Use of ribonuclease rnase h in repairing growth defects of neuron dendrites, and method therefor

Abstract

Provided are the use of a ribonuclease RNase H in repairing growth defects of neuron dendrites, and a method therefor. Specifically, the ribonuclease RNase H can be used as an R-loop degradation agent for treating and / or preventing growth defects of neuron dendrites and related diseases thereof. By means of utilizing a Rett syndrome cell model, it is found that expressing the mammalian ribonuclease RNase H can restore the growth of dendrites, thus providing means of great significance for treatment and prevention of the Rett syndrome and other diseases related to growth defects of neuron dendrites.

Description

Application and method of ribonuclease RNase H in repairing neuronal dendritic growth defects Technical Field

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

[0002] Dendrites are highly specialized nerve cell structures that play a crucial role in how neurons gather and process information. Dendrites typically protrude from the neuron's cell body, forming tree-like branches. Most dendrites are over 2 microns long and contain numerous ribosomes, smooth endoplasmic reticulum, Golgi apparatus, and cytoskeletal structures. They are highly active in protein synthesis during signal transmission. Dendrites are the primary sensors of neurons, with 75% of their surface area participating in synaptic transmission. The primary function of dendrites is to receive signals from other neurons and transmit them to the neuron's cell body.

[0003] Rett syndrome is a severe neurodevelopmental disorder that primarily affects girls. Clinical manifestations include intellectual disability, poor learning and memory, stereotypic hand movements, autism, social impairments, and respiratory and skeletal abnormalities. Currently, there is no effective treatment for this disorder. In 1992, Adrian P. Bird's laboratory in the UK first cloned the MeCP2 (Methyl-CpG binding protein 2) gene and discovered that MeCP2 binds to methylated DNA. In 1999, Huda Y. Zoghbi's laboratory in the US reported that mutations in the MeCP2 gene cause Rett syndrome. The MeCP2 protein consists of an N-terminal domain (NTD), a methylated CpG binding domain (MBD), a middle domain (ID), a transcriptional repression domain (TRD), and a C-terminal domain (CTD). Rett syndrome patients harbor approximately 200 MeCP2 mutations, which are densely distributed within the MBD and TRD. Mutations in the N-terminus are associated with more severe symptoms than those in the C-terminus. MeCP2 is ubiquitously expressed in many tissues, but its expression in the brain is extremely high, reaching the level of histones. MeCP2 is a multifunctional protein that, by binding to different protein partners, regulates chromatin structure, transcription, alternative splicing, and miRNA processing. Rett syndrome mouse models have revealed that the loss of MeCP2 in neuronal cells is primarily responsible for the symptoms of Rett syndrome. Rett syndrome patients exhibit reduced brain volume, smaller neuronal cell bodies, and abnormal dendritic complexity and spine density. To date, the pathogenic mechanism of Rett syndrome remains unclear, and there is no effective treatment.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide the use of ribonuclease H (RNase H) in treating neuronal dendritic growth defect diseases.

[0006] 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.

[0007] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or an active fragment thereof, a recombinant protein containing RNase H or an active fragment thereof, or an RNase H promoter.

[0008] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or an active fragment thereof, a recombinant protein containing RNase H1 or an active fragment thereof, or a promoter of RNase H1.

[0009] In another preferred embodiment, the RNase H1 is wild-type RNase H1.

[0010] In another preferred embodiment, the amino acid sequence of the wild-type RNase H1 is shown in SEQ ID NO:9.

[0011] In another preferred embodiment, the wild-type RNase H1 gene sequence can be found in Genbank accession number NM_001286834.3.

[0012] 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 degradation activity of the catalytic domain of the RNase H1 protein.

[0013] In another preferred embodiment, the RNase H1 active fragment comprises the amino acid sequence shown in SEQ ID NO:10.

[0014] In another preferred embodiment, the recombinant protein containing RNase H or an active fragment thereof includes: a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H or an active fragment thereof with other polypeptides.

[0015] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof includes: a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or an active fragment thereof with other polypeptides.

[0016] In another preferred embodiment, the recombinant protein containing RNase H or an active fragment thereof is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H or an active fragment thereof with MeCP2 or an active fragment thereof.

[0017] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or an active fragment thereof with MeCP2 or an active fragment thereof.

[0018] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a chimeric protein formed by replacing the N-terminal hybrid binding domain (HBD) of RNase H1 with MeCP2 or an active fragment thereof.

[0019] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a fusion protein formed by linking MeCP2 or an active fragment thereof to the N-terminus or C-terminus of RNase H1 or an active fragment thereof.

[0020] In another preferred embodiment, the connection is a direct connection or a connection through a connecting element; preferably, the connecting element is a flexible peptide linker, such as (GS)n, (GGGS)n, (GGGGS)n, (GSGGS)n, (GSSGSS)n, wherein n is a positive integer of 1-4.

[0021] In another preferred embodiment, the amino acid sequence of MeCP2 is shown in SEQ ID NO: 18.

[0022] In another preferred embodiment, the MeCP2 gene sequence is referred to Genbank accession number NM_004992.4.

