WO2023238948A1 - Novel brain neurogenesis promoter containing mirna - Google Patents

Abstract

The subject's brain neurogenesis promoter of the present invention contains miR-30a, etc. or a vector which is for expressing miR-30a, etc. in the brain nerves of young and elderly subjects. The present invention also provides an agent for improving higher nervous function, an agent for improving memory learning function, a method for promoting brain neurogenesis, a method for improving higher nervous function, and a method for improving memory learning function. In the subject's brain neurogenesis promoter, etc. of the present invention, the subject may be an elderly subject.

Description

Novel brain neurogenesis promoter containing miRNA

The present invention relates to a brain neurogenesis promoting agent for subjects. Furthermore, the present invention relates to an agent for improving higher nervous function in a test subject and an agent for improving memory learning function in a test subject.

In the brains of mammals, including humans, neurogenesis in the brain becomes inactive with aging, leading to a decline in memory and other higher neural functions. The choroid plexus is the largest secreted factor-releasing tissue in the brain and releases secreted factors into the cerebrospinal fluid. Among the secretory factors released by the choroid plexus, miRNA is encapsulated in extracellular vesicles and released into the cerebrospinal fluid, and is taken up by neural stem cells exposed to the cerebrospinal fluid, where they promote proliferation, differentiation, and other functions of neural stem cells. Involved in regulation. However, the expression levels of all miRNAs expressed in the choroid plexus do not decrease with aging (Non-Patent Document 1). Therefore, the inventors of the present invention found that among the miRNAs expressed in the choroid plexus, those whose expression levels change most markedly with aging are involved in suppressing neurogenesis in the brain; We considered that neurogenesis in the brain could be promoted by adding the miRNAs whose decrease was most significant to the cerebrospinal fluid.

miRNA can simultaneously regulate the expression of multiple genes involved in the same or similar functions within cells (Patent Document 1). Therefore, by using miRNA as a clue, it is possible to develop drugs that regulate cell functions more efficiently than individual proteins or genes encoding proteins.

Special Publication No. 2013-504542

Tietje, A. , et al. , PLOS ONE, DOI:10.1371/journal. bone. 0113116 November 24, 2014.

The purpose of the present invention is to provide miRNAs that can promote the regeneration of cranial nerves and improve memory learning performance in young and elderly people by comparing the choroid plexus of young people and the choroid plexus of elderly people. be.

In order to achieve the above objective, the present inventors carried out studies using mice, and among the miRNAs expressed in the choroid plexus of young mouse brains, we discovered miRNAs whose expression level significantly decreased in old mice, Furthermore, the present inventors discovered that aged mice in which the miRNA was overexpressed in the choroid plexus tended to have better memory and learning performance than control aged mice, leading to the completion of the present invention.

The present invention provides an agent for promoting brain neurogenesis for a subject. The subject brain neurogenesis promoting agent of the present invention is
miRNA miR-30a or its precursor,
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the miR-30a base sequence and has substantially the same function as miR-30a is administered to the subject. containing an effective amount of the vector for expression in the cranial nerves of the brain.

In the agent for promoting brain neurogenesis for subjects of the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof.

In the agent for promoting brain neurogenesis for a subject of the present invention, the precursor of miR-30a may be pri-miRNA or pre-miRNA of miR-30a.

The agent for promoting brain neurogenesis for a subject of the present invention may be administered into the brain of the subject.

The cerebral neurogenesis promoting agent for a subject of the present invention may be administered into the lateral ventricle of the subject.

In the subject cranial neurogenesis promoting agent of the present invention, the vector for expression in the subject's cranial nerves may be a vector for expression in the subject's choroid plexus.

In the subject cranial neurogenesis promoting agent of the present invention, the subject may be an elderly subject.

The present invention provides an agent for improving higher nervous function in a subject. The agent for improving higher nervous function in subjects of the present invention includes:
miR-30a or its precursor,
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, or inserted in the base sequence of miR-30a and having substantially the same function as miR-30a is administered to the cranial nerves of a subject. Contains an effective amount of vector for expression.

In the agent for improving higher nervous function of a subject of the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof.

In the agent for improving higher nervous function of a subject of the present invention, the precursor of miR-30a may be pri-miRNA or pre-miRNA of miR-30a.

The agent for improving higher nervous function of a subject of the present invention may be administered into the brain of the subject.

The cerebral neurogenesis promoting agent for a subject of the present invention may be administered into the lateral ventricle of the subject.

In the agent for improving higher nervous function of a subject of the present invention, the vector for expression in the cranial nerve of the subject may be a vector for expression in the choroid plexus of the subject.

In the agent for improving higher nervous function of a subject of the present invention, the subject may be an elderly subject.

The present invention provides an agent for improving the memory learning function of a subject. The agent for improving the memory learning function of the subject of the present invention is
miR-30a or its precursor,
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, or inserted in the base sequence of miR-30a and having substantially the same function as miR-30a is administered to the cranial nerves of a subject. Contains an effective amount of vector for expression.

In the agent for improving the memory and learning function of a subject of the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof.

In the agent for improving memory learning function of a subject of the present invention, the precursor of miR-30a may be pri-miRNA or pre-miRNA of miR-30a.

The agent for improving memory learning function of a subject of the present invention may be administered into the brain of the subject.

The agent for improving the memory and learning function of a subject of the present invention may be administered into the lateral ventricle of the subject.

In the subject's memory learning function improving agent of the present invention, the vector for expression in the subject's cranial nerves may be a vector for expression in the subject's choroid plexus.

In the agent for improving memory and learning function of a subject of the present invention, the subject may be an elderly subject.

The present invention provides a method for promoting cranial nerve regeneration in a subject. The method of promoting cranial nerve regeneration in a subject of the present invention includes:
miR-30a or its precursor, or
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a, or
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or containing a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, or , and includes the step of administering to the subject an effective amount of a vector for expressing in the cranial nerves of the subject a nucleic acid having substantially the same function as miR-30a.

In the method of promoting cranial nerve regeneration in a subject of the present invention, the vector to be expressed in the cranial nerve of the subject may be a vector to be expressed in the choroid plexus of the subject.

In the method of promoting cranial nerve regeneration in a subject of the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof.

In the method of promoting cranial nerve regeneration in a subject of the present invention, the miR-30a precursor may be miR-30a pri-miRNA or pre-miRNA.

In the method of promoting cranial nerve regeneration in a subject of the present invention, the step of administering to the subject may include administering into the brain of the subject.

In the method of promoting cranial nerve regeneration in a subject of the present invention, the step of administering to the subject may include administering into the lateral ventricle of the subject.

In the method of promoting cranial nerve regeneration in a subject of the present invention, the subject may be an elderly person.

The present invention provides a method for improving higher neurological function in a subject. The method of improving the higher nervous function of a subject according to the present invention includes:
miR-30a or its precursor, or
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a, or
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or containing a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, or , and includes the step of administering to the subject an effective amount of a vector for expressing in the cranial nerves of the subject a nucleic acid having substantially the same function as miR-30a.

In the method of improving higher nervous function of a subject of the present invention, the vector for expression in the cranial nerve of the subject may be a vector for expression in the choroid plexus of the subject.

In the method of improving higher nervous function of a subject according to the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof.

In the method of improving higher nervous function of a subject of the present invention, the precursor of miR-30a may be pri-miRNA or pre-miRNA of miR-30a.

In the method for improving higher nervous function of a subject of the present invention, the step of administering to the subject may include administering into the brain of the subject.

In the method of improving higher nervous function of a subject of the present invention, the step of administering to the subject may include administering into the lateral ventricle of the subject.

