula 1b) (In the above structural formula, X is selected from the group consisting of O, S, and NR, R is H or an alkyl group having 1 to 4 carbon atoms, and Y is CH2). In the above structural formulas 1a and 1b, when X is S, it is called a "thio LNA unit," when X is NR, it is called an "amino LNA unit," and when X is O, it is called an "oxy LNA unit."

[0039] As used herein, the term "gapmer antisense oligonucleotide" refers to a short, single-stranded antisense oligonucleotide containing a central DNA sequence between two terminal LNA sequences, which is absorbed into cells via a process called gymnosis, even without a transcatheter-infecting agent, and inhibits target mRNA expression by inducing RNase H activation (Hvam et al., Mol.Ther.25:1710-1717, 2017). Therefore, it is a type of hybrid nucleic acid molecule in which LNA, a type of RNA, and DNA are linked. The LNA at both ends provides resistance to nucleases and high affinity to the target sequence, while the internal DNA sequence acts as an RNase H activation site that induces RNA-specific degradation in a DNA / RNA hybrid. Typically, the LNA at both ends is 3-5 nt long, and the central DNA is about 10 nt long. However, in one embodiment of the present invention, the RNA at both ends of the antisense oligonucleotide may use thymine (T) as the base instead of uracil (U).

[0040] Detailed description of the invention: According to one aspect of the present invention, an antisense oligonucleotide for repressing the expression of the gene encoding Cav3.1, targeting Sequence ID No. 1, is provided.

[0041] The antisense oligonucleotide is a nucleic acid molecule that can hybridize with the nucleic acid sequence shown in Sequence ID No. 1, and may have a length of, for example, 11 to 30 nt or 12 to 29 nt. In a preferred embodiment, the antisense oligonucleotide of the present invention has a length of 13 to 28 nt, but more preferably, it may have a length of 14 to 27 nt, 15 to 26 nt, 16 to 25 nt, 17 to 24 nt, 18 to 23 nt or 19 to 22 nt. More specifically, an antisense oligonucleotide according to one embodiment of the present invention may have a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nt.

[0042] The antisense oligonucleotide according to one embodiment of the present invention is a nucleic acid molecule having a length of 7 to 30 nt. The nucleic acid sequence shown in SEQ ID NO: 1 includes exon 38 of the gene encoding Cav3.1 and a portion of the 3' end of the adjacent intron 37. Preferably, the antisense oligonucleotide according to one embodiment of the present invention may include the nucleic acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 21.

[0043] The antisense oligonucleotide is a DNA, RNA, or DNA / RNA hybrid nucleic acid molecule in which part or all of the phosphodiester (PO) backbone is substituted with another form, such as phosphorothioate (PS), phosphoro dithioate, or methylphosphonate, and part or all of the 2'-OH of the sugar moiety may be modified. Examples of such 2'-OH modifications include nucleotide analogs substituted with -O-CH3(-OMe), -O-CH2-CH3(-OEt), -O-CH2-CH2-O-CH3(-MOE), -O-CH2-CH2-CH2-NH2, -O-CH2-CH2-CH2-OH, -OF, or -F.

[0044] Selectively, an antisense oligonucleotide according to one embodiment of the present invention may also contain nucleotide analogs in which the sugar moiety has been modified, for example, a so-called LNA (locked nucleic acid) unit in which an additional ring has been formed on the sugar moiety. The LNA is as described above. At least about 30% of the total nucleotides are nucleotide analogs, for example, at least about 33%, at least about 40%, at least about 50%, at least about 60%, at least about 66%, at least about 70%, at least about 80%, or at least about 90% are nucleotide analogs. An antisense oligonucleotide according to one embodiment of the present invention may consist of a nucleotide base sequence consisting only of nucleotide analog sequences.

[0045] The purpose of modifying the oligonucleotide backbone and sugar moiety in this way is to improve drug stability and pharmacokinetic properties by enhancing the stability of hybridization with the target sequence or by suppressing the phenomenon in which the half-life in the body is significantly reduced due to degradation by nucleases in the body.

[0046] Desirable modifications of the present invention include chimeric oligonucleotides. An oligonucleotide comprises at least two chemically distinct regions (each containing one or more nucleotides). In particular, these consist of one or more regions containing modified nucleotides that confer one or more advantageous properties, such as better biological stability, increased bioavailability, increased cellular fire resistance, or increased affinity to target RNA.

[0047] The degree of complementarity between two nucleic acid sequences of the same length is determined by aligning the two sequences and then comparing the sequences complementary to the first and second sequences. The degree of complementarity is calculated by determining the number of nucleotides at the same position between the two sequences being compared, dividing the number of identical positions by the total number of positions, and multiplying the result by 100 to obtain the degree of complementarity between these two sequences.

