KCC2 truncated body or coding sequence and application thereof

Abstract

The invention discloses a K < + >-CL <-> cotransporter protein (KCC2) truncation and a coding sequence thereof, and also discloses a recombinant nucleic acid molecule which comprises an operably connected expression regulation sequence and a nucleic acid sequence for coding the KCC2 truncation. 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. The KCC2 truncated body disclosed by the invention has good chloride ion efflux activity and has a treatment effect on spinal cord injury. When the KCC2 truncation and the coding sequence thereof disclosed by the invention are used for gene therapy, the range of selectable gene therapy delivery vectors is wider, selectable promoters and other regulatory elements have larger selectable space, and different requirements can be more flexibly and widely met.

Description

Technical Field

[0001] The present invention relates to the field of biological therapy, and to the treatment of spinal cord injury, and in particular to a KCC2 truncate or its coding sequence, a recombinant adeno-associated virus vector carrying the coding sequence of the KCC2 truncate, and applications thereof. Background Art

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

[0003] For SCI, the most prominent problem in current treatment is how to effectively reconstruct the neural circuits on both sides of the injury plane and restore the patient's limb movement and sensory functions. However, the current treatment methods for SCI are difficult to meet clinical needs and the requirements for neural circuit reconstruction. The small molecule drugs that have been marketed, such as glucocorticoid receptor agonists, phospholipase A inhibitors, α2 adrenergic receptor agonists, etc., are all aimed at the pathological cascade reactions secondary to primary SCI, and play anti-inflammatory, anti-ischemic, immune response inhibition and neuroprotective effects; surgical treatment is also aimed at secondary injuries, and is performed within 24 hours after the vital signs of SCI patients are stable, to reduce bleeding and remove necrotic tissue. Among the drugs under development, the stem cell combined with neurotrophic factor therapy in phase II and III can only structurally reconstruct the neural circuits on both sides of the injury but cannot reconstruct the functional circuits, and still cannot meet the clinical needs of SCI.

[0004] Although, current research has used recombinant viral vectors to carry the full-length K + -Cl -There are literature reports on the use of nucleic acid sequences of co-transporter proteins (full-length KCC2) to treat spinal cord injury (WO2019226643A1). However, due to the large molecular weight of the full-length KCC2 protein (for example, one of its subtypes, KCC2a, is 1139 aa in length), its coding sequence length is about 3.4 kb. When selecting gene delivery vectors, only delivery vectors with larger capacity can be selected for delivery, resulting in a limited range of delivery vector selection. On the other hand, since the coding sequence is too long, even if it can be loaded into the delivery vector, it may also lead to compression of the selection space of other regulatory elements. For example, when selecting AAV vector as the delivery vector in the prior art, the promoter and the target gene alone have almost reached the upper limit of the capacity of the delivery vector, and other regulatory elements cannot be added. Therefore, it is difficult to flexibly respond to different needs. Although there are some KCC2 truncations in the prior art (e.g. Gregory et al., 2011, PNAS, 108(37): 15474-15479; Li et al., 2007, Neuron, 56(6): 1019-1033), these truncations do not show the desired biological activity and therefore cannot be used to treat spinal cord injury.

[0005] Based on the above, there is an urgent need to explore innovative therapeutic drugs and methods for spinal cord injury, especially the need to explore KCC2 truncation drugs that are both biologically active and more flexible and extensive in use (for example, in gene therapy). Summary of the invention

[0006] The present invention provides a K + -Cl - A truncated body of a cotransporter (KCC2), wherein the truncated body has biological activity in vivo and / or in vitro, and thus has the ability to treat spinal cord injury diseases. The technical solution of the present invention is based on the following unexpected discovery: the amino acid sequence at positions 667-684 (SEQ ID NO: 5, a total of 18 amino acids, referred to as "C-Helices" in this application) in the amino acid sequence of human wild-type KCC2a (SEQ ID NO: 10) is located at the N-terminus of the truncate body, which is critical for the biological activity of the KCC2 truncate body. Therefore, if the truncate body is truncated starting from the above-mentioned amino acid residue at position 667 (i.e., the amino acid residues at positions 1-666 are deleted) and at the same time contains at least these 18 amino acid residues, regardless of their length, these truncated bodies retain biological activity, and even a short peptide having only these 18 amino acids can produce corresponding biological activity. Based on the above discovery, the inventor proposed the present invention.

[0007] In a first aspect, the present invention provides a KCC2 truncate, whose amino acid sequence is selected from an amino acid sequence consisting of at least 18 consecutive amino acid residues starting from amino acid residue 667 in the amino acid sequence of wild-type human KCC2a (SEQ ID NO: 10).

[0008] In some preferred embodiments, the amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 18 consecutive amino acid residues starting from amino acid residue 667 in the amino acid sequence of human wild-type KCC2a (SEQ ID NO: 10). In some preferred embodiments, the amino acid sequence of the KCC2 truncate consists of amino acid residues 667-684 in the amino acid sequence of human wild-type full-length KCC2a. In other preferred embodiments, the amino acid sequence of the KCC2 truncate is as shown in SEQ ID NO: 5.

[0009] In other preferred embodiments, the amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 473 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10). In some preferred embodiments, the amino acid sequence of the KCC2 truncate consists of the 667th to 1139th amino acid residues in the human wild-type full-length KCC2a amino acid sequence. In other preferred embodiments, the amino acid sequence of the KCC2 truncate is as shown in SEQ ID NO: 3.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned KCC2 truncation.

[0011] In a third aspect, the present invention provides a recombinant nucleic acid molecule, which comprises the above-mentioned nucleic acid molecule encoding the KCC2 truncate and a promoter operably linked thereto.

[0012] In some specific embodiments, the promoter is selected from cytomegalovirus (CMV) promoter, CAG promoter, desmin (DES) promoter, synapsin I (SYN) promoter or muscle creatine kinase (MCK) promoter. In some preferred embodiments, the promoter is synapsin I (SYN) promoter.

[0013] In some specific embodiments, the recombinant nucleic acid molecule further comprises one or more of a polyadenylic acid sequence (polyA), a Kozak sequence, a WPRE sequence, a stuffer sequence and / or a post-transcriptional regulatory element.

[0014] In some preferred embodiments, the recombinant nucleic acid molecule comprises:

[0015] (1) the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12;

[0016] (2) a polynucleotide sequence comprising a substitution, deletion or addition of one or more nucleotides in the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12; or

[0017] (3) A polynucleotide sequence having a sequence identity of more than 80% with the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, preferably a polynucleotide sequence having an identity of more than 85%, 90%, 95%, 96%, 97%, 98%, or 99%; more preferably, a polynucleotide sequence having an identity of more than 98% or 99%.

[0018] In some specific embodiments, the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence. In some preferred embodiments, the AAV inverted terminal repeat sequence is selected from AAVs of different serotypes. In a more preferred embodiment, the AAV inverted terminal repeat sequence is selected from any serotype of AAV in clade AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or its hybrid / chimeric type. In some further preferred embodiments, the AAV inverted terminal repeat sequence is from AAV2.

[0019] In a fourth aspect, the present invention further provides a recombinant vector comprising the aforementioned recombinant nucleic acid molecule.

[0020] In some embodiments, the recombinant vector is selected from a plasmid vector, a phage vector or a viral vector. In some embodiments, the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector or a hybrid viral vector.

[0021] In a fifth aspect, the present invention also provides a recombinant adeno-associated virus, comprising an AAV capsid and a vector genome, wherein the vector genome comprises the above-mentioned recombinant nucleic acid molecule. In some preferred embodiments, the AAV capsid is selected from any serotype of AAV in the evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types. In other preferred embodiments, the capsid of the recombinant adeno-associated virus is AAVPHP.B or AAV9 (whose amino acid sequence is shown in SEQ ID NO: 13). In some preferred embodiments, the recombinant adeno-associated virus is a double-stranded recombinant adeno-associated virus.

