Human MeCp2 promoter and application thereof

Abstract

Disclosed are a nucleic acid molecule, and a recombinant nucleic acid molecule comprising the nucleic acid molecule, a nucleic acid sequence encoding a foreign protein, and a polyadenylic acid sequence (i.e. PolyA). The invention also discloses a recombinant adeno-associated virus, which comprises an AAV capsid and a vector genome, and the vector genome comprises the recombinant nucleic acid molecule. Also disclosed is the use of the recombinant adeno-associated virus for more effective treatment of spinal cord injury.

Description

A human MeCp2 promoter and its use Technical Field

[0001] The present invention relates to the biological field, in particular to a human promoter, and in particular to a recombinant adeno-associated virus vector for treating spinal cord injury constructed by using the human promoter and its application. Background Art

[0002] Spinal cord injury (SCI) is the most serious complication of spinal cord injury, often leading to sensory and motor dysfunction in the limbs below the injured segment. The persistent existence of pathogenic factors and the high incidence worldwide make SCI a serious threat to human health and impose a heavy economic burden on society and families.

[0003] The most prominent challenge in current SCI treatment is how to effectively reconstruct neural circuits on both sides of the injury plane and restore limb motor and sensory function. However, current SCI treatments struggle to meet clinical needs and the requirements for neural circuit reconstruction. Marketed small molecule drugs, such as glucocorticoid receptor agonists, phospholipase A inhibitors, and α2-adrenergic receptor agonists, target the pathological cascade secondary to primary SCI, exerting anti-inflammatory, anti-ischemic, immune-suppressing, and neuroprotective effects. Surgical treatment also targets secondary injuries and is performed within 24 hours after the SCI patient's vital signs stabilize, to reduce bleeding and remove necrotic tissue. Among the drugs under development, Phase II and III stem cell-based neurotrophic factor therapies can only structurally reconstruct neural circuits on both sides of the injury, but not functionally. Although there are reports of using recombinant viral vectors carrying a nucleic acid sequence encoding the full-length K+-Cl- cotransporter (full-length KCC2) to treat spinal cord injury (WO2019226643A1), there is still room for further improvement to achieve better efficacy. In preclinical studies of gene therapy for neurological diseases, neuron-specific promoters are often used to enhance the expression efficiency of target genes in specific cell types and reduce immune responses or side effects caused by nonspecific expression of target genes. However, currently marketed gene therapy products for neurological diseases all use broadly expressed promoters. There is still a need to explore promoters with high neuronal expression efficiency and low immunogenicity and to translate them into clinical practice. Current research still cannot meet the clinical needs of SCI.

[0004] Therefore, there is an urgent need to explore innovative, safer and effective drugs and methods for the treatment of spinal cord injury.

[0005] Summary of the Invention

[0006] In the first aspect, the present invention provides a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7, or a nucleotide sequence having more than 80% (for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 6 or 7.

[0007] In some embodiments, the nucleic acid molecule is a promoter that can initiate transcription of a nucleic acid sequence encoding an exogenous protein in a cell. In some embodiments, the nucleic acid molecule is a human MeCp2 promoter.

[0008] In a second aspect, the present invention provides a recombinant nucleic acid molecule comprising the above-mentioned nucleic acid molecule, a nucleic acid sequence encoding an exogenous protein, and a polyadenylic acid sequence (ie, polyA).

[0009] In some specific embodiments, the polyA is BGH polyA, preferably, the sequence of which is shown in SEQ ID NO: 8.

[0010] In some specific embodiments, the exogenous protein is KCC2 protein, preferably, its amino acid sequence is shown in SEQ ID NO: 3.

[0011] In some specific embodiments, the coding sequence of the KCC2 protein has a nucleotide sequence selected from the following:

[0012] 1) the nucleotide sequence shown in SEQ ID NO: 2;

[0013] 2) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 2; or

[0014] 3) a nucleotide sequence having more than 80% sequence identity with SEQ ID NO: 2, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or more; more preferably, a sequence having 98% or 99% identity or more; in a more preferred embodiment, the polynucleotide sequence is a codon-optimized sequence.

[0015] In some embodiments, the recombinant nucleic acid molecule further comprises one or more of a Kozak sequence, a WPRE, and a post-transcriptional regulatory element.

[0016] In some preferred embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

[0017] 1) the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13;

[0018] 2) a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13; or

[0019] 3) A nucleotide sequence having more than 80% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or more; more preferably, a sequence having 98% or 99% identity or more.

[0020] In certain specific embodiments, the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence; preferably, the AAV inverted terminal repeat sequence is selected from AAV of different serotypes; preferably, the AAV inverted terminal repeat sequence is selected from AAV of any serotype in evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types; more preferably, the AAV inverted terminal repeat sequence is from AAV2.

[0021] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule, wherein the vector is selected from a plasmid vector, a phage vector and a viral vector, wherein the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector and a hybrid viral vector.

[0022] In a fourth aspect, the present invention provides a recombinant adeno-associated virus, comprising an AAV capsid and a vector genome, wherein the vector genome comprises the aforementioned nucleic acid molecule or recombinant nucleic acid molecule; in certain specific embodiments, the recombinant nucleic acid molecule comprises an AAV inverted terminal repeat sequence, a nucleic acid sequence encoding a KCC2 protein, and an expression control sequence and a PolyA sequence for directing the expression of the nucleic acid sequence of the KCC2 protein in a host cell; in some embodiments, the AAV capsid is selected from any serotype of AAV in evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types; in certain preferred embodiments, the capsid of the recombinant adeno-associated virus is AAV9, and its amino acid sequence is shown in SEQ ID NO: 14.

[0023] In a fifth aspect, the present invention provides an isolated host cell comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule or the aforementioned recombinant vector or recombinant adeno-associated virus.

[0024] In some embodiments, the cells are neuronal cells. In some embodiments, the cells are cholinergic neuronal cells, noradrenergic neuronal cells, dopaminergic neuronal cells, serotonergic neuronal cells, gamma-aminobutyric acid neuronal cells. In some embodiments, the cells are neural cell lines, including NG108-15, N1E115, PC12, SHSY5Y, F11, ND7-23, N27, and SN4741 cell lines.

[0025] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule, recombinant vector or recombinant adeno-associated virus and / or the aforementioned host cell and a pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, intraspinal injection, intrathoracic injection, oral administration, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and / or other parenteral administration. In some preferred embodiments, the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.

[0026] In a seventh aspect, the present invention provides use of the aforementioned nucleic acid molecules, recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells, and / or pharmaceutical compositions in the preparation of a medicament for preventing or treating spinal cord injury. In certain embodiments, the recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells, and / or pharmaceutical compositions may be administered in combination with another therapy.

[0027] In an eighth aspect, the present invention provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell, and / or the aforementioned pharmaceutical composition to a subject in need thereof. In certain preferred embodiments, the composition is administered intravenously; in certain preferred embodiments, the composition is administered intrathecally; in certain preferred embodiments, the composition is administered intraspinal; in other preferred embodiments, the subject is a mammal; and in more preferred embodiments, the subject is a human. In some embodiments, the treatment method may also be used in combination with other therapies.

[0028] The beneficial effects of the present invention are:

[0029] The nucleic acid molecules provided herein (particularly, the human MeCp2 promoter) can initiate and / or regulate more efficient transcription and / or expression of exogenous proteins (particularly, KCC2 proteins) in cells (particularly, neurons). Compared to other promoters used in the nervous system in the prior art, the nucleic acid molecules or promoters of this application can be used in KCC2 gene therapy drugs to more safely and effectively treat spinal cord injuries and have lower immunological risks compared to the murine MeCp2 promoter.

[0030] The present disclosure is further described below with reference to the accompanying drawings and specific embodiments, but is not intended to limit the present disclosure. Any equivalent replacement in the field made in accordance with the content of the present disclosure shall fall within the scope of protection of this patent. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1. The main structure of the vector genome containing different promoters in the recombinant adeno-associated virus.

[0032] Figure 2. Plasmid map of the construction process of the shuttle plasmid pSNAV2.0-K-Syn-KCC2-BGH.

[0033] Figure 3. Map of shuttle plasmids containing different promoters.

[0034] Figure 4. Immunofluorescence detection results after different recombinant viruses infected neuronal cells.

[0035] Figure 5. In vitro biological activity test results of different recombinant viruses.

[0036] Figure 6. In vivo efficacy test results (BMS score) of different recombinant viruses.

[0037] Figure 7. In vivo efficacy test results (survival rate) of different recombinant viruses.