[0048] An antisense oligonucleotide according to one embodiment of the present invention is a gapmer, which is a nucleic acid molecule containing 3-5 nt long RNA units at both ends and 8-12 nt long DNA between them and LNA. In this case, the RNA units may contain at least one LNA.

[0049] According to one aspect of the present invention, a pharmaceutical composition for the treatment of Parkinson's disease is provided, comprising the antisense oligonucleotide as an active ingredient.

[0050] The correlation between Parkinson's disease and Cav3.1 has been well described in prior literature (Park et al., Front. Neural Circuits, 7:172, 2013; Rubit et al., Eur. J. Neurosci., 36(2):2213-2228, 2012; Xiang et al., ASC Chem. Neurosci. 2(12):730-742, 2012). Furthermore, the present inventors have experimentally demonstrated that an antisense oligonucleotide according to one embodiment of the present invention, which specifically binds to Cav3.1, particularly the region containing intron 37-exon 38, and suppresses Cav3.1 expression without other side effects, can significantly alleviate abnormal muscle symptoms in Parkinson's disease model animals. Therefore, an antisense oligonucleotide according to one embodiment of the present invention, which can specifically suppress Cav3.1 expression, could be a very efficient therapeutic agent in the treatment of Parkinson's disease.

[0051] According to one aspect of the present invention, a pharmaceutical composition for the treatment of depression is provided, comprising the antisense oligonucleotide as an active ingredient.

[0052] Recent research has reported a correlation between T-type calcium channels and anxiety-related behaviors. Specifically, it has been reported that when T-type calcium channels are opened, anxiety-related behaviors are induced, and conversely, anxiety symptoms are reduced when T-type calcium channel blockers are administered (Kaur et al., J. Basic Clin. Physiol. Pharmacol., 31(3):20190067, 2020). Furthermore, the inventors have experimentally confirmed that depression-related symptoms are alleviated in transformed mice in which the Cav3.1 gene is targeted. Therefore, an antisense oligonucleotide according to one embodiment of the present invention, targeting the Cav3.1 gene, can also be used to treat depression. Moreover, the inventors have demonstrated through animal experiments that an antisense oligonucleotide according to one embodiment of the present invention is effective in treating depression.

[0053] According to one aspect of the present invention, a pharmaceutical composition for the treatment of essential tremor is provided, comprising the antisense oligonucleotide as an active ingredient.

[0054] The inventors have discovered that essential tremor can be treated by specifically inhibiting Cav3.1 (the α1G subunit of the T-type calcium channel) (Korean Patent No. 958286). Therefore, an antisense oligonucleotide that can specifically inhibit the expression of Cav3.1 according to one embodiment of the present invention can be used to treat essential tremor. Furthermore, the inventors have demonstrated through animal experiments that an antisense oligonucleotide according to one embodiment of the present invention can be used to treat essential tremor.

[0055] According to one aspect of the present invention, a pharmaceutical composition for the treatment of epilepsy is provided, comprising the antisense oligonucleotide as an active ingredient.

[0056] It is known that T-type calcium channel blockers, which specifically inhibit T-type calcium channels, reduce thalamic rapid firing, thereby suppressing seizure symptoms (Powell et al., Br.J.Clin.Pharmacol., 77:729-739, 2014; Casillas-Espinosa et al., PLoS One, 10:30130012, 2015). Therefore, an antisense oligonucleotide according to one embodiment of the present invention can be used to treat epilepsy. Moreover, the inventors have demonstrated through animal experiments that an antisense oligonucleotide according to one embodiment of the present invention can be used to treat epilepsy.

[0057] In the aforementioned pharmaceutical composition, the epilepsy is an absence seizure.

[0058] According to another aspect of the present invention, a pharmaceutical composition for the treatment of anxiety disorders is provided, comprising the antisense oligonucleotide as an active ingredient.

[0059] The present inventors have confirmed that when mibefladil, a T-type calcium channel selective blocker, is administered to mice in which the phospholipase β4 (PLCβ4) gene, a model animal for anxiety disorders, is knocked out, the reduced memory erasure ability in the mice returns to normal. Furthermore, when mibefladil and efonidipine are administered to normal mice with MD, fear memories are erased more quickly, and the ability to recall the erased memories decreases (Korean Patent No. 958291). Therefore, an antisense oligonucleotide that can specifically suppress the expression of Cav3.1 according to one embodiment of the present invention can be used in the treatment of anxiety disorders.

[0060] According to another aspect of the present invention, a pharmaceutical composition for restoring consciousness is provided, comprising the antisense oligonucleotide as an active ingredient. The inventors of the present invention have experimentally demonstrated that when an antisense oligonucleotide according to one embodiment of the present invention is administered to experimental animals in which unconsciousness has been induced, the restoration of consciousness is promoted. Therefore, the antisense oligonucleotide according to one embodiment of the present invention can be used as a drug for restoring consciousness in patients in unconscious states, including comatose states.