[0022] In a sixth aspect, the present invention further provides an isolated host cell, wherein the host cell comprises the aforementioned KCC2 truncation, nucleic acid molecule, recombinant nucleic acid molecule, recombinant vector or recombinant adeno-associated virus.

[0023] In some embodiments, the cell is a neuron cell. In some embodiments, the cell is a cholinergic neuron cell, a noradrenergic neuron cell, a dopaminergic neuron cell, a 5-hydroxytryptamine neuron cell or a γ-aminobutyric acid neuron cell. In some embodiments, the cell is a neural cell line, including NG108-15, N1E115, PC12, SHSY5Y, F11, ND7-23, N27, SN4741 cell lines.

[0024] In the seventh aspect, the present invention provides a pharmaceutical composition, which comprises the aforementioned KCC2 truncate, nucleic acid molecule, recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus or isolated host cell and a pharmaceutically acceptable carrier and / or other conventional pharmaceutical ingredients (such as preservatives and / or stabilizers). In some preferred embodiments, the pharmaceutical composition is used to prevent or treat spinal cord injury. In some preferred embodiments, the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, spinal injection, thoracic injection, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and / or other parenteral routes for administration. In some preferred embodiments, the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.

[0025] In an eighth aspect, the present invention provides the use of the aforementioned KCC2 truncate, nucleic acid molecule, recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and / or the aforementioned pharmaceutical composition in the preparation of a drug for preventing or treating spinal cord injury. In some embodiments, the aforementioned pharmaceutical composition can be administered in combination with other drugs for treating spinal cord injury.

[0026] In the ninth aspect, the present invention provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned KCC2 truncate, nucleic acid molecule, 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 some preferred embodiments, the subject is a mammal. In some more preferred embodiments, the subject is a human. In some embodiments, the treatment method can also be used in combination with other methods for treating spinal cord injury.

[0027] The positive effects of the present invention are:

[0028] The present invention provides a group of KCC2 truncations and coding sequences thereof, as well as recombinant adeno-associated viruses carrying the coding sequences thereof. The KCC2 truncations provided by the present invention have good chloride ion efflux activity and have a therapeutic effect on spinal cord injury caused by bilateral misplaced truncation.

[0029] Compared with the wild-type full-length KCC2, the DNA coding sequence of the KCC2 truncate provided by the present invention is shorter, so when gene therapy is used, the range of gene therapy delivery vectors available for selection is wider, for example, the DNA coding sequence of the KCC2 truncate provided by the present invention can be packaged into a double-stranded AAV viral vector, and can also be packaged into a single-stranded AAV viral vector, while the wild-type full-length KCC2 can only be packaged into a single-stranded AAV viral vector due to length restrictions. At the same time, the selectable promoters and other regulatory elements in the gene therapy delivery vector also have a large optional space, and can be more flexible and widely used to respond to different needs. For example, after the coding sequence (3.4kb) of the wild-type full-length KCC2 and the promoter are packaged into a single-stranded AAV viral vector, the upper limit of the packaging capacity of the viral vector has almost been reached, making it difficult to add other expression regulatory elements to the viral vector, and the DNA coding sequence length of the KCC2 truncate of the present invention does not exceed 1.5kb at most, even with the addition of a promoter, it is far from the upper limit of the capacity of the single-stranded AAV viral vector, and other expression regulatory elements can still be added to the viral vector based on different needs.

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

[0031] Figure 1 . Map of shuttle plasmids carrying the coding sequences of various KCC2 truncations.

[0032] Figure 2 . Main structure diagram of the AAV vector genome containing the coding sequences of various KCC2 truncations.

[0033] Figure 3. Map of the shuttle plasmid carrying the wild-type full-length KCC2 protein coding sequence.

[0034] Figure 4 .In vitro biological activity test results of various KCC2 truncations.

[0035] Figure 5 .The effects of various KCC2 truncations on KCC2 levels on neuronal membranes. DETAILED DESCRIPTION

[0036] definition

[0037] 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 ed.); Handbook of Experimental Immunology, VoIs. I-FV (DM Weir and CC. Blackwell ed., Blackwell Scientific Publications); TECreighton, 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 ed., Academic Press, Inc.).

[0038] To facilitate understanding of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0039] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is known to cause no disease and to elicit 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's genome. These features make AAV an attractive viral vector for gene therapy.

[0040] Administration / administration: Providing or administering an agent, such as a therapeutic agent (e.g., a 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.

[0041] 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 change the amino acid sequence of the encoded protein.

[0042] Enhancer: A nucleic acid sequence that increases transcription efficiency by increasing promoter activity.

[0043] Intron: A stretch of DNA in a gene that does not contain protein coding information. Introns are removed before the messenger RNA is translated. Hybrid intron: A combined intron that includes sequences from more than one natural intron.

[0044] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences in the adeno-associated virus genome required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as replication origins for viral DNA synthesis and are essential cis elements for the production of AAV integrative vectors.

[0045] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from cells or tissues of a naturally occurring 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.

[0046] 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. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0047] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) that may 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 drug delivery of one or more therapeutic compounds, molecules or agents.

[0048] In general, the properties of the carrier depend on the specific mode of administration used. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as solvents. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may be included, such as pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may 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.

[0049] Prevent, treat, or ameliorate a disease: "Preventing" a disease means inhibiting the full onset of a disease. "Treatment" means therapeutic intervention to improve the signs or symptoms of a disease or pathological condition after the disease has begun to develop. "Amelioration" means reducing the number or severity of signs or symptoms of a disease.

[0050] Promoter: A region of DNA that directs / causes transcription of a nucleic acid (e.g., a gene). A promoter includes essential nucleic acid sequences near the transcription start site. Typically, a promoter is located near the gene it transcribes. The promoter region also optionally includes a distal enhancer or repressor element, which may be located thousands of base pairs away from the transcription start site.

[0051] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule that has a sequence that does not occur naturally, or a sequence that is prepared by the artificial combination of two sequence fragments that would otherwise be separate. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid molecule fragments (such as by genetic engineering techniques).

[0052] Likewise, a recombinant virus is a virus comprising a sequence that is not naturally present or a sequence that is 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.

[0053] Serotype: A group of closely related microorganisms (eg, viruses) distinguished by a characteristic set of antigens.

[0054] Subject: A living multicellular vertebrate organism, including humans and non-human mammals. Specifically, the subject of the present invention is an individual with spinal cord injury, who may have residual endogenous KCC2 activity, or no measurable activity.

[0055] Synthetic: Produced in a laboratory by artificial means, e.g. synthetic nucleic acids can be chemically synthesized in a laboratory.

[0056] Therapeutically effective amount: The amount of a specific drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve the desired effect in a subject or cell 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. Specifically, the "therapeutically effective amount" described in the present invention refers to an amount that effectively achieves the desired therapeutic result in terms of dosage and within the necessary time period, such as increasing the level of functional KCC2 in the subject, so as to cause functional KCC2 to be produced to an amount sufficient to improve one or more symptoms of spinal cord injury. The effective amount of a given substance will vary with factors such as the nature of the substance, the route of administration, the age of the subject, and the purpose of administering the substance. The effective amount in various individual cases can be determined empirically by a technician according to methods established in the art.

[0057] Vector: A vector is a nucleic acid molecule that allows the insertion of exogenous nucleic acid without destroying the ability of the vector to replicate and / or integrate in a host cell. The vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also contain one or more selective 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.

[0058] 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 from more distantly related species (e.g., human and C. elegans sequences).

[0059] The length of sequence identity comparison can be on 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, usually 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.

[0060] The percent identity of an amino acid sequence can be readily determined over a full-length protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof, or a 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 an "aligned" sequence. 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.

[0061] The alignment can be performed using any public or commercially available multiple sequence alignment program. Sequence alignment programs can be used 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 the default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use another algorithm or computer program that at least provides the identity or alignment level 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).