[0038] Detailed Description of the Invention

[0039] definition

[0040] Unless otherwise noted, the practice of the present invention will adopt conventional chemistry, biochemistry, recombinant DNA technology and immunological methods in the art. Such technology is fully explained in the literature (see, for example, Fundamental Virology, Second Edition, vol. I & II (BN Fields and DMKnipe compile); Handbook of Experimental Immunology, VoIs. I-FV (DM Weir and CC. Blackwell compile, Blackwell Scientific Publications); T Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan compile, Academic Press, Inc.).

[0041] To facilitate understanding of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0042] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Recombinant AAV vectors can infect both dividing and quiescent cells and can remain extrachromosomal without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy.

[0043] Administration / Given: Providing or administering an agent, such as a therapeutic agent (e.g., recombinant AAV), to a subject by an effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intracerebroventricular, intrathecal, intraspinal), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0044] Codon-Optimized: A "codon-optimized" nucleic acid refers to a nucleic acid sequence that has been altered so that the codons are optimal for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species (e.g., human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

[0045] Enhancer: A nucleic acid sequence that increases transcription efficiency by increasing promoter activity.

[0046] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences within the adeno-associated virus genome that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as origins of replication for viral DNA synthesis and are essential cis-elements for the production of AAV integrative vectors.

[0047] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from naturally occurring cells or tissues of an organism or from other biological components of the organism itself (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells). Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0048] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are placed in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0049] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for the drug delivery of one or more therapeutic compounds, molecules or agents.

[0050] In some embodiments, the parenteral formulation comprises an injectable fluid, which comprises a pharmaceutically and physiologically acceptable fluid, such as water, normal saline, balanced salt solution, aqueous dextrose, glycerol, etc., as a solvent. For solid compositions (such as, powders, pills, tablets or capsule forms), conventional non-toxic solid carriers such as pharmaceutical grade mannitol, lactose, starch or magnesium stearate can be included. In addition to biological neutral carriers, pharmaceutical compositions to be administered can also include a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0051] Prevent, Treat, or Ameliorate a Disease: "Preventing" a disease means inhibiting the full development of the disease. "Treatment" refers to therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after the disease has begun. "Amelioration" refers to reducing the number or severity of signs or symptoms of a disease.

[0052] Promoter: A region of DNA that directs / causes transcription of a nucleic acid (e.g., a gene). A promoter includes the necessary nucleic acid sequences near the transcription start site. Typically, a promoter is located near the gene it transcribes. The promoter region also optionally includes distal enhancer or repressor elements, which can be located thousands of base pairs away from the transcription start site.

[0053] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule that has a sequence that does not occur in nature, or a sequence that is produced by the artificial combination of two sequence fragments that would otherwise be separate. This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of isolated nucleic acid molecule fragments (e.g., through genetic engineering techniques).

[0054] Likewise, a recombinant virus is a virus comprising a non-naturally occurring sequence or a sequence prepared by an artificial combination of sequences from at least two different sources. The term "recombinant" also includes nucleic acids, proteins, and viruses that are altered only by the addition, substitution, or deletion of a portion of a natural nucleic acid molecule, protein, or virus. "Recombinant AAV" as used herein refers to an AAV particle in which a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding G6Pase-α) is packaged.

[0055] Serotype: A group of closely related microorganisms (eg, viruses) distinguished by a characteristic set of antigens.

[0056] Subject: A living multicellular vertebrate organism, a class including humans and non-human mammals. Specifically, the subject described herein is an individual with spinal cord injury, who may have residual endogenous KCC2 protein activity, or no measurable activity.

[0057] Synthetic: Produced artificially in a laboratory, e.g., synthetic nucleic acids can be chemically synthesized in a laboratory.

[0058] Therapeutically effective amount: The amount of a particular drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve the desired effect in a subject or cell being treated with the agent. The effective amount of the agent depends on a variety of factors, including, but not limited to, the subject or cell being treated, and the mode of administration of the therapeutic composition.

[0059] Vector: A vector is a nucleic acid molecule that allows the insertion of exogenous nucleic acid without disrupting the vector's ability to replicate and / or integrate in a host cell. A vector may contain nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of the inserted gene. In some embodiments herein, the vector is an AAV vector.

[0060] Sequence identity: The identity or similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percent similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more pronounced when orthologous proteins or cDNAs are from more closely related species (e.g., human and mouse sequences) than when they are from more distantly related species (e.g., human and C. elegans sequences).

[0061] The length of sequence identity comparison can be over the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides is desirable. However, identity in smaller fragments (e.g., having at least about 9 nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides) can also be desirable.

[0062] The percent identity of an amino acid sequence can be readily determined over the full length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid coding sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 700 amino acids in length. Typically, when referring to "identity," "homology," or "similarity" between two different sequences, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences, which typically contain corrections for missing or additional bases or amino acids compared to a reference sequence.

[0063] The alignment can be performed using any public or commercially available multiple sequence alignment program. Sequence alignment programs are available for amino acid sequences, such as "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" programs. Typically, any of these programs is used with default settings, although those skilled in the art can change these settings as needed. Alternatively, one skilled in the art can employ another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithm or program. See, for example, JD Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13): 2682-2690 (1999).

[0064] Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "ClustalW", "CAPSequence Assembly", "BLAST", "MAP" and "MEME", which can be accessed through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the VectorNTI application can also be used. There are also many algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the above programs. As another example, FastA TM (a program in GCG Version 6.1) to compare polynucleotide sequences. TM Provides the alignment and percent sequence identity of the best overlapping region between the query and search sequences. For example, Fasta TM Percent sequence identity between nucleic acid sequences is determined using its default parameters as provided in GCG Version 6.1 (incorporated herein by reference) (a word length of 6, and a NOPAM factor for the scoring matrix).

[0065] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial virus particle in which an expression cassette containing the relevant gene is packaged in a viral capsid or envelope, wherein any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective, i.e., they cannot produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome can be engineered to be "contentless" - containing only relevant transgenes flanking the signals required for amplification and packaging of the artificial genome), but these genes can be provided during the production process. Therefore, it is considered safe for use in gene therapy because replication and infection by progeny virions do not occur except in the presence of the viral enzymes required for replication. Such replication-defective viruses can be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating) or another suitable viral source.

[0066] "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for the cell-mediated synthesis of the second chain, the two complementary halves of the scAAV will associate to form a double-stranded DNA (dsDNA) unit ready for immediate replication and transcription, see, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAV is described in, for example, US6,596,535B1, US7,125,717B2, and US7,456,683B2, each of which is incorporated herein by reference in its entirety.

[0067] The term "expression" is used herein in its broadest sense and includes the production of DNA or RNA and proteins. With respect to RNA, the term "expression" or "translation" particularly relates to the production of peptides or proteins. Expression can be transient or can be stable.

[0068] The term "translation" in the context of the present invention relates to the process of processing at the ribosome whereby an mRNA chain is assembled into a controlled sequence of amino acids to produce a protein or peptide.

[0069] The term "a" or "an" refers to one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0070] The word "comprising" should be interpreted as inclusive rather than exclusive. The word "consisting of" and its variations should be interpreted as exclusive rather than inclusive. Although various embodiments in the specification appear to use the language of "comprising", in other cases, the relevant embodiments are also intended to be interpreted and described using the language of "consisting of" or "consisting essentially of".

[0071] As used herein, the term "about" refers to a difference of 10% (±10%) from a given reference value, unless otherwise specified.

[0072] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0073] promoter

[0074] In a first aspect, the present invention provides a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 6 or 7, or a nucleotide sequence having 80% or greater (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity to SEQ ID NO: 6 or 7. In particular, the nucleic acid molecule is a promoter; more particularly, the nucleic acid molecule is a human MeCp2 promoter.

[0075] MeCp2 is a gene encoding murine methyl-CpG-binding protein 2, which is highly expressed in neurons of the central nervous system and plays an important role in maintaining synaptic connections between neurons.

[0076] The MeCp2 promoter is the promoter of the methyl CpG binding protein 2 (murine methyl-CpG-binding protein 2), which is located upstream of the start codon of the MeCp2 gene in the genome and can regulate and initiate the transcription of the gene DNA sequence.

[0077] In some preferred embodiments of the present invention, the promoter is the mMeCp2 promoter, and the mMeCp2 is the natural promoter of the mouse MeCp2 gene.