[0061] According to another aspect of the present invention, a method for treating Parkinson's disease in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from Parkinson's disease.

[0062] According to another aspect of the present invention, a method for treating depression in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from depression.

[0063] According to another aspect of the present invention, a method for treating essential tremor in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from essential tremor.

[0064] According to another aspect of the present invention, a method for treating epilepsy in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual suffering from epilepsy.

[0065] In the above method, the epilepsy is an absence seizure.

[0066] According to another aspect of the present invention, a method for treating an anxiety disorder in an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual having the anxiety disorder.

[0067] According to another aspect of the present invention, a method for restoring consciousness of an individual is provided, comprising the step of administering a therapeutically effective amount of the antisense oligonucleotide to the individual in an unconscious state.

[0068] The composition may contain a pharmaceutically acceptable carrier, and may further contain pharmaceutically acceptable auxiliaries, excipients, or diluents in addition to the carrier.

[0069] As used herein, the term "pharmaceutically acceptable" means a composition that is physiologically acceptable and, when administered to humans, does not cause typical gastrointestinal disturbances, allergic reactions such as dizziness, or similar reactions. Examples of carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. They may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0070] Furthermore, a pharmaceutical composition according to one embodiment of the present invention may be formulated using methods known to those skilled in the art to enable rapid release, or sustained or delayed release of the active ingredient upon administration to a mammal. Dosage forms include powder, granules, tablets, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution, and sterile powder.

[0071] A composition according to one embodiment of the present invention can be administered via various routes, preferably intrathecal injection, intracranial injection, or intracerebral injection (ICV) through an Ommaya reservoir. Given the somewhat invasive nature of the administration method, automated administration at predetermined intervals is also possible through an administration device including a reservoir and pump appropriately connected to a catheter inserted at the administration site. Alternatively, instead of an automated administration device, it can be administered manually once or several times a day at appropriate intervals, such as once a week, twice a week, or once a month, and the duration of administration is not particularly limited.

[0072] A composition according to one embodiment of the present invention is formulated in a suitable form with a commonly used pharmaceutically acceptable carrier. Suitable carriers include, for example, water, a suitable oil, saline solution, parenteral carriers such as aqueous glucose and glycol, and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Furthermore, depending on the method of administration and dosage form, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, etc. Suitable pharmaceutically acceptable carriers and formulations for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0073] The dosage of the composition to a patient varies depending on many factors, including the patient's height, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The pharmaceutically active antisense oligonucleotide is administered in amounts of 100 ng / kg to 10 mg / kg, more preferably 1 to 500 μg / kg, and most preferably 5 to 50 μg / kg, but the dosage is adjusted considering the aforementioned factors.

[0074] As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease in a reasonable benefit-to-risk ratio applicable to medical treatment. The effective dose level may be determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention is 0.5 μg / kg to 1 g / kg, more preferably 10 μg / kg to 500 mg / kg, but the effective dose is appropriately adjusted depending on the patient's age, sex, and condition.

[0075] As used herein, the term “treatment” means a systematic process and activities planned to treat, cure, or alleviate a disease, disorder, or problem. Fundamentally, the ultimate goal is to eliminate the etiology of the disease or restore the lesion to its pre-disease state, but even if it is not possible to eliminate the etiology of the disease or restore the lesion to its original state, symptom relief, which eliminates or alleviates the symptoms of the disease, is also included in the category of treatment. Furthermore, preventive actions, such as administering a drug according to one embodiment of the present invention before symptoms appear, to prevent the symptoms from appearing or, if they do appear, to alleviate them so that the patient does not significantly interfere with their daily life, also fall under the category of treatment. Accordingly, a pharmaceutical composition according to one embodiment of the present invention is used for disease prevention, and a treatment method according to one embodiment of the present invention includes a method for preventing the symptoms of a disease.

[0076] The present invention will be described in more detail below through examples and experimental cases. However, the present invention is not limited to the examples and experimental cases disclosed below, and can be embodied in a variety of different forms. The following examples and experimental cases are provided to fully disclose the present invention and to fully inform those skilled in the art of the scope of the invention. [Examples]

[0077] Example: Design of a Cacna 1g-targeted antisense oligonucleotide The inventors devised 20 candidate antisense oligonucleotides (ASOs) targeting cacna1g, the gene encoding the α1G subunit (Cav3.1), from among the three isomorphs of T-type calcium channels, taking into consideration target specificity and stability (Figure 1 and Table 1). Furthermore, the inventors used a gapmer containing a 10nt DNA sequence and 5nt RNA sequences each having a 2'-MOE modified skeleton at the 5' and 3' ends, respectively, which has been conventionally known to be stable yet effective as an antisense oligonucleotide structure. In the gapmer, the DNA portion used phosphorothioate bonds (PS bonds) instead of the usual phosphodiester bonds (PO bonds) for linking sugars, while the RNA portion used PS bonds only at both ends of the last sequence, and PO bonds for the remaining RNA portion (Figure 2). On the other hand, all uracil (U) molecules in the RNA portion are replaced with thymine (T), and all cytosine (C) molecules are replaced with methylated cytosine, which further enhances the stability of the antisense oligonucleotide.