[0023] In another preferred embodiment, the amino acid sequence of the MeCP2 active fragment is shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0024] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a chimeric protein formed by replacing the N-terminal hybrid chain binding domain (HBD) of wild-type RNase H1 as shown in SEQ ID NO: 9 with a MeCP2 active fragment as shown in SEQ ID NO: 19 or SEQ ID NO: 20. In another preferred embodiment, the RNase H1 promoter can enhance the enzymatic activity of RNase H1;

[0025] 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 is ≥1.5, preferably ≥2, and most preferably ≥3;

[0026] Wherein, the enzymatic activity is the activity of degrading R-loop.

[0027] In another preferred embodiment, the RNase H1 promoter can increase the expression level of RNase H1;

[0028] 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 is ≥1.5, preferably ≥2, and most preferably ≥3.

[0029] In another preferred embodiment, the expression level of RNase H1 includes RNA expression level and protein expression level.

[0030] In another preferred embodiment, the RNase H1 promoter includes small molecule compounds, nucleic acids, polypeptides, proteins, etc.

[0031] 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:

[0032] (1) The number of dendrites of neurons decreases, the length of dendrites shortens, and / or the complexity of neuronal dendrites decreases;

[0033] (2) R-loop levels are significantly elevated in cerebral cortical tissue; and / or

[0034] (3)MeCP2 gene mutation.

[0035] In another preferred embodiment, the MeCP2 gene mutation causes the inactivation and functional loss or down-regulation of the MeCP2 protein, thereby leading to an increase in the R-loop level in neurons.

[0036] In another preferred embodiment, about 95% of patients suffering from the disease associated with defective neuronal dendritic growth have MeCP2 gene mutations.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In another preferred embodiment, the diseases associated with neuronal dendritic growth defects include neurodevelopmental and neurodegenerative diseases.

[0041] In another preferred embodiment, the diseases related to neuronal dendritic growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0042] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0043] In another preferred embodiment, the RNase H1 or its active fragment, the recombinant protein containing RNase H1 or its active fragment, or the promoter of RNase H1 repairs the dendritic growth defect of neurons by restoring (or reducing) the R-loop level in neurons with MeCP2 deficiency in the patient's cerebral cortex tissue, thereby treating related diseases (such as Rett syndrome).

[0044] In another preferred embodiment, the “repairing the dendrite growth defect of neurons” includes restoring the number and / or length of dendrites of neurons.

[0045] In another preferred embodiment, the patient includes a human or a non-human mammal.

[0046] The second aspect of the present invention provides a drug combination, comprising:

[0047] (a) a detection reagent for detecting neuronal dendrite growth defects; and

[0048] (b) R-loop degraders;

[0049] The drug combination is used for precise treatment and / or prevention of neuronal dendritic growth defects and related diseases.

[0050] 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.

[0051] In another preferred embodiment, the detection includes (but is not limited to) gene sequencing, qPCR, immunoblotting, immunohistochemistry, immunofluorescence, radiographic imaging, etc.

[0052] 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.

[0053] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or an active fragment thereof, a recombinant protein containing RNase H or an active fragment thereof, or an RNase H promoter.

[0054] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or an active fragment thereof, a recombinant protein containing RNase H1 or an active fragment thereof, or a promoter of RNase H1.

[0055] In another preferred embodiment, the RNase H1 is wild-type RNase H1.

[0056] In another preferred embodiment, the amino acid sequence of the wild-type RNase H1 is shown in SEQ ID NO:9.

[0057] In another preferred embodiment, the wild-type RNase H1 gene sequence can be found in Genbank accession number NM_001286834.3.

[0058] 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 degradation activity of the catalytic domain of the RNase H1 protein.

[0059] In another preferred embodiment, the RNase H1 active fragment comprises the amino acid sequence shown in SEQ ID NO:10.

[0060] In another preferred embodiment, the recombinant protein containing RNase H or an active fragment thereof includes: a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H or an active fragment thereof with other polypeptides.

[0061] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof includes: a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or an active fragment thereof with other polypeptides.

[0062] In another preferred embodiment, the recombinant protein containing RNase H or an active fragment thereof is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H or an active fragment thereof with MeCP2 or an active fragment thereof.

[0063] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or an active fragment thereof with MeCP2 or an active fragment thereof.

[0064] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a chimeric protein formed by replacing the N-terminal hybrid binding domain (HBD) of RNase H1 with MeCP2 or an active fragment thereof.

[0065] In another preferred embodiment, the recombinant protein containing RNase H1 or an active fragment thereof is a fusion protein formed by linking MeCP2 or an active fragment thereof to the N-terminus or C-terminus of RNase H1 or an active fragment thereof.

[0066] In another preferred embodiment, the connection is a direct connection or a connection through a connecting element; preferably, the connecting element is a flexible peptide linker, such as (GS)n, (GGGS)n, (GGGGS)n, (GSGGS)n, (GSSGSS)n, wherein n is a positive integer of 1-4.

[0067] In another preferred embodiment, the amino acid sequence of MeCP2 is shown in SEQ ID NO: 18.

[0068] In another preferred embodiment, the MeCP2 gene sequence is referred to Genbank accession number NM_004992.4.

[0069] In another preferred embodiment, the amino acid sequence of the MeCP2 active fragment is shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0070] In another preferred embodiment, the recombinant protein containing RNase H1 or its active fragment is a chimeric protein formed by replacing the N-terminal hybrid chain binding domain (HBD) of the wild-type RNase H1 as shown in SEQ ID NO: 9 with the MeCP2 active fragment as shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0071] In another preferred embodiment, the RNase H1 promoter can enhance the enzymatic activity of RNase H1;

[0072] 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 is ≥1.5, preferably ≥2, and most preferably ≥3.

