Application and method of ribonuclease RNase H in repairing growth defects of neuronal dendrites

Abstract

The invention provides application and a method for repairing growth defects of neuronal dendrites by using ribonuclease RNase H. In particular, ribonuclease RNase H can be used as an R-loop degradation agent for treating and / or preventing neuron dendritic growth defects and related diseases thereof. A Rett syndrome cell model is utilized, and it is found that growth of dendrites can be restored by expressing ribonuclease RNase H of mammals. The method disclosed by the invention provides a very significant means for treating and preventing the Rett syndrome and other neuronal dendritic growth defect related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an application and method of repairing neuron dendrite growth defects by utilizing ribonuclease RNase H. Background Art

[0002] Dendrites are a very special structure of nerve cells, which play an important role in collecting and processing information for neurons. Dendrites usually protrude from the cell body of neurons, forming tree-like branches. Most dendrites are more than 2um long, contain many ribosomes, smooth endoplasmic reticulum, Golgi bodies and cytoskeleton structures, and have high protein synthesis activity during signal transmission. Dendrites are the main sensors of neurons, and 75% of the dendritic surface participates in synaptic transmission. The main function of dendrites is to receive signals from other neurons and transmit the signals to the neuron cell body.

[0003] Rett syndrome is a severe neurodevelopmental disorder that mainly affects girls. Its clinical manifestations include intellectual disability, poor learning and memory, stereotyped hand movements, autism, social disorders, respiratory and skeletal abnormalities, etc. Currently, there is no effective treatment for this disease [1]. In 1992, Adrian P. Bird's laboratory in the UK first cloned the MeCP2 (Methyl-CpG binding protein 2) gene and found that the MeCP2 protein binds to methylated DNA [2]. In 1999, Huda Y. Zoghbi's laboratory in the United States reported that MeCP2 gene mutations cause Rett syndrome [3]. The MeCP2 protein contains an N-terminal domain (NTD), a methylated CpG binding domain (MBD), an intermediate domain (ID), a transcriptional repression domain (TRD), and a C-terminal domain (CTD). There are approximately 200 mutations in the MeCP2 protein in patients with Rett syndrome, which are densely distributed in the MBD and TRD. Mutations occurring at the N-terminus are more severe than those occurring at the C-terminus. MeCP2 is ubiquitously expressed in many tissues, but its expression in the brain is very high, reaching the level of histones [4]. MeCP2 is a multifunctional protein that can regulate chromatin structure, transcription, alternative splicing, and miRNA processing by binding to different protein partners [2,5]. The Rett syndrome mouse model revealed that the loss of MeCP2 in neuronal cells is mainly responsible for the symptoms of Rett syndrome [6,7]. Rett syndrome patients show reduced brain volume, reduced neuronal cell bodies, abnormal dendritic complexity and dendritic spine density [8]. To date, the pathogenic mechanism of Rett syndrome is still not very clear, and there is no effective treatment. Summary of the invention

[0004] The purpose of the present invention is to provide the use of ribonuclease H (RNase H) in treating neuron dendrite growth defect diseases.

[0005] In a first aspect, the present invention provides the use of an R-loop degrader in the preparation of a medicament for treating and / or preventing neuronal dendritic growth defects and related diseases.

[0006] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or its active fragment, or its promoter.

[0007] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or its active fragment, or its promoter.

[0008] In another preferred embodiment, the RNase H1 is wild-type RNase H1.

[0009] In another preferred example, the amino acid sequence of the wild-type RNase H1 is shown in SEQ ID NO:9.

[0010] In another preferred embodiment, the wild-type RNase H1 gene sequence is referred to Genbank accession number NM_001286834.3.

[0011] In another preferred embodiment, the RNase H1 active fragment refers to a protein fragment having the activity of degrading R-loop; and the active fragment has at least 50%, preferably at least 80%, more preferably at least 90%, and most preferably 100% of the R-loop degrading activity of the catalytic domain of the RNase H1 protein.

[0012] In another preferred example, the RNase H1 active fragment comprises the amino acid sequence shown in SEQ ID NO:10.

[0013] In another preferred embodiment, the RNase H1 promoter can enhance the enzymatic activity of RNase H1;

[0014] Specifically, before applying the accelerator, the enzymatic activity of RNase H1 is recorded as Z0, and after applying the accelerator, the enzymatic activity of RNase H1 is recorded as Z1, wherein Z1 / Z0≥1.5, preferably ≥2, and most preferably ≥3;

[0015] Wherein, the enzyme activity is the activity of degrading R-loop.

[0016] In another preferred embodiment, the RNase H1 promoter can increase the expression level of RNase H1;

[0017] Specifically, before the administration of the promoter, the expression level of RNase H1 is recorded as P0, and after the administration of the promoter, the expression level of RNase H1 is recorded as P1, wherein P1 / P0≥1.5, preferably ≥2, and most preferably ≥3.

[0018] In another preferred embodiment, the expression level of RNase H1 includes RNA expression level and protein expression level.

[0019] In another preferred embodiment, the RNase H1 promoter includes small molecule compounds, nucleic acids, polypeptides, proteins, etc.