[0078] [Table 1]

[0079] Experimental Example 1: Primary Screening As shown in Table 1 above, the inventors devised candidate antisense oligonucleotides targeting cacna1g mRNA, and then commissioned Mycrosynth (Germany) to manufacture them. A549 cells were transfected with the antisense oligonucleotides at a concentration of 200 nM using lipofectamine reagent. After 48 hours, the cells were lysed using a lysis and RT kit (TOYOBO, Japan), and reverse transcription was performed. Subsequently, qRT-PCR was performed using a Taqman probe and qRT-PCR premix (TOYOBO, Japan) to measure the cacna1g gene expression level. Specific probe and primer information for qRT-PCR is shown in Table 1 below. As a result, as can be seen in Figure 3, eight antisense oligonucleotides, including a80, a82, a91, and a92, were selected, each showing repeated knockdown of 50% or more of gene expression.

[0080] Experimental Example 2: Secondary Screening Subsequently, the inventors investigated whether intracellular absorption and knockdown of the cacna1g gene were possible using lipofectamine reagent with eight antisense oligonucleotides selected from Experimental Example 1 at two concentrations (100 nM and 400 nM).

[0081] As a result, as can be seen in Figure 4, all eight antisense oligonucleotides showed excellent knockdown efficiency at a concentration of 400 nM. However, in the case of a80, a82, and a84, the knockdown efficiency at a concentration of 100 nM was lower than that of the other antisense oligonucleotides.

[0082] Experimental Example 3: Toxicity Analysis and In vivo Gene Expression Regulation Analysis Based on the results of cell experiments in Experimental Example 1 and Experimental Example 2, the inventors conducted animal experiments to verify the stability and pharmacological effects of eight antisense oligonucleotide candidates according to one embodiment of the present invention.

[0083] Specifically, the inventors divided male C57BL / 6J wild-type mice at P0 week of age into groups of 4 to 6 mice each. They then administered 2 μl each of the antisense oligonucleotides (1 mM) of each example to each hemisphere of the brain, for a total of 4 μl (25 μg), via intraventricular (ICV) injection. After 7 days, the survival rate was measured, and brain tissue was extracted from the surviving mice. The expression level of the cacna1g gene in the brain tissue was measured using qRT-PCR as described in Experimental Example 1 above (Figure 5A).

[0084] As a result, as can be seen in Figure 5B, all mice administered a88 died within 6 hours. In the case of a86, 4 out of 6 mice died; in the case of a82, 2 out of 5 mice died; and in the case of a84, 2 out of 6 mice died, confirming that a88 is considerably toxic. On the other hand, a89, a91, and a92 either resulted in the death of 1 out of 6 mice, or no experimental animals died from the start, confirming that they are relatively safe antisense oligonucleotide candidate substances. On the other hand, AS-Cav3.1_20mer was used as a positive control group to knock down Cav3.1 in previous literature (Bourinet et al., EMBO J.24:315-324, 2005). However, its sequence is complementary only in mice and rats, and not in primate genes including monkeys and humans. Due to its high probability of cross-reactivity with other genes, it was used as a positive control group only in the initial screening experiment and subsequently excluded in in vivo screening.

[0085] Furthermore, when the gene expression inhibitory effect was confirmed in actual animal experiments, as can be seen in Figure 5C, in the case of a82 and a84, unlike in cell experiments, the efficiency of suppressing cacna1g gene expression was low under in vivo conditions, while the gene expression suppression efficiency of a86, a89, a91, and a92 was confirmed to be superior.

[0086] Based on the results from Figures 5B and 5C, the inventors selected a91 and a92 as the final candidate substances.

[0087] Experimental Example 4: Analysis of Cross-Reactions The α-subunit of T-type calcium channels has three isotypes, and the homology between them is very high. In particular, the α-subunits of these three isotypes are expressed in different organs and tissues, and it is known that non-discriminatory blockers, when administered systemically, can block T-type calcium channels in locations other than the target organ, leading to a variety of side effects. Due to this cross-reactivity, T-type calcium channel blockers developed to date are known to exhibit serious side effects in actual clinical use.

[0088] Therefore, developing antisense oligonucleotides that can selectively suppress the expression of only the α subunit (Cav3.1) without suppressing the expression of other isomorphs is a priority requirement for developing safer drugs.