[0062] 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 is also 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 can be used. TM (a program in GCG Version 6.1) to compare polynucleotide sequences. Fasta TM Alignment and percent sequence identity of the best overlapping region between the query and search sequences are provided. For example, Fasta TM The percent sequence identity between nucleic acid sequences is determined using the default parameters provided in GCG Version 6.1 (incorporated herein by reference) (a word length of 6, and a NOPAM factor for the scoring matrix).

[0063] "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" - only containing relevant transgenes flanking the required signals 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 will not occur except in the presence of viral enzymes required for replication. Such replication-defective viruses can be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrated or non-integrated), or another suitable viral source.

[0064] "Self-complementary adeno-associated virus" ("scAAV") refers to a construct in which the coding region carried by the recombinant AAV is designed to form an intramolecular double-stranded DNA template. During infection, rather than waiting for 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.

[0065] The term "expression" is used in this article in its broadest sense and includes the production of DNA or RNA and proteins. In the case of RNA, the term "expression" or "translation" particularly relates to the production of peptides or proteins. Expression can be transient, or can be stable. The term "translation" in the context of the present invention relates to the process of ribosome processing, wherein the mRNA chain controls the assembly of the amino acid sequence to generate a protein or peptide.

[0066] 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.

[0067] The words "comprise, comprises and comprising" should be interpreted as inclusive rather than exclusive. The words "consist, consisting" and their 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..."

[0068] As used herein, the term "about" refers to a difference of 10% (±10%) from a given reference value, unless otherwise specified.

[0069] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0070] K + -Cl - Co-transporter (KCC2) truncated form and nucleic acid sequence encoding the same

[0071] KCC2 is K + -Cl - Cotransporter 2, also known as solute carrier family 12 member 5, is a neuron-specific K + -Cl - Cotransporter that mediates neuron-specific K + -Cl - Transport. KCC2 protein has 12 transmembrane regions, 6 extracellular loops, a shorter intracellular amino terminus (NTD) and a longer intracellular carboxyl terminus (CTD). KCC2 has two subtypes: KCC2a and KCC2b, the difference between which 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 and other KCC2 proteins. 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 Ref Seq: NP_001128243.1 (SEQ ID NO: 10), and its mRNA sequence number is NCBI RefSeq: NM_001134771.2.

[0072] The first aspect of the present invention provides a KCC2 truncate, whose amino acid sequence is selected from an amino acid sequence consisting of at least 18 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10). In some preferred embodiments, the amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 18 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10). In some preferred embodiments, the amino acid sequence of the KCC2 truncate consists of amino acid residues at positions 667-684 in the human wild-type full-length KCC2a amino acid sequence. In other preferred embodiments, the amino acid sequence of the KCC2 truncate is as shown in SEQ ID NO: 5. In other preferred embodiments, the amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 473 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10). In some preferred embodiments, the amino acid sequence of the KCC2 truncate consists of amino acid residues 667-1139 in the wild-type full-length KCC2a amino acid sequence of human. In other preferred embodiments, the amino acid sequence of the KCC2 truncate is shown in SEQ ID NO:3.

[0073] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned KCC2 truncation. In some embodiments, the nucleic acid molecule is DNA or RNA.

[0074] In one embodiment, the invention provides a modified nucleic acid sequence encoding a KCC2 truncate having a modified or optimized polynucleotide sequence. In one embodiment, the sequence is a codon-optimized sequence optimized for expression in a subject species. As used herein, a "subject" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cattle, 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 the human body.

[0075] Codon optimized coding region can be designed by various methods.Can use the method (such as GeneArt) available 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, for example, US2014 / 0032186A1 and US2006 / 0136184A1.Suitably, modify the whole length of the open reading frame (ORF) of this product.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 encoding.

[0076] Many selections can be used to actually change codons or to synthesize the codon-optimized coding region designed as described herein. Such modifications or synthesis can be performed using standard and conventional molecular biological 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 nucleotides 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, such as synthesizing each oligonucleotide in the pair to extend 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to another oligonucleotide in the pair. The single-stranded ends of each oligonucleotide pair are designed to anneal with the single-stranded ends of another oligonucleotide pair. This oligonucleotide pair is annealed, and approximately five to six of these double-stranded fragments are annealed together via sticky single-stranded ends subsequently, and they are linked together and cloned into standard bacterial cloning vectors subsequently, such as those available from Invitrogen Corporation, Carlsbad, Calif. Vector. The construct is then sequenced by standard methods. Several of these 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.

[0077] The sequences encoding KCC2 truncations described herein can be synthesized using techniques well known in the art. For example, the PCR-based accurate synthesis (PAS) of long DNA sequences can be used, 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. May 2010; 81(2):147-52. Epub 2010 Mar 10; see also the following patents on oligonucleotide synthesis and gene synthesis, GeneSeq. April 2012; 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 plasmids containing KCC2-related sequences 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 (SigmaAldrich). Other techniques available in this article include sequence-specific isothermal amplification methods, which eliminate the need for thermal cycling. These methods typically use strand displacement 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.

[0078] Recombinant nucleic acid molecules

[0079] The third aspect of the present invention provides a recombinant nucleic acid molecule, which comprises the above-mentioned nucleic acid molecule encoding the KCC2 truncate, and a promoter operably linked thereto.

[0080] In some specific embodiments, the promoter is selected from cytomegalovirus (CMV) promoter, CAG promoter, desmin (DES) promoter, synapsin I (SYN) promoter or muscle creatine kinase (MCK) promoter. In some preferred embodiments, the promoter is synapsin I (SYN) promoter.

[0081] In some embodiments, the recombinant nucleic acid molecule further comprises one or more of polyadenylic acid, a Kozak sequence, a WPRE sequence, a stuffer sequence and / or a post-transcriptional regulatory element.

[0082] In some preferred embodiments, the recombinant nucleic acid molecule comprises:

[0083] (1) the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12;

[0084] (2) a polynucleotide sequence comprising a substitution, deletion or addition of one or more nucleotides in the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12; or

[0085] (3) A polynucleotide sequence having a sequence identity of more than 80% with the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, preferably a polynucleotide sequence having an identity of more than 85%, 90%, 95%, 96%, 97%, 98%, or 99%; more preferably, a polynucleotide sequence having an identity of more than 98% or 99%.

[0086] In some specific embodiments, the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence. In some preferred embodiments, the AAV inverted terminal repeat sequence is selected from AAVs of different serotypes. In some more preferred embodiments, the AAV inverted terminal repeat sequence is selected from any serotype of AAV in clade AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or its hybrid / chimeric type. In some further preferred embodiments, the AAV inverted terminal repeat sequence is from AAV2.

[0087] In some specific embodiments, the recombinant nucleic acid molecule can use constitutive promoters of different strengths, including but not limited to herpes simplex virus (HSV) promoter, thymidine kinase (TK) promoter, Rous sarcoma virus (RSV) promoter, simian virus 40 (SV40) promoter, mouse mammary tumor virus (MMTV) promoter, cytomegalovirus (CMV) promoter, β-actin promoter or CAG promoter. Inducible promoters can also be used in the present invention. Suitable inducible promoters include promoters from genes (e.g., cytochrome P450 genes, heat shock protein genes, metallothionein genes) and from hormone-inducible genes, such as estrogen gene promoters. Another example of an inducible promoter is the tetVP16 promoter in response to tetracycline. In some specific embodiments, the promoters used in the present invention also include but are not limited to: neuron-specific promoters, such as synaptic protein I (SYN) promoter, muscle creatine kinase (MCK) promoter, CBA promoter or desmin (DES) promoter. Illustrative plasmids and vectors described herein utilize the Synapsin I (SYN) promoter.