[0020] In another preferred embodiment, the subject or patient suffering from or suspected of suffering from neuronal dendritic growth defects and related diseases has the following characteristics:

[0021] (1) The number of dendrites of neurons decreases, the length of dendrites shortens, and / or the complexity of neuronal dendrites decreases;

[0022] (2) R-loop levels in cerebral cortex tissue are significantly increased; and / or

[0023] (3)MeCP2 gene mutation.

[0024] In another preferred embodiment, the MeCP2 gene mutation leads to inactivation and functional loss or down-regulated expression of the MeCP2 protein, thereby leading to increased R-loop levels in neurons.

[0025] In another preferred embodiment, about 95% of patients suffering from the neuronal dendrite growth defect-related disease have MeCP2 gene mutations.

[0026] In another preferred embodiment, the “reduction in the number of dendrites of neurons” means that the number of dendrites of neurons with dendritic growth defects is reduced by at least 20%, such as 20% to 70%, relative to the number of dendrites of normal neurons.

[0027] In another preferred embodiment, the “shortening of dendrite length of neurons” means that the dendrite length of neurons with dendrite growth defects is reduced by at least 20%, such as 20% to 70%, relative to the dendrite length of normal neurons.

[0028] In another preferred embodiment, the "significantly increased R-loop level" means that the R-loop level R1 in the patient's cerebral cortex tissue is compared with the R-loop level R0 in the normal cerebral cortex tissue, R1 / R0 ≥ 1.5, preferably ≥ 2, and optimally ≥ 3.

[0029] In another preferred embodiment, the diseases associated with neuronal dendrite growth defects include neurodevelopmental and neurodegenerative diseases.

[0030] In another preferred embodiment, the diseases related to neuronal dendrite growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0031] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0032] In another preferred embodiment, the RNase H1, or its active fragment, or its promoter repairs the dendritic growth defect of neurons by restoring (or reducing) the R-loop level in neurons lacking MeCP2 in the patient's cerebral cortex tissue, thereby treating related diseases (such as Rett syndrome).

[0033] In another preferred embodiment, the "repairing the dendrite growth defect of neurons" includes restoring the number and / or length of dendrites of neurons.

[0034] In another preferred embodiment, the patient includes a human or a non-human mammal.

[0035] The second aspect of the present invention provides a drug combination, which comprises:

[0036] (a) a detection reagent for detecting a defect in neuronal dendrite growth; and

[0037] (b) R-loop degraders;

[0038] The drug combination is used for precise treatment and / or prevention of neuronal dendrite growth defects and related diseases.

[0039] In another preferred embodiment, the detection reagent for detecting neuronal dendritic growth defects is selected from the following group: a detection reagent for detecting MeCP2 gene mutations, a detection reagent for detecting R-loop levels in cerebral cortical tissue, a detection reagent for detecting the number and / or length of neuronal dendrites, or a combination thereof.

[0040] In another preferred embodiment, the detection includes (but is not limited to) gene sequencing, qPCR, immunoblotting, immunohistochemistry, immunofluorescence, radiological imaging, etc.

[0041] In another preferred embodiment, the detection reagent for detecting MeCP2 gene mutation is selected from: primers, probes, gene chips, protein chips, antibodies, enzymes, or a combination thereof.

[0042] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or its active fragment, or its promoter.

[0043] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or its active fragment, or its promoter.

[0044] In another preferred embodiment, the RNase H1 is wild-type RNase H1.

[0045] In another preferred example, the amino acid sequence of the wild-type RNase H1 is shown in SEQ ID NO:9.

[0046] In another preferred embodiment, the wild-type RNase H1 gene sequence is referred to Genbank accession number NM_001286834.3.

[0047] In another preferred embodiment, the RNase H1 active fragment refers to a protein fragment having the activity of degrading R-loop; and the active fragment has at least 50%, preferably at least 80%, more preferably at least 90%, and most preferably 100% of the R-loop degrading activity of the catalytic domain of the RNase H1 protein.

[0048] In another preferred example, the RNase H1 active fragment comprises the amino acid sequence shown in SEQ ID NO:10.

[0049] In another preferred embodiment, the RNase H1 promoter can enhance the enzymatic activity of RNase H1;

[0050] Specifically, before applying the accelerator, the enzymatic activity of RNase H1 is recorded as Z0, and after applying the accelerator, the enzymatic activity of RNase H1 is recorded as Z1, wherein Z1 / Z0≥1.5, preferably ≥2, and most preferably ≥3.

[0051] In another preferred embodiment, the RNase H1 promoter can increase the expression level of RNase H1;

[0052] Specifically, before the administration of the promoter, the expression level of RNase H1 is recorded as P0, and after the administration of the promoter, the expression level of RNase H1 is recorded as P1, wherein P1 / P0≥1.5, preferably ≥2, and most preferably ≥3.

[0053] In another preferred embodiment, the expression level of RNase H1 includes RNA expression level and protein expression level.

[0054] In another preferred embodiment, the RNase H1 promoter includes small molecule compounds, nucleic acids, polypeptides, proteins, etc.

[0055] In another preferred embodiment, the "neuronal dendrite growth defect" is manifested as:

[0056] (1) The number of dendrites of neurons decreases, the length of dendrites shortens, and / or the complexity of dendrites decreases;

[0057] (2) R-loop levels in cerebral cortex tissue are significantly increased; and / or

[0058] (3)MeCP2 gene mutation.