In the method of improving higher nervous function of a subject of the present invention, the subject may be an elderly subject.

The present invention provides a method of improving memory learning function in a subject. The method of improving the memory learning function of a subject according to the present invention includes:
miR-30a or its precursor, or
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a, or
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or containing a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, or , and includes the step of administering to the subject an effective amount of a vector for expressing in the cranial nerves of the subject a nucleic acid having substantially the same function as miR-30a.

In the method of improving the memory learning function of a subject of the present invention, miR-30a may be RNA consisting of the base sequence of SEQ ID NO: 1, or a modified version thereof.

In the method of improving the memory learning function of a subject of the present invention, the precursor of miR-30a may be pri-miRNA or pre-miRNA of miR-30a.

In the method of improving the memory learning function of a subject of the present invention, the step of administering to the subject may be administering into the brain of the subject.

In the method of improving the memory learning function of a subject of the present invention, the step of administering to the subject may include administering into the lateral ventricle of the subject.

In the method of improving the memory learning function of a subject of the present invention, the vector to be expressed in the subject's cranial nerves may be a vector to be expressed in the choroid plexus of the subject.

In the method of improving the memory learning function of a subject of the present invention, the subject may be an elderly subject.

According to the present invention, it is possible to promote cranial neurogenesis in a subject by administering miRNA whose expression level in the choroid plexus is significantly reduced. Thereby, higher nervous functions can be improved. Furthermore, memory learning function can be improved by administering the miRNA.

Names of the top 12 miRNAs arranged in descending order of expression count in the choroid plexus of young mice, gene expression count in the choroid plexus of young and old mice, gene expression count in the choroid plexus of old mice logarithm of the ratio divided by the gene expression count in the choroid plexus of young mice, the p value, and the significance of the decrease in gene expression counts in the choroid plexus of old mice compared to the choroid plexus of young mice. A table showing the presence or absence of differences. Similar to Figure 1, the top 30 miRNAs expressed in the mouse choroid plexus are arranged in descending order of expression count, the 20 miRNAs detected in human infant cerebrospinal fluid, and the cerebrospinal fluid of Alzheimer's disease patients. Venn diagram showing the correlation between 17 miRNAs (biomarker candidates) whose detection amount decreases. miR-30a is expressed in the top 30 in the mouse choroid plexus, is among the 20 miRNAs detected in the cerebrospinal fluid of human infants, and is among the 17 miRNAs whose amount is decreased in the cerebrospinal fluid of Alzheimer's disease patients. included. Graph showing the results of an experiment in which the effect of promoting the proliferation of mouse embryonic neural stem cells by forced expression of miRNA into neural stem cells was evaluated. The vertical axis shows the percentage of cells that took up EdU among the cells that expressed each miRNA. Culture dish of neural stem cells derived from mouse fetuses infected with lentivirus expressing miR-204, miR-191, miR-143, miR-30a, and miR-1298, per field of view (0.075 mm ² ). The percentage of cells that have taken up EdU is represented by one ●, ○, ◆, ▼, ▲, and □, plotted for 6 fields, and the average and standard deviation are shown. Results showing the same trend were obtained at least three times. Graph showing the results of an experiment in which the growth promoting effect of mouse fetal neural stem cells was evaluated by exposure to a culture supernatant containing miRNA. The vertical axis shows the percentage of cells that took up EdU among the cells exposed to the culture supernatant containing each miRNA. Each field (0.075 mm ² ) of a culture dish of neural stem cells derived from mouse fetuses exposed to culture supernatant containing miR-204, miR-191, miR-143, miR-30a, and miR-1298. The percentage of cells that have taken up EdU is represented by one ●, ○, ◆, ▼, ▲, and □, plotted for 6 fields, and the average and standard deviation are shown. After injecting AAV virus expressing miR-30a shRNA (shmiR-30a, lower row) or ZsGreen1 (control, upper row) into the lateral ventricle of 8-week-old mice, BrdU was administered intraperitoneally to the hippocampal dentate gyrus. Panel of immunohistochemically stained microscopic images. The left side is a fluorescence microscope image of Hoechst 33342, and the right side is a fluorescence microscope image of BrdU, in which cell nuclei in the dentate gyrus and cell nuclei that have taken up BrdU as they proliferate are stained, respectively. An AAV virus that expresses miR-30a shRNA (shmiR-30a) or an AAV virus that expresses ZsGreen1 (control) was injected into the lateral ventricle of 8-week-old mice to express the proliferation-promoting effect of miR-30a in the adult mouse brain. A graph showing the results of a quantitatively evaluated experiment. The vertical axis indicates the total number of cells that have incorporated BrdU in the entire region of the hippocampal dentate gyrus, which was measured by preparing serial sections. The asterisk (*) indicates a significant difference (T test, p<0.05). After infecting the human neural stem cell line AF22 in culture with a lentivirus that expresses miRNA-30a (miR-30a, lower row) and a control lentivirus that does not express miRNA (pLLX, upper row), EdU was applied. Panel of fluorescence microscopy images of the results of addition to the culture medium. The left side (Hoechest) is a fluorescence microscopy image in which all cell nuclei were stained with Hoechst 33342. The center (EdU) is a fluorescence microscopy image of cells that have taken up EdU as they proliferate. On the right is a fluorescence microscopy image of green fluorescent protein (GFP) contained in the viral vector pLLX. Graph showing the results of an experiment in which the growth-promoting effect of AF22 on human neural stem cell lines was evaluated by forced expression of miRNA. The vertical axis shows the percentage of cells (EdU(+)) that took up EdU as they proliferated among the cells (GFP(+)) that expressed each miRNA. Culture dish of human neural stem cell line AF22 infected with lentivirus that does not express miRNA (pLLX) and lentivirus that expresses miR-10a, miR-30a, and miR-204 miRNA (10a, 30a, and 204, respectively) Percentage of cells (EdU(+)) that took up EdU during proliferation among cells (GFP(+)) that expressed each miRNA per one field of view (0.075 mm ² ) (EdU(+)/GFP(+) )) are represented by one ●, ■, ▲, and ▼, plotted for 8 fields of view, and the average and standard deviation are shown. The asterisk (*) indicates that there is a significant difference (ANOVA, p<0.05) in the proliferation promoting effect of AF22 by miR-30a compared to the negative control pLLX. According to Tukey's multiple comparison test, the AF22 proliferation promoting effect by miR-30a was significantly different from the AF22 proliferation promoting effect by miR-204 (q=4.312). A photograph showing the Barnes maze experimental setup used for quantitative analysis of hippocampus-dependent spatial memory. There are 16 holes in the edge of the 90cm diameter disk, one of which can be used to install an escape box. Schematic diagram showing the schedule of Barnes maze test in aged mice overexpressing miRNA-30a in the choroid plexus. On the first day of training, the animals are acclimatized twice for 5 minutes each time, and trained to enter the escape box. If the mouse does not enter the escape box within 5 minutes, use the guide frame to encourage the mouse to enter the escape box and stay in the escape box for 30 seconds. Starting from the next day, a 5-minute Barnes maze test was performed three times each day for 4 days. A 3 minute probe test was conducted on the 6th day. No escape box was installed in the probe test. Line graph showing the change in success rate of the 5 minute Barnes maze test during the training period. The dashed line shows the change in success rate of old mice overexpressing miRNA-30a (miRNA30a-OE), and the solid line shows the change in success rate of control old mice (Cont). The number of mice in each group was 6. The vertical axis of the graph represents the success rate in 5 minutes (unit: percentage), and the horizontal axis represents the day of training. Error bars represent standard errors. A line graph showing changes in exploration time (time taken until the mouse enters the escape box) in a 5-minute Barnes maze test during the training period. The dashed line shows the change in search time in old mice overexpressing miRNA-30a (miRNA30a-OE), and the solid line shows the change in search time in control old mice (Cont). The number of mice in each group was 6. The vertical axis of the graph represents the search time (in seconds), and the horizontal axis represents the training day. Error bars represent standard errors. Bar graph showing cognitive scores for the probe test the day after the training period. The white bar graph on the left shows the cognitive score of control old mice, and the black bar graph on the right shows the cognitive score of old mice overexpressing miRNA-30a. The number of mice in each group was 6. The vertical axis is defined as the ratio of the time spent in the goal (the hole where the escape box was installed) to the total exploration time, and represents the recognition score. Error bars represent standard errors.