[0073] In another preferred embodiment, the RNase H1 promoter can increase the expression level of RNase H1;

[0074] 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 is ≥1.5, preferably ≥2, and most preferably ≥3.

[0075] In another preferred embodiment, the expression level of RNase H1 includes RNA expression level and protein expression level.

[0076] In another preferred embodiment, the RNase H1 promoter includes small molecule compounds, nucleic acids, polypeptides, proteins, etc.

[0077] In another preferred embodiment, the “neuronal dendritic growth defect” is manifested as:

[0078] (1) The number of dendrites of neurons decreases, the length of dendrites shortens, and / or the complexity of neuronal dendrites decreases;

[0079] (2) R-loop levels are significantly elevated in cerebral cortical tissue; and / or

[0080] (3)MeCP2 gene mutation.

[0081] In another preferred embodiment, the MeCP2 gene mutation causes the inactivation and functional loss or down-regulation of the MeCP2 protein, thereby leading to an increase in the R-loop level in neurons.

[0082] In another preferred embodiment, the diseases related to neuronal dendritic growth defects include neurodevelopmental and neurodegenerative diseases.

[0083] In another preferred embodiment, the diseases related to neuronal dendritic growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0084] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0085] A third aspect of the present invention provides a medicine kit, comprising:

[0086] (C1) a detection reagent for detecting neuronal dendritic growth defects;

[0087] (C2) R-loop degraders; and

[0088] (C3) instructions, which indicate that the drug kit is used for precise treatment and / or prevention of neuronal dendritic growth defects and related diseases.

[0089] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or an active fragment thereof, a recombinant protein containing RNase H or an active fragment thereof, or an RNase H promoter.

[0090] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or an active fragment thereof, a recombinant protein containing RNase H1 or an active fragment thereof, or a promoter of RNase H1.

[0091] A fourth aspect of the present invention provides a method for repairing neuronal dendritic growth defects, the method comprising: administering an R-loop degrader to neurons with dendritic growth defects; wherein the dendritic growth defect is caused by a MeCP2 gene mutation.

[0092] In another preferred embodiment, the R-loop level in the neurons with dendritic growth defects is significantly increased compared with that in normal neurons.

[0093] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or an active fragment thereof, a recombinant protein containing RNase H or an active fragment thereof, or an RNase H promoter.

[0094] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or an active fragment thereof, a recombinant protein containing RNase H1 or an active fragment thereof, or a promoter of RNase H1.

[0095] In another preferred embodiment, the method is an in vitro method.

[0096] In another preferred embodiment, the method is a non-therapeutic method.

[0097] In another preferred embodiment, the “repairing the dendrite growth defect of neurons” includes restoring the number and / or length of dendrites of neurons.

[0098] 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 test animal, wherein if the test neuronal cell or test animal exhibits the following characteristics, the candidate therapeutic agent is considered to be a therapeutic agent for neuronal dendritic growth defects and related diseases:

[0099] (i) The number and / or length of dendrites of neurons are restored;

[0100] (ii) reduced R-loop levels; and / or

[0101] (iii) The enzymatic activity of RNase H1 is increased and / or the expression level of RNase H1 is increased.

[0102] In another preferred embodiment, the test neuronal cells or test animals have MeCP2 gene deletion.

[0103] In another preferred embodiment, the test neuronal cells are primary neuronal cells.

[0104] In another preferred embodiment, the test animals include (but are not limited to) mice, rats, rabbits, monkeys, and dogs.

[0105] 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.

[0106] In another preferred embodiment, the R-loop degrading agent includes ribonuclease RNase H or an active fragment thereof, a recombinant protein containing RNase H or an active fragment thereof, or an RNase H promoter.

[0107] In another preferred embodiment, the R-loop degrading agent includes ribonuclease H1 (RNase H1) or an active fragment thereof, a recombinant protein containing RNase H1 or an active fragment thereof, or a promoter of RNase H1.

[0108] 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.

[0109] In another preferred embodiment, the subject has the following characteristics:

[0110] (1) The number of dendrites of neurons decreases and / or the length of dendrites shortens, and / or the complexity of neuronal dendrites decreases;

[0111] (2) R-loop levels are significantly elevated in cerebral cortical tissue; and / or

[0112] (3)MeCP2 gene mutation.

[0113] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0114] In another preferred embodiment, the subject suffers from or is suspected of suffering from a disease associated with defective neuronal dendritic growth.

[0115] In another preferred embodiment, the diseases related to neuronal dendritic growth defects include neurodevelopmental and neurodegenerative diseases.

[0116] In another preferred embodiment, the diseases related to neuronal dendritic growth defects include Rett syndrome, schizophrenia, Down syndrome, chronic anxiety disorder, autism, etc.

[0117] In another preferred embodiment, the neuronal dendritic growth defect-related disease is associated with MeCP2 gene deletion.

[0118] In another preferred embodiment, the neuronal dendritic growth defect-related disease is Rett syndrome caused by MeCP2 gene mutation in neuronal cells.

[0119] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 shows protein detection after knockdown of MeCP2 in primary cultured neuronal cells.

[0121] Figure 2 shows that knocking down Mecp2 in primary neurons leads to dendritic 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 shortening of neuronal dendrite length.

[0122] FIG3 shows the detection of overexpression of wild-type and enzyme-active mutant RNase H1 proteins in primary neuronal cells.