[0059] In another preferred embodiment, the MeCP2 gene mutation leads to inactivation and functional loss or down-regulated expression of the MeCP2 protein, thereby leading to increased R-loop levels in neurons.

[0060] In another preferred embodiment, the diseases related to neuronal dendrite growth defects include neurodevelopmental and neurodegenerative diseases.

[0061] In another preferred embodiment, the diseases related to neuronal dendrite growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0062] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0063] The third aspect of the present invention provides a medicine kit, comprising:

[0064] (C1) a detection reagent for detecting neuronal dendrite growth defects;

[0065] (C2) R-loop degraders; and

[0066] (C3) instructions, which indicate that the drug kit is used for precise treatment and / or prevention of neuronal dendritic growth defects and related diseases.

[0067] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or its active fragment, or its promoter.

[0068] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or its active fragment, or its promoter.

[0069] In a fourth aspect, the present invention provides a method for repairing neuronal dendritic growth defects, the method comprising: administering an R-loop degrader to neurons having dendritic growth defects; wherein the dendritic growth defect is caused by a MeCP2 gene mutation.

[0070] In another preferred embodiment, the R-loop level in the neurons with dendritic growth defects is significantly increased relative to that in normal neurons.

[0071] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or its active fragment, or its promoter.

[0072] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or its active fragment, or its promoter.

[0073] In another preferred embodiment, the method is an in vitro method.

[0074] In another preferred embodiment, the method is a non-therapeutic method.

[0075] In another preferred embodiment, the "repairing the dendrite growth defect of neurons" includes restoring the number and / or length of dendrites of neurons.

[0076] In a fifth aspect, the present invention provides a method for screening therapeutic agents for neuronal dendritic growth defects and related diseases, comprising the steps of: administering a candidate therapeutic agent to a test neuronal cell or a test animal, and if the test neuronal cell or the test animal shows the following characteristics, it is proved that the candidate therapeutic agent can be used as a therapeutic agent for neuronal dendritic growth defects and related diseases:

[0077] (i) The number and / or length of dendrites of neurons are restored;

[0078] (ii) reduced R-loop levels; and / or

[0079] (iii) The enzymatic activity of RNase H1 is increased and / or the expression level of RNase H1 is increased.

[0080] In another preferred embodiment, the test neuronal cells or test animals have MeCP2 gene deletion.

[0081] In another preferred embodiment, the test neuronal cells are primary neuronal cells.

[0082] In another preferred embodiment, the test animals include (but are not limited to) mice, rats, rabbits, monkeys, and dogs.

[0083] In a sixth aspect, the present invention provides a method for treating and / or preventing neuronal dendritic growth defects and related diseases, the method comprising: administering an R-loop degrader to a subject in need thereof.

[0084] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or its active fragment, or its promoter.

[0085] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or its active fragment, or its promoter.

[0086] In another preferred embodiment, the method further comprises: before administering the R-loop degrader, detecting the MeCP2 gene mutation status of the subject, detecting the R-loop level in the subject's cerebral cortex tissue, and / or detecting the number and / or length of dendrites of neurons.

[0087] In another preferred embodiment, the subject has the following characteristics:

[0088] (1) The number of dendrites of neurons decreases and / or the length of dendrites shortens, and / or the complexity of neuronal dendrites decreases;

[0089] (2) R-loop levels in cerebral cortex tissue are significantly increased; and / or

[0090] (3)MeCP2 gene mutation.

[0091] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0092] In another preferred embodiment, the subject suffers from or is suspected of suffering from a disease associated with defective neuronal dendrite growth.

[0093] In another preferred embodiment, the diseases related to neuronal dendrite growth defects include neurodevelopmental and neurodegenerative diseases.

[0094] In another preferred embodiment, the diseases related to neuronal dendrite growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0095] In another preferred embodiment, the neuronal dendrite growth defect-related disease is associated with MeCP2 gene deletion.

[0096] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0097] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Shown is protein detection after knockdown of MeCP2 in primary cultured neuronal cells.

[0099] Figure 2 The results show that knocking down Mecp2 in primary neuronal cells leads to dendrite growth defects. A shows that knocking down Mecp2 leads to a decrease in the number of neuronal dendrites; B shows that knocking down Mecp2 leads to a shortened length of neuronal dendrites.

[0100] Figure 3 Shown are the detection of overexpression of wild-type and enzymatically active mutant RNASEH1 proteins in primary neuronal cells.

[0101] Figure 4 It shows that wild-type RNASEH1 can restore the dendrite growth defects caused by Mecp2 knockdown. A shows that wild-type RNASEH1 can restore the number of neuronal dendrites; B shows that wild-type RNASEH1 can restore the length of neuronal dendrites. DETAILED DESCRIPTION

[0102] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that abnormal neuronal dendritic growth is related to R-loop formation, and proposed for the first time a scheme for treating and / or preventing diseases of abnormal neuronal dendritic growth using R-loop degraders (such as RNase H1 or its active fragments, or its promoters).

