Gene therapy for Rett syndrome

Abstract

The invention provides a gene expression cassette containing a polynucleotide sequence for coding human MeCP2 protein, a viral vector carrying the gene expression cassette and application of the viral vector in treatment of Rett syndrome. According to the present invention, the provided gene expression cassette introduces one or more miRNA binding sites, such that the overexpression of the MeCP2 in the brain tissue is prevented, the expression of the MeCP2 in the DRG and the liver is reduced, and the safety problem possibly caused by the gene therapy is solved;

Description

A gene therapy for Rett syndrome Technical Field

[0001] The present application relates to the field of gene therapy, and in particular to a gene expression cassette comprising a polynucleotide sequence encoding human MeCP2 protein, a viral vector carrying the gene expression cassette, and use thereof in the treatment of Rett syndrome. Background Art

[0002] Rett syndrome (RTT) is a severe neurological disorder with a prevalence of 1 in 15,000 to 1 in 10,000 children. It is caused by mutations or deletions in the X-linked gene Mecp2. Because the mutated Mecp2 gene is primarily located on the paternal X chromosome, female patients are far more likely to be affected than males. Clinical features include progressive intellectual decline, autistic behaviors, stereotyped movements, and gait abnormalities, with symptoms typically becoming apparent 6-18 months after birth. Most patients with Rett syndrome survive to adulthood but are unable to care for themselves. Currently, there is no effective cure for RTT; interventions to improve symptoms, such as physical therapy, surgery, and anti-epileptic treatment, are available based on the patient's clinical stage.

[0003] Animal experiments have found that reactivating the Mecp2 gene in conditional Mecp2 knockout mice can effectively reverse RTT-related symptoms. Therefore, introducing a normal Mecp2 gene via a vector to replace the defective Mecp2 gene may be a potential treatment for Rett syndrome. However, low-level Mecp2 gene expression may not successfully rescue symptoms, and overexpression of Mecp2 in brain tissue can cause toxicity. Therefore, the challenge facing this gene therapy approach is how to transduce a sufficient number of neurons in brain tissue while avoiding harmful overexpression of Mecp2.

[0004] Over the past few decades, gene therapy based on adeno-associated virus (AAV) vectors has shown great potential in treating genetic diseases. Therefore, it is possible to introduce an exogenous MeCP2 gene via AAV vectors, thereby expressing normal MeCP2 protein in the body and treating Rett syndrome from the root cause. However, previous studies have found that AAV vectors enter the central nervous system of nonhuman primates (NHPs) through the blood or cerebrospinal fluid and cause toxicity in the dorsal root ganglion (DRG), with the most severe manifestation of toxicity being ataxia. Traditional immunosuppressive regimens are unable to prevent this toxicity, possibly because it may be caused by high transduction rates, which in turn can cause cellular stress due to excessive transgene production in target cells. Therefore, there is an urgent need to develop safer and more effective gene therapy drugs for the treatment of Rett syndrome.

[0005] It should be noted that the approaches described in this section are not necessarily approaches that have been previously conceived or employed. Unless otherwise indicated, it should not be assumed that any approach described in this section is prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art.

[0006] Summary of the Invention

[0007] To address the above-mentioned issues, the present application provides a gene expression cassette comprising a polynucleotide sequence encoding a human MeCP2 protein, characterized in that the gene expression cassette comprises at least one first miRNA binding site, wherein the first miRNA binding site comprises one or more of a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, or a miR-130 binding site. The gene expression cassette in the present application, by carrying the polynucleotide sequence encoding the human MeCP2 protein, can correctly express the MeCP2 protein in brain tissue. Furthermore, by incorporating one or more first miRNA binding sites, the gene expression cassette utilizes the characteristic of miRNAs inhibiting gene expression at specific sites to further regulate the expression level of the MeCP2 gene at the post-transcriptional level, thereby preventing overexpression of MeCP2 in brain tissue and avoiding toxicity. The gene expression cassette provided in this application ensures that the MeCP2 protein has a certain expression in brain tissue while controlling the expression level of the MeCP2 protein in the brain tissue to not be too high. Since too low or too high expression levels of the MeCP2 protein in the brain tissue cannot effectively treat Rett syndrome, the gene expression cassette provided in this application has high technical difficulty and good application prospects.

[0008] In some embodiments, the gene expression cassette further comprises a second miRNA binding site and / or a third miRNA binding site. The gene expression cassette in the present application, by adding one or more second miRNA binding sites and / or third miRNA binding sites, reduces the expression of MeCP2 protein in DRG and liver, thereby alleviating DRG toxicity and liver toxicity caused by AAV vectors and reducing the body's adverse reactions to AAV vectors and transgenic products.

[0009] In some embodiments, the gene expression cassette further comprises a promoter comprising at least one of a MeCP2 core promoter, a MeCP2 silencer element, or a CNS regulatory element. In some embodiments, the promoter comprises MeP549, MeP426, MeP322, or MeP223. By selecting a specific promoter, the expression level of MeCP2 protein in brain tissue can be further regulated to achieve a more appropriate expression level to meet the treatment needs of Rett syndrome.

[0010] In some embodiments, the gene expression cassette is no longer than about 2.2 kb. The gene expression cassette in the present application has a shorter length, thereby avoiding exceeding the upper limit of the AAV vector's carrying capacity due to the longer gene length when delivered using the AAV vector.

[0011] According to one embodiment of the present application, a recombinant vector is further provided, characterized in that the recombinant vector comprises the gene expression cassette described in the claims of the present application.

[0012] According to one embodiment of the present application, a pharmaceutical composition is further provided, characterized in that the pharmaceutical composition comprises the gene expression cassette described in the present application and the recombinant vector described in the present application.

[0013] According to one embodiment of the present application, further provided is the use of the gene expression cassette, the recombinant vector, and the pharmaceutical composition described herein for expressing MeCP2 protein in cells.

[0014] According to one embodiment of the present application, there is also provided the use of the gene expression cassette, the recombinant vector, and the pharmaceutical composition described herein in the preparation of drugs for detecting, preventing or treating diseases.

[0015] According to one embodiment of the present application, a method for detecting, preventing or treating a disease is also provided, comprising administering the gene expression cassette, the recombinant vector, and the pharmaceutical composition described herein to a subject in need thereof.

[0016] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings illustrate exemplary embodiments and constitute a part of the specification. Together with the description of the specification, they serve to explain exemplary implementation of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals designate similar, but not necessarily identical, elements.

[0018] Figure 1 shows a diagram of the elements of the vector scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130 provided in Example 1 of the present application. The miRNA binding sites contained in the vector include: a miR-22 binding site located in pA1 (proximal termination sequence), a miR-132 binding site and a miR-124 binding site located in pA2 (distal termination sequence), and a miR-183 binding site, a miR-483 binding site, a miR-122 binding site, and a miR-130 binding site located between pA1 and pA2.

[0019] Figure 2 shows the results of MeCP2 protein expression in Neuro-2a cells after transfection of different vectors in Example 2.1. Different labels in the figure represent different vectors used for transfection: #1 represents scAAV-MeP223-Mecp2-pA1-pA2, #2 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122, #3 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-1244, and #4 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122 -130, #5 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-132, #6 represents scAAV-MeP322-Mecp2-pA1-pA2, #7 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122, #8 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-124, and #9 represents scAAV-MeP322- Mecp2-pA1-pA2-183-483-122-130, #10 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132, #11 represents scAAV-MeP426-Mecp2-pA1-pA2, #12 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132, #13 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132 -483-122-124, #14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130, #15 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132, EGFP represents scAAV-MeP426-EGFP(c-myc)-pA49, #549 represents scAAV-MeP549-Mecp2-pA49, and blank represents the untransfected blank group.

[0020] Figure 3 shows the results of MeCP2 protein expression in Neuro-2a cells after transfection with different vectors in Example 2.2. The different labels in the figure represent the use of different vectors for transfection: blank represents the untransfected blank group, PC represents scAAV-MeP426-Mecp2-pA1-pA2, 14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130, and Let-7a-1 represents scAAV-MeP426-Mecp2-pA1-pA2-let-7a -1,802 represents scAAV-MeP426-Mecp2-pA1-pA2-802, 99a-5p represents scAAV-MeP426-Mecp2-pA1-pA2-99a-5p, 100-5p represents scAAV-MeP426-Mecp2-pA1-pA2-100-5p, and 1200 represents scAAV-MeP426-Mecp2-pA1-pA2-1200.

[0021] FIG4 shows the results of Mecp2 protein expression after Neuro-2a cells were infected with different vectors packaged with serotype AAV9 in Example 2.3. Among them, different labels in the figure represent different vectors used for infection: #6 represents scAAV-MeP322-Mecp2-pA1-pA2, #7 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122, #8 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-124, #9 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-130, #10 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132, #11 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132 2, #12 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122, #13 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-124, #14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130, #15 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132, EGFP represents scAAV-MeP426-EGFP(c-myc)-pA49, #549 represents scAAV-MeP549-Mecp2-pA49, and blank represents the untransfected blank group.