[0078] recombinant nucleic acid molecules

[0079] The second aspect of the present invention provides a recombinant nucleic acid molecule, which comprises the above-mentioned nucleic acid molecule (or, also referred to as a promoter), a nucleic acid sequence encoding an exogenous protein and a polyadenylic acid sequence (ie, polyA).

[0080] In one embodiment, the exogenous protein is KCC2 protein.

[0081] KCC2 protein and nucleic acid sequence encoding the same

[0082] KCC2 is K + -Cl - Cotransporter 2, also known as solute carrier family 12 member 5, is a neuron-specific K + -Cl - Cotransporter, mainly mediates neuron-specific K + -Cl - Transport. The KCC2 protein has 12 transmembrane regions, 6 extracellular loops, a short intracellular amino terminus (NTD), and a longer intracellular carboxyl terminus (CTD). KCC2 has two isoforms: KCC2a and KCC2b. The difference between them is that the NTD of KCC2a has 23 more amino acids than the NTD of KCC2b. KCC2 proteins have been found in many species, including human KCC2 protein, mouse KCC2 protein, rat KCC2 protein, rabbit, dog, pig, etc. The amino acid sequences and nucleotide sequences of various KCC2 proteins are known and can be found in various biological information databases such as NCBI. For example, the NCBI Gene ID of the full-length human KCC2a is 57468, its amino acid sequence number is NCBI RefSeq: NP_001128243.1 (SEQ ID NO: 3), and its mRNA sequence number is NCBI RefSeq: NM_001134771.2.

[0083] The KCC2 protein of the present invention can be derived from different species. In one embodiment, the KCC2 protein of the present invention is a naturally occurring mature KCC2 protein. In another embodiment, the KCC2 protein of the present invention includes various mutants or derivatives that retain biological activity.

[0084] In one embodiment, a modified or optimized KCC2 protein coding sequence is provided. In one embodiment, the KCC2 protein coding sequence is a codon-optimized sequence optimized for expression in the subject species. As used herein, a "subject" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, or gorilla. In a preferred embodiment, the subject is a human. In one embodiment, the sequence is codon-optimized for expression in humans.

[0085] Codon optimized coding region can be designed by various methods.Can use the method (such as GeneArt) that can obtain online, disclosed method or the company that provides codon optimization service, such as DNA2.0 (Menlo Park, CA) to carry out this optimization.For example, in International Patent Publication No. WO 2015 / 012924A2, a kind of codon optimization method is described, and it is incorporated herein by reference in its entirety.Also referring to such as US2014 / 0032186A1 and US2006 / 0136184A1.Suitably, the whole length of the open reading frame (ORF) of this product is revised.But, in some embodiments, can only change the fragment of ORF.By using one in these methods, can apply described frequency to any given peptide sequence, and produce the codon optimized encoding sequence of this polypeptide of coding.

[0086] Many selections can be used for actually changing codon or for synthesizing the codon-optimized coding region of design as described herein.Such modification or synthesis can be carried out using standard and conventional molecular biology operations well known to those of ordinary skill in the art.In one method, a series of complementary oligonucleotide pairs having a length of 80-90 nucleotide and spanning a desired sequence length are synthesized by standard methods.These oligonucleotide pairs are synthesized so that they form double-stranded fragments of 80-90 base pairs containing sticky ends when annealed, for example, each oligonucleotide in the pair is extended by 3,4,5,6,7,8,9,10 or more bases beyond the region complementary to another oligonucleotide in the pair.The single-stranded end of each oligonucleotide pair is designed to anneal with the single-stranded end of another oligonucleotide pair.This oligonucleotide pair is annealed, and subsequently about five to six of these double-stranded fragments are annealed together via sticky single-stranded ends, and subsequently they are linked together and cloned into standard bacterial cloning vectors, for example, available from Invitrogen Corporation, Carlsbad, California. Vector. The construct is then sequenced by standard methods. Several constructs consisting of 5 to 6 fragments of 80 to 90 base pairs (i.e., fragments of about 500 base pairs) linked together are prepared so that the entire desired sequence is displayed as a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and linked together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be apparent to those skilled in the art. In addition, gene synthesis is readily available.

[0087] The nucleotide sequences encoding the KCC2 proteins described herein can be synthesized using techniques well known in the art. For example, PCR-based accurate synthesis of long DNA sequences (PAS) can be employed, as described by Xiong et al., PCR-based accurate synthesis of long DNA sequences, Nature Protocols 1, 791-797 (2006). A method combining double asymmetric PCR and overlap extension PCR is described by Young and Dong, Two-step total gene synthesis method, Nucleic Acids Res. 2004; 32(7): e59. See also Gordeeva et al., J Microbiol Methods. Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification. 2010 May;81(2):147-52. Epub 2010 Mar 10; see also the following patents on oligonucleotide synthesis and gene synthesis, Gene Seq. 2012 Apr;6(1):10-21; US ​​8008005 and US 7985565. Each of these documents is incorporated herein by reference. In addition, kits and protocols for generating DNA via PCR are commercially available. These include the use of polymerases, including but not limited to Taq polymerase, (New England Biolabs), High-Fidelity DNA polymerase (New England Biolabs) and G2 polymerase (Promega). DNA can also be generated from cells transfected with a plasmid containing the KCC2 protein sequence described herein. Kits and protocols are known and commercially available and include, but are not limited to, QIAGEN plasmid kits, Pro Filter Plasmid Kit (Invitrogen) and GenElute TMPlasmid kit (Sigma Aldrich). Other techniques available in this article include sequence-specific isothermal amplification methods, which eliminate the need for thermal cycling. These methods typically use strand-displacing DNA polymerases such as Bst DNA polymerase, Large Fragment (New England Biolabs) rather than heating to separate duplex DNA. DNA can also be generated from RNA molecules by amplification using reverse transcriptase (RT), which is an RNA-dependent DNA polymerase. RT polymerizes a DNA strand complementary to the original RNA template and is referred to as cDNA. This cDNA can then be further amplified by PCR or isothermal methods as described above. Custom DNA can also be generated commercially by companies including, but not limited to, GenScript, (Life Technologies) and Integrated DNA Technologies.

[0088] In some specific embodiments of the present invention, the nucleic acid molecule encoding the KCC2 protein is DNA or RNA. In some specific embodiments of the present invention, the nucleic acid molecule encoding the KCC2 protein has a polynucleotide sequence selected from the following:

[0089] 1) the polynucleotide sequence shown in SEQ ID NO: 2;

[0090] 2) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the polynucleotide sequence shown in SEQ ID NO: 2; or

[0091] 3) a polynucleotide sequence having more than 80% sequence identity with SEQ ID NO: 2, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or more; more preferably, a sequence having 98% or 99% identity or more; in a more preferred embodiment, the polynucleotide sequence is a codon-optimized sequence.

[0092] Polyadenylation sequence (PolyA)

[0093] In some embodiments of the present invention, the polyadenylation sequence (polyA) is BGH polyA.

[0094] BGH polyA is a bovine growth hormone polyadenylation signal, which is a transcription termination signal in eukaryotic organisms. In a specific embodiment of the present invention, the sequence of BGH polyA is shown in SEQ ID NO:8.

[0095] Other regulatory elements

[0096] In some specific embodiments of the present invention, the recombinant nucleic acid molecule further comprises other expression control elements, such as one or more of a Kozak sequence, a WPRE, and a post-transcriptional regulatory element. The Kozak sequence can enhance translation efficiency, and the WPRE sequence can enhance and stabilize mRNA expression.

[0097] In some preferred embodiments of the present invention, the recombinant nucleic acid molecule comprises a polynucleotide sequence selected from the group consisting of:

[0098] 1) the polynucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13;

[0099] 2) a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13; or

[0100] 3) A polynucleotide sequence having more than 80% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or more; more preferably, a sequence having 98% or 99% identity or more.

[0101] The polynucleotide sequence shown in SEQ ID NO: 12 comprises the following elements: an hMeCp2 promoter, a nucleic acid sequence encoding full-length KCC2, and a BGH polyA sequence. The polynucleotide sequence shown in SEQ ID NO: 13 comprises the following elements: a short hMeCp2 promoter, a nucleic acid sequence encoding full-length KCC2 protein, and a BGH polyA sequence.

[0102] In certain specific embodiments, the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence; preferably, the AAV inverted terminal repeat sequence is selected from AAV of different serotypes; preferably, the AAV inverted terminal repeat sequence is selected from AAV of any serotype in evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types; more preferably, the AAV inverted terminal repeat sequence is from AAV2.

[0103] Recombinant vector or recombinant adeno-associated virus

[0104] The third aspect of the present invention further provides a recombinant vector comprising the aforementioned nucleic acid molecule (or, also referred to as a promoter) or a recombinant nucleic acid molecule.