[0123] Figure 4 shows that wild-type RNase H1 can restore the dendrite growth defects caused by Mecp2 knockdown. A shows that wild-type RNase H1 can restore the number of neuronal dendrites; B shows that wild-type RNase H1 can restore the length of neuronal dendrites.

[0124] Figure 5 shows in vitro enzymatic activity assays of wild-type RNase H1, an active fragment of RNase H1, and chimeric proteins recombined with an active fragment of MeCP2. A shows that the chimeric proteins MILH and ILH, constructed by replacing part of the amino acid sequence of wild-type RNase H1 with the amino acid sequence of the active fragment of MeCP2, exhibit higher enzymatic activity than RNase H1. B shows a statistical analysis of the results in A. DETAILED DESCRIPTION

[0125] 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 for the first time proposed 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).

[0126] 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 RNase H1 were overexpressed respectively. Wild-type RNase H1 can inhibit R-loop formation, but the mutant type cannot. More interestingly, wild-type RNase H1 can restore the growth of neuronal dendrites. Therefore, the present invention discovered for the first time that overexpression of RNase H1 can repair the abnormal dendritic growth caused by MeCP2 deficiency, indicating that increasing the expression or activity of RNase H1 can be used to treat or prevent diseases related to abnormal neuronal dendritic growth, including Rett syndrome.

[0127] On this basis, the present invention was completed.

[0128] the term

[0129] 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.

[0130] 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.).

[0131] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0132] As used herein, the terms "subject" and "subject in need thereof" refer 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, and goats.

[0133] RNase H

[0134] As used herein, the terms "ribonuclease H," "RNase H," and "RNASEH" are used interchangeably; and "ribonuclease H1," "RNase H1," and "RNASEH1" are used interchangeably.

[0135] The RNase H family is a class of metal-ion-dependent endonucleases that specifically hydrolyze the RNA strand within RNA:DNA hybrids. There are two main types of RNase H family enzymes, RNase H1 and RNase H2, which share a common structural feature of a catalytic domain but have different substrate types. An R-loop is a triple-stranded nucleic acid structure. Most R-loops are generated during transcription, where the RNA transcript forms a double-stranded RNA:DNA hybrid with the template DNA, while the non-template DNA strand remains single-stranded. Both RNase H1 and RNase H2 can unwind R-loops, preventing their excessive accumulation, and play important roles in regulating gene expression, maintaining genome stability, and mitochondrial DNA replication. RNase H2's unique function is to remove mismatched single nucleotides from genomic DNA, thus participating in ribonucleotide repair.

[0136] 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.

[0137] Diseases related to defective neuronal dendritic growth

[0138] As used herein, the terms "neuronal dendritic growth defect-related diseases" and "neuronal dendritic growth abnormality diseases" can be used interchangeably and 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 disorder, and autism.

[0139] 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. This process is crucial for information exchange between neurons. Abnormal dendritic growth and development is one of the key pathogenic factors of many neurodevelopmental diseases, neurodegenerative diseases, and psychiatric diseases. 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, branching, and dendritic spines. Therefore, repairing defects in neuronal dendritic growth is a key means of treating and preventing these diseases.

[0140] R-loop degraders

[0141] The present invention provides the use of an R-loop degrader for preparing a drug for treating and / or preventing neuronal dendritic growth defects and related diseases.

[0142] As used herein, the term "R-loop degrader" refers to an agent capable of cleaving and degrading R-loops, or disrupting R-loop structures. In some embodiments of the present invention, the R-loop degrader is RNase H1 or its active fragment, or a promoter thereof. In some embodiments of the present invention, the R-loop degrader is a recombinant protein containing RNase H1 or its active fragment. In some embodiments of the present invention, the R-loop degrader comprises a coding sequence for RNase H1 or its active fragment. RNase H1 or its active fragment exerts its endonuclease activity to specifically hydrolyze RNA chains within R-loops, thereby degrading R-loops. RNase H1 promoters enhance RNase H1 enzymatic activity or expression levels, thereby promoting RNase H1's specific hydrolysis of RNA chains within R-loops, thereby degrading R-loops. RNase H1 or its active fragments or promoters, by restoring R-loop levels in neurons, partially offset or complement the increase in R-loops caused by MeCP2 mutations, thereby restoring normal neuronal function.

[0143] In one embodiment of the present invention, RNase H1 is wild-type RNase H1, and the RNase H1 active fragment is an 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 RNase H1).

[0144] In one embodiment of the present invention, the recombinant protein containing RNase H1 or its active fragment is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H or its active fragment with other polypeptides. In a preferred embodiment, the other polypeptide is MeCP2 or its active fragment. In a preferred embodiment, the recombinant protein containing RNase H1 or its active fragment is a chimeric protein formed by replacing the N-terminal hybrid binding domain (HBD) of RNase H1 with MeCP2 or its active fragment. In a preferred embodiment, the recombinant protein containing RNase H1 or its active fragment is a fusion protein formed by connecting MeCP2 or its active fragment to the N-terminus or C-terminus of RNase H1 or its active fragment.

[0145] The MeCP2 described in the present invention includes MeCP2 derived from various species, including (but not limited to) humans, mice, rats, rabbits, monkeys, dogs, etc. Furthermore, the MeCP2 includes wild-type MeCP2 or mutants thereof.