[0088] In some specific embodiments, in addition to the promoter, the recombinant nucleic acid molecule may contain one or more other suitable transcription initiation, termination, enhancer sequences, effective RNA processing signals such as splicing and polyadenylation sequence (polyA) signals, sequences for stabilizing cytoplasmic mRNA such as WPRE, sequences for enhancing translation efficiency (i.e., Kozak consensus sequences), sequences for enhancing protein stability, and sequences for enhancing secretion of the encoded product when necessary. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (BGH), human growth hormone, and synthetic polyA. An example of a suitable enhancer is the CMV enhancer. In one embodiment, the recombinant nucleic acid molecule comprises one or more expression enhancers. In one embodiment, the recombinant nucleic acid molecule contains two or more expression enhancers. These enhancers may be the same, or may be different from each other. For example, an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies positioned adjacent to each other. Or the double copies of the enhancer may be separated by one or more sequences. In another embodiment, the recombinant nucleic acid molecule further contains an intron, such as a chicken beta actin intron. Other suitable introns include those known in the art, for example, described in WO 2011 / 126808. Optionally, one or more sequences can be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be engineered upstream of the polyA sequence and downstream of the coding sequence (see, for example, MA Zanta-Boussif et al., Gene Therapy (2009) 16: 605-619).

[0089] In some specific embodiments, for AAV recombinant vectors, when the exogenous gene is short, the recombinant nucleic acid molecule may also contain one or more filler sequences, which can make the sequence length between AAV-ITR close to the length of the wild-type AAV genome, thereby ensuring the virus packaging yield and increasing the expression of the exogenous gene. The filler sequence can be selected from human non-coding sequences. Optionally, the human non-coding sequence is inert or harmless and has no function or activity. In various special aspects, the human non-coding sequence is not a sequence encoding a protein or polypeptide, and is different from any of the following substances: shRNA, AAV terminal inverted repeat (ITR) sequence, promoter, replication point, polyadenylation sequence, etc. In some embodiments of the present invention, the human non-coding sequence is selected from the intron sequence of human factor IX (GenBank: K02402.1), the intron I fragment of human factor IX, the sequence of human cosmid C346, or a sequence or a combination of multiple sequences of HPRT-intron sequence 1704-14779 (GenBank: M26434.1); or even a sequence of HPRT-intron sequence 1704-14779 (GenBank: M26434.1). The length of the human non-coding sequence can be adjusted according to the packaging capacity of different serotypes of AAV or other requirements, and is not limited to a fixed length.

[0090] In some specific embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence as shown in SEQ ID NO: 11 or 12. The polynucleotide sequence shown in SEQ ID NO: 11 comprises the following elements: a synapsin I (SYN) promoter, a coding sequence of amino acids 667-1139 of KCC2; the polynucleotide sequence shown in SEQ ID NO: 12 comprises the following elements: a synapsin I (SYN) promoter, a coding sequence of amino acids 667-684 of KCC2.

[0091] Recombinant vector or recombinant adeno-associated virus

[0092] The fourth aspect of the present invention also provides a recombinant vector, which comprises the aforementioned recombinant nucleic acid molecule.

[0093] In some specific embodiments, the recombinant vector is selected from a plasmid vector, a phage vector or a viral vector. In some specific embodiments, the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector or a hybrid viral vector.

[0094] In some specific embodiments, the nucleic acid molecules encoding KCC2 truncations described herein are engineered to be 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, free genes, etc., which transmit the nucleic acid molecules encoding KCC2 truncations carried thereon. The selected vector 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. The method for making such constructs is known to those skilled in the art of nucleic acid manipulation, and includes 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.

[0095] The fifth aspect of the present invention also provides a recombinant adeno-associated virus (rAAV), which comprises an AAV capsid and a vector genome, and the vector genome comprises the above-mentioned recombinant nucleic acid molecule.

[0096] 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 icosahedral symmetry at a ratio of about 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.

[0097] 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, January 2008, 1190: 15-22; Petrosyan et al., Gene Therapy, December 2014, 21(12): 991-1000; Holehonnur et al., BMC Neuroscience, 2014, 15: 28; and Cearley et al., Mol Ther. October 2008, 16(10): 1710–1718, each of which is incorporated herein by reference.

[0098] 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 the capsid of AAV viral vector (DNase-resistant virions), other AAV serotypes can 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 / 042397A2 (AAV7 and other simian AAVs), US7790449B2 and US7282199B2 (AAV8), WO2005 / 033321A2 and US 7,906,111B2 (AAV9) and WO 2006 / 110689A2 and WO 2003 / 042397A2 (AAVrh.10). Alternatively, a recombinant AAV based on any of the AAVs described 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 by reference herein. In some embodiments, the AAV capsid for the viral vector can be produced by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV capsids or its encoding nucleic acid.

[0099] In some specific embodiments, the AAV capsid is chimeric, comprising 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 the AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made to 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.

[0100] In some preferred embodiments, the AAV capsid is selected from AAV of any serotype selected from clade AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric forms. In other preferred embodiments, the capsid of the recombinant adeno-associated virus is AAVPHP.B or AAV9 (whose amino acid sequence is shown in SEQ ID NO: 13). In some preferred embodiments, the recombinant adeno-associated virus is a double-stranded adeno-associated virus.

[0101] 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 used to generate viral vectors contain the nucleic acid sequence encoding the KCC2 truncate described herein, the packaging signal of the viral genome flanking it, and other expression control sequences, such as those described herein. For example, for AAV viral vectors, the packaging signal is 5' reverse terminal repeats (ITR) and 3' ITR. When packaged into the AAV capsid, the recombinant nucleic acid molecule and the ITRs associated therewith may also be referred to herein as "recombinant AAV (rAAV) genome" or "vector genome".

[0102] In some specific embodiments, the recombinant adeno-associated virus vector (rAAV) provided by the present invention comprises an AAV capsid and a vector genome. In some specific embodiments, the vector genome comprises a nucleic acid molecule encoding a KCC2 truncate, and a regulatory sequence for guiding and / or regulating the expression of the above nucleic acid molecule in a host cell. In some specific embodiments, the vector genome comprises the recombinant nucleic acid molecule described in the present invention. In other specific embodiments, the vector genome also comprises an ITR sequence of AAV. In one embodiment, the ITRs are from an AAV serotype different from the capsid provided. In a preferred embodiment, the ITR sequence is from AAV2, or a deletion variant thereof (ΔITR), which may be used for convenience and accelerate regulatory approval. However, ITRs from other AAV sources can be selected. When the source of 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 AAV 5'ITR and AAV 3'ITR. However, other configurations of these elements may be suitable. Deletion variants of the 5'ITR in which the D-sequence and the terminal resolution site (trs) are deleted (referred to as ΔITR) have been described. In some embodiments, full-length AAV 5' and 3' ITRs are used. The sequences of the ITRs or ΔITRs of each serotype of AAV are well known to those skilled in the art.

[0103] In one embodiment, the capsid is an AAV9 capsid (whose amino acid sequence is shown in SEQ ID NO: 13) or a variant thereof.

[0104] In one embodiment, double-stranded AAV or self-complementary AAV (scAAV) may be selected.

[0105] 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 / 042397A2, WO 2005 / 033321 A2, WO 2006 / 110689A2 and US7588772B2. In one system, a production cell line is transiently transfected with a construct encoding a transgenic flanking ITR and a construct encoding rep and cap. In a second system, a packaging cell line stably providing rep and cap is transiently transfected with a construct encoding a transgenic flanking ITR. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, wherein rAAV needs to be separated from contaminating viruses. Later, a system was developed that did not require infection with a helper virus to recover 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 which the auxiliary functions can be provided by transiently transfecting the cell with a construct encoding the required auxiliary function, or the cell can be engineered to stably contain a gene encoding the auxiliary function, the expression of which can be controlled at the transcriptional or post-transcriptional level. 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-associatedvirus 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; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0106] Optionally, the nucleic acid molecules encoding KCC2 truncations 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, slow virus, retrovirus, etc. Suitably, when one of these other vectors is generated, it is generated as a replication-defective viral vector.

[0107] cell

[0108] The sixth aspect of the present invention also provides an isolated host cell, which comprises the aforementioned KCC2 truncation, nucleic acid molecule, recombinant nucleic acid molecule, recombinant vector or recombinant adeno-associated virus.

[0109] 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, 5-hydroxytryptamine 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.