[0105] In some embodiments, the vector is selected from a plasmid vector, a phage vector or a viral vector, wherein the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector or a hybrid viral vector.

[0106] In some embodiments, the nucleic acid molecules encoding KCC2 proteins described herein can be engineered into suitable genetic elements (vectors) that can be used to generate viral vectors and / or delivered to host cells, such as naked DNA, bacteriophages, transposons, cosmids, episomes, etc., which transmit the nucleic acid molecules encoding KCC2 proteins carried thereon. The selected vectors can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated beads, viral infection, and protoplast fusion. Methods for making such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0107] The fourth aspect of the present invention also provides a recombinant adeno-associated virus, which comprises an AAV capsid and a vector genome, wherein the vector genome comprises the aforementioned nucleic acid molecule (or, also referred to as a promoter) or a recombinant nucleic acid molecule.

[0108] Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles that can package exogenous nucleic acid sequences into AAV protein capsids for delivery to target cells. The AAV capsid is composed of 60 capsid protein subunits VP1, VP2, and VP3, which are arranged in an icosahedral symmetry at a ratio of approximately 1:1:10 to 1:1:20 depending on the AAV subtype. The AAV capsid can be selected from those AAV capsids known in the art, including variants thereof.

[0109] In one embodiment, the AAV capsid is selected from those AAV capsids that can effectively transduce neuronal cells. In one embodiment, the AAV capsid is selected from AAV1, AAV2, AAV7, AAV8, AAV9, AAVrh.10, AAV5, AAVhu.11, AAV8DJ, AAVhu.32, AAVhu.37, AAVpi.2, AAVrh.8, AAVhu.48R3 and variants thereof, see Royo et al., Brain Res, 2008 Jan, 1190: 15-22; Petrosyan et al., Gene Therapy, 2014 Dec, 21(12): 991-1000; Holehonnur et al., BMC Neuroscience, 2014, 15: 28; and Cearley et al., Mol Ther. 2008 Oct, 16(10): 1710-1718, each of which is incorporated herein by reference.

[0110] Other AAV capsids available herein include AAVrh.39, AAVrh.20, AAVrh.25, AAV10, AAVbb.1, and AAVbb.2, and variants thereof. As the source of capsids for AAV viral vectors (DNase-resistant virions), other AAV serotypes may be selected, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64R1, AAVrh64R2, AAVrh8, AAVrh.10, any known or mentioned AAV variants, or AAVs yet to be discovered, see, for example, US20070036760A1, US20090197338A1, EP 1310571A2. See also WO 2003 / 042397 A2 (AAV7 and other simian AAVs), US7790449 B2 and US7282199 B2 (AAV8), WO2005 / 033321 A2 and US 7,906,111 B2 (AAV9), and WO 2006 / 110689 A2 and WO 2003 / 042397 A2 (AAVrh.10). Alternatively, recombinant AAV based on any of the described AAVs can be used as a source of AAV capsids. These documents also describe other AAVs that can be selected for generating AAVs and are incorporated herein by reference. In some embodiments, the AAV capsid for the viral vector can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV capsids or its encoding nucleic acid.

[0111] In some specific embodiments, the AAV capsid is chimeric and comprises domains from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a chimera of Vpl, Vp2, and Vp3 monomers from two or three different AAVs or recombinant AAVs. As used herein, with respect to AAV, the term variant refers to any AAV sequence derived from a known AAV sequence, including sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity in amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant comprising up to about 10% variation with any of the described or known AAV capsid sequences. That is, the AAV capsid shares about 90% to 99.9% identity, about 95% to 99% identity, or about 97% to 98% identity with the AAV capsids provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made for any variable protein (e.g., vp1, vp2, or vp3). In another embodiment, a self-complementary AAV (scAAV) is used. In another embodiment, a single-stranded AAV (ssAAV) is used.

[0112] In some preferred embodiments, the AAV capsid is selected from any serotype of AAV in clades AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or hybrid / chimeric forms thereof. In certain preferred embodiments, the capsid of the recombinant adeno-associated virus is AAV9, the amino acid sequence of which is shown in SEQ ID NO: 14. In some preferred embodiments, the recombinant adeno-associated virus is a single-stranded recombinant adeno-associated virus.

[0113] In some specific embodiments, the recombinant nucleic acid molecules described herein can be packaged into the capsid of a viral vector (e.g., virion). Typically, such recombinant nucleic acid molecules for generating viral vectors contain the coding sequence of the KCC2 protein described herein, the packaging signal of the viral genome on its side, and other expression control sequences, such as those described herein. For example, for AAV viral vectors, the packaging signal is 5' inverted terminal repeats (ITR) and 3' ITR. When packaged into the AAV capsid, the recombinant nucleic acid molecule and the ITRs associated therewith are referred to herein as "recombinant AAV (rAAV) genome" or "vector genome."

[0114] In some embodiments, the recombinant adeno-associated virus vector (rAAV) provided by the present invention comprises an AAV capsid and a vector genome as described herein. In some embodiments, the vector genome comprises a nucleic acid molecule encoding a KCC2 protein and a regulatory sequence (such as a nucleic acid molecule described herein (or, alternatively, a promoter)) that guides and / or regulates the expression of the above nucleic acid molecule in a host cell. In some embodiments, the vector genome comprises the recombinant nucleic acid molecule described in the present invention. In other embodiments, the vector genome further comprises an ITR sequence of AAV. In one embodiment, the ITRs are from an AAV serotype different from the one providing the capsid. In a preferred embodiment, the ITR sequence is from AAV2, or a deletion variant thereof (ΔITR), which may be used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources can be selected. When the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as a pseudotype. Typically, the AAV vector genome comprises an AAV 5'ITR and an AAV3'ITR. Deletion variants of the 5' ITR (referred to as ΔITR) in which the D-sequence and terminal resolution site (trs) are deleted have been described. In some embodiments, the 5' and 3' ITRs of full-length AAV are used. The ITR or ΔITR sequences of each serotype are well known to those skilled in the art.

[0115] In one embodiment, the capsid is an AAV9 capsid (the amino acid sequence of which is shown in SEQ ID NO: 14) or a variant thereof.

[0116] In one embodiment, single-stranded AAV (ssAAV) can be selected.

[0117] In one embodiment, double-stranded AAV, or self-complementary AAV (scAAV) can be selected.

[0118] The method for generating and separating AAV viral vectors suitable for delivery to a subject is known in the art. See, for example, US2007 / 0036760A1 (February 15, 2007), US7790449B2, US7282199B2, WO 2003 / 042397 A2, WO 2005 / 033321 A2, WO 2006 / 110689A2, and US7588772B2. In one system, a transgenic construct encoding side ITRs and a construct encoding rep and cap are transiently transfected to produce a cell line. In a second system, a transgenic construct encoding side ITRs is transiently transfected to stably provide a packaging cell line of rep and cap. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, wherein it is necessary to separate rAAV from contaminating viruses. Later, a system was developed that did not require infection with a helper virus to recycle AAV, by which the required auxiliary functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29 and herpesvirus polymerase) were provided in trans. In these systems, the auxiliary functions can be provided by transiently transfecting the cells with a construct encoding the required auxiliary functions, or the cells can be engineered to stably contain genes encoding the auxiliary functions, the expression of which can be controlled at the transcriptional or post-transcriptional levels. In another system, transgenic and rep / cap genes flanking ITRs are introduced into insect cells by infection with a baculovirus-based vector. For an overview of these production systems, see, for example, Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Patent Nos. 5,139,941;

[0119] Optionally, the recombinant nucleic acid molecules described herein can also be used to generate other viral vectors. Such other viral vectors can include any virus suitable for gene therapy that can be used, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc. Suitably, when one of these other vectors is generated, it is generated as a replication-defective viral vector.

[0120] cell

[0121] The fifth aspect of the present invention further provides an isolated host cell, which comprises the aforementioned nucleic acid molecule (or, also referred to as a promoter) or recombinant nucleic acid molecule or the aforementioned recombinant vector or recombinant adeno-associated virus.

[0122] In some specific embodiments, the cells are neuronal cells; in some specific embodiments, the cells are cholinergic neuronal cells, noradrenergic neuronal cells, dopaminergic neuronal cells, serotonergic neuronal cells, γ-aminobutyric acid neuronal cells; in some specific embodiments, the cells are neural cell lines, including NG108-15, N1E115, PC12, SHSY5Y, F11, ND7-23, N27, SN4741 cell lines.