[0022] Figure 5 shows the results of Mecp2 protein expression in the brain, spinal cord and liver of mice after mice were infected with the vector scAAV-pMeP549-Mecp2-pA49 packaged with three serotypes of AAV1, AAV8 and AAV9 in Example 2.4.

[0023] Figure 6 shows the survival results of the Rett model mice in Example 3.3. Different labels in the figure represent the use of different vectors for treatment: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 8245 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 45 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT group represents wild-type mice that were not administered with medication, hemi group represents model mice that were not administered with medication, and vehicle represents Rett model mice that were administered with drug vehicle.

[0024] Figure 7 shows the stride length results of the Rett model mice in Example 3.4. Different labels in the figure represent the use of different vectors for treatment: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 8245 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 45 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT group represents wild-type mice that were not administered, hemi group represents model mice that were not administered, and vehicle represents Rett model mice that were administered the drug vehicle. RF represents the right forelimb of the mouse, RH represents the right hindlimb, LF represents the left forelimb, and LH represents the left hindlimb.

[0025] Figure 8 shows the swing speed results of the Rett model mice in Example 3.4. Different labels in the figure represent the use of different vectors for treatment: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, and 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130. 130, 8245 represent scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT group represents wild-type mice that were not treated with the drug, hemi group represents model mice that were not treated with the drug, and vehicle represents Rett model mice that were administered with the drug vehicle. RF represents the right forelimb of the mouse, RH represents the right hindlimb, LF represents the left forelimb, and LH represents the left hindlimb.

[0026] Figure 9 shows the results of the regularity index (RI) of the Rett model mice in Example 3.4. The different labels in the figure represent the treatment with different vectors: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 8245 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 45 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT group represents wild-type mice that were not administered, hemi group represents model mice that were not administered, and vehicle represents Rett model mice that were administered the drug vehicle. RF represents the right forelimb of the mouse, RH represents the right hindlimb, LF represents the left forelimb, and LH represents the left hindlimb.

[0027] FIG10 shows the survival results of Rett model mice in Example 4.1. The different labels in the figure represent different doses of vector 8244 used for treatment: Dose 1 represents 3.5×10 11 vg / each, Dose 2 represents 1.75×10 11 vg / piece, Dose 3 represents 8.75×1010 vg / piece, Dose 4 represents 4.38×10 10 vg / piece, Dose 5 represents 1.75×10 10 vg / mouse, WT represents wild-type mice that were not given medication, and vehicle represents Rett model mice that were given drug vehicle.

[0028] FIG11 shows the results of the rotarod test of Rett model mice in Example 4.2. The different labels in the figure represent different doses of vector 8244 used for treatment: Dose 1 represents 3.5×10 11 vg / each, Dose 2 represents 1.75×10 11 vg / piece, Dose 3 represents 8.75×10 10 vg / piece, Dose 4 represents 4.38×10 10 vg / piece, Dose 5 represents 1.75×10 10 vg / mouse, WT represents wild-type mice that were not given medication, and vehicle represents Rett model mice that were given drug vehicle.

[0029] FIG12 shows the results of Mecp2 protein expression in the mouse brain and spinal cord in Example 5.1. Among them, different labels in the figure represent the use of different vectors for treatment: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 8245 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT represents wild-type mice that were not administered medication, and vehicle represents Rett model mice that were administered drug solvent.

[0030] Figures 13, 14 and 15 show the staining results of pathological sections of mouse liver tissue in Example 5.2. Among them, different labels in the figure represent the use of different vectors for treatment: 8241 represents scAAV9-MeP549-Mecp2-pA49, 8243 represents scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130, 8244 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130, 8245 represents scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132, 8246 represents scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132, WT represents wild-type mice that were not administered medication, and vehicle represents Rett model mice that were administered drug solvent.

[0031] Figure 16 shows a schematic diagram of miRNA binding sites in the vector scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130 provided in Example 1 of the present application. Among them, miR-22 represents a miR-22 binding site, miR-132 represents a miR-132 binding site, miR-124 represents a miR-124 binding site, miR-183 represents a miR-183 binding site, miR-483 represents a miR-483 binding site, miR-122 represents a miR-122 binding site, miR-130 represents a miR-130 binding site, and spacer represents a spacer sequence. DETAILED DESCRIPTION

[0032] Unless otherwise indicated, all numbers used in this specification and claims to represent content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" under any circumstances. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values. Unless otherwise indicated, the terms used herein have the usual understanding meanings for those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through this application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0033] Although the numerical ranges and parameters setting forth the broad scope of this application are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. Every numerical range given herein will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0034] Unless otherwise specified or contradicted by the context, the terms or expressions used in this article should be read in conjunction with the entire content of this article and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0036] As used herein, "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0037] As used herein, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included within the definition of polypeptide.

[0038] It should be noted that in the context of this application, upstream refers to the 5' end of the gene or the N-terminus of the protein, and downstream refers to the 3' end of the gene or the C-terminus of the protein, and from upstream to downstream is from 5' end to 3' end or N-terminus to C-terminus.

[0039] As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, phages, and insertable DNA segments.

[0040] As used herein, "adeno-associated viral vectors" are derived from adeno-associated viruses. An "adeno-associated viral vector" is derived from the wild-type genome of the virus by removing all or part of the wild-type genome from the adeno-associated viral genome using molecular methods and replacing it with a heterologous (non-natural) nucleic acid (e.g., a nucleic acid encoding a therapeutic protein or polynucleotide sequence). Typically, for adeno-associated viral vectors, one or two inverted terminal repeats (ITRs) in the adeno-associated viral genome are retained in the adeno-associated viral vector. Adeno-associated viral vectors can be used as gene therapy vectors because they can introduce nucleic acid / genetic material into cells so that the nucleic acid / genetic material can remain in the cells.

[0041] When used herein, " serotype " refers to an adeno-associated virus with a capsid that is serologically different from other adeno-associated virus serotypes. Serological specificity is based on the cross-reactivity between the lack of antibodies to a kind of adeno-associated virus compared with another adeno-associated virus to determine. Such cross-reactivity differences are usually due to the difference in capsid protein sequence / antigenic determinants (for example, due to VP1, VP2 and / or VP3 sequence differences of adeno-associated virus serotypes). Under the traditional definition, serotype means that the virus of interest has been tested for neutralizing activity with serum specific for all existing and characterized serotypes, and no antibodies to neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, there may or may not be serological differences with any one of the currently existing serotypes. Therefore, in the case where a new virus (such as an adeno-associated virus) does not have serological differences, this new virus (such as an adeno-associated virus) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not been performed on mutant viruses with modified capsid sequences to determine whether they are of another serotype according to the traditional serotype definition. Therefore, for convenience and to avoid repetition, the term "serotype" is used broadly to refer to serologically distinct viruses (e.g., adeno-associated viruses) as well as viruses that are not serologically distinct (e.g., adeno-associated viruses) that may be within a subgroup or variant of a given serotype.

[0042] As used herein, "miRNA (microRNA)" refers to a single-stranded non-coding RNA of a certain length. The RNA-induced silencing complex formed by miRNA and protein factors can recognize target sequences in mRNA and reduce the expression level of mRNA by degrading mRNA molecules, promoting 3'-end deadenylation of mRNA molecules, and inhibiting translation, thereby regulating gene expression at the post-transcriptional level (Kim VN. Nat Rev Mol Cell Biol. 2005; 6(5): 376-385.).

[0043] As used herein, "miRNA target sequence" and "miRNA binding site" are used interchangeably to refer to a nucleotide sequence that can bind to a specific miRNA. In some non-limiting embodiments of the present application, the miRNA target sequence is designed within the 3' transcription termination sequence of the Mecp2 gene, which can bind to a specific miRNA to inhibit the expression level of the Mecp2 gene.

[0044] As used herein, "operably linked" refers to the connection of multiple nucleic acid segments in a functional relationship. A nucleic acid is "operably linked" when it forms a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence (or gene), and the promoter is connected in such a way that it can induce transcription of the gene. Operably linked means that the nucleotide sequences being linked may be contiguous or discontinuous.

[0045] As used herein, the terms "relieve," "treat," and their synonyms refer to the improvement of a disease, disorder, and / or condition. "Relieve," "treat," can be an improvement in at least one measurable physical parameter, which is not necessarily recognizable by the patient. "Relieve," "treat," can also be the inhibition of the development of a disease, disorder, and / or condition physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both. "Relieve," "treat," can also be the slowing down of the development or reversal of a disease, disorder, and / or condition.