[0146] In a preferred embodiment, the amino acid sequence of MeCP2 is shown in SEQ ID NO: 18, and its gene sequence is shown in NM_004992.4 (Genbank accession number). In a preferred embodiment, the MeCP2 active fragment comprises the amino acid sequence shown in SEQ ID NO: 19 (derived from a partial amino acid sequence of the MeCP2 protein). In a preferred embodiment, the MeCP2 active fragment comprises the amino acid sequence shown in SEQ ID NO: 20 (derived from a partial amino acid sequence of the MeCP2 protein).

[0147] In a specific embodiment of the present invention, the recombinant protein containing RNase H1 or its active fragment is a chimeric protein formed by replacing the N-terminal hybrid chain binding domain (HBD) of the wild-type RNase H1 as shown in SEQ ID NO: 9 with the MeCP2 active fragment as shown in SEQ ID NO: 19 or SEQ ID NO: 20, i.e., the chimeric proteins MILH and ILH constructed in the examples of the present invention.

[0148] SEQ ID NO:9

[0149] SEQ ID NO: 10

[0150] SEQ ID NO: 18

[0151] SEQ ID NO: 19

[0152] SEQ ID NO:20

[0153] Drug combination and medicine kit of the present invention

[0154] The present invention also provides a drug combination comprising: (a) a detection reagent for detecting neuronal dendritic growth defects, and (b) the R-loop degrader of the present invention, for use in the 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 group consisting of: 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.

[0155] When using the drug combination of the present invention for treatment, the subject is first detected using the detection reagent to determine whether the subject has a neuronal dendritic growth defect; if so, the R-loop degrader is administered to the subject; wherein the subject suffers from a neurological disease.

[0156] In a preferred embodiment of the present invention, a test kit for MeCP2 gene mutations is first used to confirm whether the subject has a MeCP2 gene mutation. If the subject has a MeCP2 gene mutation, an R-loop degrader is administered to the subject. The MeCP2 gene mutation status of the subject can be confirmed by methods such as gene sequencing, qPCR, and immunoblotting (e.g., Western blotting).

[0157] The pharmaceutical combination of the present invention includes a safe and effective amount of an R-loop degrader. The term "safe and effective amount" refers to an amount of the active ingredient sufficient to significantly improve the condition or symptoms without causing serious side effects.

[0158] In addition, the pharmaceutical combination of the present invention can also be used together with other therapeutic agents.

[0159] Furthermore, the drug combination can be packaged into a suitable container and placed together with instructions to form a medicine kit, wherein the instructions describe how to use the drug combination and indicate that the medicine kit is used for the precise treatment and / or prevention of diseases associated with defects in neuronal dendritic growth.

[0160] The main advantages of the present invention include:

[0161] (1) The present invention discovered for the first time that elevated R-loop can lead to abnormal neuronal dendritic growth, and this mechanism is associated with a variety of neurological diseases, such as Rett syndrome.

[0162] (2) The present invention proposes for the first time that R-loop degraders (e.g., RNase H1 or its active fragments, recombinant proteins containing RNase H1 or its active fragments, or promoters of RNase H1) can be used to treat and / or prevent neuronal dendritic growth defects and related diseases. By administering the R-loop degrader RNase H1, the increase in R-loop caused by MeCP2 mutations can be partially offset or compensated, thereby restoring the normal function of neurons. The present invention reveals a new mechanism of abnormal neuronal dendritic growth and provides a new target for the treatment and prevention of related diseases.

[0163] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally 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.

[0164] Materials and Methods

[0165] 1.1 Primary culture of mouse cerebral cortical neurons

[0166] (1) Place a cover glass (Fisherbrand TM 12-545-100) were sequentially immersed in acetone, double-distilled water, and anhydrous ethanol and sonicated for 30 min. The pretreated coverslips were stored in anhydrous ethanol.

[0167] (2) 12-well cell culture plate per well ( CLS3512) were placed on each of the pretreated coverslips and 500 μL of poly-D-lysine (Gibco TM A3890401), and placed in a 37°C cell culture incubator overnight;

[0168] (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;

[0169] (4) Repeat step (3) once, remove all residual liquid, and air-dry for later use;

[0170] (5) P0 male C57BL / 6 mice (Shanghai Lingchang Biotechnology Co., Ltd.) were disinfected with 75% ethanol, the heads of the mice were cut off, the cortex was isolated and placed in pre-cooled 1× HBSS (0.14 g / L CaCl2, 0.1 g / L MgCl2·6H2O, 0.1 g / L MgSO4·7H2O, 0.4 g / L KCl, 0.06 g / L KH2PO4, 8 g / L NaCl, 0.09 g / L Na2HPO4·7H2O, 1 g / L D-Glucose), and two pieces of intact cortical tissue were isolated under a stereoscope and the hippocampus was peeled off;

[0171] (6) Add 1 mL of pre-cooled digestion solution, DMEM (Gibco) containing 10 μg / mL deoxyribonuclease I (Sigma-Aldrich DN25-10MG) and 1 U / μL papain (Worthington LS003126), to a 1.5 mL centrifuge tube. TM C11965500BT), the two peeled cortex pieces were placed in a tube and digested in a 37°C water bath for 20 min;

[0172] (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;

[0173] (8) Repeat step (7) once and remove the supernatant;

[0174] (9) Add 1 mL of Neurobasal medium (Gibco) preheated at 37°C containing 5% FBS. TM 21103049), pipette 1 mL 15-20 times to fully resuspend the digested tissue, and transfer it to a new centrifuge tube;

[0175] (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.