[0103] Specifically, the inventors used primary cultured cerebral cortical neurons as a research model and found that after knocking down Mecp2 in primary neuronal cells using shRNA technology, dendritic growth was defective, which can simulate the dendritic abnormalities in Rett syndrome. At the same time, the inventors also observed that after knocking down Mecp2 in primary neuronal cells, the level of R-loop increased. In primary neuronal cells with Mecp2 knocked down, wild-type and mutant RNASEH1 were overexpressed respectively. Wild-type RNASEH1 can inhibit R-loop formation, but mutants cannot. More interestingly, wild-type RNASEH1 can restore the growth of neuronal dendrites. Therefore, the present invention first discovered that when RNASEH1 is overexpressed, the abnormal dendritic growth caused by MeCP2 deficiency can be repaired, indicating that increasing the expression or activity of RNASEH1 can be used for the treatment or prevention of diseases related to abnormal neuronal dendritic growth, including Rett syndrome.

[0104] On this basis, the present invention has been completed.

[0105] the term

[0106] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0107] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0108] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0109] As used herein, the term "subject", "subject in need" refers to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

[0110] RNase H

[0111] As used herein, the terms "ribonuclease H", "RNase H" and "RNASEH" can be used interchangeably; "ribonuclease H1", "RNase H1" and "RNASEH1" can be used interchangeably.

[0112] The RNase H family is a class of metal ion-dependent nucleases that can specifically hydrolyze RNA chains in RNA:DNA hybrids. There are two main types of the RNase H family, RNase H1 and RNase H2, which have the common structural feature of carrying a catalytic domain but have different types of substrates. R-loop is a nucleic acid structure formed by three strands. Most R-loops are produced with transcription, where the RNA transcript forms an RNA:DNA double-stranded hybrid with the template DNA, and the non-template DNA strand remains single-stranded [9]. Both RNase H1 and RNase H2 can unwind R-loops and avoid excessive accumulation of R-loops, playing an important role in regulating gene expression, maintaining genome stability, and replicating mitochondrial DNA. The unique function of RNase H2 is to remove mismatched single nucleotides in genomic DNA, so it participates in ribonucleotide repair

[10] .

[0113] The RNase H described in the present invention includes RNase H derived from various species, including (but not limited to) humans, mice, rats, rabbits, monkeys, dogs, etc.

[0114] Diseases related to defective neuronal dendrite growth

[0115] As used herein, the terms "neuronal dendritic growth defect-related diseases" and "neuronal dendritic growth abnormal diseases" can be used interchangeably, and both refer to diseases that exhibit abnormal neuronal dendritic growth (including a decrease in the number of neuronal dendrites and / or a shortening of dendrite length and / or a decrease in the complexity of neuronal dendrites). Typical neuronal dendritic growth defect-related diseases include neurodevelopmental, neurodegenerative, and psychiatric diseases. Specific types of neuronal dendritic growth defect diseases include (but are not limited to) Rett syndrome, schizophrenia, Down syndrome, chronic anxiety, and autism.

[0116] Dendrites are key structures for neurons to receive information, maintain intercellular connections, and form neural circuits. Dendritic morphogenesis is a complex and strictly controlled process, including the development of dendritic branches and dendritic spines, which is essential for information exchange between neurons. Abnormal dendritic growth and development is one of the key pathogenic factors of many neurodevelopmental diseases, neurodegenerative diseases, and mental illnesses. For example: autism spectrum disorder, Alzheimer's disease, schizophrenia, Down syndrome, Rett syndrome, anxiety, depression, intellectual disability and other neurological diseases. In these diseases, dendrites often show phenomena such as reduced length, branches, and dendritic spines. Therefore, repairing neuronal dendritic growth defects is a key means of treating and preventing these diseases.

[0117] R-loop degraders

[0118] The present invention provides an application of an R-loop degrader for preparing a drug for treating and / or preventing neuronal dendrite growth defects and related diseases.

[0119] As used herein, the term "R-loop degrader" refers to an agent that can cut and degrade R-loop, or destroy the structure of R-loop. In some embodiments of the present invention, the R-loop degrader is RNase H1 or its active fragment, or its promoter. In some embodiments of the present invention, the R-loop degrader includes a coding sequence of RNase H1 or its active fragment. Among them, RNase H1 or its active fragment exerts its endonuclease to specifically hydrolyze the RNA chain in the R-loop, thereby degrading the R-loop. The RNase H1 promoter promotes RNase H1 to specifically hydrolyze the RNA chain in the R-loop by increasing the enzymatic activity of RNase H1 or increasing the expression level of RNase H1, thereby degrading the R-loop. RNase H1 or its active fragment, or its promoter, partially offsets or complements the increase in R-loop caused by MeCP2 mutation by restoring the R-loop level in neurons, thereby restoring the normal function of neurons.

[0120] In one embodiment of the present invention, RNase H1 is wild-type RNase H1, and the RNase H1 active fragment is the active fragment of wild-type RNase H1. In a preferred embodiment of the present invention, the amino acid sequence of the wild-type RNase H1 is shown in SEQ ID NO:9, and its gene sequence is shown in NM_001286834.3 (Genbank accession number). In a preferred embodiment, the RNase H1 active fragment comprises the amino acid sequence shown in SEQ ID NO:10 (catalytic domain of RNaseH1).