[0046] As used herein, the term "prevent," "prevent," and its synonyms refer to delaying the onset of or reducing the risk of acquiring a particular disease, disorder, and / or condition, or symptoms associated with such disease, disorder, and / or condition.

[0047] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below.

[0048] Gene expression cassette

[0049] According to one embodiment of the present application, a gene expression cassette comprising a polynucleotide sequence encoding a human MeCP2 protein is provided, wherein the gene expression cassette comprises at least one first miRNA binding site. In some non-limiting embodiments, the human MeCP2 protein comprises the amino acid sequence shown in SEQ ID NO: 1. In some non-limiting embodiments, the MeCP2 protein may additionally comprise a heterologous (i.e., non-MeCP2) sequence (e.g., at the C-terminus of the molecule), such as an epitope tag C-myc that facilitates isolation or identification. The sequence of the MeCP2 protein after adding C-myc is as follows (the underlined portion is the heterologous sequence other than MeCP2): MAAAAAAAPSGGGGGGEEERLEEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGR PKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSK ESSPKGRSSSSASSPPKKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVSGSSSGSSGEQKLISEEDL.

[0050] In some embodiments, the first miRNA binding site comprises at least one or more of a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, or a miR-130 binding site. In some preferred embodiments, the first miRNA binding site comprises at least one of the following groups: (1) a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, and a miR-483 binding site; and / or (2) a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, and a miR-130 binding site. In some more preferred embodiments, the first miRNA binding site includes at least one of the following groups: (1) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, and 1 miR-483 binding site; (2) 1 miR-22 binding site, 1 miR-132 binding site, 2 miR-124 binding sites, and 1 miR-483 binding site; (3) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, 1 miR-483 binding site, and 1 miR-130 binding site; and / or (4) 1 miR-22 binding site, 2 miR-132 binding sites, 1 miR-124 binding site, and 1 miR-483 binding site. In some embodiments, the first miRNA binding site can reduce the expression efficiency of the polynucleotide sequence encoding the human MeCP2 protein in human brain tissue. The presence of multiple miRNA binding sites can lead to the cooperative action of multiple RISCs and provide efficient expression inhibition.

[0051] The length of the 3' transcription termination sequence in brain tissue mainly includes 200bp and 8.5kb, and the mRNA content of 8.5kb is more than 80%. In order to better play the role of the two transcription termination signals, the two transcription termination sequences are combined. In some embodiments, the first miRNA binding site is located in the 3' transcription termination sequence of the gene expression frame, and the 3' transcription termination sequence includes a distal termination sequence and / or a proximal termination sequence. In some embodiments, the proximal termination sequence (as shown in SEQ ID NO: 2) includes a miR-22 binding site. In some non-limiting embodiments, the following is a proximal termination sequence, wherein the underlined portion is a binding site for miRNA: acaagaataaaggcagctgttgtctcttctccttatgggtagggctctgacaaagcttccc. In some embodiments, the distal termination sequence (as shown in SEQ ID NO: 3) comprises at least one of a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, or a miR-130 binding site.

[0052] In some embodiments, the distal termination sequence further comprises an upstream sequence element (USE) and a core upstream element (CSE). In the distal termination sequence, a length of 5-50 bp is maintained between the upstream sequence element (USE) and the core upstream element (CSE), wherein multiple miRNA binding sites are integrated to regulate the expression of MeCP2. In some non-limiting embodiments, the following is a distal termination sequence, wherein the underlined portion is the upstream sequence element (USE) and the core upstream element (CSE): atatcaccaggactgttaatatatttaaaaacaaaaggcaatttattaaggaaatttgtaccatttcagtaaacctgtctgaatgtacctgtatacgtttcaaaaacaccccccccccactgaatccctgtaacctatttattatataaagagtttgccttataaatttacataaaaatgtccgtttgtgtcttttgttg In some embodiments, the miR-483 binding site (as shown in SEQ ID NO: 4) and the miR-130 binding site (as shown in SEQ ID NO: 5) are located between the upstream sequence element and the core upstream element. In some embodiments, the miR-132 binding site (as shown in SEQ ID NO: 6) and the miR-124 binding site (as shown in SEQ ID NO: 7) are not located between the upstream sequence element and the core upstream element.

[0053] In some embodiments, at least one thymine nucleotide in the miR-483 and miR-130 binding sites is replaced with an adenine nucleotide. In some non-limiting embodiments, in order to enhance the inhibitory effect of the miRNA binding site, based on the characteristics of the Ago2 protein, the nucleotide at the first position at the 3' end of the miR-483 and miR-130 binding sites is replaced with an adenine nucleotide. In some non-limiting embodiments, the underlined portion in the following sequence is the nucleotide position at which miR-483 is replaced with an adenine nucleotide: CTCCCTTCTTTCCTCCCGTCTa. In some non-limiting embodiments, the underlined portion in the following sequence is the nucleotide position at which miR-130 is replaced with an adenine nucleotide: ATGCCCTTTTAACATTGCACTa. In some preferred embodiments, the nucleotide sequence of the miR-22 binding site includes GGCAGCT; the miR-132 binding site includes the nucleotide sequence shown in SEQ ID NO:6; the miR-124 binding site includes the nucleotide sequence shown in SEQ ID NO:7; the miR-483 binding site includes the nucleotide sequence shown in SEQ ID NO:4; and / or the miR-130 binding site includes the nucleotide sequence shown in SEQ ID NO:5.

[0054] In some embodiments, the gene expression cassette further comprises a second miRNA binding site and / or a third miRNA binding site. In some embodiments, the second miRNA binding site can reduce the expression efficiency of the polynucleotide sequence encoding the human MeCP2 protein in human dorsal root ganglia, and the third miRNA binding site can reduce the expression efficiency of the polynucleotide sequence encoding the human MeCP2 protein in human liver tissue. The presence of multiple miRNA binding sites can lead to the cooperative action of multiple RISCs and provide efficient expression inhibition. In some embodiments, the second miRNA binding site and / or the third miRNA binding site include one or more of the miR-183 binding site, miR-182 binding site, miR-96 binding site, miR23b binding site, miR-145 binding site, miR-148a binding site, miR-22 binding site, miR-122 binding site, miR-143 binding site, miR-21 binding site, or miR-192 binding site. In some preferred embodiments, the second miRNA binding site includes the miR-183 binding site. In some preferred embodiments, the third miRNA binding site comprises a miR-122 binding site. In some preferred embodiments, the miR-183 binding site comprises the nucleotide sequence set forth in SEQ ID NO: 9. In some preferred embodiments, the miR-122 binding site comprises the nucleotide sequence set forth in SEQ ID NO: 10.

[0055] In some embodiments, the first miRNA binding site, the second miRNA binding site, and / or the third miRNA binding site are operably connected via a spacer sequence. In some embodiments, the spacer sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 11-13. In some non-limiting examples, endogenous sequences of 22bp, 20bp, and 18bp are selected to be used as spacers to improve the synergistic effect of different miRNAs.

[0056] In the gene expression cassette of the present application, the first miRNA binding site, the second miRNA binding site, and the third miRNA binding site can be combined in any suitable manner. In some preferred embodiments, the gene expression cassette includes at least one of the following groups: (1) a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, a miR-183 binding site, and a miR-122 binding site; and / or (2) a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, a miR-130 binding site, a miR-183 binding site, and a miR-122 binding site. In some more preferred embodiments, the gene expression cassette includes at least one of the following groups: (1) one miR-22 binding site, one miR-132 binding site, one miR-124 binding site, one miR-483 binding site, one miR-183 binding site, and one miR-122 binding site; (2) one miR-22 binding site, one miR-132 binding site, two miR-124 binding sites, one miR-483 binding site, one miR-183 binding site, and one miR- (3) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, 1 miR-483 binding site, 1 miR-130 binding site, 1 miR-183 binding site, and 1 miR-122 binding site; and / or (4) 1 miR-22 binding site, 2 miR-132 binding sites, 1 miR-124 binding site, 1 miR-483 binding site, 1 miR-183 binding site, and 1 miR-122 binding site.

[0057] In some embodiments, the gene expression cassette further comprises a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein or polypeptide expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to the host cell. In some non-limiting embodiments, the host cell is a neural cell, and the promoter can be a nucleic acid sequence that is transcriptionally active in neural cells. In some embodiments, the promoter comprises at least one of the MeCP2 core promoter, the MeCP2 silencer element, or the CNS regulatory element. In some preferred embodiments, the promoter is selected from at least one of MeP549 (as shown in SEQ ID NO: 14), MeP426 (as shown in SEQ ID NO: 15), MeP322 (as shown in SEQ ID NO: 16), and MeP223 (as shown in SEQ ID NO: 17).