[0089] Based on this, the inventors investigated through animal experiments whether a91 and a92, which were selected as the final candidate substances, selectively inhibit only Cav3.1.

[0090] To this end, the inventors administered 2 μl each of a91 and a92 (1 mM) to each hemisphere of the brain of male C57BL / 6J wild-type mice at P0 week of age, for a total of 4 μl (25 μg), via intraventricular (ICV) injection. After 7 days, brain tissue was extracted from the mice, and the expression levels of the cacna1g (Cav3.1), cacna1h (Cav3.2), and cacna1i (Cav3.3) genes in the brain tissue were measured using the primers and probes listed in Table 1 and the qRT-PCR described in Experimental Example 1 (Figure 5A).

[0091] [Table 2]

[0092] As a result, as can be seen from Figures 6A and 6B, it was confirmed that the antisense oligonucleotides (a91 and a92) according to one embodiment of the present invention specifically suppress the expression of cacna1g without suppressing the expression of genes encoding other isomorphs. Therefore, it can be seen that the antisense oligonucleotides according to one embodiment of the present invention can be efficiently used to treat a variety of diseases involving the α1G subunit by selectively suppressing only the expression of the α1G subunit without affecting the expression of other isomorphs.

[0093] Experimental Example 5: Analysis of the therapeutic effects of Parkinson's disease treatment Based on the results of Experimental Examples 1 to 4 described above, the inventors investigated whether an antisense oligonucleotide according to one embodiment of the present invention shows a therapeutic effect against Parkinson's disease, which is known as a representative disease among cacana 1g-related diseases.

[0094] 5-1: Verification of treatment effectiveness To this end, the inventors first prepared mice administered with 6-OHDA (6-hydroxydopamine) as a Parkinson's disease model animal. Then, 200 μg of antisense oligonucleotide (a91) according to one embodiment of the present invention was administered to 7-8 week old male C57BL / 6J wild-type mice via intracerebroventricular injection, and after one week, open-field analysis was performed (Figure 7A).

[0095] As a result, as can be seen in Figure 7B, analysis of the open field one week after injection of antisense oligonucleotide according to one embodiment of the present invention showed a decrease in ipsilateral rotation when antisense oligonucleotide (a91) according to one embodiment of the present invention was administered.

[0096] In Parkinson's disease model mice induced by unilateral injection of 6-OHDA into the striatum (unilaterally), a characteristic phenotype of repeated rotation in the direction of injection was observed (Thiele et al., J.Vis.Exp., 60:e3234, 2012). In the rats used in the experiments shown in Figures 7B and 7C, 6-OHDA was injected into the striatum located in the left hemisphere, resulting in a phenotype of rotation to the left. This type of rotation, where the rotation is in the direction of injection, is called ipsilateral rotation. In Figure 7B, in the group of mice injected only with 6-OHDA, the ratio of coaxial rotation increased compared to the distance traveled, while in the group of mice that received an additional 200 μg intracerebroventricular injection of Cav3.1-ASO(a91) in addition to the 6-OHDA model, the ratio of ipsilateral rotation decreased. This means that the Parkinson's disease-related pathological phenotype caused by 6-OHDA was reduced through Cav3.1-ASO 200 μg injection.

[0097] In Figure 7C, the distance traveled by the mouse in 30 minutes was measured. In rats given an additional 200 μg of Cav3.1-ASO, movement appeared to decrease to the level of the negative control group, but statistically, there was no significant difference in travel distance across any group. Similarly, in Figure 7D, the movement speed of the mouse was measured. As with the distance traveled, in rats given an additional 200 μg of Cav3.1-ASO, movement appeared to decrease to the level of the negative control group, but statistically, there was no significant difference in movement speed across any group.

[0098] The inventors subsequently performed accelerated rotorod testing as an evaluation index for motor ability. Accelerated rotorod testing was performed on the experimental animals at daily intervals one week after injection of antisense oligonucleotides.

[0099] As a result, as can be seen in Figure 7E, the antisense oligonucleotide according to one embodiment of the present invention showed that motor performance improved in a concentration-dependent manner, and as the number of test days increased. On the other hand, in the negative control group administered only 6-OHDA, the ataxia phenomenon did not improve at all even as time passed.

[0100] 5-2: Confirmation of the mechanism The inventors confirmed whether the antisense oligonucleotide action mechanism according to one embodiment of the present invention actually acted after the death of dopamine neurons.