In the present invention, the age until the new generation of cranial nerves is limited to the hippocampal region is referred to as young or young, and all ages after the new generation of cranial nerves is limited to the hippocampal region are referred to as old or old age.

Creation of cranial nerves includes activation of dormant neural stem cells, proliferation of activated neural stem cells, and differentiation of neural stem cells into nerve cells.

Therefore, the subject cranial neurogenesis promoting agent of the present invention activates dormant neural stem cells, proliferates activated neural stem cells, and activates neural stem cells after cranial nerve generation is limited to the hippocampal region. A composition that has the effect of promoting at least one of the following types of differentiation into nerve cells.

In one aspect of the present invention, the active ingredient of the agent for promoting brain neurogenesis in a subject is miRNA.

miRNA is a microRNA that is an endogenous non-coding RNA of about 20 to 25 bases encoded on the genome.More than 2,000 types are known in humans, and each of them has the ability to control multiple target genes. It regulates expression and is involved in various biological phenomena such as cell proliferation and differentiation.

miR-30a is an miRNA reported by Lagos-Quintana M et al. (Science. 294:853-858 (2001)). A portion of the 5' arm sequence (21 bases long) of mature miR-30a is shown in SEQ ID NO: 1. The nucleotide sequence of miR-30a, for example, the sequence of SEQ ID NO: 1, is the same in humans, chimpanzees, cows, dogs, rats, and mice. In vivo, miR-30a has a hairpin structure as a result of intracellular processing of single-stranded RNA (pri-miRNA), which is the primary transcript transcribed from the chromosomal DNA of the gene encoding miR-30a. It becomes single-stranded RNA (pre-miRNA), and the paired double-stranded portions of the hairpin structure are cleaved to finally form miR-30a as mature miRNA. pri-miRNA usually has a length of several hundred to several thousand bases. Pre-miRNA usually has a length of 70 to 110 bases. Furthermore, a single-stranded nucleic acid in which, for example, the base sequence represented by SEQ ID NO: 1 (first sequence) and its complementary sequence (second sequence) are linked via a loop portion, - An artificial nucleic acid in which the first sequence and the second sequence form a double-stranded structure by adopting a loop-type structure is also one of the preferred embodiments of the nucleic acid of the present invention. In this case, the loop portion can be any base sequence of about 3 to 10 bases.

Therefore, the precursor of miRNA including miR-30a is pri-miRNA, that is, the single-stranded RNA of the primary transcript transcribed from the chromosomal DNA of the miRNA-encoding gene, and It refers to RNA that is longer than mature miRNA, including, but not limited to, single-stranded RNA that is being processed and pre-miRNA that is formed as a result of processing, that is, single-stranded RNA that has a hairpin structure.

Below,
miR-30a or its precursor, or
The base sequence of miR-30a, for example, contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the sequence of SEQ ID NO: 1, and is substantially the same as miR-30a. A functional nucleic acid is referred to as a "nucleic acid of the present invention".

In one aspect of the present invention, the active ingredient of the agent for promoting brain neurogenesis in a subject is a nucleic acid of the present invention other than miRNA.

The nucleic acid of the present invention is RNA, a chimeric nucleic acid of RNA and DNA (hereinafter referred to as chimeric nucleic acid), or a hybrid nucleic acid. Here, chimeric nucleic acid refers to a single-stranded or double-stranded nucleic acid that contains RNA and DNA in one nucleic acid, and hybrid nucleic acid refers to a double-stranded nucleic acid in which one strand is RNA or DNA. A chimeric nucleic acid refers to a nucleic acid in which the other strand is DNA or a chimeric nucleic acid.

The nucleic acid having substantially the same function as miR-30a of the present invention is single-stranded or double-stranded. Double-stranded embodiments include double-stranded RNA, double-stranded chimeric nucleic acids, RNA/DNA hybrids, RNA/chimeric nucleic acid hybrids, chimeric nucleic acids/chimeric nucleic acid hybrids, and chimeric nucleic acids/DNA hybrids. The nucleic acid of the present invention is preferably a single-stranded RNA, a single-stranded chimeric nucleic acid, a double-stranded RNA, a double-stranded chimeric nucleic acid, an RNA/DNA hybrid, an RNA/chimeric nucleic acid hybrid, a chimeric nucleic acid/chimeric nucleic acid hybrid, or a chimeric nucleic acid. /DNA hybrid, more preferably single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA/DNA hybrid, chimeric nucleic acid/chimeric nucleic acid hybrid, or RNA/chimeric nucleic acid hybrid. .

There is no upper limit to the length of the nucleic acid of the present invention as long as it has the activity of promoting the proliferation of mammalian (preferably human) brain nerve cells, preferably neural stem cells. However, in consideration of ease of synthesis, antigenicity, etc., the length of the nucleic acid of the present invention is, for example, about 200 bases or less, preferably about 130 bases or less, more preferably about 50 bases or less, and most preferably It is 30 bases or less. The lower limit is, for example, 15 bases or more, preferably 17 bases or more. In addition, when a nucleic acid forms a hybrid by forming a stem-loop secondary structure, the length of the nucleic acid is considered to be the length of one strand of the hybrid.

The nucleic acid of the present invention can be obtained by isolation from mammalian cells (human cells, etc.) using conventionally known techniques, by chemical synthesis, or by production using genetic recombination technology. be able to. It is also possible to use appropriately commercially available nucleic acids.

The nucleic acid of the present invention can also be provided in a form that is biosynthesized by being introduced into cells and transcribed by inserting it downstream of the promoter of an appropriate expression vector. As the expression vector, one containing a promoter that is functional in the host cell is used. Examples of promoters include RNA polymerase II (pol II) promoters and RNA polymerase III (pol III) promoters. Examples of the pol II promoter include the cytomegalovirus (human CMV) IE (immediate early) gene promoter and the SV40 early promoter. Examples of expression vectors using these include pCDNA6.2-GW/miR (manufactured by Invitrogen) and pSilencer 4.1-CMV (manufactured by Ambion). Examples of pol III promoters include U6 RNA, H1 RNA, and tRNA promoters. Examples of expression vectors using these include pSINsi-hH1 DNA (manufactured by Takara Bio), pSINsi-hU6 DNA (manufactured by Takara Bio), and pENTR/U6 (manufactured by Invitrogen).