[0058] In some embodiments, the 3' transcription termination sequence further comprises an AU-rich element. The AU-rich element can be used to regulate the stability of the mRNA. In some embodiments, the 3' transcription termination sequence further comprises a termination signal. Any transcription termination sequence known in the art can be used in the present application. In some non-limiting embodiments, the termination signal is a synthetic termination signal (as shown in SEQ ID NO: 8).

[0059] In some embodiments, the gene expression cassette is no longer than about 2.2 kb. The gene expression cassette is no longer than the maximum carrying capacity of the AAV vector, facilitating delivery using a variety of vectors including the AAV vector.

[0060] Recombinant vector, pharmaceutical composition

[0061] According to one embodiment of the present application, a recombinant vector is also provided, characterized in that the recombinant vector comprises the gene expression cassette described in the claim of the present application. Any suitable vector can be used to deliver the gene expression cassette. In some embodiments, the recombinant vector comprises a recombinant plasmid expression vector or a recombinant viral vector. In some embodiments, the recombinant viral vector comprises a recombinant adenoviral vector, a recombinant adeno-associated viral vector, a recombinant retroviral vector, a recombinant herpes simplex viral vector, or a recombinant vaccinia viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated viral vector. The recombinant adeno-associated viral vector can be of any suitable serotype. In some embodiments, the recombinant adeno-associated viral vector serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12, AAV13, AAV-PHP.B, AAV-PHP.EB, AAVhu37, AAVrh74, or AAVhu68. In some embodiments, the recombinant adeno-associated viral vector serotype includes AAV1, AAV8, or AAV9. In some non-limiting embodiments, the recombinant viral vector is a serotype that targets neural cells. The nucleotide sequence of the genome of the AAV serotype is known in the art. In some embodiments, the gene expression cassette is located downstream of the 5' inverted terminal repeat sequence of the adeno-associated viral vector.

[0062] Virion particles containing the gene expression cassettes described herein are typically produced in packaging cells that are capable of replicating the viral genome, expressing viral proteins, and assembling virion particles. Techniques for producing AAV vector particles in packaging cells are well known in the art. In some non-limiting embodiments, packaging cells can be produced by simply transforming suitable cells with one or more plasmids encoding the AAV genome, AAV proteins, and any desired helper virus functions, a so-called "triple transfection" approach: using three plasmids, each of which carries a set of such genes. See Grieger et al., Nature Protocols, 2006, 1(3): 1412-28.

[0063] According to one embodiment of the present application, a pharmaceutical composition is also provided, characterized in that the pharmaceutical composition includes the gene expression cassette described in the present application and the recombinant vector described in the present application. In some embodiments, the pharmaceutical composition is in the form of a drug for intracerebroventricular injection, intravenous injection or intrathecal injection. The pharmaceutical composition of the present application, in addition to the gene expression cassette and / or recombinant vector provided herein, may also include a pharmaceutically, nutritionally or physiologically acceptable carrier, such as a liquid, gel or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, a suspending agent, a dispersant, a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, and other components known in the art or various combinations thereof.

[0064] use

[0065] According to one embodiment of the present application, further provided is the use of the gene expression cassette, the recombinant vector, and the pharmaceutical composition described herein for expressing MeCP2 protein in cells. The cell can be any cell of interest. In some embodiments, the cell is an animal cell. In some embodiments, the animal cell includes a Neuro-2a cell.

[0066] According to one embodiment of the present application, further provided is the use of the gene expression cassette, the recombinant vector, and the pharmaceutical composition described herein in the preparation of a medicament for detecting, preventing, or treating a disease. In some embodiments, the disease is a MeCP2-related disease. In some embodiments, the MeCP2-related diseases include: diseases associated with insufficient MeCP2 protein expression, diseases associated with MeCP2 protein deficiency, diseases associated with MeCP2 gene deletion, and diseases associated with MeCP2 gene mutation. In some embodiments, the MeCP2-related disease is Rett syndrome. In some embodiments, the medicament comprises an intraventricular, intravenous, or intrathecal injection.

[0067] Disease treatment methods

[0068] According to one embodiment of the present application, a method for detecting, preventing, or treating a disease is also provided, comprising administering the gene expression cassette, the recombinant vector, or the pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the disease is a MeCP2-related disease. In some embodiments, the MeCP2-related disease includes: diseases associated with insufficient MeCP2 protein expression, diseases associated with MeCP2 protein deficiency, diseases associated with MeCP2 gene deletion, and diseases associated with MeCP2 gene mutation. In some embodiments, the MeCP2-related disease is Rett syndrome.

[0069] The various embodiments and preferences for the present application described above can be combined with each other (as long as they are not inherently contradictory to each other) and are applicable to the purposes of the present application. The various embodiments formed by such combination are all considered as part of the present application.

[0070] Example

[0071] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0072] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0073] Example 1: Construction of expression vector

[0074] 1.1 Construction of plasmid vector

[0075] We commissioned Qingke Biotechnology to synthesize the relevant components and construct them into the puc57 ampicillin-resistant cloning vector. The synthesis steps of the relevant components are as follows:

[0076] The CDS sequence of the Mecp2 gene encoding the MeCP2 protein (amino acid sequence shown in SEQ ID NO:1) was combined with the endogenous 3'UTR to generate Mecp2-pA1-pA2 (nucleotide sequence shown in SEQ ID NO:18). The endogenous 3'UTR includes a proximal terminator sequence (pA1) and a distal terminator sequence (pA2). pA1 contains a miR-22 binding site, while pA2 contains miR-132 and miR-124 binding sites. The MeCP2 protein can contain a C-myc tag at the C-terminus for subsequent expression efficiency evaluation. 4 μg of the synthesized plasmid was diluted with 40 μL of ddH2O to a concentration of 100 ng / μL for subsequent PCR experiments.

[0077] 1.2 Construction of scAAV vectors

[0078] The Mecp2-pA1-pA2 constructed in 1.1, as well as the neural cell-specific promoters MeP549 (sequence shown in SEQ ID NO: 14), MeP426 (sequence shown in SEQ ID NO: 15), MeP322 (sequence shown in SEQ ID NO: 16), and MeP223 (sequence shown in SEQ ID NO: 17) were used to construct the following plasmid vectors: scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2.

[0079] Meanwhile, scAAV-MeP549-Mecp2-pA49 was synthesized using a 49 bp terminator sequence (pA49) without a miRNA binding site as a control.

[0080] The detailed construction steps are as follows:

[0081] (1) The scAAV backbone plasmid (PackGene Biotech, EC021) was digested with ScaI-HF (NEB, R3122V).

[0082] (2) The fast and high-fidelity premixed PCR enzyme KOD One PCR Master Mix (TOYOBO, KMM-201) was used for cloning experiments. Microhomology sequences of 18–20 bp were introduced between different fragments or between fragments and vectors by PCR to enable subsequent seamless cloning experiments.

[0083] (3) Construction of scAAV-MeP549-Mecp2-pA49: MeP549, Mecp2, and pA49 were amplified by PCR, and the templates and primers are shown in Table 1. The amplified products were ligated to the scAAV linear backbone by seamless cloning (Clone Smarter, C5891-50) to construct the scAAV-MeP549-Mecp2-pA49 plasmid (the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 20). The ligated products were transformed into recombinase-deficient Escherichia coli competent stabl3 (Full Gold, CD521-01) for amplification.

[0084] Table 1 PCR primer sequences for scAAV-MeP549-Mecp2-pA49

[0085] (4) Construction of scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2: MeP426, MeP322, MeP226, and Mecp2-pA1-pA2 were amplified by PCR using the templates and primers shown in Table 2. The amplified products were ligated into the scAAV linear backbone by seamless cloning (Clone Smarter, C5891-50) to construct scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2.

[0086] Table 2 PCR primer sequences for scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2

[0087] (5) Construction of scAAV-MeP426-EGFP(c-myc)-pA49:

[0088] The scAAV backbone plasmid (PackGene Biotech, EC021) was digested with NheI-HF (NEB, R3131V) and HindIII-HF (NEB, R3104V) to remove the CMV promoter. MeP426 was amplified by PCR using the template and primers shown in Table 3. The amplified product was ligated to the scAAV linear backbone using seamless cloning (Clone Smarter, C5891-50). The ligated product was then transformed into recombinase-deficient Escherichia coli stabl3 (Full Gold, CD521-01) for amplification to construct scAAV-MeP426-EGFP-WPRE-pA(SV40).

[0089] The constructed scAAV-MeP426-EGFP-WPRE-pA(SV40) plasmid was digested with BsrGI-HF (NEB, R3575V) and NotI-HF (NEB, R3189V) to remove the WPRE-pA(SV40) element. c-myc-pA49 was amplified by PCR using the template and primers shown in Table 3. The amplified product was ligated into the scAAV linear backbone using seamless cloning (Clone Smarter, C5891-50). The ligated product was then transformed into recombinase-deficient Escherichia coli stabl3 (Full Gold, CD521-01) for amplification to construct scAAV-MeP426-EGFP(c-myc)-pA49.