[0110] Pharmaceutical composition

[0111] The seventh aspect of the present invention also provides a pharmaceutical composition, which comprises the aforementioned KCC2 truncate, nucleic acid molecule, recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus or isolated host cell and a pharmaceutically acceptable carrier and / or other conventional pharmaceutical ingredients (such as preservatives and / or stabilizers). In some preferred embodiments, the pharmaceutical composition is used to prevent or treat spinal cord injury. In some preferred embodiments, the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, spinal injection, thoracic injection, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and / or other parenteral routes for administration. In some preferred embodiments, the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.

[0112] In some specific embodiments, the pharmaceutical composition described herein is 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 is directly delivered to the CNS and 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 (e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and other parenteral routes) can be selected.

[0113] In some embodiments, the KCC2 truncates, nucleic acid molecules, recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses or isolated host cells described herein can be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs can be delivered (see, e.g., 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; combined 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 carriers as described herein, see, for example, X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; online publication: March 21, 2011; WO2013 / 182683A1, WO 2010 / 053572A2 and WO 2012 / 170930A1, all of which are incorporated herein by reference, and such non-viral KCC2 delivery vectors can be administered by the aforementioned routes.

[0114] In some specific embodiments, the above-mentioned viral vector or non-viral DNA or RNA transfer part can be formulated with a physiologically acceptable carrier for gene transfer and gene therapy. In some specific embodiments, the delivery vector can select a variety of suitable purification methods. Describe the example of a purification method suitable for separating empty capsids from carrier particles, such as International Patent Application No. PCT / US16 / 65976 and its priority document filed on December 9, 2016, U.S. Patent Provisional Application No. 62 / 322,098 filed on April 13, 2016 and U.S. Patent Provisional Application No. 62 / 266,341 filed on December 11, 2015 and entitled "Scalable Purification Method for AAV8", which is incorporated herein by reference. See also the purification methods described in the following documents: 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 filed on December 11, 2015 (AAV1); International Patent Application No. PCT / US16 / 66013 filed on December 9, 2016 and Its priority documents, U.S. Provisional Application Nos. 62 / 322,055 filed on April 13, 2016 and 62 / 266,347 filed on December 11, 2015 (AAVrh10); and International Patent Application No. PCT / US16 / 65970 filed on December 9, 2016 and its priority applications, U.S. Provisional Application Nos. 62 / 266,357 and 62 / 266,357 (AAV9), which are hereby incorporated by reference herein. In one embodiment, purification comprises a two-step purification scheme that selectively captures and separates rAAV vector particles containing the genome from the clarified concentrated supernatant of the rAAV production 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, and the like.

[0115] 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, 1×10 10vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×l0 14 vg, 1×10 15 vg, 1×10 16 vg). 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.

[0116] These above-mentioned doses can be administered in various volumes of carrier, excipient or buffer formulations, the volume being in the range of about 25 microliters to about 15 milliliters, including all numbers within this range, depending on the size of the area to be treated, the viral titer 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 another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is about 800 μL. In another embodiment, the volume is about 900 μL. In 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.

[0117] The rAAV is preferably suspended in a physiologically compatible carrier and can be administered to a human or non-human mammal 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, including saline, it 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 attaching to the infusion tube but do not interfere with the binding activity of rAAV in vivo.

[0118] Optionally, the composition of the present invention may contain other conventional pharmaceutical ingredients in addition to rAAV and a pharmaceutically acceptable carrier, such as preservatives or chemical stabilizers. 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.

[0119] The compositions of the 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, which are designed to be delivered to a subject via injection, an osmotic pump, an intrathecal catheter, or by another device or route of delivery.

[0120] The viral vectors described herein can be used to prepare drugs so that nucleic acid molecules encoding KCC2 truncations are delivered to subjects (e.g., human patients) in need thereof, providing functional KCC2 to subjects for the treatment of spinal cord injury. The treatment process can optionally include repeated administration of the same viral vector (e.g., 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.

[0121] Pharmaceutical Uses and Treatment Methods

[0122] In an eighth aspect, the present invention provides the use of the above-mentioned KCC2 truncate, nucleic acid molecules encoding the same, recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and / or the above-mentioned pharmaceutical compositions in the preparation of drugs for preventing or treating spinal cord injury. In some embodiments, the above-mentioned pharmaceutical compositions can be administered in combination with other drugs for treating spinal cord injury.

[0123] The ninth aspect of the present invention provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned KCC2 truncate, a nucleic acid molecule encoding it, a recombinant nucleic acid molecule, a recombinant vector, a recombinant adeno-associated virus, a host cell and / or the aforementioned pharmaceutical composition to a subject in need thereof. In some preferred embodiments, the subject is a mammal. In some more preferred embodiments, the subject is a human. In some embodiments, the treatment method can also be used in combination with other methods for treating spinal cord injury.

[0124] In some specific embodiments, the above-mentioned KCC2 truncate, nucleic acid molecule encoding it, recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and / or the aforementioned pharmaceutical composition can be administered repeatedly. Optionally, more than one repeated administration is allowed. Such repeated administration can have the same type of vector, different viral vectors, 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 truncate and a secondary treatment is required, rAAV10 carrying the above-mentioned nucleic acid molecule can be subsequently administered, and vice versa.

[0125] Treatment of patients with spinal cord injury may require combined therapy, such as in some embodiments of the present invention, the pharmaceutical composition of the present invention can be used in combination with any 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 treatments may include taking steroids (e.g., prednisone) once a day in decreasing doses for 7 days, starting with an amount of about 60 mg, and reducing 10 mg per day (no dose on the 7th day). Other doses and immunosuppressants can be selected.

[0126] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and by reference to the disclosure, which provides a general guide to many of the terms used in this application. DETAILED DESCRIPTION

[0128] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0129] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0130] The KCC2 truncations involved in the examples of the present application are as follows:

[0131] Truncated form 1 (hereinafter referred to as CTD): It consists of amino acid residues 667-1139 of human wild-type full-length KCC2a, and its sequence is shown in SEQ ID NO: 3, and its coding sequence is shown in SEQ ID NO: 2.

[0132] Truncated form 2 (hereinafter referred to as C Helices): It consists of amino acid residues 667-684 of the human wild-type full-length KCC2a, and its amino acid sequence is shown in SEQ ID NO: 5, and its coding sequence is shown in SEQ ID NO: 4.

[0133] Truncated form 3 (hereinafter referred to as Rat-CTD): It consists of amino acids 637-1116 of rat wild-type full-length KCC2b (see Li et al., 2007, Neuron, 56(6): 1019-1033), its amino acid sequence is shown in SEQ ID NO: 8, and its coding sequence is shown in SEQ ID NO: 7. After sequence comparison, it is equivalent to the sequence consisting of amino acid residues 660-1139 of human wild-type full-length KCC2a, and can be used as a control in the examples of this application.

[0134] Example 1 Construction of shuttle plasmids carrying coding sequences of various KCC2 truncations

[0135] 1.1 Construction of shuttle plasmid carrying CTD coding sequence

[0136] 1. Use pSC-L-orS1 vector (see Chinese patent application CN113755524A) and replace the resistance gene Amp with Kan (its structure is as follows Figure 1 A) was double-digested with XhoⅠ and NheⅠ, and the large fragment was recovered to obtain the backbone vector.

[0137] 2. Synthesize Syn promoter (SEQ ID NO: 1) and its seamless connection primers (forward primer: 5'-GAGGGAGTGGAATTCGAGCTCGAGCAGTGCAAGTGGGTTTTAGGACCA-3'; reverse primer: 5'-CATGGTGGCGGCGATATCTG-3').

[0138] 3. Synthesize the CTD coding sequence (SEQ ID NO: 2, whose amino acid sequence is shown in SEQ ID NO: 3) and its seamless connection primers (forward primer: 5'-GATATCGCCGCCACCATGGGAATCAGAGGCCTGAGCC-3'; reverse primer: 5'-CTAGAAGGCACAGGCTAGCTCAGGAGTAGATTGTAATGACCTCTCTGCC-3') (synthesized by Beijing Nosai Genome Research Center Co., Ltd.).