[0123] Pharmaceutical composition

[0124] The sixth aspect of the present invention further provides a pharmaceutical composition comprising the aforementioned nucleic acid molecule (or, also referred to as a promoter) or recombinant nucleic acid molecule, a recombinant vector or a recombinant adeno-associated virus and / or the aforementioned host cell and a pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, intraspinal injection, intrathoracic injection, oral administration, inhalation, intranasal administration, intratracheal administration, intraarterial administration, intraocular administration, intramuscular administration, intraperitoneal administration and / or other parenteral administration. In some preferred embodiments, the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.

[0125] In some specific embodiments, the pharmaceutical composition of the present invention can be designed to be delivered to a subject in need thereof by any suitable route or a combination of different routes. In one embodiment, the composition is delivered via intravenous injection. In one embodiment, the pharmaceutical composition is delivered via intraspinal injection. In one embodiment, it can be carried out via intrathecal injection. In one embodiment, the composition is delivered via intraventricular injection. In another embodiment, the composition is delivered via thoracic injection. In another embodiment, the in situ injection of the target tissue can be carried out. Alternatively, other routes of administration (such as oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and other parenteral routes) can be selected.

[0126] In some embodiments, the recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, or isolated host cells described herein can be delivered as a single composition or multiple compositions. Optionally, two or more different AAVs can be delivered (see, for example, WO 2011 / 126808A2 and WO 2013 / 049493A1). In another embodiment, such multiple viruses can contain different replication-defective viruses (e.g., AAV, adenovirus, and / or lentivirus). Alternatively, delivery can be mediated by non-viral vectors, such as "naked DNA," "naked plasmid DNA," RNA, and mRNA; in combination with various delivery compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions and other polymers, lipid-based and / or cholesterol-nucleic acid conjugates, and other constructs as described herein, see, for example, X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; online publication: Mar. 21, 2011; WO 2013 / 182683 A1, WO 2010 / 053572 A2, and WO 2012 / 170930 A1, all of which are incorporated herein by reference, such non-viral KCC2 delivery vectors can be administered by the aforementioned routes.

[0127] In some embodiments, the viral vector or non-viral DNA or RNA transfer moiety can be formulated with a physiologically acceptable carrier for gene transfer and gene therapy. In some embodiments, the delivery vector can be prepared using a variety of suitable purification methods, and examples of purification methods suitable for separating empty capsids from vector particles are described, such as those described in International Patent Application No. PCT / US16 / 65976, filed on December 9, 2016, and its priority documents, U.S. Provisional Application No. 62 / 322,098, filed on April 13, 2016, and U.S. Provisional Application No. 62 / 266,341, filed on December 11, 2015, and entitled "Scalable Purification Method for AAV8," which are incorporated herein by reference. See also the purification methods described in International Patent Application No. PCT / US16 / 65974 filed on December 9, 2016 and its priority documents, U.S. Provisional Application No. 62 / 322,083 filed on April 13, 2016, and 62 / 266,351 (AAV1) filed on December 11, 2015, International Patent Application No. PCT / US16 / 66013 filed on December 9, 2016 and its priority documents, and 62 / 266,357 (AAV9), which are incorporated herein by reference. In one embodiment, purification comprises a two-step purification scheme that selectively captures and isolates rAAV vector particles containing the genome from the clarified, concentrated supernatant of the rAAV producer cell culture. The method utilizes an affinity capture method performed at high salt concentrations, followed by an anion exchange resin method performed at high pH to provide rAAV vector particles that are substantially free of rAAV intermediates. In another embodiment, purification comprises affinity chromatography, ultracentrifugation, molecular sieve chromatography, or the like.

[0128] In the case of AAV viral vectors, quantification of viral genomes (vg) can be used as a measure of the dose contained in the formulation. The dose of rAAV administered in the methods disclosed herein will vary depending on, for example, the specific rAAV, the mode of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. The dose can be expressed in units of viral genomes (vg) (i.e., 1×10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×1010 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×l0 14 vg, 1×10 15 vg, 1×10 16 Doses can also be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1×10 6 vg / kg, 1×10 7 vg / kg, 1×10 8 vg / kg, 1×10 9 vg / kg, respectively 1×10 10 vg / kg, 1×10 11 vg / kg, 5×10 11 vg / kg, 1×10 12 vg / kg、5×10 12 vg / kg, 1×10 13 vg / kg, 1×10 14 vg / kg, 1×10 15 vg / kg, 1×10 16 vg / kg). Methods for titrating AAV are described in Clark et al., Hum. Gene Ther. 1999; 10: 1031-1039.

[0129] These aforementioned doses can be administered in various volumes of carrier, excipient, or buffer formulations ranging from about 25 microliters to about 15 milliliters, including all numbers within that range, depending on the size of the area to be treated, the titer of the virus used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 500 μL. In yet another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 700 μL. In yet another embodiment, the volume is about 800 μL. In yet another embodiment, the volume is about 900 μL. In yet another embodiment, the volume is about 1 mL. In yet another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 6 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 10 mL. In another embodiment, the volume is about 15 mL.

[0130] The rAAV is preferably suspended in a physiologically compatible carrier and can be administered to a human or non-human mammalian patient. In another embodiment, the composition includes a pharmaceutically acceptable carrier, a diluent, an excipient and / or an adjuvant. Suitable carriers can be easily selected by those skilled in the art in view of the indications for which the transfer virus is directed. For example, saline is included, which can be prepared with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The buffer / carrier should include components that prevent rAAV from adhering to the infusion tube but do not interfere with the binding activity of rAAV in vivo.

[0131] Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and a pharmaceutically acceptable carrier. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0132] The compositions of the present invention may comprise a pharmaceutically acceptable carrier as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient, and are designed to be delivered to a subject via injection, an osmotic pump, an intrathecal catheter, or by another device or route of delivery.

[0133] The viral vectors described herein can be used to prepare a medicament for delivering a nucleic acid molecule encoding a KCC2 protein to a subject (e.g., a human patient) in need thereof, or to provide a KCC2 protein to a subject for the treatment of spinal cord injury. The treatment process can optionally include repeated administration of the same viral vector (e.g., an AAV9 vector) or different viral vectors (e.g., AAV9 and AAV10). Other combinations can still be selected using the viral vectors and non-viral delivery systems described herein.

[0134] Pharmaceutical uses and treatments

[0135] A seventh aspect of the present invention further provides the use of the aforementioned nucleic acid molecule (or promoter), recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell, and / or pharmaceutical composition in the preparation of a medicament for preventing or treating spinal cord injury. In certain embodiments, the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell, and / or pharmaceutical composition may be administered in combination with another therapy.

[0136] An eighth aspect of the present invention further provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell, and / or the aforementioned pharmaceutical composition to a subject in need thereof; in certain preferred embodiments, the composition is administered intravenously; in certain preferred embodiments, the composition is administered intrathecally; in certain preferred embodiments, the composition is administered intraspinal; in other preferred embodiments, the subject is a mammal; in more preferred embodiments, the subject is a human. In some embodiments, the treatment method may also be used in combination with other therapies.

[0137] In some embodiments, the above-mentioned recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and / or the aforementioned pharmaceutical compositions can be administered repeatedly. Optionally, more than one repeated administration is allowed. Such repeated administration can be with the same type of vector, a different viral vector, or via non-viral delivery as described herein. For example, if a patient is treated with rAAV9 carrying a nucleic acid molecule encoding a KCC2 protein and a secondary treatment is required, rAAV10 carrying the aforementioned nucleic acid molecule can be subsequently administered, and vice versa.

[0138] The treatment of patients with spinal cord injury may require combination therapy, such as in some embodiments of the present invention, the pharmaceutical composition of the present invention can be used in combination with any one of substances including but not limited to CLP290, osteopontin, growth factors or 4-aminopyridine. As in other specific embodiments of the present invention, before, during and / or after treatment with the composition of the present invention, immunosuppressants are used for transient co-treatment. Immunosuppressants used for such co-therapy include but are not limited to steroids, antimetabolites, T cell inhibitors and alkylating agents. For example, such transient treatment may include taking a steroid (e.g., prednisone) once daily in decreasing doses for 7 days, starting with an amount of about 60 mg and reducing by 10 mg per day (no dose on the 7th day). Other dosages and immunosuppressants can be selected.

