WO2024255792A1 - Promoteur mecp2 humain et son utilisation - Google Patents

Promoteur mecp2 humain et son utilisation Download PDF

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WO2024255792A1
WO2024255792A1 PCT/CN2024/098967 CN2024098967W WO2024255792A1 WO 2024255792 A1 WO2024255792 A1 WO 2024255792A1 CN 2024098967 W CN2024098967 W CN 2024098967W WO 2024255792 A1 WO2024255792 A1 WO 2024255792A1
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nucleic acid
recombinant
acid molecule
sequence
vector
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Chinese (zh)
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韩荣荣
刘洪洋
李忠
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Staidson Beijing Biopharmaceutical Co Ltd
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Staidson Beijing Biopharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

Definitions

  • the present invention relates to the biological field, and in particular to a human promoter, and in particular to a recombinant adeno-associated virus vector for treating spinal cord injury constructed by using the human promoter, and an application thereof.
  • the present invention provides a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7, or a nucleotide sequence having more than 80% (for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 6 or 7.
  • the nucleic acid molecule is a promoter that can initiate transcription of a nucleic acid sequence encoding an exogenous protein in a cell. In some specific embodiments, the nucleic acid molecule is a human MeCp2 promoter.
  • the present invention provides a recombinant nucleic acid molecule, which comprises the above-mentioned nucleic acid molecule, a nucleic acid sequence encoding an exogenous protein and a polyadenylic acid sequence (ie, polyA).
  • a recombinant nucleic acid molecule which comprises the above-mentioned nucleic acid molecule, a nucleic acid sequence encoding an exogenous protein and a polyadenylic acid sequence (ie, polyA).
  • the polyA is BGH polyA, preferably, its sequence is shown in SEQ ID NO: 8.
  • the exogenous protein is KCC2 protein, preferably, its amino acid sequence is as shown in SEQ ID NO: 3.
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 2; or
  • the recombinant nucleic acid molecule further comprises one or more of a Kozak sequence, a WPRE and a post-transcriptional regulatory element.
  • the recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; or
  • the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence; preferably, the AAV inverted terminal repeat sequence is selected from AAV of different serotypes; preferably, the AAV inverted terminal repeat sequence is selected from AAV of any serotype in evolutionary branch AF or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid/chimeric types; more preferably, the AAV inverted terminal repeat sequence is from AAV2.
  • the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule, wherein the vector is selected from a plasmid vector, a phage vector and a viral vector, wherein the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector and a hybrid viral vector.
  • the present invention provides a recombinant adeno-associated virus, comprising an AAV capsid and a vector genome, wherein the vector genome comprises the aforementioned nucleic acid molecule or recombinant nucleic acid molecule; in certain specific embodiments, the recombinant nucleic acid molecule comprises an AAV inverted terminal repeat sequence, a nucleic acid sequence encoding a KCC2 protein, and an expression control sequence and a PolyA sequence for directing the expression of the nucleic acid sequence of the KCC2 protein in a host cell; in some embodiments, the AAV capsid is selected from any serotype of AAV in evolutionary branches A-F or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid/chimeric types; in certain preferred embodiments, the capsid of the recombinant adeno-associated virus is AAV9, and its amino acid sequence is shown in SEQ ID
  • the present invention provides an isolated host cell comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule or the aforementioned recombinant vector or recombinant adeno-associated virus.
  • the cell is a neuronal cell. In some specific embodiments, the cell is a cholinergic neuronal cell, a noradrenergic neuronal cell, a dopaminergic neuronal cell, a 5-hydroxytryptamine neuronal cell, a ⁇ -aminobutyric acid neuronal cell. In some specific embodiments, the cell is a neural cell line, including NG108-15, N1E115, PC12, SHSY5Y, F11, ND7-23, N27, SN4741 cell lines.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned nucleic acid molecule or recombinant nucleic acid molecule, recombinant vector or recombinant adeno-associated virus and/or the aforementioned host cell and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, intraspinal injection, thoracic injection, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and/or other parenteral administration.
  • the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.
  • the present invention provides the use of the aforementioned nucleic acid molecules, recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and/or the aforementioned pharmaceutical compositions in the preparation of a medicament for preventing or treating spinal cord injury.
  • the recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and/or pharmaceutical compositions can be administered in combination with another therapy.
  • the present invention provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or the aforementioned drug combination to a subject in need thereof.
  • the composition is administered intravenously; in certain preferred embodiments, the composition is administered intrathecally; in certain preferred embodiments, the composition is administered intraspinal; in other preferred embodiments, the subject is a mammal; in more preferred embodiments, the subject is a human.
  • the treatment method can also be used in combination with other therapies.
  • the nucleic acid molecule provided by the present invention can initiate and/or regulate the more efficient transcription and/or expression of the encoded exogenous protein (particularly, KCC2 protein) in cells (particularly, nerve cells).
  • the nucleic acid molecule or promoter of the present application can be used in KCC2 gene therapy drugs to treat spinal cord injury more safely and effectively, and has a lower immune risk than the mouse MeCp2 promoter.
  • Figure 1 The main structure of the vector genome containing different promoters in the recombinant adeno-associated virus.
  • Figure 2 Plasmid map of the construction process of the shuttle plasmid pSNAV2.0-K-Syn-KCC2-BGH.
  • FIG. 1 Map of shuttle plasmids containing different promoters.