Alternatively, a recombinant viral vector is created by inserting the nucleic acid of the present invention downstream of the promoter in a viral vector, the vector is introduced into packaging cells to produce a recombinant virus, and the virus is infected into a host cell. By doing so, the nucleic acid of the present invention can be introduced and expressed in the host cell.
The packaging cell may be any cell that can supply the defective protein of a recombinant viral vector that is defective in any of the genes encoding proteins necessary for viral packaging, such as human cells. Kidney-derived HEK293 cells, mouse fibroblasts NIH3T3, and the like can be used. In the case of retrovirus vectors, proteins such as gag, pol, and env derived from mouse retroviruses are used as proteins to be supplied in packaging cells, and in the case of lentivirus vectors, proteins such as gag, pol, env, vpr, vpu, and so on derived from HIV virus are used. Proteins such as vif, tat, rev, nef, etc. In the case of adenovirus vectors, proteins such as E1A and E1B derived from adenovirus, and in the case of adeno-associated virus vectors, Rep (p5, p19, p40), Vp (Cap) Proteins such as can be used.

As used herein, the vector for expression in the cranial nerves of a subject can be expressed by linking miR-30a or a nucleic acid having a function equivalent to miR-30a downstream of a nerve-specific promoter. Here, the neuron-specific promoters are synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, platelet-derived growth factor beta chain, 67 kDa glutamate decarboxylase (GAD67), homeobox Dlx5/ 6, glutamate receptor 1 (GluR1), preprotachykinin 1 (Tac1), neuron-specific enolase (NSE), and dopaminergic receptor 1 (Drd1a) gene promoters. A vector for expressing miR-30a or a nucleic acid having a function equivalent to miR-30a, for example, Christensen, IB et al., Am J Physiol Cell Physiol 314: C519-C533, 2. Explained to 018 Expression can be achieved by linking downstream of the promoter of a gene encoding P-cadherin or other protein expressed in the choroid plexus.

As explained in the Examples herein, for example, lentiviral vectors for overexpressing miRNA can be prepared using specific restriction enzymes of pLLX (Lois, C., et al. (2002). Science 295, 868-872.). It can be created by inserting a miRNA precursor sequence into the cleavage site. Lentiviruses can be used, for example, as described by Tsujimura K. (Cell Rep. 2015; 12: 1887-1901) using HEK293 cells. Furthermore, an AAV virus (adeno-associated virus) vector that expresses miR-30a shRNA (short hairpin RNA) can be created using AAVpro (registered trademark) Helper Free System (Takara Bio Inc., Shiga). can.

A nucleic acid having substantially the same function as miR-30a consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a. Here, one to several refers to 1 to 6 (preferably 1 to 4, more preferably 1 or 2, even more preferably 1) in the base sequence represented by SEQ ID NO: 1.

In miRNA, the 2nd to 8th base sequence (seed region) on the 5' end mainly contributes to hybridization with target mRNA and translational suppression (Current Biology, 15, R458-R460 (2005)), so SEQ ID NO: 1 It is desirable that the 2nd to 8th nucleotide sequences in the nucleotide sequence represented by are conserved.

In the first embodiment, the base sequence in which one to several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a may be an isoform of mature miR-30a (isomiR). IsomiR of miR-30a includes a nucleic acid consisting of a base sequence entered in IsomiR Bank (Bioinformatics, 32(13): 2069-2071 (2016)) provided by the University of Science and Technology of China.

In a preferred embodiment, the nucleic acid of the present invention is incorporated into a RISC complex and acts on a target mRNA. In order for the nucleic acid of the present invention to be efficiently incorporated into the RISC complex and processed into mature miR-30a, the nucleic acid must be a double-stranded nucleic acid (one strand that forms a duplex by adopting a stem-loop structure). stranded nucleic acids).

When the nucleic acid having substantially the same function as miR-30a of the present invention is a double-stranded nucleic acid (including a single-stranded nucleic acid that forms a duplex by taking a stem-loop structure), the complementary strand (passenger The sequence may be completely complementary to the base sequence represented by SEQ ID NO: 1 or a base sequence to which one or several bases have been substituted, deleted, inserted, or added, May include mismatch. The number of mismatches is, for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6. However, for efficient target mRNA function suppression, it is believed that not only the stability of hybridization with the target mRNA but also the pairing stability of the 5'-end side of the double-stranded miRNA contributes. Therefore, it is desirable that there be no mismatch in this region.

Since natural nucleic acids are easily degraded by nucleolytic enzymes present in cells, the nucleic acids of the present invention having substantially the same function as miR-30a can be resistant to various degrading enzymes. It may be modified as a modified form. The modified product of the present invention has one or several bases substituted, deleted, inserted, or added to the base sequence represented by SEQ ID NO: 1, provided that it has the same function as miR-30a. It consists of a base sequence and includes modified versions. Examples of modifications in modified products include those in which the sugar chain moiety is modified (for example, 2'-O methylation), those in which the base moiety is modified, and those in which the phosphate moiety or hydroxyl moiety is modified. Examples include, but are not limited to, biotin, amino groups, lower alkylamine groups, acetyl groups, etc.

The nucleic acid having substantially the same function as miR-30a of the present invention may have an additional base at the 5' or 3' end. The length of the additional bases is usually 5 bases or less. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Sequences of such additional bases include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Examples include, but are not limited to, sequences such as ', uuuuu-3'.

When miR-30a is taken into nerve cells including neural stem cells in the brain, especially neural stem cells in a dormant state, it has the activity of promoting the proliferation of the cells. In the present invention, "having a function equivalent to miR-30a" means having an activity that promotes the proliferation of neural cells including neural stem cells in the brain, particularly neural stem cells in a dormant state. As long as it has the activity, the degree does not matter. miRNA forms a hybrid with target mRNA and suppresses its translation into protein or cleaves the target mRNA, thereby suppressing its function. Therefore, a nucleic acid having the same function as miR-30a should hybridize with miR-30a target mRNA under physiological conditions (for example, 0.1M phosphate buffer (pH 7.0) at 25°C). Examples include nucleic acids. The target mRNA of miR-30a can be searched using various algorithms such as TargetScan, TargetMiner, and miRDB.

The agent for promoting cranial neurogenesis for a subject, the agent for improving higher nervous function for a subject, and the agent for improving memory learning function for a subject (hereinafter referred to as "the agent of the present invention") of the present invention contain an effective amount of miR-30a or the present invention. In addition to a nucleic acid having a function equivalent to miR-30a, it can contain any carrier, such as a pharmaceutically acceptable carrier, and is applied as a medicine in the form of a pharmaceutical composition.

Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.

In order to promote the introduction of miR-30a contained in the agent of the present invention or a nucleic acid having a function equivalent to miR-30a into nerve cells, the agent of the present invention may further contain a reagent for nucleic acid introduction. Examples of the nucleic acid introduction reagent include atelocollagen; liposome; nanoparticles including lipid nanoparticles; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, Polybrene, (registered trademark, CAS registration number: 28728-55-4), or a cationic lipid such as poly(ethyleneimine) (PEI), etc. can be used.

The nucleic acid introduction reagent is, for example, an antibody against Crumbs 3 (Crb3), syntaxin-3 (Stx3), syntaxin-4 (Stx4), etc., which are membrane proteins present on the surface of the choroid plexus facing the ventricle, or an antibody thereof. By linking antigen-binding fragments, nucleic acids can be delivered specifically to the choroid plexus. For membrane proteins exposed on the ventricular-facing surface of the choroid plexus, see, for example, Christensen, IB et al., Am J Physiol Cell Physiol 314: C519-C533, 2018.

Although the agent of the present invention can be administered to mammals orally or parenterally, it is preferable to administer the agent of the present invention parenterally.