[0121] SEQ ID NO:9

[0122] mfyavrrgrktgvfltwnecraqvdrfpaarfkkfatedeawafvrksaspevseghenqhgqeseakaskrlrepldgdghesaepyakhmkpsvepappvsrdtfsymgdfvvvytdgccssngrrrp ragigvywgpghplnvgirlpgrqtnqraeihaackaieqaktqninklvlytdsmftingitnwvqgwkkngwktsagkevinkedfvalerltqgmdiqwmhvpghsgfigneeadrlaregakqsed

[0123] SEQ ID NO:10

[0124] mgdfvvvytdgccssngrrrpragigvywgpghplnvgirlpgrqtnqraeihaackaieqaktqninklvlytdsmftingitnwvqgwkkngwktsagkevinkedfvalerltqgmdiqwmhvpghsgfigneeadrlaregakqsed

[0125] Drug combination and medicine kit of the present invention

[0126] The present invention also provides a drug combination, which comprises: (a) a detection reagent for detecting neuronal dendritic growth defects, and (b) the R-loop degrader of the present invention, for precise treatment and / or prevention of neuronal dendritic growth defects and diseases related thereto. In some embodiments of the present invention, the detection reagent for detecting neuronal dendritic growth defects is selected from the following group: a detection reagent for detecting MeCP2 gene mutations, a detection reagent for detecting R-loop levels in cerebral cortical tissue, a detection reagent for detecting the number and / or length of dendrites of neurons, or a combination thereof. Any reagent that can reflect the MeCP2 gene mutation, the R-loop level in cerebral cortical tissue, and the number and / or length of dendrites of neurons can be used in embodiments of the present invention to determine whether there is a neuronal dendritic growth defect.

[0127] When using the drug combination of the present invention for treatment, the detection reagent is first used to detect whether the subject has a neuronal dendrite growth defect; if so, an R-loop degrader is administered to the subject; wherein the subject suffers from a neurological disease.

[0128] In a preferred embodiment of the present invention, a detection reagent for detecting MeCP2 gene mutation is first used to confirm whether the subject has MeCP2 gene mutation; if the subject has MeCP2 gene mutation, an R-loop degrader is administered to the subject. The MeCP2 gene mutation of the subject can be confirmed by gene sequencing, qPCR, immunoblotting (e.g., Western Blotting), and the like.

[0129] The pharmaceutical combination of the present invention comprises a safe and effective amount of an R-loop degrader. The term "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects.

[0130] In addition, the pharmaceutical combination of the present invention can also be used together with other therapeutic agents.

[0131] Furthermore, the above-mentioned drug combination can be packaged into a suitable container and placed together with instructions to obtain a medicine box, wherein the instructions record the use of the drug combination and indicate that the medicine box is used for the precise treatment and / or prevention of diseases related to defective neuronal dendrite growth.

[0132] The main advantages of the present invention include:

[0133] (1) The present invention is the first to discover that elevated R-loop can lead to abnormal growth of neuronal dendrites, and that this mechanism is associated with a variety of neurological diseases, such as Rett syndrome.

[0134] (2) The present invention proposes for the first time that R-loop degraders (such as RNase H1 or its active fragments, or its promoters) can be used to treat and / or prevent neuronal dendritic growth defects and related diseases. By administering the R-loop degrader RNaseH1, the increase in R-loop caused by MeCP2 mutation can be partially offset or compensated, thereby restoring the normal function of neurons. The present invention reveals a new mechanism for abnormal neuronal dendritic growth and provides a new target for the treatment and prevention of related diseases.

[0135] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0136] Materials and Methods

[0137] 1.1 Primary culture of mouse cerebral cortical neurons

[0138] (1) Place a cover glass (Fisherbrand TM 12-545-100) were sequentially immersed in acetone, double distilled water and anhydrous ethanol and ultrasonicated for 30 min. The pretreated coverslips were stored in anhydrous ethanol;

[0139] (2) 12-well cell culture plate per well ( CLS3512) were placed in each well and 500 μL of poly-D-lysine (Gibco TM A3890401), and placed in a 37°C cell culture incubator overnight;

[0140] (3) Aspirate the poly-D-lysine in the 12-well cell culture plate, add 1 mL of sterilized double-distilled water to each well to wash away the residual poly-D-lysine, and aspirate all the liquid in the well;

[0141] (4) Repeat step (3) once, remove all residual liquid, and air-dry for later use;

[0142] (5) P0 male C57BL / 6 mice (Shanghai Lingchang Biotechnology Co., Ltd.) were disinfected with 75% ethanol, the mouse heads were cut off, the cortex was separated and placed in pre-cooled 1× HBSS (0.14 g / L CaCl 2 , 0.1g / L MgCl 2 6H2 O, 0.1 g / L MgSO 4 7H 2 O, 0.4 g / L KCl, 0.06 g / L KH 2 PO 4 , 8g / L NaCl, 0.09g / L Na 2 HPO 4 7H 2 O, 1g / L D-Glucose), two pieces of complete cortical tissue were separated under a stereoscope and the hippocampus was peeled off;

[0143] (6) Add 1 mL of pre-cooled digestion solution, i.e., DMEM (Gibco Biosystems, Inc.) containing 10 μg / mL deoxyribonuclease I (Sigma-Aldrich DN25-10MG) and 1 U / μL papain (Worthington LS003126), into a 1.5 mL centrifuge tube. TM C11965500BT), put the two peeled cortexes into a tube and digest them in a 37℃ water bath for 20min;