[0139] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art by reference to the disclosure, which provides a general guide to many of the terms used in this application. DETAILED DESCRIPTION

[0140] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0141] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0142] Example 1. Design of expression cassette and construction of shuttle plasmid carrying the expression cassette

[0143] 1. Design of expression cassettes containing different promoters

[0144] To validate the efficacy of the human promoter (SEQ ID NO: 6, hereinafter referred to as the hMeCp2 promoter; or SEQ ID NO: 7, hereinafter referred to as the short hMeCp2 promoter) described herein, this example employed multiple promoters (Syn, mMeCp2, short mMeCp2, hMeCp2, or short hMeCp2) to design multiple expression cassettes containing these different promoters. Furthermore, using an adeno-associated virus (AAV) vector as a delivery vehicle, the effects of these different promoters on the expression of wild-type, full-length human KCC2 protein in the nervous system were validated.

[0145] The expression cassette containing the combination and its nucleic acid sequence are shown below (the schematic diagram of its structure and composition can be seen in Figure 1, which also shows the ITRs located on both sides of the expression cassette, and the ITRs are derived from AAV2):

[0146] (1) Syn-KCC2-BGH poly A (SEQ ID NO: 9);

[0147] (2) mMeCp2-KCC2-BGH polyA (SEQ ID NO: 10);

[0148] (3) short mMeCp2-KCC2-BGH polyA (SEQ ID NO: 11);

[0149] (4) hMeCp2-KCC2-BGH polyA (SEQ ID NO: 12);

[0150] (5) short hMeCp2-KCC2-BGH polyA (SEQ ID NO: 13).

[0151] Among them, Syn-KCC2-BGH poly A (SEQ ID NO: 9) is an expression cassette that has been disclosed in existing literature (see WO2019226643A1) and is used as a positive control in this example.

[0152] The nucleotide sequences of each promoter and poly A are shown in Table 1 below:

[0153] Table 1

[0154] 2. Construction of the shuttle plasmid carrying the above expression cassette

[0155] Based on the above expression cassettes, shuttle plasmids carrying the above expression cassettes are constructed for packaging the corresponding recombinant adeno-associated virus vectors.

[0156] 2.1 Construction of the shuttle plasmid carrying the expression cassette Syn-KCC2-BGH poly A

[0157] (1) The pSNAV2.0-Syn-EGFP vector (Figure 2A) was double-digested with restriction endonucleases Sal I and Xho I, and the large fragment (approximately 6329 bp) was recovered.

[0158] (2) The human Syn promoter (human synapsin I promoter, human synapsin I promoter, SEQ ID NO: 1) and its seamless primers (forward primer: 5'-TGGTGGCGGCGATATCCTGCGCTCTCAGGCACGACAC-3', reverse primer: 5'-GGGTTCCTCTGCTCGAGCAGTGCAAGTGGGTTTTAGG-3') and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, the amino acid sequence of which is shown in SEQ ID NO: 3) and its seamless primers (forward primer: 5'-TCGAGGCAGATCTGTCGAC-3', reverse primer: 5'-CAGGATATCGCCGCCACCATGAGCCGCAGGTTCACGGTC-3') were synthesized respectively (synthesized by Beijing Nosai Genome Research Center Co., Ltd.).

[0159] (3) The Syn promoter and KCC2 protein coding sequence were inserted between the 5'ITR sequence and the BGH polyadenylation of the backbone vector by seamless cloning PCR. The constructed plasmid was named pSNAV2.0-Syn-KCC2 (Figure 2B).

[0160] (4) The plasmid pSNAV2.0-Syn-KCC2 was double-digested with SwaI and NruI to recover a vector fragment of approximately 6.2 kb.

[0161] (5) Use seamless ligation primer pair 1 (forward primer:

[0162] 5'-CAGCTGAACCAACTCGCGAATTCCACAGCTGCCTCGCGC-3', reverse primer:

[0163] 5'-GGTGGCGGCGATATCCTGCGCTCTCAGGCACGAC-3') and primer pair 2 (forward primer:

[0164] 5'-CCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTA-3', reverse primer:

[0165] 5'-AGGTTAAGTCCTCATTTAAATTAGGCAA-3') was used to amplify fragment 1 (about 1.27 kb) and fragment 2 (about 270 bp) on the original vector by PCR using the above-mentioned plasmid pSNAV2.0-Syn-KCC2 as a template.

[0166] (6) The kana resistance-encoding gene was amplified by PCR using the seamless primer pair 3 (forward primer: 5′-TTAGAAAAACTCATCGAGCATCAAATG-3′, reverse primer: 5′-ATGAGCCATATTCAACGGGA-3′) and the synthetic 1902-Kan (containing the kana resistance-encoding gene) as a template, and was named fragment 3.

[0167] (7) PCR was used to add homology arms between the vector and fragment 1, fragment 1 and fragment 3, fragment 3 and fragment 2, and fragment 2 and the vector. Fragment 1, fragment 2, fragment 3, and the vector fragment of pSNAV2.0-Syn-KCC2 were connected by seamless connection. The resulting shuttle plasmid was named pSNAV2.0-K-Syn-KCC2-BGH ( Figure 2C , the main structure of its vector genome is shown in Figure 1A ).

[0168] 2.2 Construction of shuttle plasmid carrying the mMeCp2-KCC2-BGH polyA expression cassette

[0169] (1) The pSNAV2.0-K-Syn-KCC2-BGH vector (as shown in Figure 3A) was double-digested with restriction endonucleases SalI and XhoI, and the large fragment (about 4.5 kb) was recovered.

[0170] (2) The mMeCp2 promoter (SEQ ID NO: 4) and its seamless primer (forward primer: 5′-TGGTGGCGGCGATATCTCCGCTCGGCGCGGCGGC-3′, reverse primer: 5′-CTAGGGGTTCCTCTGCTCGAGCACACACCATCTGCCCATTATAAACGTC-3′) and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is as SEQ ID NO: 3) and its seamless primer (forward primer: same as Section 2.1, reverse primer: 5′-GATATCGCCGCCACCATGAGC-3′) were synthesized by Beijing Nosai Genome Research Center Co., Ltd.

[0171] (3) The mMeCp2 promoter and KCC2 protein coding sequence were inserted between the 5' ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR. The resulting shuttle plasmid was named pSNAV2.0-K-mMeCp2-KCC2-BGH ( Figure 3B , the main structure of the vector genome is shown in Figure 1B ).

[0172] 2.3 Construction of a shuttle plasmid carrying the shortmMeCp2-KCC2-BGH expression cassette

[0173] (1) The pSNAV2.0-K-Syn-KCC2-BGH vector (as shown in Figure 3A) was double-digested with restriction endonucleases Sal I and Xho I, and the large fragment (about 4.5 kb) was recovered.

[0174] (2) The short mMeCp2 promoter (SEQ ID NO: 5) and its seamless primer (forward primer: same as in Section 2.2, reverse primer: 5'-CTAGGGGTTCCTCTGCTCGAGCACAACCAATGAAGGGTAATCTCGACAAAG-3') and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is as SEQ ID NO: 3) and its seamless primer (forward primer: same as in Section 2.2, reverse primer: same as in Section 2.2) were synthesized by Beijing Nosai Genome Research Center Co., Ltd.

[0175] (3) The short mMeCp2 promoter and KCC2 coding sequence were inserted between the 5' ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR. The resulting shuttle plasmid was named pSNAV2.0-K-short mMeCp2-KCC2-BGH ( Figure 3C , the main structure of the vector genome is shown in Figure 1C ).

[0176] 2.4 Construction of a shuttle plasmid carrying the hMeCp2-KCC2-BGH polyA expression cassette

[0177] (1) The pSNAV2.0-K-Syn-KCC2-BGH vector (as shown in Figure 3A) was digested with restriction endonucleases Sal I and Xho I, and the large fragment (approximately 4.5 kb) was recovered.

[0178] (2) The hMeCp2 promoter (SEQ ID NO: 6) and its seamless primer (forward primer: 5′-CATGGTGGCGGCGATATCCGCGGCGGCGGCGGC-3′, reverse primer: 5′-CTAGGGGTTCCTCTGCTCGAGCAAGCCAGGGTTGCGATTTGTTG-3′) and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is as SEQ ID NO: 3) and its seamless primer (forward primer: 5′-GTCGAGGCAGATCTGTCGACTCAGGAGTAGATGGTGATGACCTC-3′, reverse primer: 5′-GATATCGCCGCCACCATGAGCC-3′) were synthesized by Beijing Nosai Genome Research Center Co., Ltd.

[0179] (3) The hMeCp2 promoter and KCC2 protein coding sequence were inserted between the 5' ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR. The resulting shuttle plasmid was named pSNAV2.0-K-hMeCp2-KCC2-BGH (as shown in FIG3D , and the main structure of the vector genome is shown in FIG1D ).