Suitable formulations for parenteral administration (e.g., subcutaneous, intramuscular, local, intraperitoneal, etc.) include aqueous and non-aqueous isotonic sterile injectable solutions containing antioxidants. , a buffer, a bacteriostatic agent, a tonicity agent, etc. may be included. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like. The formulation can be packaged in units or multiple doses in containers such as ampoules and vials. Alternatively, the active ingredient and the pharmaceutically acceptable carrier can be lyophilized and stored by dissolving or suspending them in a suitable sterile vehicle immediately before use.

The content of the nucleic acid of the present invention in the agent of the present invention is, for example, about 0.01 to 100% by weight of the entire agent of the present invention. Since the nucleic acid of the present invention is the active ingredient of the present invention, the proportion of the active ingredient contained in the agent of the present invention can be appropriately set within a range that can achieve the desired effect, but is usually 0. 01 to 100% by weight, preferably 0.1 to 99.9% by weight, more preferably 0.5 to 99.5% by weight.

The dosage of the agent of the present invention varies depending on the purpose of administration, the method of administration, and the condition of the recipient (sex, age, body weight, etc.), but when locally administered to adults by injection, the amount of the nucleic acid of the present invention is usually For systemic administration, it is preferably 2 nmol/kg or more and 50 nmol/kg or less. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times.

The agent of the present invention can be safely administered to a subject so that its active ingredient, miR-30a of the present invention or a nucleic acid having a function equivalent to miR-30a, is delivered into the brain, for example, to the choroid plexus. be done.

The method of promoting the regeneration of cranial nerves in a subject of the present invention includes the step of administering to the subject an effective amount of the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject. Here, regarding the nucleic acid of the present invention, the vector for expressing the nucleic acid of the present invention in the cranial nerves of a subject, the effective amount, etc., all the contents of the agent of the present invention may be referred to. In the method of promoting cranial nerve regeneration in a subject of the present invention, the step of administering to the subject an effective amount of the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject. It refers to administration into the ventricle of the brain, preferably into the ventricle of the brain near the choroid plexus. Preferably, the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject is administered together with a reagent for introducing the nucleic acid. More preferably, it is administered together with a nucleic acid introduction reagent bound to an antibody or antigen-binding fragment thereof against a protein localized on the surface of the choroid plexus facing the ventricle.

The effectiveness of the agent for promoting cranial neurogenesis in a subject of the present invention and the method of promoting cranial neurogenesis in a subject of the present invention is determined by the effectiveness of the agent for promoting cranial neurogenesis in a subject of the present invention. This can be verified by analyzing the presence and extent of proliferation of brain nerve stem cells. For example, using the incorporation of isotope-labeled monodeoxyribonucleotides and image diagnostic techniques such as MRI, the brain nerve cells, more specifically the brain nerve cells of the hippocampus, and more specifically the brain nerve system associated with the proliferation of hippocampal brain nerve stem cells, It can be analyzed by morphological changes.

The method of improving the higher nervous function of a subject of the present invention includes administering to the subject an effective amount of the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject, such as the choroid plexus. Contains steps. Here, regarding the nucleic acid of the present invention, the vector for expressing the nucleic acid of the present invention in the cranial nerves of a subject, such as the choroid plexus, the effective amount, etc., all the contents of the agent of the present invention may be referred to. In the method of improving the higher nervous function of a subject of the present invention, the step of administering to the subject the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject, such as the choroid plexus. It refers to administering an effective amount into the ventricles of a subject, preferably into the ventricles of the brain near the choroid plexus. Preferably, the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the subject's cranial nerves, such as the choroid plexus, is administered together with a reagent for introducing the nucleic acid. More preferably, it is administered together with a nucleic acid introduction reagent bound to an antibody or antigen-binding fragment thereof against a protein localized on the surface of the choroid plexus facing the ventricle.

The method of improving the memory learning function of a subject of the present invention includes the step of administering to a subject an effective amount of the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject, such as the choroid plexus. including. Here, regarding the nucleic acid of the present invention, the vector for expressing the nucleic acid of the present invention in the cranial nerves of a subject, such as the choroid plexus, the effective amount, etc., all the contents of the agent of the present invention may be referred to. In the method of improving the memory learning function of a subject according to the present invention, the step of administering to the subject means administering the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject, such as the choroid plexus. Refers to administering an amount into the ventricles of a subject, preferably into the ventricles of the brain near the choroid plexus. Preferably, the nucleic acid of the present invention or a vector for expressing the nucleic acid of the present invention in the cranial nerves of the subject is administered together with a reagent for introducing the nucleic acid. More preferably, it is administered together with a nucleic acid introduction reagent bound to an antibody or antigen-binding fragment thereof against a protein localized on the surface of the choroid plexus facing the ventricle.

The method of promoting the regeneration of cranial nerves in a subject of the present invention, the method of improving the higher nervous function of a subject, and the method of improving the memory learning function of a subject of the present invention are hereinafter referred to as "the method of the present invention".

As used herein, a subject refers to an individual mammal to whom the agent of the present invention and/or the method of the present invention is applied. The subject may be a human, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, dog, cat, monkey, human, preferably human.

In the present specification, an elderly subject refers to a human subject who is 65 years of age or older, 70 years of age or older, or 75 years of age or older (Ouchi, Y., et al., Geriatr Gerontol Int 17.7 (2017 ): 1045-1047.). Moreover, in this specification, a young subject refers to a subject who is less than 65 years old after sexual maturity. A non-human elderly subject refers to a subject who corresponds to a human age of 65 years or older, 70 years or older, or 75 years or older when converted to human age using any conversion method. A young non-human subject refers to a subject who is equivalent to a human being of less than 65 years of age after sexual maturity when converted to human age using any of the conversion methods described above. Here, the age conversion between humans and non-human mammals is described, for example, by Wang, T. Any conversion method known to those skilled in the art can be applied, including, but not limited to, Cell Syst.

The agent for improving higher nervous function in a test subject of the present invention, the method for improving higher nervous function in a test subject of the present invention, the agent for improving memory learning function in a test subject of the present invention, and the improving memory learning function in a test subject of the present invention The effectiveness of the method can be quantitatively verified by the Barnes maze test, the MMSE test, and other tests related to memory learning functions described in the Examples of the present application. The test for the memory learning function is described, for example, by Larner, A.; J. ed. , Cognitive Screening Instruments A Practical Approach, Second Edition, 2016, Springer International Publishing AG, Cham, Switzerland) and are well known to those skilled in the art.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

In this specification, the adjunctive word "about" modifying a numerical value means a numerical range of 90% or more and within 110% of the numerical value. For example, "about 40 bases" refers to a numerical range of 36 bases to 44 bases.

All documents mentioned herein are incorporated by reference in their entirety.

The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Changes in the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.

A. Materials and Methods (1) Comprehensive Identification of miRNAs Expressed in the Choroid Plexus The lateral ventricular choroid plexus was isolated from three young and old mice (4 and 25 months old, respectively) and treated with Sepasol (registered trademark). Total RNA was collected using RNA I Super G (Nacalai Tesque Co., Ltd., Kyoto). A library was prepared according to the recommended protocol of TruSeq Small RNA Library Prep Kit (Illumina Corporation, Tokyo), and sequenced using Hiseq3000 (Illumina Corporation, Tokyo). The sequence results were mapped to the mouse genome (mm10), and differential expression analysis was performed using the free software Cuffdiff (Trapnell C, et al. Nat Biotechnol. 2010 May; 28 (5): 511-5., https://github.com /cole-trapnell-lab/cufflinks) was used to perform a significant difference test in young and old mice.