[0139] 4. The above-synthesized Syn promoter sequence and CTD coding sequence were inserted between the 5'ITR sequence and the BGH polyadenylation sequence of the backbone vector by seamless cloning PCR method. The constructed shuttle plasmid was named pSC-K-Syn-CTD plasmid vector ( Figure 1 B), used to package an adeno-associated virus vector expressing a truncated KCC2-CTD. The structure (main structure) between the two ITRs in the plasmid vector is shown in Figure 2 A.

[0140] 1.2 Construction of shuttle plasmid carrying C-Helices coding sequence

[0141] 1. Obtain the backbone vector using the method in Section 1.1.

[0142] 2. Synthesize Syn promoter (SEQ ID NO: 1) and its seamless connection primers (forward primer: 5'-GAGGGAGTGGAATTCGAGCTCGAGCAGTGCAAGTGGGTTTTAGGACCA-3'; reverse primer: 5'-CATGGTGGCGGCGATATCTG-3').

[0143] 3. Synthesize the C-helices coding sequence (SEQ ID NO: 4, whose amino acid sequence is shown in SEQ ID NO: 5) and its seamless connection primers (forward primer: 5'-GATATCGCCGCCACCATGGGAATCAGAGGCCTGAGCC-3'; reverse primer: 5'-ACGTAAAGCCGAACAAGCTCACTCCAGCCTCAGCAGGGC-3').

[0144] 4. Synthesize the stuffing sequence (an intron sequence of the HPRT gene, whose sequence is shown in SEQ ID NO: 6) and its seamless connection primer (forward primer: 5'-GCTTGTTCGGCTTTACGTCAC-3'; reverse primer: 5'-GCAACTAGAAGGCACAGGCTAGCGACCTAGGGTATGTCTGTTAGCCTC-3') (synthesized by Beijing Nosai Genome Research Center Co., Ltd.).

[0145] 5. The above-synthesized Syn promoter sequence, C-helices coding sequence and stuffer sequence were inserted between the 5'ITR sequence and the BGH polyadenylation sequence of the backbone vector by seamless cloning PCR method. The shuttle plasmid obtained was named pSC-K-Syn-C helices plasmid vector ( Figure 1 C), used to package an adeno-associated virus vector expressing Chelices truncations. The structure (main structure) between the two ITRs in the plasmid vector is shown in Figure 2 In B.

[0146] 1.3 Construction of shuttle plasmid carrying Rat-CTD coding sequence

[0147] 1. Obtain the backbone vector using the method in Section 1.1.

[0148] 2. Synthesize Syn promoter (SEQ ID NO: 1) and its seamless connection primers (forward primer: 5'-GAGGGAGTGGAATTCGAGCTCGAGCAGTGCAAGTGGGTTTTAGGACCA-3'; reverse primer: 5'-CATGGTGGCGGCGATATCTG-3').

[0149] 3. Synthesize the coding sequence of rat KCC2b CTD (637-1116aa) (Rat-CTD) (SEQ ID NO: 7, the amino acid sequence of which is shown in SEQ ID NO: 8, and see Li et al., 2007, Neuron, 56(6): 1019-1033) and its seamless primers (forward primer: 5'-TATCGCCGCCACCATGGCAGAGAAGGAGTGGGGGGAT-3'; reverse primer: 5'-CAACTAGAAGGCACAGGCTAGCTCAGGAGTAGATGGTGATGACCTCTC-3')

[0150] 4. The above-synthesized Syn promoter sequence and Rat-CTD coding sequence were inserted between the 5'ITR sequence and the BGH polyadenylation sequence of the backbone vector by seamless cloning PCR method. The shuttle plasmid obtained was named pSC-K-Syn-rat-CTD plasmid vector ( Figure 1 D), used to package an adeno-associated virus vector expressing a truncated KCC2-rat-CTD. The structure (main structure) between the two ITRs in the plasmid vector is shown in Figure 2 C.

[0151] 1.4 Construction of shuttle plasmid carrying full-length KCC2 coding sequence

[0152] 1. Double digest the pSNAV2.0-Syn-EGFP vector (such as Figure 3A As shown), large fragments were recovered.

[0153] 2. Synthesize Syn promoter (SEQ ID NO: 1) and its seamless connection primers (forward primer: 5'-TGGTGGCGGCGATATCCTGCGCTCTCAGGCACGACAC-3'; reverse primer: 5'-GGGTTCCTCTGCTCGAGCAGTGCAAGTGGGTTTTAGG-3').

[0154] 3. Synthesize the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 9, the amino acid sequence encoded by it is shown in SEQ ID NO: 10) and seamless connection primers (forward primer: 5'-TCGAGGCAGATCTGTCGAC-3'; reverse primer: 5'-CAGGATATCGCCGCCACCATGAGCCGCAGGTTCACGGTC-3') (synthesized by Beijing Nosai Genome Research Center Co., Ltd.).

[0155] 4. The Syn promoter and full-length KCC2 coding sequence were inserted between the 5'ITR sequence and the BGH polyadenylation sequence of the backbone vector by seamless cloning PCR. The resulting plasmid was named pSNAV2.0-Syn-KCC2 (eg Figure 3B shown).

[0156] 5. The above-mentioned pSNAV2.0-Syn-KCC2 was double-digested with SwaI and NruI to recover a vector fragment of about 6.2 kb.

[0157] 6. Use seamless ligation primer pair 1 (forward primer:

[0158] 5'-CAGCTGAACCAACTCGCGAATTCCACAGCTGCCTCGCGC-3'; reverse primer: 5'-GGTGGCGGCGATATCCTGCGCTCTCAGGCACGAC-3') and primer pair 2 (forward primer:

[0159] 5'-CCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTA-3'; reverse primer: 5'-AGGTTAAGTCCTCATTTAAATTAGGCAA-3') was used as a template for PCR amplification to obtain fragment 1 (about 1.27 Kb) and fragment 2 (about 270 bp).

[0160] 7. Use seamless primer pair 3 (forward primer: 5'-TTAGAAAAACTCATCGAGCATCAAATG-3'; reverse primer: 5'-ATGAGCCATATTCAACGGGA-3') and synthetic 1902-Kan (containing the kana resistance encoding gene) as a template to synthesize the kana resistance encoding gene by PCR, which is fragment 3.

[0161] 8. Use seamless connection to connect fragment 1, fragment 2, fragment 3 and the vector, and the resulting shuttle plasmid is named pSNAV2.0-K-Syn-KCC2 (eg Figure 3C), used to package an adeno-associated virus vector expressing full-length KCC2.

[0162] Example 2 Virus packaging, purification and genome titer detection

[0163] In this example, HEK293T cells were used as production cell lines to produce AAV virus vectors using a conventional three-plasmid packaging system. The experimental methods used were all conventional methods in the art (e.g., 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).

[0164] The shuttle plasmids obtained in Example 1 (pSC-K-Syn-CTD, pSC-K-Syn-C Helices, pSC-K-Syn-rat-CTD or pSNAV2.0-K-syn-KCC2) were used to package recombinant adeno-associated viruses that can be used to express the above-mentioned KCC2 truncation and full-length KCC2, and were named scAAV2 / 9-syn-CTD, scAAV2 / 9-syn-C helices, scAAV2 / 9-syn-Rat-CTD, and ssAAV2 / 9-syn-KCC2, respectively.

[0165] The packaged virus was purified by AAV9 affinity chromatography (Thermo Scientific POROSCaptureSelectAAV9 affinity resin A27359)-ultracentrifugation-G25 chromatography, and then the genome titer of the virus vector was detected by Q-PCR. The primer probe sequences used in the PCR detection are shown in Table 1, and the results of the genome titer detection of each AAV recombinant virus are shown in Table 2.