[0176] (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 TM 15140163) of Neurobasal medium.

[0177] 1.2 Oligonucleotide sequences

[0178] The following oligonucleotides were designed and synthesized

[0179] 1.3 Construction of shRNA Lentiviral Plasmids

[0180] Forward and reverse primers for control shRNA (SEQ ID NO:1 and SEQ ID NO:2) and Mecp2-targeting shRNA (SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6) were mixed in equimolar amounts in a 1.5 mL centrifuge tube, placed in boiling water, and allowed to anneal naturally at room temperature. The annealed products were inserted into the pLSAR lentiviral vector using the BamHI and XbaI restriction sites to generate the pLSAR-shLuc-EV-RFP, pLSAR-shMecp2-1-EV-RFP, and pLSAR-shMecp2-2-EV-RFP plasmids, respectively.

[0181] 1.4 Construction of lentiviral expression plasmids for combined shRNA overexpression of wild-type and mutant RNase H1

[0182] PCR amplification was performed using the ppyCAG_RNaseH1_WT plasmid (Addgene plasmid #111906) as a template and RNH1_F (SEQ ID NO: 7) and RNH1_R (SEQ ID NO: 8) as primers. The resulting PCR product was inserted into pLSAR-shMecp2-1-EV-RFP via the NheI and BsrGI restriction sites to obtain a lentiviral expression plasmid for shRNA combined with overexpression of RNase H1 wild type, namely pLSAR-shMecp2-1-RNaseH1-WT-RFP. PCR was performed using the 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 RNase H1 mutant, namely pLSAR-shMecp2-1-RNaseH1-D210N-RFP.

[0183] 1.5 Lentiviral infection of primary mouse neurons

[0184] (1) Plate HEK293T cells in a 6 cm cell culture dish 12 h in advance to ensure a cell density of 40-50% at the time of transfection;

[0185] (2) HEK293T cells were transfected with the corresponding lentiviral expression plasmid, 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;

[0186] (3) 8-12 h after transfection, change the medium and add 3 mL of Neurobasal medium;

[0187] (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 long-term storage;

[0188] (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 the neurons and the cells were returned to the 37°C cell culture incubator.

[0189] (6) 24 h after viral infection, the virus was aspirated and preheated fresh primary neuron culture medium and cell culture medium collected before virus infection were mixed at a ratio of 1:1 and added;

[0190] (7) Change half of the medium of primary neurons every two days until DIV7.

[0191] 1.6 Detection of protein expression using immunoblotting

[0192] (1) Aspirate the culture medium of the primary neurons to be tested and directly add 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). Collect the cells into a 1.5 mL centrifuge tube and fully denature them in a 95°C metal bath.

[0193] (2) The prepared protein samples were electrophoresed using 10% SDS-PAGE gel in a buffer solution (3.029 g / L Tris-base, 18.768 g / L Glycine, 1 g / L SDS);

[0194] (3) Separate the electrophoresed SDS-PAGE gel with a nitrocellulose membrane of the same size (Amersham TM 10600002) and filter paper were soaked 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 apparatus ( SD Semi-Dry Transfer Cell) transfer, transfer conditions are 12V, 60min;

[0195] (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.

[0196] (5) After blocking, discard the blocking solution and add the corresponding primary antibody diluted with blocking solution and incubate at room temperature for 2 hours 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 wash the membrane twice with blocking buffer at room temperature for 15 minutes each time. After washing, add the corresponding secondary antibody diluted 1:2000 in blocking buffer and incubate at room temperature for 1 hour. After the secondary antibody binds, discard the antibody and wash the membrane twice with blocking buffer at room temperature for 15 minutes each time.

[0197] (6) After washing the membrane, use color developing solution (Millipore WBKLS0500) to develop the color. TM Imager 680 was used for detection and analysis.

[0198] 1.7 Detection of dendritic growth

[0199] (1) Primary neurons were cultured in vitro until DIV7. The coverslips containing 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 cells were slowly washed on a horizontal shaker for 5 minutes.

[0200] (2) Aspirate PBS and add 4% PFA prepared with PBS as solvent to each well and fix at room temperature for 10 min;

[0201] (3) Aspirate 4% PFA, add PBS, and wash the coverslips three times on a horizontal shaker, each time for 8 minutes;

[0202] (4) Add a small amount of fluorescent mounting medium to 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 in the dark overnight;

[0203] (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;

[0204] (6) The number and length of dendrites of the captured neurons were analyzed using the NeuronJ 1.4.3 plug-in of Fiji (Fiji Is Just ImageJ). The raw data were statistically analyzed using Prism 8.0 (Graphpad) and Excel (Microsoft).

[0205] (7) The double-blind principle was followed during dendritic growth detection and data analysis.