[0090] Table 3 PCR primer sequences for scAAV-MeP426-EGFP(c-myc)-pA49

[0091] 1.3 Construction of scAAV vectors containing miRNA binding sites

[0092] The detailed construction steps are as follows:

[0093] (1) The scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2 plasmids constructed in 1.2 were linearized by BsaI-HFv2 (NEB, R3733S).

[0094] Table 4 PCR primer sequences of miRNA clusters

[0095] (2) The miRNA cluster sequences 183-483-122, 183-483-122-124, 183-483-122-130, and 183-483-122-132 were synthesized in the form of long primers. The primer sequences are shown in Table 4. The two primers served as templates for each other to amplify the desired fragments.

[0096] (3) The amplified fragments were seamlessly ligated into the linearized vectors scAAV-MeP426-Mecp2-pA1-pA2, scAAV-MeP322-Mecp2-pA1-pA2, and scAAV-MeP223-Mecp2-pA1-pA2. The ligated products were then transformed into recombinase-deficient Escherichia coli competent stabl3 (Full Gold, CD521-01) for amplification. The resulting AAV vectors are shown below, and the sequences of the exogenous nucleic acid fragments between their ITR sequences are shown below:

[0097] scAAV-MeP426-Mecp2-pA1-pA2 (as shown in SEQ ID NO:42): scAAV-MeP426-Mecp2-pA1-pA2-183-483-122 (as shown in SEQ ID NO:43), scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-124 (as shown in SEQ ID NO:44), scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130 (as shown in SEQ ID NO:45), scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132 (as shown in SEQ ID NO:46).

[0098] scAAV-MeP322-Mecp2-pA1-pA2 (as shown in SEQ ID NO:47): scAAV-MeP322-Mecp2-pA1-pA2-183-483-122 (as shown in SEQ ID NO:48), scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-124 (as shown in SEQ ID NO:49), scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-130 (as shown in SEQ ID NO:50), scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132 (as shown in SEQ ID NO:51).

[0099] scAAV-MeP223-Mecp2-pA1-pA2 (shown in SEQ ID NO: 52), scAAV-MeP223-Mecp2-pA1-pA2-183-483-122 (shown in SEQ ID NO: 53), scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-124 (shown in SEQ ID NO: 54), scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-130 (shown in SEQ ID NO: 55), scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-132 (shown in SEQ ID NO: 56).

[0100] Taking scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130 as an example, the sequence of its elements is shown in FIG1 , and FIG16 is a schematic diagram of the miRNA binding sites contained in the vector.

[0101] Example 2: Expression efficiency evaluation of expression vector

[0102] 2.1 Evaluation of in vitro expression efficiency of vector transfection

[0103] The vector constructed in Example 1 was used to transfect Neuro-2a cells (Zhongqiao Xinzhou, ZQ0207) with the transfection reagent PEIpro (Polyplus, 101000026). Western blot analysis was performed using an antibody against the C-myc tag (cell signaling technology, 2276S) to detect the expression of the target gene, thereby detecting the expression level of Mecp2 protein. An antibody against the internal control protein tubulin (Proteintech, 66240-1-Ig) was also used to detect the expression of the target gene.

[0104] The steps were as follows: Incubate with the primary antibody overnight at 4°C with shaking. After the primary antibody incubation was complete, incubate with the horseradish peroxidase-conjugated secondary antibody, Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, 115-035-003). After incubation, the cells were developed using ECL chemiluminescent solution (Shenger Biotechnology, SB-WB012).

[0105] The results are shown in Figure 2, where different labels represent the use of different vectors for transfection:

[0106] #1 represents scAAV-MeP223-Mecp2-pA1-pA2,

[0107] #2 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122,

[0108] #3 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-124

[0109] #4 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-130,

[0110] #5 represents scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-132,

[0111] #6 represents scAAV-MeP322-Mecp2-pA1-pA2,

[0112] #7 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122,

[0113] #8 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-124,

[0114] #9 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-130,

[0115] #10 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132,

[0116] #11 represents scAAV-MeP426-Mecp2-pA1-pA2,

[0117] #12 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122,

[0118] #13 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-124,

[0119] #14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130,

[0120] #15 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132,

[0121] EGFP represents scAAV-MeP426-EGFP(c-myc)-pA49,

[0122] #549 represents scAAV-MeP549-Mecp2-pA49,

[0123] Blank represents the untransfected blank group.

[0124] The results in Figure 2 show that compared with the untransfected group (blank) and the experimental control group (scAAV-MeP426-EGFP(c-myc)-pA49), the vectors constructed in Example 1 all expressed MeCP2 protein to varying degrees. Among them, compared with scAAV-MeP549-Mecp2-pA49, which does not contain a miRNA binding site, all 15 vectors in this application reduced the expression of MeCP2 protein in nerve cells, indicating that when the vector contains a first miRNA binding site (including one or more of the miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, or miR-130 binding site), the MeCP2 protein can be expressed in nerve cells, and the expression level of the MeCP2 protein is not too high. In addition, among these 15 vectors, 4 of them can more significantly reduce the expression of MeCP2 protein in nerve cells (including scAAV-MeP223-Mecp2-pA1-pA2-183-483-122-130, scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-130, scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-124, scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130), especially the vector scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130.

[0125] 2.2 Comparison of in vitro expression effects of vectors

[0126] According to the vector construction method in Example 1, a scAAV vector containing a control miRNA binding site was constructed. The construction method is briefly described as follows:

[0127] (1) The vector scAAV-MeP426-Mecp2-pA1-pA2 obtained in Example 1 was subjected to P-cycling and a control miRNA binding site was introduced. The PCR primer sequences of the control miRNA binding site are shown in Table 5 below.

[0128] Table 5 PCR primer sequences for control miRNA

[0129] (2) Amplification primers for control miRNAs hsa-let-7a-1, hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-802, and hsa-miR-1200 were synthesized in the form of long primers. The primer sequences are shown in Table 5. Circular PCR was performed on the vector scAAV-MeP426-Mecp2-pA1-pA2 to introduce miRNA binding sites.

[0130] (3) The amplified fragments were circularized by seamless cloning, and the ligated products were transformed into recombinase-deficient Escherichia coli stabl3 (Full Gold, CD521-01) for amplification. The resulting AAV vector is shown below, and the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown below:

[0131] scAAV-MeP426-Mecp2-pA1-pA2-let-7a-1 (as shown in SEQ ID NO: 71),

[0132] scAAV-MeP426-Mecp2-pA1-pA2-99a-5p (as shown in SEQ ID NO: 72),

[0133] scAAV-MeP426-Mecp2-pA1-pA2-100-5p (as shown in SEQ ID NO: 73),

[0134] scAAV-MeP426-Mecp2-pA1-pA2-802 (shown in SEQ ID NO: 74),

[0135] scAAV-MeP426-Mecp2-pA1-pA2-1200 (shown in SEQ ID NO: 75).

[0136] After the construction was completed, the five scAAV vectors containing the control miRNA binding sites obtained above were transfected into Neuro-2a cells according to the method in Example 2.1. The vector scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130 in Example 2.1 was also used for the experiment. The expression level of MeCP2 protein was then detected by Western blot. The results are shown in Figure 3. Different labels in the figure represent different vectors used for transfection:

[0137] Blank represents the untransfected blank group.

[0138] PC stands for scAAV-MeP426-Mecp2-pA1-pA2,

[0139] 14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130,

[0140] Let-7a-1 represents scAAV-MeP426-Mecp2-pA1-pA2-let-7a-1,

[0141] 802 represents scAAV-MeP426-Mecp2-pA1-pA2-802,

[0142] 99a-5p represents scAAV-MeP426-Mecp2-pA1-pA2-99a-5p,

[0143] 100-5p represents scAAV-MeP426-Mecp2-pA1-pA2-100-5p,

[0144] 1200 represents scAAV-MeP426-Mecp2-pA1-pA2-1200.

[0145] The results in Figure 3 show that compared with the five first miRNA binding sites provided in this application (miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, or miR-130 binding site), other miRNA binding sites (such as miR-let-7a-1, miR-802, miR-99a-5p, miR-100-5p, miR-1200, etc.) cannot effectively reduce the expression of the target gene.

[0146] In addition, in addition to miR-let-7a-1, miR-802, miR-99a-5p, miR-100-5p, and miR-1200, the applicant also discovered a large number of miRNA binding sites that could not effectively reduce the expression efficiency of the target gene during the screening process, confirming that the strategy of reducing the expression of the target gene through miRNA binding sites will be affected by multiple factors such as the type of miRNA, the length and type of the target gene, and the species type, and is unpredictable. Therefore, the five first miRNA binding sites provided in this application are the most preferred when used to reduce the expression level of the Mecp2 gene in human brain tissue.