[0166] Table 1 Primer and probe sequences used in genomic titer detection

[0167] Primer name Sequence (5'—3') Syn-1-Forward Primer GCACCCAACCCCCATTC Syn-1-Reverse Primer TCCCCTCCCCCTCTCTGA Syn-1-probe ATTGCGCATCCCCT

[0168] Table 2 Genome titer detection results

[0169] Plasmid name Viral vector name Genome titer (vg / ml) pSC-K-syn-CTD scAAV2 / 9-syn-CTD 3.89E+12 pSC-K-syn-C-helices scAAV2 / 9-syn-Chelices 8.06E+12 pSC-K-syn-rat-CTD scAAV2 / 9-syn-Rat-CTD 5.32E+12 pSNAV2.0-K-syn-KCC2 ssAAV2 / 9-syn-KCC2 6.63E+12

[0170] Example 3 In vitro efficacy evaluation

[0171] 3.1 In vitro biological activity detection of truncated forms

[0172] In this example, the intracellular chloride ions (Cl - ) levels to evaluate the in vitro activity of the above KCC2 truncations. The biological function of KCC2 is to convert 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 and its truncated forms. The SuperClomeleon (see Grimley et al., 2013, Journal of Neuroscience, 33(41): 16297–16309) technology 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 marker for 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, it can be reflected that SuperClomeleon and Cl - The FRET efficiency is characterized by the ratio of 535nm / 485nm, which is used as an indicator of intracellular chloride ion levels. The specific experimental process is as follows:

[0173] The mouse neuroblastoma cell / rat glioma cell hybrid cell line NG108-15 (ATCC, HB-12317) was used to construct a stable cell line NG108-15-Cl expressing SuperClomeleon: specifically, the recombinant lentivirus expressing SuperClomeleon was used to infect NG108-15 cells at an MOI of 1. After 48 hours, blasticidin was added for screening. After 7 days, the surviving cells were obtained by limiting dilution to obtain monoclonal cells. After the monoclonal cells were amplified and passaged, their sensitivity to chloride ions was tested by solutions with different chloride ion concentrations. If the ratio of 535nm / 485nm was between 1-4, it was a stable cell line that could be used for detection (see Grimley et al., 2013, Journal of Neuroscience, 33(41): 16297-16309).

[0174] The recombinant adeno-associated viruses (scAAV2 / 9-syn-CTD, scAAV2 / 9-syn-Chelices, scAAV2 / 9-syn-Rat-CTD) packaged in Example 2 were used to infect the above-mentioned stable cell line NG108-15-Cl at the same MOI (MOI=2E+6). Among them, the negative control group (NC) was a stable cell line NG108-15-Cl in which the same volume of virus solvent was added. After 48 hours of infection, the fluorescence intensity was detected by an ELISA instrument, and the ratio of 535nm / 485nm was calculated to compare the chloride ion efflux activity of each truncated form of KCC2 (the change relative to NC, expressed as a percentage, a positive value indicates that the chloride ion efflux activity is higher than that of the NC group, and a negative value is the opposite).

[0175] Test results such as Figure 4 As shown, the KCC2 truncations expressed in the recombinant viruses of the present invention all showed chloride ion efflux activity. In comparison, the chloride ion efflux activity was the highest in the scAAV2 / 9-syn-CTD group infected with the scAAV2 / 9-syn-Chelices group, and the chloride ion efflux activity was slightly lower in the scAAV2 / 9-syn-Rat-CTD group infected with the recombinant virus expressing the KCC2 truncations constructed according to the prior art, and there was almost no chloride ion efflux activity.

[0176] The above results show that the KCC2 truncations of the present application can enhance the chloride ion efflux activity of cells, that is, they still have corresponding biological activity. However, the truncations (Rat-CTD) recorded in the prior art did not show biological activity, which is consistent with the results reported in the literature (see Gregory et al., 2011, PNAS, 108(37): 15474-15479). 3.2 Effect of truncations on the level of KCC2 on the cell membrane in primary neurons

[0177] Literature reports that BDNF can promote KCC2 ubiquitination and then enter the degradation pathway (see Lee-Hotta, S et al.,

[0178] (2019). Neurochemistry International, 128: 32–38), thereby reducing the level of KCC2 on the neuronal cell membrane. In this experiment, primary neurons infected with scAAV2 / 9-syn-CTD and scAAV2 / 9-syn-C helices recombinant viruses and stably expressing KCC2 truncated proteins were treated with BDNF, and then the expression of KCC2 on the membrane was detected by immunofluorescence to confirm whether the KCC2 truncated protein of the present application can inhibit the decrease in KCC2 protein levels caused by BDNF or even increase the level of KCC2 on the neuronal membrane.

[0179] 3.2.1 Western-Blot detection of recognition and binding of KCC2 antibodies to KCC2 truncations and full-length KCC2

[0180] The scAAV2 / 9-syn-CTD, scAAV2 / 9-syn-C helices and ssAAV2 / 9-syn-KCC2 recombinant viruses were used to infect the NG108-15 cell line at the same MOI (2E+6), and the proteins were extracted 48 hours later for Western-Blot detection. Specifically, the extracted proteins were quantified by the BCA method, denatured, separated by electrophoresis, transferred to a membrane, blocked with 5% BSA, and then incubated overnight at 4°C with the primary antibody anti-KCC2 (Millipore, 07-432) at a dilution ratio of 1:500 and the internal reference anti-βactin (Cell Signaling, #4967S) at a dilution ratio of 1:2000, and then incubated at room temperature for 1 hour with the secondary antibody horseradish peroxidase-labeled goat antirabbit IgG at a dilution ratio of 1:2000 (Invitrogen, 65-6120), and then developed and photographed.

[0181] According to the results of Western-blot, after the NG108-15 cell line was infected with the ssAAV2 / 9-syn-KCC2 recombinant virus, the level of full-length KCC2 was significantly higher than that of the NC group; while in the cells infected with the scAAV2 / 9-syn-CTD group and the scAAV2 / 9-syn-Chelices group, no bands of CTD and Chelices were detected (results not shown). This indicates that the KCC2 antibody (anti-KCC2, Millipore, 07-432) used in this experiment can recognize and bind to the full-length KCC2 protein, but not to the truncated form of the present application.

[0182] 3.2.2 Immunofluorescence detection of primary neurons

[0183] This experiment was designed to verify the effect of the truncated form of the present application on the level of KCC2 on the cell membrane of primary neurons. The experimental groups were NC group, NC+BDNF group, CTD+BDNF group, and C-helices+BDNF group. Among them, the NC+BDNF group treated neurons that were not infected with the virus with BDNF; the CTD+BDNF group treated neurons that were infected with the scAAV2 / 9-syn-CTD virus with BDNF; and the C-helices+BDNF group treated neurons that were infected with the scAAV2 / 9-syn-C helices virus with BDNF.

[0184] Isolation of primary cortical neurons of P0 rats for in vitro culture: Specifically, SD rats within 12 hours after birth were placed under a microscope and the pia mater tissue was carefully removed. The cortex was digested with papain and then blasted into a single cell suspension. The isolated neuronal cells were cultured in Neurobasal medium (Invitrogen, 21103-049) containing 1% FBS and 2% B27. scAAV2 / 9-syn-CTD and scAAV2 / 9-syn-C helices recombinant viruses were used to infect neuronal cells at the same MOI (1E+6), and the negative control group (negative control, NC) was added with the same volume of virus solvent and cultured in a 37°C, 5% CO2 incubator for 7 days. Then BDNF was added to the neuronal culture medium of each group at 50 ng / mL, and treated in a 37°C incubator for 3 hours. Immunofluorescence detection was performed in all groups. Specifically, neurons were fixed with 4% PFA for 10 min, then blocked with 1% BSA, 0.1% TritonX-100 blocking solution at room temperature for 1 h, and the primary antibody anti-KCC2 (Millipore, 07-432) was diluted 1:500 and incubated overnight at 4°C. The next day, the secondary antibody Goat anti-Rabbit IgG (H+L) Alexa FluorTMPlus555 (Invitrogen, A32732) was added at a dilution ratio of 1:1000, incubated at room temperature for 1 h, and sealed with anti-fading sealing agent containing DAPI, and observed and photographed with a fluorescence microscope (Olympus, IX71). The average optical density was counted using Fiji ImageJ software to calculate the changes in the expression level of KCC2 on the neuronal membrane.