[0206] 1.8 Construction of prokaryotic expression plasmids for RNase H1 and chimeric proteins

[0207] Using the aforementioned pLSAR-shMecp2-1-RNaseH1-WT-RFP plasmid as a template and His-RNH1_F (SEQ ID NO: 11) and His-RNH1_R (SEQ ID NO: 12) as primers, PCR amplification was performed, and the resulting PCR product was inserted into the pET28a vector via the HindIII and XhoI restriction sites to obtain the RNase H1 prokaryotic expression plasmid, i.e., pET28a-RNH1; using the pCMV-3*FLAG-MeCP2 plasmid as a template and MILH_F (SEQ ID NO: 13) and mid_R (SEQ ID NO: 14) as primers, PCR amplification was performed to obtain fragment 1; using the pET28a-RNH1 plasmid as a template and mid_F (SEQ ID NO: 15) and CAT_R (SEQ ID NO: 16) as primers, PCR amplification was performed to obtain fragment 2. Overlap extension PCR amplification was performed using fragment 1 and fragment 2 as templates and MILH_F and CAT_R as primers. The resulting PCR product was inserted into the pET28a vector via the HindIII and XhoI restriction sites to obtain the prokaryotic expression plasmid of MeCP2 and RNase H1 chimeric protein 1, namely, pET28a-MILH. PCR amplification was performed using pET28a-MILH as a template and ILH_F (SEQ ID NO: 17) and CAT_R as primers. The resulting PCR product was inserted into the pET28a vector via the HindIII and XhoI restriction sites to obtain the prokaryotic expression plasmid of MeCP2 and RNase H1 chimeric protein 2, namely, pET28a-ILH.

[0208] 1.9 Expression and purification of RNase H1 and chimeric proteins in prokaryotes

[0209] Prokaryotic protein expression and purification were performed using conventional methods in the art. The exemplary steps are as follows:

[0210] (1) The prokaryotic expression plasmids of RNase H1 and chimeric protein were transformed into competent cells of Escherichia coli Rosetta (DE3) strain, single clones were picked and cultured in 200 mL LB medium at 37°C in a shaking incubator until OD 600 When the pH reached 0.7, 0.5 mM IPTG was added for induction, and the cells were cultured in a shaking incubator at 18 °C overnight;

[0211] (2) The induced bacterial liquid was collected by high-speed centrifugation at 6,000 g at 4°C for 15 minutes, the supernatant was discarded, and the liquid was quickly frozen at -80°C.

[0212] (3) Resuspend and lyse the bacterial pellet using lysis buffer, perform ultrasonic disruption at a power of 200 W, centrifuge the fully disrupted lysate at 14,000 rpm at 4°C for 10 min, and collect the supernatant;

[0213] (4) The supernatant after lysis was mixed with 200 μL Ni-NTA Agarose Beads (Qiagen) and rotated at 4°C for 3 h;

[0214] (5) Centrifuge the bound beads at 800 rpm at 4°C for 5 minutes and discard the supernatant. Wash the beads three times with wash buffer, spinning for 5 minutes each time.

[0215] (6) Add 200 μL of elution buffer to the beads and rotate at 4°C to elute the protein for 2-3 times, each time for 20 minutes;

[0216] (7) The protein eluate was dialyzed in a dialysis bag in a storage buffer at 4°C for 4 hours. After dialysis, the protein sample was quickly frozen at -80°C.

[0217] (8) Take an appropriate amount of dialyzed protein sample and a BSA protein standard of known concentration and mix them with 2×SDS loading buffer respectively. After full denaturation in a 95°C metal bath, perform 10% SDS-PAGE gel electrophoresis and stain the gel with Coomassie Brilliant Blue staining solution. After decolorization with a decolorizing solution, determine the molecular weight and specificity of the protein, and determine the protein concentration by comparison with the BSA sample.

[0218] 1.10 In vitro enzyme activity assay of RNase H1 and chimeric proteins

[0219] (1) An RNA oligonucleotide labeled with FAM at its 3' end and an unlabeled DNA oligonucleotide complementary thereto were mixed in annealing buffer at an equal molar ratio, denatured at 95°C for 5 minutes, and then slowly cooled to room temperature to prepare an RNA-DNA hybrid chain as a substrate for the enzyme activity reaction;

[0220] (2) 4 pmol of RNA-DNA hybrid chain was used as substrate, 1 μL of 10× Reaction Buffer (25 mM Tris-HCl pH 7.5, 1 mM DTT, 5 mM MgCl2, 50 μg / mL BSA, 50 mM KCl) was added, and different amounts of RNase H1 or chimeric protein were added to the reaction system with a total volume of 10 μL. The enzyme activity reaction was carried out at 37°C for 5 minutes;

[0221] (3) After the enzyme activity reaction, add 10 μL of 2× RNA loading buffer and denature at 98°C for 10 minutes. Electrophorese the enzyme activity reaction samples using 15% Urea PAGE.

[0222] (4) The fluorescence detection module (488 nm laser) in Typhoon FLA-9000 (GE) was used to image the Urea PAGE after electrophoresis;

[0223] (5) Use ImageJ to perform grayscale quantitative analysis of enzyme activity reaction products.

[0224] Example 1 Knockdown of Mecp2 in primary neuronal cells leads to dendritic growth defects

[0225] Mouse primary neurons were infected with shRNA lentivirus, with shLuc serving as a control and shMecp2-1 and shMecp2-2, two shRNAs targeting Mecp2 with different sequences. Compared to shLuc, both shMecp2-1 and shMecp2-2 significantly reduced Mecp2 protein expression in primary neurons (Figure 1).