(2) Promoting proliferation of mouse embryonic neural stem cells by miRNA To evaluate the proliferation promoting effect of forced expression on neural stem cells, we created a lentiviral vector that overexpresses miRNA, and used it to stimulate neural stem cells derived from 14-day-old mouse embryos. and cultured in the presence of 10 μM FGF. As miRNAs, miR-204 is ranked first, miR-191 is ranked second, and miR-143 is ranked third, in order of the highest expression level counts in both young and old mice. miR-1298, which ranked first, and miR-30a, which ranked second, were selected in order of the smallest logarithm of the ratio divided by the gene expression level count of old mice. The lentiviral vector for overexpressing miR-204, miR-191, miR-143, miR-30a, and miR-1298 is pLLX (Lois, C., et al. (2002). Science 295, 868-872.) was created by inserting the precursor sequences of miR-204-5p, miR-191-5p, miR-143-3p, miR-30a-5p, and miR-1298-5p into the HpaI and XhoI sites of . The lentivirus was developed by Tsujimura K. (Cell Rep. 2015; 12: 1887-1901) using HEK293 cells. Neural stem cells derived from a 14-day-old mouse fetus were obtained from Nakashima H. (J. Neurosci. 2018; 38: 4791-4810). The DNA analog EdU (5-ethynyl-2'-deoxyuridine) was added to the medium of neural stem cells 2 days after viral vector infection at a final concentration of 10 μM, cultured for 1 hour, fixed, and placed in one field of view (0.075 ^mm2) using a microscope. ) The percentage of cells that had taken up EdU was measured.

To evaluate the growth promoting effect of neural stem cells exposed to culture supernatant containing miRNA, the same lentiviral expression vectors of miR-204, miR-191, miR-143, miR-30a and miR-1298 as above were used. On the day after transfecting HEK293 cells with polyethyleneimine (PEI; Polyscineces, 23966-1) and inducing production of each lentivirus, the HEK293 cell medium was replaced with neural stem cell medium, and each lentivirus was transfected with medium for neural stem cells. Culture of HEK293 cells was continued for an additional 2 days under conditions that induce virus production. Neural stem cell medium containing each lentivirus was collected, 10 μM FGF was added, and the medium was used for culturing neural stem cells. After culturing for one day in a neural stem cell medium containing each lentivirus, the DNA analog EdU was added to the medium at a final concentration of 10 μM, and after culturing for 1 hour, it was fixed, and a microscopic field of the EdU-incorporated cells (0. The percentage per 0.075 mm ² ) was measured.

(3) Functional evaluation of miR-30a in adult mouse brain An AAV virus (adeno-associated virus) vector that expresses miR-30a shRNA (short hairpin RNA) or a control AAV vector that expresses ZsGreen1 was AAVpro (registered (trademark) Helpaer Free System (Takara Bio Inc., Shiga). A vector plasmid expressing miR-30a shRNA or ZsGreen1 was transfected into HEK293 cells together with a packaging plasmid containing AAV Rep gene and serotype 5 Cap gene, and a packaging plasmid containing adenovirus-derived E2A, E4, and VA. AAV virus expressing miR-30a shRNA (shmiR-30a) or ZsGreen1 expressing AAV virus (control) was collected using the cells after 3 days of infection according to the protocol recommended by the kit.

4 μL (3.01 x 10 ⁹ vg/ml) of AAV virus that expresses miR-30a shRNA or control AAV virus that expresses ZsGreen1 was injected into the lateral ventricle of 8-week-old mice, and injected for 7 days starting 21 days later. The DNA analog BrdU (50 mg/kg) was administered intraperitoneally, and 28 days later, BrdU uptake in the hippocampal dentate gyrus was evaluated by immunohistochemical method.

(4) Evaluation of proliferation promoting function of miRNA-30a in human neural stem cells A lentivirus overexpressing miR-10a-5p was created using pLLX in the same manner as in (2) above. The lentivirus that overexpresses miR-10a-5p, the lentivirus that overexpresses miR-204-5p and miR-30a-5p created in (2) above, and the viral vector pLLX that does not express miRNA. , respectively, were infected with the human neural stem cell line AF22. EdU at a final concentration of 10 μM was added to the human neural stem cell culture medium 2 days after virus infection, fixed after 1 hour, and the effect on the proliferation ability of each miRNA was evaluated using the percentage of cells that had taken up EdU as an index.

(5) Evaluation of memory learning improvement effect by forced expression of miRNA-30a in the choroid plexus of aged mice (trademark) Helpaer Free System (Takara Bio Inc., Shiga). A vector plasmid expressing miR-30a-5p or ZsGreen1 was transfected into HEK293 cells together with a packaging plasmid containing AAV Rep gene and serotype 5 Cap gene, and a packaging plasmid containing adenovirus-derived E2A, E4, and VA. AAV virus that expresses miR-30a-5p (miR-30aOE) or AAV virus that expresses ZsGreen1 (control) was collected using the cells after 3 days of infection according to the protocol recommended by the kit.

4 μL (3.01 x 10 ⁹ vg/ml) of AAV virus expressing miR-30a-5p or control AAV virus expressing ZsGreen1 was injected into the lateral ventricle of 65- or 71-week old mice, and 14 days later. Hippocampal-dependent spatial memory ability was evaluated using the Barnes maze.

B. Results (1) Comprehensive identification of miRNAs expressed in the choroid plexus Figure 1 shows the names of miRNAs arranged in descending order of expression counts in young mice, gene expression counts in young and old mice, and gene expression counts in old mice. Table showing the logarithm of the ratio of the gene expression count divided by the gene expression count of young mice, the p value, and the presence or absence of a significant difference in the decrease in gene expression count in old mice compared to young mice. It is.

Among the 154 miRNAs whose gene expression count RPM (normalized count converted to the number of reads when the total number of reads mapped to the genome is set to 1 million) in the choroid plexus exceeds 10,000, The gene expression counts in the mice were higher than those in young mice, that is, only eight miRNAs were up-regulated in old mice, and all others were down-regulated in old mice.

miR-204, miR-191, and miR-143 were ranked first, second, and third in terms of expression levels in young mice, respectively. miR-1298, miR-30a and miR-99b have a significant degree of downward regulation in old mice, expressed as the logarithm of the ratio of gene expression counts in old mice divided by gene expression counts in young mice. They were ranked first, second and third, respectively. miR-1298, miR-30a, and miR-99b were ranked first, second, and third, respectively, in descending order of the p-value of the significant difference in the decrease in gene expression counts in old mice compared to young mice. It was 3rd place.

Next, the profiles of the top 30 miRNAs (hereinafter referred to as "group 1") when the miRNAs expressed in the mouse choroid plexus are arranged in descending order of expression level count in the same manner as in Figure 1 were determined according to Non-Patent Document 1. The 20 miRNAs reported in human infant cerebrospinal fluid (hereinafter referred to as "Group 2") and the miRNAs reported by Lusardi, TA et al. (Alzheimers Dis. 2017; 55(3): 1223-1233) A comparison was made with 17 miRNAs (biomarker candidates) whose expression decreases in Alzheimer's disease patients (hereinafter referred to as "Group 3"). The results are shown in the Venn diagram of FIG. From FIG. 2, 14 of the 30 items in group 1 were included in the 20 items in group 2. Four of the group 1 items were included in the 17 items of group 3. Three of Group 1 were also included in both Group 2 and Group 3 at the same time. miR-30a was included in these three miRNAs.