[0147] 2.3 Identification of in vitro expression efficiency of vector infection

[0148] The vector constructed in Example 1 was used to package the AAV9 virus according to the method disclosed in the literature (Mol Ther. 2013, 21(1): 18-30.), and Neuro-2a cells were infected. The Mecp2 expression level after cells were infected with different vectors was determined using the method in 2.1.

[0149] The results are shown in Figure 4, where different labels represent different vectors used for infection:

[0150] #6 represents scAAV-MeP322-Mecp2-pA1-pA2,

[0151] #7 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122,

[0152] #8 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-124,

[0153] #9 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-130,

[0154] #10 represents scAAV-MeP322-Mecp2-pA1-pA2-183-483-122-132,

[0155] #11 represents scAAV-MeP426-Mecp2-pA1-pA2,

[0156] #12 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122,

[0157] #13 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-124,

[0158] #14 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130,

[0159] #15 represents scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-132,

[0160] EGFP represents scAAV-MeP426-EGFP(c-myc)-pA49,

[0161] #549 represents scAAV-MeP549-Mecp2-pA49,

[0162] Blank represents the uninfected blank group.

[0163] The results in Figure 4 show that, compared to the uninfected group (blank) and the experimental control group (scAAV-MeP426-EGFP(c-myc)-pA49), the vectors constructed in Example 1 all expressed MeCP2 protein to varying degrees, further confirming the results in Example 2.1. Furthermore, compared to the vector using the MeP322 promoter (corresponding to lanes #7, #8, #9, and #10 in Figure 4 ), the vector using the MeP426 promoter was able to further reduce the expression of MeCP2 protein in neurons (corresponding to lanes #12, #13, #14, and #15 in Figure 4 ). These results demonstrate that, based on the selection of a specific first miRNA binding site (miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, or miR-130 binding site), the use of the MeP426 promoter in the gene expression cassette can further reduce the expression of MeCP2 protein in neurons, demonstrating superior efficacy.

[0164] 2.4 In vivo expression efficiency evaluation of vector infection

[0165] The scAAV-pMeP549-Mecp2-pA49 constructed in Example 1 was used to package the three serotypes of AAV1, AAV8, and AAV9 according to the method disclosed in the literature (Mol Ther. 2013, 21(1): 18-30.) and injected into the P1 ventricle of wild-type C57 mice. Each mouse was injected into the left and right ventricles at a dose of 5×10 10 vg, three animals were injected with each serotype. Four weeks after injection, brain, spinal cord, and liver samples were collected and the expression of MeCP2 was determined by Western blot using the method described in 2.1.

[0166] The results are shown in Figure 5. The results show that the three serotypes of scAAV-pMeP549-Mecp2-pA49 can express MeCP2 protein to varying degrees in the mouse brain, so it may have a better therapeutic effect on Rett syndrome (Brain. 2021, 144(10): 3005-3019).

[0167] Example 3: Therapeutic effects of different expression vectors on Rett model mice

[0168] 3.1 Method for constructing Rett model mice

[0169] B6.129P2(C)-Mecp2 tm1.1Bird / J transgenic mice (Jax.003890) were purchased from Jackson laboratory and were induced by Mecp2 + / - The female mice were bred with wild-type C57BL / 6J mice to obtain the genotype Mecp2 - / y The hemi mice are the Rett model mice used in the experiment.

[0170] 3.2 Vector construction method

[0171] The vector constructed in Example 1 was packaged into two serotypes, AAV1 and AAV9, according to the method disclosed in the literature (Mol Ther. 2013, 21(1): 18-30.), wherein:

[0172] 8241 represents the vector scAAV9-MeP549-Mecp2-pA49 of AAV9 serotype,

[0173] 8243 represents the vector scAAV9-MeP322-Mecp2-pA1-pA2-183-483-122-130 of AAV9 serotype,

[0174] 8244 represents the vector scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-130 of AAV9 serotype,

[0175] 8245 represents the vector scAAV9-MeP426-Mecp2-pA1-pA2-183-483-122-132 of AAV9 serotype,

[0176] 8246 represents the vector scAAV1-MeP426-Mecp2-pA1-pA2-183-483-122-132 with the AAV1 serotype.

[0177] 3.3 Survival detection

[0178] After the birth of Rett model mice, Mecp2- / y mice were selected for the experiment through genotyping. The vehicle (N=5) or 5.0×10 10 vg / mouse dose of 8244 (N=7), 8241 (N=6), 8243 (N=6), 8245 (N=6), and 8246 (N=7); in addition, 5.0×10 10 8245 (N=6) was administered at a dose of vg / mouse. The administration volume for mice was 2.5 μL per intracerebroventricular injection.

[0179] The results of the survival curve are shown in Figure 6. The median survival of Rett model mice in the untreated vehicle group was 66 days; 10 At the dose level of vg / mouse, the median survival of the model mice in the 8244 treatment group was 175 days, the median survival of the model mice in the 8241 treatment group was 78.5 days, the median survival of the model mice in the 8243 treatment group was 166.5 days, the median survival of the model mice in the 8245 treatment group was 134.5 days, and the median survival of the model mice in the 8246 treatment group was 108 days, indicating that the vectors provided in this application can effectively prolong the survival of Rett model mice, among which 8244 can better prolong the median survival of model mice. In addition, the median survival of PND7 model mice treated with 8245 (administered at 7 days of birth) was 108 days, and the median survival of PND1 model mice was 134.5 days, indicating that administration within 24 hours of birth of the mouse is more effective in improving the survival period.

[0180] 3.4 Behavioral testing

[0181] After the birth of Rett model mice, Mecp2- / y mice were selected for the experiment by genotyping. The vehicle (N=5) or 5.0×10 10 vg / mouse dose of 8244 (N=6), 8241 (N=4), 8243 (N=6), 8245 (N=5), and 8246 (N=6); in addition, 5.0×10 10 8245 (N=6) was administered at a dose of vg / mouse. 50 days after administration, gait analysis was performed on the administered model mice and wild-type mice.

[0182] Gait and locomotion of mice were evaluated using a gait tester (Catwalk, Noldus, CatWalk XT 10.1). The specific test methods are as follows:

[0183] (1) Experimental animals need to be moved into the experimental room in advance to adapt to the dark environment;

[0184] (2) Set the instrument runway length to 3-6 times the animal's body length, ensure that the animal takes 4-6 steps, adjust the track position to make it parallel, and then set the scale under this parameter;

[0185] (3) The animal is placed in from the starting side. The system will automatically detect that the animal has entered the monitoring range and start recording. Each time an animal completes a run, an experimental record will be generated. After each animal has three successful experimental records, the system will stop and move on to the next animal's experiment.

[0186] (4) If there are stains on the runway, it must be cleaned in time to ensure the quality of the collected data;

[0187] (5) Statistical analysis of all animal gait data.

[0188] The results of gait analysis are shown in Figures 7 to 9. The results show that the vectors provided in this application can effectively improve the regularity index (RI) of the Rett model mice, and their swing speed (Swing Speed) and stride length (Stride Length) are improved to varying degrees, where RF represents the right forelimb, RH represents the right hindlimb, LF represents the left forelimb, and LH represents the left hindlimb. The above results indicate that all the vectors provided in this application can effectively alleviate the symptoms associated with Rett syndrome.

[0189] In addition, among all the vectors provided in this application, the gait regularity index of all treated mice in group 8244 (N=6) returned to normal levels, while there were still mice with low gait regularity index in other vector groups; at the same time, the swing speed (Swing Speed) and stride (Stride Length) of the mice in group 8244 during exercise reached the same level as those of wild-type mice, indicating that the therapeutic effect of 8244 is the best.

[0190] Example 4: Therapeutic effects of different doses of expression vector on Rett model mice

[0191] The construction methods of Rett model mice and expression vector 8244 were the same as those in Example 3.

[0192] 4.1 Survival detection

[0193] After the transgenic mice were born, the Rett model mice were selected for experiment through genotyping. 8244 was given to the mice at different doses: 1.75×10 10 vg / piece (Dose 5), 4.38×10 10 vg / each (Dose 4), 8.75×10 10 vg / each (Dose 3), 1.75×10 11 vg / (Dose 2) and 3.5×10 11 vg / each (Dose 1).

[0194] The results of the survival curve are shown in Figure 10. The results showed that the median survival of Rett model mice in the vehicle group without 8244 vehicle treatment was 54 days, 1.75×10 10 The median survival of mice in the vg / dose group was 63 days, and 4.38×10 10 The median survival of mice in the vg / dose group was 89.5 days (the longest survival period was more than 185 days), and the 8.75×10 10The median survival of mice in the vg / dose group was 135.5 days, and the 1.75×10 11 The median survival of mice in the vg / dose group was 100.5 days. 10 vg / piece~1.75×10 11 Within the dosage window of 1.5 vg / mouse, the survival time of Rett model mice was significantly prolonged with the continuous increase of the dosage.