[0226] 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, specifically a significant decrease in the number of dendrites per neuron and a significant decrease in the sum of the dendrite lengths of each neuron (Figure 2). In Figure 2A, the number of dendrites in neurons of the control group (shLuc) and Mecp2 knockdown (shMecp2-1, shMecp2-2) was counted. Each data point represents the number of dendrites in a neuron, and the horizontal line on the graph represents the average number of dendrites in each group of neurons. The number of dendrites in 35 neurons was detected and counted in each group. Figure 2B shows the summed dendritic lengths of neurons in the control group (shLuc) and Mecp2 knockdown (shMecp2-1, shMecp2-2). Each data point represents the summed dendritic length of a single neuron, and the horizontal line represents the mean summed dendritic length for each group (in μm). The summed dendritic lengths of 35 neurons were examined and counted in each group. Mann-Whitney test: ***p < 0.001.

[0227] Example 2 Wild-type RNASEH1 can restore dendritic growth defects caused by Mecp2 knockdown

[0228] Through R-loop Dot-Blot and ssDRIP-seq experiments, the inventors found that R-loop levels were significantly upregulated in the cortical tissue of mice with neuronal-specific Mecp2 knockout. To determine whether restoring R-loop levels in Mecp2-deficient neurons could repair the defects in dendritic growth, they overexpressed wild-type and enzyme-active mutant RNase H1 in primary neurons with Mecp2 knockdown.

[0229] As shown in Figure 3, the Mecp2 protein level in neurons infected with shMecp2-1 was significantly reduced compared with the control group, and the protein levels of RNase H1 overexpressing wild type or enzyme-active mutant were similar.

[0230] The dendritic growth of primary neurons infected with different lentiviruses was detected and analyzed at DIV7 (Figure 4). After overexpressing wild-type RNase H1 in Mecp2-knockdown neurons, the total number of dendrites and dendritic length of each neuron was 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 RNase H1 in Mecp2-knockdown neurons failed to restore the dendritic growth defect. In Figure 4A, the number of dendrites of neurons in the control group (shLuc), Mecp2 knockdown (shMecp2-1), and Mecp2 knockdown and overexpression of wild-type RNase H1 (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 graph represents the average number of dendrites in each group of neurons. The number of dendrites of 35 neurons in each group was detected and counted. Figure 4B shows the summed dendritic lengths of neurons in the control group (shLuc), Mecp2 knockdown (shMecp2-1), and Mecp2 knockdown with simultaneous overexpression of wild-type RNase H1 (shMecp2-1+RNH1-WT) or an enzyme-activated mutant (shMecp2-1+RNH1-D210N). Each data point represents the summed dendritic length of a single neuron, and the horizontal line represents the mean summed dendritic length for each group (in μm). The summed dendritic lengths of 35 neurons were examined and counted for each group. Mann-Whitney test: ***p < 0.001; ns p > 0.05.

[0231] Table 1

[0232] Example 3 RNase H1 and MeCP2 recombinant proteins can promote RNase H1 R-loop degradation activity

[0233] To further enhance the R-loop degradation activity of RNase H1, the present inventors modified RNase H1 and found that the chimeric proteins MILH and ILH, composed of the active fragment of RNase H1 and a partial protein sequence of MeCP2, showed higher activity in degrading RNA-DNA hybrid chains in in vitro biochemical experiments ( FIG5 ).

[0234] In Figure 5A, RNase H1, MILH, and ILH all have the activity of degrading RNA-DNA hybrid chains. At the same protein concentration, compared with RNase H1, MILH and ILH have significantly higher degradation activity on RNA-DNA hybrid chains. In Figure 5B, the degradation ratios of RNA-DNA hybrid chain substrates at different concentrations of RNase H1, MILH, and ILH are statistically analyzed (Two-way ANOVA test: ***p<0.001). It can be seen that the chimeric proteins MILH and ILH exhibit high degradation activity on RNA-DNA hybrid chains at low protein concentrations, which can reach about 3 times the degradation activity of RNase H1.

[0235] Example 4 RNase H1 and MeCP2 recombinant proteins restore dendritic growth defects caused by Mecp2 knockdown

[0236] Similar to Example 2, wild-type RNase H1 and chimeric proteins MILH and ILH were overexpressed in Mecp2-knockdown primary neurons, respectively, and the chimeric proteins MILH and ILH were tested for their ability to restore neuronal dendritic growth defects caused by Mecp2 knockdown. The number and length of dendrites in primary neurons infected with different lentiviruses were detected and analyzed at DIV7. The results showed that both chimeric proteins MILH and ILH were able to effectively restore dendritic growth defects caused by Mecp2 knockdown, and exhibited better restoration effects than wild-type RNase H1.

[0237] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 (RNase H1) 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 The R-loop degrading agent includes a recombinant protein containing RNase H1 or an active fragment thereof.

5. The use according to claim 4, characterized in that The recombinant protein containing RNase H1 or its active fragment includes: a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or its active fragment with other polypeptides.

6. The use according to claim 4, characterized in that The recombinant protein containing RNase H1 or its active fragment is a chimeric protein or fusion protein having R-loop degradation activity formed by the recombination of RNase H1 or its active fragment and MeCP2 or its active fragment.

7. The use according to claim 2, characterized in that The RNase H1 promoter includes small molecule compounds, nucleic acids, polypeptides and proteins.

8. The use according to claim 1, characterized in that The subject or patient suffering from or suspected of suffering from the neuronal dendritic growth defect and its related diseases has 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.

9. The use according to claim 1, 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.

10. 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.

11. The pharmaceutical composition according to claim 10, 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.

12. 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.

13. 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.

14. 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.

15. 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.

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