[0101] To address this, the inventors specifically administered a 6-OHDA drug, known as a Parkinson's disease-inducing drug, to the striatum of the brain in the experimental animals of Experimental Example 5-1, and administered an antisense oligonucleotide together with it on the day of administration. Since 6-OHDA administration induces cell death within one week, and the effect of the antisense oligonucleotide takes about one week, it is difficult to expect an inhibitory effect on dopamine neuron death using this experimental method. To measure dopamine neuron death, after 5 weeks, the experimental animals were sacrificed, the brains were extracted, and tissue sections were prepared using a cryotome. Histochemical analysis of the striatum and substantia nigra was then performed using an antibody that specifically binds to tyrosine hydroxylase (TH), a dopaminergic neuron-specific marker. As a result, as shown in Figure 7F, dopamine neurons in the control group, which received no drug, appeared normally, but dopamine neuron death was observed in all groups that received 6-OHDA. This indicates that, in the experimental method used in this embodiment, the antisense oligonucleotide did not affect the death of dopamine neurons, but it did work after the dopamine neurons died by blocking the abnormal motor neuron signaling that occurs due to the disappearance of dopamine neurons.

[0102] Experimental Example 6: Analysis of the therapeutic effect of essential tremor The inventors subsequently investigated whether an antisense oligonucleotide according to one embodiment of the present invention is effective in treating essential tremor by conducting animal experiments using a harmaline-induced essential tremor model mouse.

[0103] To this end, specifically as shown in Figure 8A, 200 μg of an antisense oligonucleotide (a91) according to one embodiment of the present invention was intracerebroventricularly administered to 7-8 week old male C57BL / 6J wild-type mice. Two weeks later, harmaline, a tremor-inducing substance, was administered intraperitoneally at a dose of 9 mg / kg, and after 8-10 minutes, a tremor test was performed using the apparatus shown in Figure 8B.

[0104] As a result, as shown in Figures 8C and 8D, the antisense oligonucleotide according to one embodiment of the present invention significantly suppressed tremors compared to the control group (wild-type) and the comparison group (control ASO-administered group).

[0105] The above results demonstrate that an antisense oligonucleotide according to one embodiment of the present invention is effective in treating essential tremor.

[0106] Experimental Example 7: Analysis of the effectiveness of treatment for depression The inventors subsequently investigated whether an antisense oligonucleotide according to one embodiment of the present invention is effective in treating depression by conducting animal experiments using stress-induced depression model mice.

[0107] 7-1: Effects in the Chronic Social Stress Model First, the inventors investigated the measure of depression using the experimental schedule shown in Figure 9A and the specific method shown in Figure 9B. Specifically, 200 μg of antisense oligonucleotide (a91) according to one embodiment of the present invention was injected intracerebroventricularly into 7-8 week old male C57BL / 6J wild-type mice. Then, from day 5 to day 10, other aggressive individuals were housed in the same cage as the experimental animals to induce social defeat stress. On day 17, the degree of depression due to stress was measured by measuring social interaction.

[0108] As a result, as shown in Figure 9C, experimental animals administered with the antisense oligonucleotide according to one embodiment of the present invention showed decreased Cav3.1 expression, while social interactions were restored to almost the control group level. This indicates that the antisense oligonucleotide according to one embodiment of the present invention is a highly effective substance for reducing social stress.

[0109] 7-2: Effects in a Short-Term Acute Stress Model Subsequently, the inventors conducted a forced swimming test to confirm whether the antisense oligonucleotide according to one embodiment of the present invention has a therapeutic effect on acute depression.

[0110] Specifically, 200 μg of antisense oligonucleotide (a91) according to one embodiment of the present invention was administered intracerebroventricularly to 7-8 week old male C57BL / 6J wild-type mice. After 3 weeks, as shown in Figure 10A, the mice were placed in a water tank, and the stress index was determined by measuring the immobility time that appeared due to stress.

[0111] As a result, as can be seen in Figure 10B, the antisense oligonucleotide according to one embodiment of the present invention not only significantly reduced the expression of Cav3.1 in the brain, but also significantly reduced immobility time, a sign of stress. This result indicates that the antisense oligonucleotide according to one embodiment of the present invention is a substance that provides resistance not only to chronic stress but also to short-term stress.

[0112] Example 8: Therapeutic effect on epilepsy The inventors investigated whether an antisense oligonucleotide according to one embodiment of the present invention can also be used for the treatment of epilepsy.

[0113] Specifically, as shown in Figure 11A, the inventors administered 200 μg of an antisense oligonucleotide (a91) according to one embodiment of the present invention intracerebroventricularly to 7-8 week old male C57BL / 6J wild-type mice. After 12 days, an EEG probe was inserted, and after 14 days, 70 mg / kg of γ-butyrolactone (GBL), which induces absence seizures, was administered. Electroencephalograms (EEGs) were measured 30 minutes before and after GBL administration. 42 days after antisense oligonucleotide administration, the experimental animals were sacrificed, their brains were removed, and the Cav3.1 mRNA expression level was measured.

[0114] As a result, as can be seen in Figure 11B, the antisense oligonucleotide according to one embodiment of the present invention significantly reduced both the frequency and number of spikes and wave discharges (SWDs) per 10 minutes.