[0144] (7) Remove the supernatant and add 1 mL of 37°C preheated digestion stop solution containing 10% FBS ( HB-FBS-500) in DMEM, gently invert 5 times;

[0145] (8) Repeat step (7) once and remove the supernatant;

[0146] (9) Add 1 mL of Neurobasal medium (Gibco) preheated to 37°C containing 5% FBS. TM 21103049), pipette 1 mL for 15-20 times to fully resuspend the digested tissue, and transfer it to a new centrifuge tube;

[0147] (10) After centrifugation at 2000 rpm for 5 min, the supernatant was removed and the pellet was resuspended in Neurobasal medium containing 5% FBS preheated at 37°C. Each piece of cortical tissue was divided into 7 wells of a 12-well cell culture plate and transferred to a 37°C cell culture incubator.

[0148] (11) After 4-6 hours of culture, the culture medium was replaced with primary neuron culture medium preheated at 37°C, containing 1× B-27 TM Additives (Gibco TM 17504044), 1×GlutaMAX TM Additives (Gibco TM 35050061) and 1% penicillin-streptomycin (Gibco TM15140163) of Neurobasal medium.

[0149] 1.2 Oligonucleotide sequences

[0150] The following oligonucleotides were designed and synthesized

[0151]

[0152]

[0153] 1.3 Construction of shRNA lentiviral plasmid

[0154] The control shRNA and the shRNA targeting Mecp2 forward primer and reverse primer were mixed in 1.5 mL centrifuge tubes in equal molar numbers, placed in boiling water, and cooled naturally at room temperature for annealing. The annealed products were inserted into the pLSAR lentiviral vector with BamHI and XbaI restriction sites to obtain pLSAR-shLuc-EV-RFP, pLSAR-shMecp2-1-EV-RFP and pLSAR-shMecp2-2-EV-RFP plasmids, respectively.

[0155] 1.4 Construction of lentiviral expression plasmids for combined shRNA overexpression of wild-type and mutant RNASEH1

[0156] PCR amplification was performed using ppyCAG_RNaseH1_WT plasmid (Addgene plasmid #111906) as a template and RNH1_F and RNH1_R as primers. The resulting PCR product was inserted into pLSAR-shMecp2-1-EV-RFP using NheI and BsrGI restriction sites to obtain a lentiviral expression plasmid for shRNA combined with overexpression of RNASEH1 wild type, i.e., pLSAR-shMecp2-1-RNaseH1-WT-RFP. PCR was performed using ppyCAG_RNaseH1_D210N plasmid (Addgene plasmid #111904) as a template, and the same steps were followed to obtain a lentiviral expression plasmid for shRNA combined with overexpression of RNASEH1 mutant, i.e., pLSAR-shMecp2-1-RNaseH1-D210N-RFP.

[0157] 1.5 Lentivirus infection of primary mouse neurons

[0158] (1) 12 h in advance, HEK293T cells were plated in a 6 cm cell culture dish so that the cell density at the time of transfection was 40-50%;

[0159] (2) HEK293T cells were transfected with the corresponding lentiviral expression plasmid and lentiviral packaging plasmid psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) at a mass ratio of 4:3:1 using the calcium phosphate method;

[0160] (3) 8-12 h after transfection, change the medium and add 3 mL of Neurobasal medium;

[0161] (4) 48 h after transfection, the cell culture medium containing viral particles was collected and filtered through a 0.45 μm filter membrane for immediate use or stored at -80°C for a long time;

[0162] (5) Primary neurons were cultured in vitro until DIV2. Before adding the virus, a small amount of culture medium from the cell culture plate was collected and stored at 4°C for later use. Then, an appropriate amount of virus was added to infect neurons and the plate was returned to the 37°C cell culture incubator.

[0163] (6) 24 h after virus infection, the virus was aspirated and preheated fresh primary neuron culture medium and cell culture medium collected before virus infection were mixed in a 1:1 ratio and added;

[0164] (7) Change the medium of primary neurons every two days until DIV7.

[0165] 1.6 Detection of protein expression by immunoblotting

[0166] (1) The culture medium of the primary neurons to be tested was aspirated, and an appropriate amount of 2×SDS protein loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 200 mM DTT, 20% Glycerol, 0.2% Bromophenol blue) was directly added. The cells were collected into a 1.5 mL centrifuge tube and fully denatured in a 95°C metal bath;

[0167] (2) The prepared protein samples were electrophoresed using 10% SDS-PAGE gel in a buffer (3.029 g / L Tris-base, 18.768 g / L Glycine, 1 g / L SDS);

[0168] (3) Separate the SDS-PAGE gel after electrophoresis with a nitrocellulose membrane of the same size (Amersham TM 10600002) and filter paper were immersed in transfer solution (14.4 g / L Glycine, 3.03 g / L Tris-base, 200 mL / L Methanol) for 5 min and then transferred using a semi-dry transfer instrument (Trans- SD Semi-Dry Transfer Cell) transfer, transfer conditions are 12V, 60min;

[0169] (4) After transfer, the membrane was immersed in NET blocking solution (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.05% Triton X-100, 0.25% Gelatin) and incubated on a shaker at room temperature for 60 min;