[0195] However, the results also showed that at 3.5×10 11 At the high dose level of 100 vg / mouse, the median survival time of the model mice was only 30.5 days, and the survival period was not extended. This confirmed that excessive injection doses would express excessive MeCP2 protein, which would then cause toxicity and affect the therapeutic effect of the mice.

[0196] 4.2 Behavioral testing

[0197] (1) The detection method of the rotarod test is as follows:

[0198] Rotarod testing was performed using a rotarod apparatus (Harvard Apparatus, Panlab Rotarod, LE8205, USA). Animals underwent rotarod pretraining on P69 to acclimate to the apparatus and environment, and rotarod testing was performed on P70-P71. The rotarod rotated at a constant speed during the training phase and at a uniformly accelerated speed during the testing phase. The time the mice spent on the rotarod was recorded.

[0199] Training Phase: P69 animals were placed on a rotarod and required to maintain balance for 5 minutes while rotating at a constant speed of 4 rpm. Considering that the experimental animals in this study are models of motor dysfunction, if an animal cannot maintain balance for 5 minutes, it was trained until no further progress was achieved.

[0200] Testing phase: Animals on P70-P71 were required to perform three uniform acceleration rotarod tests (accelerating from 4 to 40 rpm over 5 minutes, with an angular acceleration of 0.72° / s²). The time from the start of the experiment to the moment the animal lost balance and fell was recorded. For animals that did not fall within the 5-minute test, the fall time was recorded according to the maximum test duration. The interval between each test round was set to 5 minutes. All equipment was cleaned with 75% alcohol and thoroughly deodorized before the next test.

[0201] (2) The results of the rotarod test are shown in Figure 11. 70-71 days after administration, the surviving mice were subjected to the rotarod test 4 times a day for two consecutive days. The time the mice persisted on the rotarod in each of the 8 experiments was counted. The results showed that the time the mice in the vehicle group (N=3) persisted on the rotarod in the 4 experiments on the second day was significantly different from that of the wild-type mice (N=10), indicating that the exercise ability and muscle endurance of the model mice were affected; 1.75×10 10 vg / (N=3), 4.38×10 10 vg / (N=5) and 8.75×10 10 In the 8 experiments of the 1.75×10 10 vg / (N=3),

[0202] 4.38×10 10 vg / (N=5) and 8.75×10 10 After the Rett model mice were treated with 8244 vector in three dose groups (vg / mouse (N=8), the motor coordination ability and muscle endurance of the model mice were effectively restored.

[0203] However, 1.75×10 11 vg / (N=7) and 3.5×10 11 In the high-dose group (vg / mouse) (N=1), the mice persisted on the rotarod for a shorter time than the mice in the vehicle group (N=3), indicating that excessive doses of virus would express excessive MeCP2 protein, thereby affecting the therapeutic effect of mice.

[0204] Example 5: Effects of expression vectors on protein expression and liver tissue damage in mice

[0205] The methods for constructing Rett model mice and expression vectors were the same as those in Example 3. Rett model mice aged 0-1 day were injected intracerebroventricularly with expression vectors 8244, 8241, 8243, 8245, and 8246. After 28 days, brain and spinal cord tissues were harvested, and the brain, spinal cord, and liver were collected. Half of the tissue was fixed in 4% paraformaldehyde for tissue damage detection; the other half was snap-frozen in liquid nitrogen and stored at -80°C for in vivo protein expression detection.

[0206] 5.1 Protein expression detection in mice

[0207] The liquid nitrogen-quick-frozen tissue was homogenized and protein extracted. The expression of MeCP2 protein was detected using the Western blot assay described in Example 2, as shown in Figure 12. The results showed that, compared to vehicle-treated Rett model mice, the vectors provided herein all expressed a certain level of MeCP2 protein in the brain and spinal cord of the central nervous system. Specifically, the level of MeCP2 expressed by expression vector 8244 was close to that of wild-type mice, suggesting that expression vector 8244 is a more preferred treatment option.

[0208] 5.2 Mouse liver tissue damage detection

[0209] Liver tissue damage is detected by staining pathological sections as follows:

[0210] (1) Sampling and fixation: Tissue blocks (generally no thicker than 0.5 cm) are taken from fresh human or animal corpses and placed in a pre-prepared fixative (10% formalin, Bouin's fixative, etc.) to denature and coagulate the proteins in the tissues and cells to prevent autolysis of the cells after death or bacterial decomposition, thereby maintaining the original morphological structure of the cells.

[0211] (2) Dehydration and transparency:

[0212] Generally, alcohol is used as a dehydrating agent, gradually removing the water from the tissue block. The tissue block is then placed in xylene, a clearing agent that is soluble in both alcohol and paraffin, to make it transparent. The xylene replaces the alcohol in the tissue block before wax embedding.

[0213] (3) Wax embedding:

[0214] Place the transparent tissue block in melted paraffin and place it in a wax melting chamber to keep it warm. Once the paraffin has completely soaked the tissue block, embed it. Prepare a container (such as a small folded cardboard box), pour the melted paraffin, and quickly place the paraffin-soaked tissue block inside. Allow to cool and solidify into a block. Once the embedded tissue block is hard, it can be cut into very thin sections on a microtome.

[0215] (4) Slicing and patching:

[0216] The embedded wax block is fixed on a microtome and cut into thin slices, generally 5-8 microns thick. The slices are often wrinkled and need to be ironed flat in heated water, then attached to a glass slide and dried in a 45°C constant temperature oven.

[0217] (5) Dewaxing and staining: HE staining is commonly used to increase the color difference of different parts of the tissue cell structure, which is convenient for observation. Hematoxylin (H) is a basic dye that can dye the cell nucleus and ribosomes in the cell into blue-purple. The structure stained by the basic dye has a basophilic appearance. Eosin (Eosin, E) is an acidic dye that can dye the cytoplasm into red or light red. The structure stained by the acidic dye has an eosinophilic appearance. Before staining, the paraffin in the section must be removed with xylene, and then the section is stained through a high concentration to a low concentration of alcohol, and finally into distilled water.

[0218] HE staining process: After soaking in distilled water, place the sections in a hematoxylin solution for several minutes. Separate the sections in acid and ammonia solutions for a few seconds each. Rinse in running water for 1 hour, then briefly soak in distilled water. Dehydrate in 70% and 90% alcohol for 10 minutes each. Soak in alcohol-eosin solution for 2-3 minutes.

[0219] (6) Dehydration and transparency: The stained sections are dehydrated with pure alcohol and then with xylene to make them transparent.

[0220] (7) Sealing: Add a drop of Canada balsam to the transparent section and cover with a cover slip. After the balsam has dried slightly, apply a label and the section is ready for use.

[0221] The results of pathological sections are shown in Figures 13-15. The results showed that compared with the control group, all vectors provided in this application did not cause obvious liver tissue damage, confirming that the addition of a third miRNA binding site (such as a miR-122 binding site) to the vector can effectively reduce the liver toxicity caused by AAV administration. In addition, all vectors provided in this application did not cause obvious dorsal root ganglion lesions (results not shown), confirming that the addition of a second miRNA binding site (such as a miR-183 binding site) to the vector can effectively reduce the dorsal root ganglion toxicity caused by AAV administration.

[0222] In addition, the results in Figures 13-15 also show that the liver tissues of animals in each group showed only minor pathological changes. For example, in animal No. 2 of group 8241, a small number of hepatocytes with vacuolar degeneration around the liver vein were observed; in animal No. 2 of group 8243, vacuolar degeneration of hepatocytes around the portal area and an increase in Kupffer cells (macrophages / histiocytes) within the sinusoids were observed; in animal No. 1 of group 8245, mild hyperplasia of bile duct epithelial cells was observed in the portal area; and in animal No. 2 of group 8245, ballooning and vacuolar degeneration of hepatocytes were observed. Furthermore, compared with groups 8241, 8243, and 8245, no minor pathological changes were observed in the liver sections of the three animals in group 8244, confirming that 8244 can further reduce the impact of AAV vectors on the liver, making it a more preferred treatment option.

[0223] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementation methods have been described, for the applicant or other skilled in the art, there may be or are currently no alternatives, modifications, changes, improvements and substantial equivalents of the above implementation methods. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important to note that as technology evolves, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A gene expression cassette comprising a polynucleotide sequence encoding human MeCP2 protein, characterized in that: The gene expression cassette comprises at least one first miRNA binding site, wherein the first miRNA binding site comprises one or more of a miR-22 binding site, a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, or a miR-130 binding site.