[0115] This result demonstrates that an antisense oligonucleotide according to one embodiment of the present invention is effective in treating epilepsy, particularly absence seizures.

[0116] Experiment Example 9: Effects of Restoring Consciousness A coma, or comatose state, is a medical term for a deep state of unconsciousness. A person in a comatose state, often referred to as a "vegetative state," cannot be awakened and does not respond to external stimuli such as pain, light, or sound. While drug intoxication, metabolic disorders, central nervous system diseases, hypoxia, and stroke are known causes of comatose states, comatose states can also be caused by trauma to the brain, such as from car accidents or falls. Unfortunately, no drugs have been reported to restore consciousness to patients in such comatose states.

[0117] The main symptom of epilepsy discussed in Experimental Example 8 is loss of consciousness. Furthermore, the correlation between epilepsy, T-type calcium channels, and absence seizures is well known, and based on the results of Experimental Example 8, which demonstrated that the symptoms of absence seizures are alleviated by suppressing the expression of the α1G subunit of T-type calcium channels, the inventors sought to confirm whether an antisense oligonucleotide according to one embodiment of the present invention is effective in restoring consciousness.

[0118] To this end, the inventors administered 200 μg of an antisense oligonucleotide (a91) according to one embodiment of the present invention intracerebroventricularly to 7-8 week old male C57BL / 6J wild-type mice, as shown in Figure 12A. After 11 days, they administered 400 mg / gk of the anesthetic Avertin intraperitoneally to induce loss of righting reflex (LORR). Subsequently, the duration of loss of righting reflex (LORR) was measured. As can be seen in Figure 12B, the duration of loss of righting reflex was significantly reduced in the group administered with the antisense oligonucleotide (a91) according to one embodiment of the present invention.

[0119] Furthermore, the inventors investigated whether the antisense oligonucleotide according to one embodiment of the present invention rapidly restores alcohol-induced loss of consciousness using an alcohol-induced loss of consciousness model animal.

[0120] Specifically, following the experimental schedule shown in Figure 12C, 200 μg of antisense oligonucleotide (a91) according to one embodiment of the present invention was intracerebroventricularly administered to 7-8 week old male C57BL / 6J wild-type mice. After 12-13 days, a 20% ethanol aqueous solution was administered intraperitoneally at a dose of 0.6 g / kg relative to ethanol. The behavior of the experimental animals was then videotaped in an open area for 30 minutes, and the movement speed, distance traveled, and rotation frequency of the experimental animals were measured using computer software (EthoVision, Noldus, Netherlands). Furthermore, after two days, a high concentration (3.5 g / kg) of ethanol was administered intraperitoneally to induce loss of consciousness, and the delay time to loss of consciousness (LORR latency) was measured. In addition, 21 days after administration of oligonucleotide (a91) according to one embodiment of the present invention, the experimental animals were sacrificed, their brains were removed, and the level of Cav3.1 gene expression was measured.

[0121] As a result, as shown in Figure 12D, more active motility was observed in the group administered antisense oligonucleotides according to one embodiment of the present invention, and in the case of the delay time of loss of consciousness, the group administered antisense oligonucleotides according to one embodiment of the present invention showed a longer duration compared to the control group.

[0122] This means that an antisense oligonucleotide according to one embodiment of the present invention can promote the recovery of consciousness in patients in a state of unconsciousness by specifically suppressing the expression of the Cav3.1 gene. This result indicates that the antisense oligonucleotide of the present invention is a promising substance that can be developed as a consciousness recovery inducer for patients in a comatose state for which no suitable therapeutic agent currently exists.

[0123] Examples 21-23: Confirmation of ASO target sites Based on the results of the above-mentioned examples and experimental cases, the inventors attempted to more accurately identify the target position of the antisense oligonucleotide according to one embodiment of the present invention.

[0124] For this purpose, as shown in Table 4 below, multiple target sequences were selected from within Sequence ID No. 1, taking into consideration the position of the cacna1g genome gene, and after determining the target positions near the antisense oligonucleotide (a91) according to one embodiment of the present invention (a4, a7, and a11), their safety and the suppression rate of Cav3.1 expression were investigated.

[0125] [Table 3]

[0126] The genomic locations in Table 3 above are based on the genomic nucleic acid sequence of the cacna1g gene described in GenBank Reference Sequence NC_000017.11 REGION:50560715..50627474.

[0127] Specifically, the inventors divided C57BL / 6J wild-type mice at P0 week of age into groups of 4 to 6 mice each. They then administered 2 μl each of the antisense oligonucleotides (1 mM) listed in Table 3 to each hemisphere of the brain, for a total of 4 μl (25 μg), via intraventricular (ICV) injection. After 7 days, they measured the survival rate, extracted brain tissue from the surviving mice, and measured the expression level of the cacna1g gene in the brain tissue using qRT-PCR as described in Experimental Example 1 (Figure 13A).