[0180] 2.5 Construction of a shuttle plasmid carrying the short hMeCp2-KCC2-BGH polyA expression cassette

[0181] (1) The pSNAV2.0-K-Syn-KCC2-BGH vector (as shown in Figure 3A) was digested with restriction endonucleases SalI+XhoI to recover the large fragment (approximately 4.5 kb).

[0182] (2) The short hMeCp2 promoter (SEQ ID NO: 7) and its seamless primers (forward primer: 5'-TCATGGTGGCGGCGATATCGGCGGCGGCGGCGGC-3', reverse primer: 5'-ACTAGGGGTTCCTCTGCTCGAGCACTCCCCGTCCCAGCCCGG-3') and the coding sequence of KCC2 protein (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is as SEQ ID NO: 3) and its seamless primers (forward primer: same as Section 2.4, reverse primer: same as Section 2.4) were synthesized by Beijing Nosai Genome Research Center Co., Ltd.

[0183] (3) The short hMeCp2 promoter and KCC2 protein coding sequence were inserted between the 5' ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR. The resulting shuttle plasmid was named pSNAV2.0-K-short hMeCp2-KCC2-BGH (as shown in Figure 3E , the main structure of the vector genome is shown in Figure 1E ).

[0184] Example 2: Virus packaging and purification

[0185] The present invention uses HEK293T cells as the production cell line and a conventional three-plasmid packaging system to produce AAV viral vectors. The experimental methods used are conventional methods in the art. (For example, see Xiao Xiao, Juan Li, and Richard Jude Samulski. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 1998, 72(3):2224).

[0186] The shuttle plasmids obtained in Example 1 (pSNAV2.0-K-Syn-KCC2-BGH, pSNAV2.0-K-mMeCp2-KCC2-BGH, pSNAV2.0-K-short mMeCp2-KCC2-BGH, pSNAV2.0-K-hMeCp2-KCC2-BGH and pSNAV2.0-K-short hMeCp2-KCC2-BGH) were used to package recombinant adeno-associated virus vectors carrying the above-mentioned different promoters and expression cassettes, respectively, and were named ssAAV 2 / 9-Syn-KCC2-BGH polyA, ssAAV 2 / 9-mMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-short mMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA and ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA.

[0187] The packaged AAV viral vectors were purified using AAV9 affinity chromatography (Thermo Scientific POROS CaptureSelect AAV9 affinity resin A27359)-ultracentrifugation-G25 chromatography, and the viral vector genome titer was then determined by Q-PCR. The primer and probe sequences used in the PCR assay are shown in Table 2, and the results of the recombinant AAV viral genome titer are shown in Table 3.

[0188] Table 2 Primer and probe sequences used in genomic titer detection

[0189] Table 3 Genome titer detection results

[0190] Example 3: In vitro efficacy evaluation

[0191] 1. Immunofluorescence detection of primary neuronal system

[0192] Primary cortical neurons from P0 rats were isolated and cultured in vitro. Specifically, SD rats within 12 hours after birth were placed under a microscope, the pia mater was carefully removed, the cortex was digested with papain, and then perforated into a single-cell suspension, which was then cultured in Neurobasal medium (Invitrogen, 21103-049) containing 1% FBS and 2% B27.

[0193] Neurons were infected with recombinant viral vectors (ssAAV 2 / 9-mMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-shortmMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-hMeCp2-KCC2-BGH polyA, and ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA) containing different promoter expression cassettes at the same MOI (1E+6). Negative controls (NCs) received the same volume of viral solvent. The cells were cultured for 7 days at 37°C in a 5% CO2 incubator before immunofluorescence analysis. Neurons were fixed with 4% PFA for 10 minutes and then blocked with 1% BSA, 0.1% Triton X-100 for 1 hour at room temperature. Primary antibodies against rabbit KCC2 (Millipore, 07-432) were incubated at 1:500 overnight at 4°C. The next day, a secondary antibody, Goat anti-Rabbit IgG (H+L) Alexa Fluor™ Plus 555 (Invitrogen, A32732), was added at a dilution of 1:1000 and incubated at room temperature for 1 hour. The slides were then mounted with antifade mounting medium containing DAPI and observed and photographed under a fluorescence microscope (Olympus, IX71). Fiji ImageJ software was used to calculate the mean optical density and calculate the changes in KCC2 protein levels in neurons of the different groups relative to the NC group.

[0194] The results are shown in Figure 4. Compared with NC, the expression levels of KCC2 protein on the neuronal cell membrane were significantly increased in all recombinant adeno-associated virus infection groups, indicating that the above promoters can effectively promote the expression of exogenous KCC2 gene in primary neurons. Among them, there was no significant difference in the expression level of KCC2 protein on the neuronal cell membrane between the ss-AAV2 / 9-hMeCp2-KCC2-BGH polyA infection group and the ss-AAV2 / 9-mMeCp2-KCC2-BGH polyA infection group, and between the ss-AAV2 / 9-short hMeCp2-KCC2-BGH polyA infection group and the ss-AAV2 / 9-short mMeCp2-KCC2-BGH polyA infection group, indicating that the human MeCp2 promoter and the mouse MeCp2 promoter are basically equivalent in promoting the transcription of exogenous genes in neurons.

[0195] 2. In vitro biological activity assay

[0196] In this example, the intracellular chloride ions (Cl -The biological function of KCC2 protein is to convert the intracellular Cl - Transported to the extracellular space, maintaining a low Cl - concentration. Therefore, Cl - The efflux activity of KCC2 is an important indicator for evaluating the biological activity of KCC2 protein. The SuperClomeleon technology (see Grimley, JS; Li, L.; Wang, W.; Wen, L.; Beese, LS; Hellinga, HW; Augustine, GJ (2013). Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation. Journal of Neuroscience, 33 (41), 16297–16309.) was used to evaluate the intracellular Cl - SuperClomeleon is a fluorescent protein composed of a CFP mutant and a YFP mutant expressed in cells, which can be used as a fluorescent protein for the detection of intracellular chloride ions (Cl - ) concentration changes of fluorescent reporter molecules. - Binding to SuperClomeleon will cause its conformational change, and the fluorescence resonance energy transfer (FRET) efficiency between the two fluorescent protein variants will change. By detecting this change, the interaction between SuperClomeleon and Cl - The FRET efficiency is measured using the 535nm / 485nm ratio, which serves as an indicator of intracellular chloride levels. The negative control (NC) result is set as 1, and the results of other recombinant adeno-associated viruses are compared relative to the negative control.

[0197] First, lentivirus expressing SuperClomeleon was packaged. Then, primary cortical neurons of P0 rats were isolated in vitro. The recombinant adeno-associated viruses ssAAV 2 / 9-mMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-short mMeCp2-KCC2-BGH polyA, ssAAV 2 / 9-hMeCp2-KCC2-BGH polyA, and ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA obtained by packaging in Example 2 were infected with neurons at the same MOI (1E+6). A negative control group (negative control group) was used. The same volume of virus solvent was added to the neurons (control, NC) and cultured in a 37°C, 5% CO2 incubator. Five days after infection, the neurons were infected with lentivirus expressing SuperClomeleon (MOI = 2.5). After 48 hours, the fluorescence intensity was measured by microplate reader, and the ratio of 535nm / 485nm was calculated to compare the chloride ion efflux activity of KCC2 viral vectors containing different promoters.

[0198] The results, as shown in Figure 5, showed increased chloride ion efflux activity in all recombinant adeno-associated virus vector groups compared with NC. There were no significant differences in chloride ion efflux activity between the recombinant adeno-associated virus ss-AAV2 / 9-hMeCp2-KCC2-BGH polyA infection group and the ss-AAV2 / 9-mMeCp2-KCC2-BGH polyA infection group, or between the ss-AAV2 / 9-short hMeCp2-KCC2-BGH polyA infection group and the ss-AAV2 / 9-short mMeCp2-KCC2-BGH polyA infection group. This result is generally consistent with the results obtained in the immunofluorescence assay.

[0199] The above results further confirmed that compared with mouse mMeCp2 or short mMeCp2, the use of human hMeCp2 or short hMeCp2 promoters in the technical solution of the present application did not affect the expression level of KCC2 protein on the neuronal cell membrane and in vitro biological activity. The above promoters have a high efficacy in promoting the expression of exogenous genes in nerve cells.