2. The gene expression cassette according to claim 1, wherein the first miRNA binding site is located in the 3' transcription termination sequence of the gene expression cassette, and the 3' transcription termination sequence includes a distal termination sequence and / or a proximal termination sequence.

3. The gene expression cassette of claim 2, wherein the proximal termination sequence comprises a miR-22 binding site.

4. The gene expression cassette of claim 2, wherein the distal termination sequence comprises at least one of a miR-132 binding site, a miR-124 binding site, a miR-483 binding site, or a miR-130 binding site.

5. The gene expression cassette of claim 1, wherein the first miRNA binding site comprises at least one of the following groups: (1) miR-22 binding site, miR-132 binding site, miR-124 binding site, and miR-483 binding site; and / or (2) miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, and miR-130 binding site.

6. The gene expression cassette of claim 5, wherein the first miRNA binding site comprises at least one of the following groups: (1) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, and 1 miR-483 binding site; (2) 1 miR-22 binding site, 1 miR-132 binding site, 2 miR-124 binding sites, and 1 miR-483 binding site; (3) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, 1 miR-483 binding site, and 1 miR-130 binding site; and / or (4) 1 miR-22 binding site, 2 miR-132 binding sites, 1 miR-124 binding site, and 1 miR-483 binding site.

7. The gene expression cassette according to any one of claims 1 to 6, wherein the first miRNA binding site is capable of reducing the expression efficiency of the polynucleotide sequence encoding human MeCP2 protein in human brain tissue.

8. The gene expression cassette according to any one of claims 1 to 6, wherein at least one thymine nucleotide in the nucleotide sequence of the miR-483 and / or miR-130 binding site is replaced by an adenine nucleotide.

9. The gene expression cassette according to claim 8, wherein: The nucleotide sequence of the miR-22 binding site includes GGCAGCT; The miR-132 binding site includes the nucleotide sequence shown in SEQ ID NO:6; The miR-124 binding site includes the nucleotide sequence shown in SEQ ID NO:7; The miR-483 binding site comprises the nucleotide sequence shown in SEQ ID NO: 4; and / or The miR-130 binding site includes the nucleotide sequence shown in SEQ ID NO:

5.

10. The gene expression cassette according to claim 1, wherein the gene expression cassette further comprises a second miRNA binding site and / or a third miRNA binding site, wherein the second miRNA binding site and / or the third miRNA binding site comprises one or more of a miR-183 binding site, a miR-182 binding site, a miR-96 binding site, a miR23b binding site, a miR-145 binding site, a miR-148a binding site, a miR-22 binding site, a miR-122 binding site, a miR-143 binding site, a miR-21 binding site, or a miR-192 binding site.

11. The gene expression cassette of claim 10, wherein the second miRNA binding site comprises a miR-183 binding site, and the third miRNA binding site comprises a miR-122 binding site.

12. The gene expression cassette according to claim 11, wherein the gene expression cassette comprises at least one of the following groups: (1) miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, miR-183 binding site, and miR-122 binding site; and / or (2) miR-22 binding site, miR-132 binding site, miR-124 binding site, miR-483 binding site, miR-130 binding site, miR-183 binding site, and miR-122 binding site.

13. The gene expression cassette of claim 11, wherein the gene expression cassette comprises at least one of the following groups: (1) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, 1 miR-483 binding site, 1 miR-183 binding site, and 1 miR-122 binding site; (2) 1 miR-22 binding site, 1 miR-132 binding site, 2 miR-124 binding sites, 1 miR-483 binding site, 1 miR-183 binding site, and 1 miR-122 binding site; (3) 1 miR-22 binding site, 1 miR-132 binding site, 1 miR-124 binding site, 1 miR-483 binding site, 1 miR-130 binding site, 1 miR-183 binding site, and 1 miR-122 binding site; and / or (4) 1 miR-22 binding site, 2 miR-132 binding sites, 1 miR-124 binding site, 1 miR-483 binding site, 1 miR-183 binding site, and 1 miR-122 binding site.

14. The gene expression cassette according to any one of claims 10 to 13, wherein the second miRNA binding site can reduce the expression efficiency of the polynucleotide sequence encoding the human MeCP2 protein in human dorsal root ganglia, and the third miRNA binding site can reduce the expression efficiency of the polynucleotide sequence encoding the human MeCP2 protein in human liver tissue.

15. The gene expression cassette according to any one of claims 10 to 13, wherein The miR-183 binding site comprises the nucleotide sequence shown in SEQ ID NO:9; and / or The miR-122 binding site includes the nucleotide sequence shown in SEQ ID NO:

10.

16. The gene expression cassette according to any one of claims 1 to 15, wherein the first miRNA binding site, the second miRNA binding site, and / or the third miRNA binding site are operably linked via a spacer sequence, and the spacer sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 11 to 13.

17. The gene expression cassette according to any one of claims 1 to 16, wherein the gene expression cassette further comprises a promoter comprising at least one of a MeCP2 core promoter, a MeCP2 silencer element, or a CNS regulatory element.

18. The gene expression cassette of claim 17, wherein the promoter comprises MeP549, MeP426, MeP322, MeP223.

19. The gene expression cassette according to claim 18, wherein: The MeP549 promoter includes the nucleotide sequence shown in SEQ ID NO: 14; The MeP426 promoter includes the nucleotide sequence shown in SEQ ID NO: 15; The MeP322 promoter comprises the nucleotide sequence shown in SEQ ID NO: 16; and / or The MeP223 promoter includes the nucleotide sequence shown in SEQ ID NO:

17.

20. The gene expression cassette of any one of claims 1-19, wherein the gene expression cassette is no more than about 2.2 kb in length.

21. A recombinant vector, characterized in that: The recombinant vector comprises the gene expression cassette according to any one of claims 1 to 20.

22. The recombinant vector according to claim 21, wherein the recombinant vector comprises a recombinant plasmid expression vector or a recombinant viral vector.

23. The recombinant vector of claim 22, wherein the recombinant viral vector comprises a recombinant adenoviral vector, a recombinant adeno-associated viral vector, a recombinant retroviral vector, a recombinant herpes simplex viral vector, or a recombinant vaccinia viral vector. The recombinant vector according to claim 23 , wherein the recombinant viral vector is a recombinant adeno-associated viral vector.

25. The recombinant vector of claim 24, wherein the recombinant adeno-associated virus vector serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12, or AAV13, AAV-PHP.B, AAV-PHP.EB, AAVhu37, AAVrh74, AAVhu68.

26. The recombinant vector according to claim 25, wherein the recombinant adeno-associated virus vector serotype comprises AAV1, AAV8, or AAV9.

27. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the gene expression cassette according to any one of claims 1 to 20 and the recombinant vector according to any one of claims 21 to 26.

28. The pharmaceutical composition according to claim 27, wherein the pharmaceutical composition is in the form of intraventricular injection of the drug, intravenous injection of the drug or intrathecal injection of the drug.

29. Use of the gene expression cassette according to any one of claims 1 to 20, the recombinant vector according to any one of claims 21 to 26, or the pharmaceutical composition according to any one of claims 27 to 28 for expressing MeCP2 protein in cells.

30. The use according to claim 29, wherein the cell is an animal cell.

31. The use according to claim 30, wherein the animal cell comprises Neuro-2a cell.

32. Use of the gene expression cassette according to any one of claims 1 to 20, the recombinant vector according to any one of claims 21 to 26, or the pharmaceutical composition according to any one of claims 27 to 28 in the preparation of a drug for preventing, alleviating or treating a disease.

33. The use according to claim 32, wherein the disease is a MeCP2-related disease.

34. The use according to claim 33, wherein the MeCP2-related diseases include: Diseases associated with insufficient expression of MeCP2 protein, diseases associated with MeCP2 protein deficiency, diseases associated with MeCP2 gene deletion, and diseases associated with MeCP2 gene mutation.

35. The use according to claim 34, wherein the MeCP2-related disease is Rett syndrome.

36. The use according to claim 32, wherein the drug comprises an intraventricular injection of a drug, an intravenous injection of a drug, or an intrathecal injection of a drug.

37. A method for preventing, alleviating or treating a disease, the method comprising administering the gene expression cassette of any one of claims 1 to 20, the recombinant vector of any one of claims 21 to 26, or the pharmaceutical composition of any one of claims 27 to 28 to a subject in need thereof.

38. The use according to claim 37, wherein the disease is a MeCP2-related disease.

39. The use according to claim 38, wherein the MeCP2-related diseases include: Diseases associated with insufficient expression of MeCP2 protein, diseases associated with MeCP2 protein deficiency, diseases associated with MeCP2 gene deletion, and diseases associated with MeCP2 gene mutation.

40. The use according to claim 39, wherein the MeCP2-related disease is Rett syndrome.

Images