[0185] Test results such as Figure 5 As shown, in the NC+BDNF group, the KCC2 level on the neuronal cell membrane was significantly decreased compared with the NC group, that is, BDNF treatment reduced the protein level of wild-type KCC2 on the neuronal membrane, which is consistent with the conclusion reported in the literature, indicating that the model in this example was successfully constructed.

[0186] Compared with the NC+BDNF group, the KCC2 level on the neuronal cell membrane in the CTD+BDNF group was significantly increased, even slightly higher than the NC group without BDNF treatment, indicating that the truncated CTD of the present application can significantly increase the level of wild-type KCC2 on the neuronal membrane. Similar results were obtained in the C-helices+BDNF group. The trend reflected in the above results is consistent with the results of in vitro activity detection. Combined with the results in 3.2.1 and 3.2.2, it is speculated that the truncated molecule of the present application may exert its biological efficacy by increasing the level of wild-type KCC2 on the cell membrane in some way.

[0187] Example 4 In vivo efficacy evaluation

[0188] In order to further verify the in vivo biological activity of the KCC2 truncate in the present application, 8-week-old female C57BL / 6 mice were selected for in vivo efficacy evaluation.

[0189] First, mice were anesthetized with 1% sodium pentobarbital at a dose of 65 mg / kg. Then, a spinal cord injury model with bilateral lower limb paralysis was created 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 thoracic vertebrae T7 and T10, with the T10 transverse hemisection ending at the midline of the spinal cord and the T7 transverse hemisection located on the contralateral side of the T10 injury, slightly beyond the midline of the spinal cord. After modeling, the experimental groups were divided into model animal group, scAAV2 / 9-syn-CTD group and scAAV2 / 9-syn-C helices group, in which the model animal group was injected with virus solvent through the tail vein as a control. The scAAV2 / 9-syn-CTD group and the scAAV2 / 9-syn-C helices group were injected with 200 μL of recombinant virus scAAV2 / 9-syn-CTD and scAAV2 / 9-syn-C helices, respectively, with an injection dose of 1E+12 vg / animal, and antibiotics were injected for 2 consecutive days after surgery to prevent infection. The recovery of animal motor function was evaluated by BMS behavioral scoring every week, and the observation period was 5 weeks. The BMS behavioral scoring criteria are shown in Table 3.

[0190] Table 3

[0191]

[0192] According to the evaluation results, the BMS behavioral scores of mice injected with sc-AAV2 / 9-syn-CTD recombinant virus or sc-AAV2 / 9-syn-C helices recombinant virus were significantly increased compared with the model animal group, indicating that the mice's mobility was gradually restored (results not shown). Based on the above results, it is shown that the truncated CTD and C-Helices constructed by the present invention have obvious recovery and treatment effects on spinal cord injury in animal models.

Claims

1. A KCC2 truncation, characterized in that: The amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of at least 18 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10).

2. The KCC2 truncation according to claim 1, characterized in that The amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 18 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10); preferably, the amino acid sequence of the KCC2 truncate consists of amino acid residues 667-684 in the human wild-type full-length KCC2a amino acid sequence.

3. The KCC2 truncation according to claim 3, characterized in that The amino acid sequence of the KCC2 truncate is shown in SEQ ID NO:

5.

4. The KCC2 truncation according to claim 1, characterized in that The amino acid sequence of the KCC2 truncate is selected from an amino acid sequence consisting of 473 consecutive amino acid residues starting from the 667th amino acid residue in the human wild-type KCC2a amino acid sequence (SEQ ID NO: 10). Preferably, the amino acid sequence of the KCC2 truncate consists of amino acid residues at positions 667-1139 in the human wild-type full-length KCC2a amino acid sequence.

5. The KCC2 truncation according to claim 4, characterized in that The amino acid sequence of the KCC2 truncate is shown in SEQ ID NO:

3.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the KCC2 truncation according to any one of claims 1 to 5.

7. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule comprises the nucleic acid molecule of claim 6 and a promoter operably linked thereto.

8. The recombinant nucleic acid molecule according to claim 7, characterized in that The promoter is selected from the group consisting of cytomegalovirus (CMV) promoter, CAG promoter, desmin (DES) promoter, synapsin I (SYN) promoter or muscle creatine kinase (MCK) promoter; preferably, the promoter is synapsin I (SYN) promoter.

9. The recombinant nucleic acid molecule according to claim 7 or 8, characterized in that The recombinant nucleic acid molecule further comprises one or more of a polyadenylic acid sequence (polyA), a Kozak sequence, a stuffer sequence, a WPRE and / or a post-transcriptional regulatory element.

10. The recombinant nucleic acid molecule according to any one of claims 7 to 9, characterized in that: The recombinant nucleic acid molecule comprises: (1) the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12; (2) a polynucleotide sequence comprising a substitution, deletion or addition of one or more nucleotides in the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12; or (3) A polynucleotide sequence having a sequence identity of more than 80% with the polynucleotide sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, preferably a polynucleotide sequence having an identity of more than 85%, 90%, 95%, 96%, 97%, 98%, or 99%; more preferably, a polynucleotide sequence having an identity of more than 98% or 99%.

11. The recombinant nucleic acid molecule according to any one of claims 7 to 10, 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; more 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; further preferably, the AAV inverted terminal repeat sequence is from AAV2.

12. A recombinant vector, characterized in that: The recombinant vector comprises the recombinant nucleic acid molecule according to any one of claims 7 to 10, and the recombinant 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.

13. A recombinant adeno-associated virus, characterized in that: The recombinant adeno-associated virus comprises an AAV capsid and a vector genome, wherein the vector genome comprises the recombinant nucleic acid molecule according to any one of claims 7 to 11, and the AAV capsid is selected from AAV of any serotype selected from evolutionary branches AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid / chimeric types. Preferably, the capsid of the recombinant adeno-associated virus is preferably AAV9.

14. An isolated host cell, characterized in that The cell comprises the KCC2 truncation according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant nucleic acid molecule according to any one of claims 7 to 11, the recombinant vector according to claim 12, or the recombinant adeno-associated virus according to claim 13.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the KCC2 truncate according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant nucleic acid molecule according to any one of claims 7 to 11, the recombinant vector according to claim 12, the recombinant adeno-associated virus according to claim 13 and / or the host cell according to claim 14, and a pharmaceutically acceptable carrier and / or other conventional pharmaceutical ingredients.

16. The pharmaceutical composition according to claim 15, characterized in that: The other conventional pharmaceutical ingredients include preservatives and / or stabilizers.

17. The pharmaceutical composition according to claim 15 or 16, characterized in that The pharmaceutical composition can be formulated for intravenous, intrathecal, intracerebroventricular, intraspinal, intrathoracic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and / or other parenteral administration.

18. The pharmaceutical composition according to any one of claims 15 to 17, characterized in that The pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.

19. Use of the KCC2 truncate according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant nucleic acid molecule according to any one of claims 7 to 11, the recombinant vector according to claim 12, the recombinant adeno-associated virus according to claim 13, the host cell according to claim 14 and / or the pharmaceutical composition according to any one of claims 15 to 18 in the preparation of a medicament for preventing or treating spinal cord injury.

20. The use according to claim 19, characterized in that The pharmaceutical composition can be administered in combination with other drugs for treating spinal cord injury.

21. A method for treating spinal cord injury in a subject, characterized in that The method comprises administering to a subject in need thereof the KCC2 truncate of any one of claims 1-5, the nucleic acid molecule of claim 6, the recombinant nucleic acid molecule of any one of claims 7-11, the recombinant vector of claim 12, the recombinant adeno-associated virus of claim 13, the host cell of claim 14 and / or the pharmaceutical composition of any one of claims 15-18; preferably, the subject is a mammal; more preferably, the subject is a human.

22. The method according to claim 21, characterized in that The method may also be used in combination with other methods for treating spinal cord injury.

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