[0200] Example 4: In vivo efficacy evaluation

[0201] In order to further evaluate and verify the in vivo effect of the recombinant adeno-associated virus vector described in the present invention, an in vivo efficacy evaluation experiment was performed.

[0202] 1. Evaluation of motor function recovery in model animals

[0203] First, 8-week-old female C57BL / 6 mice were anesthetized with 1% sodium pentobarbital at a dose of 65 mg / kg. Then, a spinal cord injury model with bilateral paralysis of the lower limbs was induced by bilateral hemisection of T7 and T10 (Chen, Bo et al., (2018), Cell, 174(6)1599.doi:10.1016). In short, two transverse hemisections were performed simultaneously at the thoracic vertebrae T7 and T10 of the mouse. The T10 transverse hemisection ended at the midline of the spinal cord, and the T7 transverse hemisection was located on the contralateral side of the T10 injury, slightly beyond the midline of the spinal cord. After modeling, the animal was injected with 200 μL of either the virus solvent (as a model control group) or the recombinant adeno-associated virus of this application (using ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA as an example) or the positive control ssAAV2 / 9-Syn-KCC2-BGH polyA via the tail vein, with an injection dose of 1E+12 vg per animal. Antibiotics were injected for two consecutive days postoperatively to prevent infection. The recovery of motor function in the animals was assessed weekly using the BMS behavioral score (see Table 4 for scoring criteria) for a period of 5 weeks.

[0204] Table 4

[0205] The evaluation results are shown in Figure 6. At 14 days, the BMS behavioral scores of mice in the ssAAV 2 / 9-short hMeCp2-KCC2-BGHpolyA recombinant virus infection group were basically equivalent to those of the positive control group (ss-AAV 2 / 9-Syn-KCC2-BGHpolyA); on days 21, 28, and 35, they were even better than those of the positive control group. During the entire observation period, the BMS behavioral scores of mice in the ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA infection group and the positive control group were significantly higher than those in the model control group (i.e., "Model" in Figure 6). The above results show that the human hMeCp2 or short hMeCp2 promoter used in the technical solution of this application can also effectively restore the motor function of mice with spinal cord injury in vivo.

[0206] 2. Animal survival rate test

[0207] The mouse model and dosing procedures were the same as described in 4.1. Mouse survival was measured and calculated weekly for a 5-week observation period. Survival rate = number of animals surviving per week / number of animals initially modeled and dosed (expressed as a percentage). This experiment also used ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA as an example.

[0208] The animal survival rate test results are shown in Figure 7. From day 21 onward, the survival rate of mice in the ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA recombinant virus infection group was higher than that of mice in the positive control group (ss-AAV 2 / 9-Syn-KCC2-BGH polyA). This result demonstrates that the use of human hMeCp2 or short hMeCp2 promoters is safer than other existing promoters, which has great advantages in future clinical applications.

[0209] Based on the above in vivo experiments, in the assessment of animal motor function recovery, the ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA recombinant virus infection group achieved results that were essentially equivalent to or even superior to the prior art positive control ss-AAV 2 / 9-Syn-KCC2-BGH polyA. In the survival rate detection experiment, the survival rate of mice in the ssAAV 2 / 9-short hMeCp2-KCC2-BGH polyA recombinant virus infection group was higher than the survival rate of mice in the prior art positive control group (ss-AAV 2 / 9-Syn-KCC2-BGH polyA). This further confirms that the human hMeCp2 or short hMeCp2 promoter used in the technical solution of this application, consistent with its in vitro effects, also has surprising and superior in vivo activity and can also bring higher safety.

Claims

1. A nucleic acid molecule, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7, or a nucleotide sequence having more than 80% sequence identity with SEQ ID NO: 6 or 7, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid is a promoter, which can initiate transcription of a nucleic acid encoding a foreign protein in a cell.

3. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule comprises the nucleic acid molecule according to claim 1 or 2, a nucleic acid sequence encoding a foreign protein and a polyadenylic acid sequence (polyA).

4. The recombinant nucleic acid molecule according to claim 3, characterized in that The polyadenylation sequence is BGH polyA. Preferably, the sequence of BGH polyA is shown in SEQ ID NO:

8.

5. The recombinant nucleic acid molecule according to claim 3 or 4, characterized in that The exogenous protein is KCC2 protein, and preferably, its amino acid sequence is shown in SEQ ID NO:

3.

6. The recombinant nucleic acid molecule according to claim 5, characterized in that The coding sequence of the KCC2 protein is selected from: 1) the nucleotide sequence shown in SEQ ID NO: 2; 2) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 2; or 3) A nucleotide sequence having more than 80% sequence identity with SEQ ID NO: 2, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, a sequence having 98% or 99% or more identity; more preferably, the nucleotide sequence is a codon-optimized sequence.

7. The recombinant nucleic acid molecule according to any one of claims 3 to 6, characterized in that The recombinant nucleic acid molecule further comprises one or more of the following regulatory elements: Kozak sequence, WPRE and post-transcriptional regulatory element.

8. The recombinant nucleic acid molecule according to any one of claims 3 to 7, characterized in that: The recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: 1) the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; 2) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; or 3) A nucleotide sequence having more than 80% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, a sequence having 98% or 99% or more identity.

9. The recombinant nucleic acid molecule according to any one of claims 3 to 8, characterized in that The recombinant nucleic acid molecule also contains an AAV inverted terminal repeat sequence; preferably, the AAV inverted terminal repeat sequence is selected from AAVs of different serotypes; preferably, the AAV inverted terminal repeat sequence is selected from AAVs of any serotype in evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types; more preferably, the AAV inverted terminal repeat sequence is from AAV2.

10. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule described in claim 1 or 2 or the recombinant nucleic acid molecule described in any one of claims 3-8, wherein the vector is selected from a plasmid vector, a phage vector and a viral vector, wherein the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector and a hybrid viral vector.

11. A recombinant adeno-associated virus, characterized in that: The recombinant adeno-associated virus comprises an AAV capsid and a vector genome, and the vector genome comprises the nucleic acid molecule of claim 1 or 2 or the recombinant nucleic acid molecule of any one of claims 3-9.

12. The recombinant adeno-associated virus according to claim 11, characterized in that: The capsid of the recombinant adeno-associated virus is derived from AAV of any serotype in AAV clade AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types, preferably, derived from AAV9.

13. An isolated host cell, characterized in that The cell comprises the nucleic acid molecule of claim 1 or 2, the recombinant nucleic acid molecule of any one of claims 3 to 9, the recombinant vector of claim 10, or the recombinant adeno-associated virus of claim 11 or 12.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleic acid molecule of claim 1 or 2, the recombinant nucleic acid molecule of any one of claims 3-9, the recombinant vector of claim 10, the recombinant adeno-associated virus of claim 11 or 12 and / or the host cell of claim 13, and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, characterized in that The pharmaceutical composition is formulated for administration by intravenous, intrathecal, intracerebroventricular, intraspinal, intrathoracic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and / or other parenteral routes.

16. The pharmaceutical composition according to claim 14 or 15, characterized in that The pharmaceutical composition further comprises other drugs that can be used to treat spinal cord injury.

17. Use of the nucleic acid molecule according to claim 1 or 2, the recombinant nucleic acid molecule according to any one of claims 3 to 9, the recombinant vector according to claim 10, the recombinant adeno-associated virus according to claim 11 or 12, the host cell according to claim 13 and / or the pharmaceutical composition according to any one of claims 14 to 16 in the preparation of a medicament for preventing or treating spinal cord injury.

18. The use according to claim 17, characterized in that The recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and / or pharmaceutical composition may be administered in combination with another therapy.

19. The use according to claim 17 or 18, characterized in that The recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and / or pharmaceutical composition can be prepared in an amount of about 1×10 6 vg / kg to about 1×10 18 vg / kg; preferably, the recombinant adeno-associated virus can be administered at a dose of about 5×10 12 vg / kg; preferably, the recombinant adeno-associated virus can be administered at a dose of about 5×10 11 vg / kg; preferably, the vector or composition can be administered more than once.

20. A method for treating spinal cord injury, characterized in that: The method comprises administering to a subject in need of treatment a recombinant nucleic acid molecule according to any one of claims 3 to 9, a recombinant vector according to claim 10, a recombinant adeno-associated virus according to claim 11 or 12, a host cell according to claim 13 and / or a pharmaceutical composition according to any one of claims 14 to 16; preferably, the composition is administered intravenously, intrathecally or intraspinally; preferably, the subject is a mammal; more preferably, the subject is a human.

21. The method according to claim 20, characterized in that The method may also be used in conjunction with other therapies.

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