(2) Promotion of proliferation of mouse embryonic neural stem cells by miRNA FIG. 3-1 is a graph showing the results of an experiment in which the effect of forced expression of miRNA in neural stem cells to promote proliferation of mouse embryonic neural stem cells was evaluated. The vertical axis shows the percentage of cells that took up EdU among the cells that expressed each miRNA. Culture dish of neural stem cells derived from mouse fetuses infected with lentivirus expressing miR-204, miR-191, miR-143, miR-30a, and miR-1298, per field of view (0.075 mm ² ). The percentage of cells that have taken up EdU is represented by one ●, ○, ◆, ▼, ▲, and □, plotted for 6 fields, and the average and standard deviation are shown. As shown in Figure 3-1, the lentivirus expressing miR-204 most strongly promoted the proliferation of mouse embryonic neural stem cells, followed by the lentivirus expressing miR-30a. was strongly promoted. Although the lentivirus expressing miR-191 and miR-143 had the second highest gene expression count in the choroid plexus after miR-204, it was more likely to be derived from mouse embryos than the control lentivirus expressing ZsGreen1. The effect of promoting proliferation of neural stem cells was weak. Although miR-1298 was down-regulated to a greater extent than miR-30a in aged mice, it had a weaker growth-promoting effect on mouse fetal neural stem cells than the control lentivirus expressing ZsGreen1.

FIG. 3-2 is a graph showing the results of an experiment in which the proliferation promoting effect of mouse fetal neural stem cells was evaluated by exposure to a culture supernatant containing miRNA. The vertical axis shows the percentage of cells that took up EdU among the cells exposed to the culture supernatant containing each miRNA. Each field (0.075 mm ² ) of a culture dish of neural stem cells derived from mouse fetuses exposed to culture supernatant containing miR-204, miR-191, miR-143, miR-30a, and miR-1298. The percentage of cells that have taken up EdU is represented by one ●, ○, ◆, ▼, ▲, and □, plotted for 6 fields, and the average and standard deviation are shown. As shown in Figure 3-2, the culture supernatant containing miR-204 most strongly promoted the proliferation of mouse fetal neural stem cells, followed by the culture supernatant containing miR-30a. was strongly promoted. Although the culture supernatant containing miR-191 and miR-143 had the second highest gene expression count in the choroid plexus after miR-204, the culture supernatant containing miR-191 and miR-143 was more sensitive to mouse embryo-derived nerves than the culture supernatant containing control RNA (control). The effect of promoting stem cell proliferation was weak.

(3) Functional evaluation of miR-30a in the adult mouse brain Figure 4-1 shows the AAV virus (shmiR-30a, lower row) expressing miR-30a shRNA in the lateral ventricle of 8-week-old mice or the control ZsGreen1 ( This is a panel of immunohistochemically stained microscopic images of the hippocampal dentate gyrus in which BrdU was intraperitoneally administered after injection of an AAV virus expressing BrdU (control, upper row). The left side is a fluorescence microscope image of Hoechst 33342, and the right side is a fluorescence microscope image of BrdU, in which cell nuclei in the dentate gyrus and cell nuclei that have taken up BrdU as they proliferate are stained, respectively.

As shown in Figure 4-1, the AAV virus that expresses miR-30a shRNA (shmiR-30a) is more effective than the control AAV virus that expresses ZsGreen1 (control) in the hippocampal dentate gyrus stained with Hoechst 33342. The number of cells was small, and the number of cells that had taken up BrdU was also small. The results shown in Figure 4-1 demonstrate that expression of miR-30a shRNA reduces the proliferation of neural stem cells in the adult mouse brain.

Figure 4-2 shows miR-30a in adult mouse brains by injecting AAV virus expressing miR-30a shRNA (shmiR-30a) or AAV virus expressing ZsGreen1 (control) into the lateral ventricle of 8-week-old mice. 2 is a graph showing the results of an experiment that quantitatively evaluated the growth promoting effect of . The vertical axis indicates the total number of cells that have incorporated BrdU in the entire region of the hippocampal dentate gyrus, which was measured by preparing serial sections. As shown in Figure 4-2, the AAV virus expressing miR-30a shRNA (shmiR-30a) significantly suppressed the proliferation of neural stem cells in the adult mouse brain. The asterisk (*) indicates a significant difference (T test, p<0.05). From the results shown in FIG. 4-2, it was quantitatively and statistically confirmed that expressing miRNA-30a shRNA reduces the proliferation of neural stem cells in the adult mouse brain.

(4) Evaluation of the growth-promoting function of miRNA-30a in human neural stem cells Figure 5-1 shows a lentivirus that expresses miR-30a (miR-30a, lower row) and a control lentivirus that does not express miRNA (pLLX, upper row). ) is a panel of fluorescence microscopy images of the results obtained by infecting the human neural stem cell line AF22 in culture and then adding EdU to the medium. The left side (Hoechest) is a fluorescence microscopy image in which all cell nuclei were stained with Hoechst 33342. The center (EdU) is a fluorescence microscopy image of cells that have taken up EdU as they proliferate. On the right is a fluorescence microscopy image of green fluorescent protein (GFP) contained in the viral vector pLLX. In both the upper and lower rows, almost the same cells are emitting fluorescence in the Hoechst staining image on the left and the GFP fluorescence image, indicating that the lentivirus infects almost all cells, regardless of whether they contain miR-30a. It can be seen that the GFP protein incorporated into the virus is expressed. However, more cells have taken up EdU in AF22 infected with miR-30a-containing lentivirus (lower panel) than in miR-30a-free lentivirus (upper panel). This means that the lentivirus containing miR-30a multiplied AF22 more than the lentivirus containing no miR-30a.

FIG. 5-2 is a graph showing the results of an experiment in which the growth-promoting effect of AF22 on human neural stem cell lines was evaluated by forced expression of miRNA. The vertical axis shows the percentage of cells (EdU(+)) that took up EdU as they proliferated among the cells (GFP(+)) that expressed each miRNA. AF22 of a human neural stem cell line infected with a control lentivirus that does not express miRNA (pLLX) and a lentivirus that expresses miR-10a, miR-30a, and miR-204 miRNAs (10a, 30a, and 204, respectively). The percentage (EdU(+)/GFP(+)) of cells (EdU(+)) that took up EdU as they proliferated among the cells (GFP(+)) that expressed each miRNA in each field of the culture dish. It is represented by one ●, ■, ▲, and ▼, plotted for 8 visual fields, and its average and standard deviation are shown. As shown in Figure 5-2, lentivirus expressing miR-30a most strongly promoted the proliferation of human neural stem cell line AF22, followed by lentivirus expressing miR-204. promoted the proliferation of AF22. A lentivirus that does not express miRNA (pLLX) is a negative control, and a lentivirus that expresses miR-10a (10a) promoted the proliferation of AF22 to the same extent as pLLX. had almost no growth-promoting effect. The asterisk (*) indicates that there is a significant difference (ANOVA, p<0.05) in the proliferation promoting effect of AF22 by miR-30a compared to the negative control pLLX. According to Tukey's multiple comparison test, the AF22 proliferation promoting effect by miR-30a was significantly different from the AF22 proliferation promoting effect by miR-204 (q=4.312).

Figure 6 is a photograph showing the Barnes maze experimental setup used for quantitative analysis of hippocampus-dependent spatial memory. There are 16 holes in the edge of the 90cm diameter disk, one of which can be used to install an escape box. Each side of the test room is decorated with different decorations, and the mice use these decorations as cues to learn direction. During the test, a mouse was placed in the space inside a cylinder installed in the center, but by pressing a switch (after 10 seconds), the cylinder lowered and the mouse could be moved. The test was conducted in a room with a brightness of 600 lux or more, and was a spatial memory test that took advantage of the habit of mice, which are sensitive to light, exploring escape boxes and escaping into dark places. In the examples of the present invention, the above-mentioned Barnes maze experimental apparatus was used.