[0128] As a result, as shown in Figure 13B, the antisense oligonucleotides newly devised in the present invention, with the exception of a4, were found to be similar to or have superior survival rates compared to the control group and the antisense oligonucleotide (a91) from one embodiment of the present invention, confirming their high safety.

[0129] Furthermore, investigations into the effects on Cav3.1 expression revealed that, as shown in Figure 13C, among the newly devised antisense oligonucleotides, a11 showed a significant suppressive ability to inhibit Cav3.1 mRNA expression, albeit somewhat weaker than a91. A4 and a7 also showed suppressive effects on Cav3.1 mRNA expression, although not statistically significant. The reason for the lack of statistical significance is that each individual in each experimental group showed an even higher gene expression rate compared to the control group. This is considered an experimental error, and it is believed that increasing the number of experimental subjects would result in a statistically significant decrease in gene expression.

[0130] As can be seen from the results above, the location on the cacna1g genomic gene selected in this invention is, contrary to expectations, a location close to the 3' end of the mRNA before splicing, and any location within Sequence ID No. 1, including intron 37 and exon 38, can be selected to efficiently reduce Cav3.1 mRNA expression and be useful in treating neurological diseases associated with α1G T-type calcium channels.

[0131] As described above, the antisense oligonucleotide according to one embodiment of the present invention not only selectively suppresses the expression of the α1G subunit without affecting the expression of other isomorphic T-type calcium channels, but also showed a remarkably effective treatment for diseases associated with the α1G subunit, particularly Parkinson's disease, without any other side effects. Therefore, the antisense oligonucleotide according to one embodiment of the present invention can be used as a therapeutic agent for a variety of diseases associated with the α1G subunit of T-type calcium channels, including not only Parkinson's disease, but also various degenerative neurological diseases such as depression, essential tremor, epilepsy, anxiety disorders, and unconsciousness.

[0132] Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely illustrative, and those skilled in the art will understand that a wider variety of modifications and equivalent other embodiments are possible. Therefore, the true scope of technical protection of the present invention must be determined by the technical idea of ​​the claims.

[0133] An antisense oligonucleotide according to one embodiment of the present invention can be used as a medicine for restoring consciousness in patients in an unconscious state, as well as for neuropsychiatric disorders such as Parkinson's disease, depression, essential tremor, epilepsy, and loss of consciousness, by selectively suppressing the expression of α1G T-type calcium channels.

Claims

1. An antisense oligonucleotide for repressing the expression of a gene encoding Cav3.1, comprising the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 and having a length of 20 to 25 nt.

2. The antisense oligonucleotide according to claim 1, wherein the DNA, RNA, or DNA / RNA chimeric nucleic acid molecule.

3. The antisense oligonucleotide according to claim 1, wherein part or all of the phosphodiester (PO) skeleton is substituted with phosphorothioate (PS), phosphorodithioate, or methylphosphonic acid.

4. The antisense oligonucleotide according to claim 1, wherein part or all of the 2'-OH group of the sugar portion is modified.

5. The 2'-OH of the sugar moiety is -O-CH ２ (-OMe), -O-CH ２ -CH ３ (-OEt), -O-CH ２ -CH ２ -O-CH ３ (-MOE), -O-CH ２ -CH ２ -CH ２ -NH ２ , -O-CH ２ -CH ２ -CH ２ The antisense oligonucleotide according to claim 4, wherein the 2'-OH of the sugar moiety is substituted with -O-CH(-OMe), -O-CH(-OEt), -O-CH(-MOE), -O-CH(-OH), -OF or -F.

6. The antisense oligonucleotide according to claim 1, comprising at least one nucleotide analog in which an additional ring is formed on the sugar portion.

7. The antisense oligonucleotide according to claim 6, wherein the nucleotide analog is Loc nucleic acid (LNA).

8. The antisense oligonucleotide according to claim 6, wherein the nucleotide analogs constitute at least 33% of the total nucleotides.

9. The antisense oligonucleotide according to claim 1, which is a gapmer.

10. The antisense oligonucleotide according to claim 9, wherein the gapmer is a nucleic acid molecule comprising RNA units of 3–7 nt length at both ends and DNA of 8–12 nt length present between the RNA units.

11. The antisense oligonucleotide according to claim 10, wherein part or all of the RNA is LNA.

12. A pharmaceutical composition for the treatment of Parkinson's disease, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

13. A pharmaceutical composition for the treatment of depression, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

14. A pharmaceutical composition for the treatment of essential tremor, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

15. A pharmaceutical composition for the treatment of epilepsy, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

16. A pharmaceutical composition for the treatment of anxiety disorders, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

17. A pharmaceutical composition for restoring unconsciousness, comprising an antisense oligonucleotide according to any one of claims 1 to 11.

Images