[0170] (5) After blocking, discard the blocking solution and add the corresponding primary antibody diluted with the blocking solution and incubate at room temperature for 2 h or at 4°C overnight. The primary antibodies are MeCP2 polyclonal antibody (Diagenode pAb-052-050), RNASEH1 polyclonal antibody ( 15606-1-AP) and GAPDH monoclonal antibody (ABclonal AC002). After incubation, discard the antibody and add blocking solution to wash the membrane twice at room temperature, 15 minutes each time. After washing, add the corresponding secondary antibody diluted with blocking solution at a ratio of 1:2000 and incubate at room temperature for 1 hour. After the secondary antibody binds, discard the antibody and add blocking solution to wash the membrane twice at room temperature, 15 minutes each time;

[0171] (6) After washing the membrane, use color developing solution (Millipore WBKLS0500) to develop the color. TM Imager 680 was used for detection and analysis.

[0172] 1.7 Detection of dendritic growth

[0173] (1) Primary neurons were cultured in vitro until DIV7. The coverslips with primary neurons were removed from the 12-well cell culture plate and transferred to new 12-well cell culture plates. Room temperature PBS was added and the plates were slowly washed on a horizontal shaker for 5 min.

[0174] (2) Aspirate PBS and add 4% PFA prepared with PBS as solvent to each well and fix at room temperature for 10 min;

[0175] (3) Remove 4% PFA, add PBS, and wash the coverslips three times on a horizontal shaker, each time for 8 min;

[0176] (4) Add a small amount of fluorescent mounting medium onto the slide, cover with a cover slip, apply a small amount of nail polish around the cover slip for further sealing, and store at room temperature away from light overnight;

[0177] (5) Use a Leica TCS SP8X White Light Laser confocal microscope with a 63x oil objective to observe and photograph the neurons in the field of view;

[0178] (6) The number and length of dendrites of the imaged neurons were analyzed using the NeuronJ 1.4.3 plug-in of Fiji (Fiji Is Just ImageJ), and the raw data were statistically analyzed using Prism 8.0 (Graphpad) and Excel (Microsoft).

[0179] (7) The double-blind principle was followed during dendritic growth detection and data analysis.

[0180] Example 1 Knockdown of Mecp2 in primary neuronal cells leads to dendritic growth defects

[0181] Mouse primary neurons were infected with shRNA lentivirus, with shLuc as the control group and shMecp2-1 and shMecp2-2 as two shRNAs with different sequences targeting Mecp2. Compared with shLuc, both shMecp2-1 and shMecp2-2 can significantly reduce the expression of Mecp2 protein in primary neurons ( Figure 1 ).

[0182] The dendritic growth of DIV7 primary neurons infected with different lentiviruses was detected and analyzed. The results showed that compared with neurons infected with shLuc, neurons infected with shMecp2-1 or shMecp2-2 showed dendritic growth defects, which were manifested by a significant decrease in the number of dendrites per neuron and a significant decrease in the sum of the dendritic lengths of each neuron ( Figure 2 ). Figure 2 In A, the number of dendrites of neurons in the control group (shLuc) and Mecp2 knockdown (shMecp2-1, shMecp2-2) were counted. Each data point represents the number of dendrites in a neuron. The horizontal line on the figure represents the average number of dendrites in each group of neurons. The number of dendrites of 35 neurons was detected and counted in each group. Figure 2 In B, the sum of dendritic lengths of neurons in the control group (shLuc) and Mecp2 knockdown (shMecp2-1, shMecp2-2) was counted. Each data point represents the sum of dendritic lengths of a neuron. The horizontal line on the graph represents the average of the sum of dendritic lengths of neurons in each group, in μm. The sum of dendritic lengths of 35 neurons was detected and counted in each group. Mann-Whitney test: ***p<0.001.

[0183] Example 2 Wild-type RNASEH1 can restore dendrite growth defects caused by Mecp2 knockdown

[0184] Through Dot-Blot and ssDRIP-seq experiments of R-loop, the inventors found that the level of R-loop was significantly upregulated in the cortical tissue of neuron-specific Mecp2 knockout mice. In order to determine whether restoring the level of R-loop in Mecp2-deficient neurons can repair the defect of dendritic growth, wild-type and enzyme-active mutant RNASEH1 were overexpressed in primary neurons with Mecp2 knockdown.

[0185] like Figure 3 As shown, the Mecp2 protein level in neurons infected with shMecp2-1 was significantly reduced compared with the control group, while the RNASEH1 protein level overexpressing wild-type or enzyme-active mutant was similar.

[0186] The dendritic growth of primary neurons infected with different lentiviruses was detected and analyzed at DIV7 ( Figure 4 ). After overexpression of wild-type RNASEH1 in Mecp2 knockdown neurons, the total number of dendrites per neuron and the length of dendrites were significantly improved compared with neurons infected with shMecp2-1, and there was no significant difference with the control group infected with shLuc. However, overexpression of enzyme-active mutant RNASEH1 in Mecp2 knockdown neurons could not restore the dendritic growth defect. Figure 4 In A, the number of dendrites of neurons in the control group (shLuc), Mecp2 knockdown (shMecp2-1), and Mecp2 knockdown and simultaneous overexpression of RNASEH1 wild type (shMecp2-1+RNH1-WT) or enzyme active mutant (shMecp2-1+RNH1-D210N) were counted. Each data point represents the number of dendrites in a neuron, and the horizontal line on the figure represents the average number of dendrites in each group of neurons. The number of dendrites of 35 neurons was detected and counted in each group. Figure 4 In B, the sum of dendritic lengths of neurons in the control group (shLuc), Mecp2 knockdown (shMecp2-1), and Mecp2 knockdown and simultaneous overexpression of RNASEH1 wild type (shMecp2-1+RNH1-WT) or enzyme active mutant (shMecp2-1+RNH1-D210N) was counted. Each data point represents the sum of dendritic lengths of a neuron, and the horizontal line on the figure represents the average of the sum of dendritic lengths of neurons in each group, in μm. The sum of dendritic lengths of 35 neurons was detected and counted in each group. Mann-Whitney test: ***p<0.001; ns p>0.05.