FIG. 7-1 is a schematic diagram showing the schedule of Barnes maze test in aged mice overexpressing miRNA-30a in the choroid plexus. On the first day of training, the animals were habituated twice for 5 minutes each time, and trained to enter the escape box. Mice that did not enter the escape box within 5 minutes were encouraged to enter the escape box using the guidance frame, and were allowed to stay in the escape box for 30 seconds. Starting from the next day, a 5-minute Barnes maze test was performed three times each day for 4 days. A 3 minute probe test was conducted on the 6th day. No escape box is installed in the probe test.

FIG. 7-2 is a line graph showing changes in the success rate of the 5-minute Barnes maze test during the training period. The dashed line shows the change in success rate of old mice overexpressing miRNA-30a (miRNA30a-OE), and the solid line shows the change in success rate of control old mice (Cont). The number of mice in each group was 6. The vertical axis of the graph represents the success rate in 5 minutes (unit: percentage), and the horizontal axis represents the day of training.

As shown in Figure 7-2, the average success rate for miRNA30a-OE was more than 20% higher than Cont for all days of the training period. Regarding changes in the average success rate during the training period, the average success rate for miRNA30a-OE was already high from the first day of the training period, at approximately 80%, and by the fourth and final day of the training period, it was around 90%. there were. On the other hand, the average success rate of Cont was about 40% on the first day of the training period, and showed a tendency to improve on the second day, but almost no change was observed on the third and fourth days, and on the final day The average value was also less than 70%.

FIG. 7-3 is a line graph showing changes in the exploration time (time until the mouse enters the escape box) in the 5-minute Barnes maze test during the training period. The dashed line shows the change in search time in old mice overexpressing miRNA-30a (miRNA30a-OE), and the solid line shows the change in search time in control old mice (Cont). The number of mice in each group was 6. The vertical axis of the graph represents the search time (in seconds), and the horizontal axis represents the training day.

As shown in Figure 7-3, for all days of the training period, the average value of search time was lower for miRNA30a-OE than for Cont by more than 40 seconds. Regarding changes during the training period, for both miRNA30a-OE and Cont, the average value of search time clearly decreased on the second day compared to the first day of the training period. The average search time for miRNA30a-OE did not change much from day 2 to day 4. The average value of search time for Cont decreased on the third day compared to the second day, but there was almost no change between the third and fourth days.

From Figures 7-2 and 7-3, in Cont, during the training period, the success rate and search time improved more from the second day onwards than on the first day, but the improvement was not as great as in miRNA30a-OE. Since the success rate of miRNA30a-OE was high from the first day, little change was observed throughout the training period, but the search time improved from the second day onwards compared to the first day.

Figure 7-4 is a bar graph showing the cognitive scores for the probe test conducted the day after the training period. The white bar graph on the left shows the cognitive score of control old mice, and the black bar graph on the right shows the cognitive score of old mice overexpressing miRNA-30a. The number of mice in each group was six. The vertical axis is defined as the ratio of the time spent in the goal (the hole where the escape box was installed) to the total exploration time, and represents the recognition score.

In the probe test, an escape box is not installed, so mice cannot escape. The more accurately a mouse has achieved spatial memory learning, the longer it spends in the hole in which the escape box was placed in the Barnes maze test during the training period than in other holes. Between miRNA30a-OE and Cont, miRNA30a-OE tended to have higher cognitive scores in the probe test.

According to the present invention, it is possible to promote the proliferation of neural stem cells, particularly human neural stem cells, and thereby promote the regeneration of cranial nerves. It also promotes the proliferation of resting neural stem cells in the adult brain, thereby making it possible to promote the regeneration of cranial nerves. Therefore, it is possible to contribute to the development of an agent and method for improving the clinically important decline in higher nervous function associated with a decrease in brain neurons in young and elderly people. Furthermore, the results of the Barnes maze show that the present invention can improve the memory learning performance of young and elderly people. Therefore, it can also contribute to the development of agents and methods for improving memory learning performance in young and elderly people.

This application is based on Japanese Patent Application No. 2022-094016 filed in Japan on June 9, 2022, and the entire content thereof is incorporated herein by reference.

Claims (21)

miR-30a or its precursor, or
A nucleic acid that contains or consists of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the miR-30a base sequence and has substantially the same function as miR-30a is administered to a subject. An agent for promoting cranial neurogenesis in a subject, comprising an effective amount of a vector for expression in cranial nerves. The brain neurogenesis promoting agent according to claim 1, wherein miR-30a is RNA consisting of the base sequence of SEQ ID NO: 1, or a modified form thereof. The brain neurogenesis promoting agent according to claim 1 or 2, wherein the precursor of miR-30a is pri-miRNA or pre-miRNA of miR-30a. The brain neurogenesis promoting agent according to any one of claims 1 to 3, which is administered into the brain of the subject. The cranial neurogenesis promoting agent according to claim 4, which is administered into the lateral ventricle of the subject. The cranial neurogenesis promoting agent according to claim 5, wherein the vector to be expressed in the cranial nerve of the subject is a vector to be expressed in the choroid plexus of the subject. The brain neurogenesis promoting agent according to any one of claims 1 to 6, wherein the subject is an elderly subject. miR-30a or its precursor,
A nucleic acid consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the miR-30a base sequence and has substantially the same function as miR-30a is administered to a subject. An agent for improving higher nervous function in a subject, comprising an effective amount of a vector for expression in cranial nerves. The agent for improving higher nervous function of a subject according to claim 8, wherein miR-30a is RNA consisting of the base sequence of SEQ ID NO: 1, or a modified version thereof. The agent for improving higher nervous function of a subject according to claim 8 or 9, wherein the precursor of miR-30a is pri-miRNA or pre-miRNA of miR-30a. The agent for improving higher nervous function of a subject according to any one of claims 8 to 10, which is administered into the brain of the subject. The higher nervous function improving agent according to claim 12, which is administered into the lateral ventricle of the subject. 13. The agent for improving higher nervous function of a subject according to claim 12, wherein the vector to be expressed in the cranial nerve of the subject is a vector to be expressed in the choroid plexus of the subject. The agent for improving higher nervous function of a subject according to any one of claims 8 to 13, wherein the subject is an elderly subject. miR-30a or its precursor,
A nucleic acid consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the base sequence of miR-30a, and has substantially the same function as miR-30a,
A vector for expressing miR-30a or its precursor in the cranial nerves of a subject, or
A nucleic acid containing or consisting of a base sequence in which one or several bases are substituted, deleted, inserted, or added to the miR-30a base sequence and has substantially the same function as miR-30a is administered to a subject. An agent for improving memory and learning functions in a test subject, comprising an effective amount of a vector for expression in cranial nerves. The agent for improving memory and learning function in a subject according to claim 15, wherein miR-30a is RNA consisting of the base sequence of SEQ ID NO: 1, or a modified version thereof. The memory learning function improving agent for a subject according to claim 15 or 16, wherein the precursor of miR-30a is pri-miRNA or pre-miRNA of miR-30a. The memory learning function improving agent for a subject according to any one of claims 15 to 17, which is administered into the brain of the subject. The memory learning function improving agent for a subject according to claim 18, which is administered into the lateral ventricle of the subject. 20. The memory learning function improving agent for a subject according to claim 19, wherein the vector to be expressed in the cranial nerve of the subject is a vector to be expressed in the choroid plexus of the subject. The memory learning function improving agent for a subject according to any one of claims 15 to 20, wherein the subject is an elderly subject.