[0187] Table 1

[0188]

[0189] References

[0190] 1. Lombardi LM et al. MECP2 disorders: from the clinic to mice and back. J Clin Invest, 125, 2914 - 2923, (2015)

[0191] 2. Lewis JD et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69, 905 - 914, (1992)

[0192] 3. Amir RE et al. Rett syndrome is caused by mutations in X - linked MECP2, encoding methyl - CpG - binding protein 2. Nat Genet, 23, 185 - 188 (1999).

[0193] 4. Skene PJ et al. Neuronal MeCP2 is expressed at near histone - octamer levels and globally alters the chromatin state. Mol. Cell 37, 457–468 (2010).

[0194] 5. Lyst et al. Rett syndrome: a complex disorder with simple roots. Nat Rev Genet, 16, 261 - 275(2015).

[0195] 6. Chen RZ et al. Deficiency of methyl - CpG binding protein - 2 in CNS neurons results in a Rett - like phenotype in mice. Nat Genet, 27, 327–331 (2001).

[0196] 7. Guy J et al. A mouse Mecp2-null mutation causes neurologicalsymptoms that mimic Rett syndrome. Nat Genet 27,322–326(2001).

[0197] 8.Armstrong D et al. Selective dendritic alterations in the cortex of Rett syndrome. J Neuropathol Exp Neurol, 54, 195–201 (1995).

[0198] 9. Niehrs C et al. Regulatory R-loops as facilitators of gene expression and genome stability. Nat Rev Mol Cell Biol, 21, 167-178 (2020).

[0199] 10. Hyjek M et al. RNases H: Structure and mechanism. DNA Repair, 84, 102672, (2019).

[0200] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of R-loop degraders in the preparation of drugs for treating and / or preventing neuronal dendritic growth defects and related diseases.

2. The use according to claim 1, characterized in that The R-loop degrading agent includes ribonuclease H1 (RNaseH1) or its active fragment, or its promoter.

3. The use according to claim 2, characterized in that The RNase H1 active fragment refers to a protein fragment having the activity of degrading R-loop; and the active fragment has at least 50%, preferably at least 80%, more preferably at least 90%, and most preferably 100% of the R-loop degrading activity of the catalytic domain of the RNase H1 protein.

4. The use according to claim 1, characterized in that Subjects or patients suffering from or suspected of suffering from neuronal dendritic growth defects and related diseases have the following symptoms: (1) The number of dendrites of neurons decreases, the length of dendrites shortens, and / or the complexity of dendrites decreases; (2) R-loop levels in cerebral cortex tissue are significantly increased; and / or (3)MeCP2 gene mutation.

5. The use according to claim 4, characterized in that The diseases associated with neuronal dendrite growth defects include neurodevelopmental and neurodegenerative diseases; Preferably, the diseases associated with neuronal dendrite growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

6. A drug combination, characterized in that The drug combination comprises: (a) a detection reagent for detecting a defect in neuronal dendrite growth; and (b) R-loop degraders; The drug combination is used for precise treatment and / or prevention of neuronal dendrite growth defects and related diseases.

7. The pharmaceutical composition according to claim 6, characterized in that The detection reagent for detecting neuronal dendrite growth defects is selected from the following group: a detection reagent for detecting MeCP2 gene mutation, a detection reagent for detecting R-loop levels in cerebral cortical tissue, a detection reagent for detecting the number and / or length of neuronal dendrites, or a combination thereof.

8. A medicine box, characterized in that: The medicine kit comprises: (C1) a detection reagent for detecting neuronal dendrite growth defects; (C2) R-loop degraders; and (C3) instructions, which indicate that the drug kit is used for precise treatment and / or prevention of neuronal dendritic growth defects and related diseases.

9. A method for repairing neuronal dendrite growth defects, the method comprising: An R-loop degrader is administered to neurons having a dendritic growth defect; wherein the dendritic growth defect is caused by a mutation in the MeCP2 gene.

10. A method for screening therapeutic agents for neuronal dendritic growth defects and related diseases, comprising the steps of: administering a candidate therapeutic agent to a test neuronal cell or a test animal, and if the test neuronal cell or the test animal shows the following characteristics, it is proved that the candidate therapeutic agent can be used as a therapeutic agent for neuronal dendritic growth defects and related diseases: (i) The number and / or length of dendrites of neurons are restored; (ii) reduced R-loop levels; and / or (iii) The enzymatic activity of RNase H1 is increased and / or the expression level of RNase H1 is increased.

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