  • Adeno-associated virus 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.
  • Administration/administration Providing or administering an agent, such as a therapeutic agent (e.g., a recombinant AAV), to a subject by an effective route.
  • a therapeutic agent e.g., a recombinant AAV
  • 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.
  • Codon optimized 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).
  • 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.
  • Enhancer A nucleic acid sequence that increases transcription efficiency by increasing promoter activity.
  • ITRs Inverted terminal repeats: 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.
  • 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.
  • 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.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Pharmaceutically acceptable carriers Pharmaceutically acceptable carriers (vehicles) that may be used in the present disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. 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.
  • 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.
  • injectable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc.
  • solvents such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc.
  • 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.
  • 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.
  • auxiliary substances such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.
  • 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.
  • 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.
  • 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).
  • 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- ⁇ ) is packaged.
  • Serotype A group of closely related microorganisms (eg, viruses) distinguished by a characteristic set of antigens.
  • Subject A living multicellular vertebrate organism, including the class of humans and non-human mammals. Specifically, the subject described herein is an individual with spinal cord injury, who may have residual endogenous KCC2 protein activity, or no measurable activity.
  • Synthetic Produced in a laboratory by artificial means, e.g. synthetic nucleic acids can be chemically synthesized in a laboratory.
  • Therapeutically effective amount The amount of a particular drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve the desired effect in a subject or cell treated with the agent.
  • the effective amount of an agent depends on a variety of factors, including but not limited to the subject or cell being treated. cells, and modes of administration of therapeutic compositions.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • sequence alignment program is available for amino acid sequences, such as "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Typically, any of these programs is used with 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 provides at least the same level of identity or alignment as provided by the reference algorithm or program. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13): 2682-2690 (1999).
  • 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 a Web server 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 contained in the above-mentioned programs. As another example, Fasta TM (a program in GCG Version 6.1) can be used to compare polynucleotide sequences. Fasta TM provides alignment and percentage sequence identity of the best overlapping region between the query and search sequences. For example, Fasta TM can be used with its default parameters provided in GCG Version 6.1 (incorporated herein by reference) (word length of 6, and NOPAM factor for scoring matrix) to determine the percentage sequence identity between nucleic acid sequences.
  • 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.
  • 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.
  • replication-defective viruses can be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrated or non-integrated), or another suitable viral source.
  • AAV adeno-associated viruses
  • adenoviruses adenoviruses
  • lentiviruses integrated or non-integrated
  • Self-complementary AAV refers to a construct in which the coding region carried by the recombinant AAV is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form a double-stranded DNA (dsDNA) unit ready for immediate replication and transcription, see, e.g., D M 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, e.g., US6,596,535B1, US7,125,717B2, and US7,456,683B2, each of which is incorporated herein by reference in its entirety.
  • expression is used in this article in its broadest sense and includes the production of DNA or RNA and proteins.
  • expression or “translation” particularly refers to the production of peptides or proteins. Expression can be transient, or can be stable.
  • translation in the context of the present invention relates to the process of ribosome processing in which an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.
  • the term "about” refers to a difference of 10% ( ⁇ 10%) from a given reference value, unless otherwise specified.
  • disease As used herein, “disease,” “disorder,” and “condition” are used interchangeably to refer to an abnormal state in a subject.
  • the first aspect of the present invention provides a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 6 or 7, or a nucleotide sequence having a sequence identity of more than 80% (e.g., more than 85%, 90%, 95%, 96%, 97%, 98%, 99%) with SEQ ID NO: 6 or 7.
  • the nucleic acid molecule is a promoter; more particularly, the nucleic acid molecule is a human MeCp2 promoter.
  • MeCp2 is a gene encoding murine methyl-CpG-binding protein 2, which is highly expressed in neurons of the central nervous system and plays an important role in maintaining synaptic connections between neurons.
  • the MeCp2 promoter is the promoter of murine methyl-CpG-binding protein 2, which is located upstream of the start codon of the MeCp2 gene in the genome and can regulate and initiate the transcription of the gene's DNA sequence.
  • the promoter is the mMeCp2 promoter, and the mMeCp2 is the natural promoter of the mouse MeCp2 gene.
  • the second aspect of the present invention provides a recombinant nucleic acid molecule, which comprises the above-mentioned nucleic acid molecule (or, also referred to as a promoter), a nucleic acid sequence encoding an exogenous protein and a polyadenylic acid sequence (ie, polyA).
  • the exogenous protein is KCC2 protein.
  • KCC2 is K + -Cl - cotransporter 2, also known as solute carrier family 12 member 5. It is a neuron-specific K + -Cl - cotransporter encoded by the SLC12A5 gene, which mainly mediates neuron-specific K + -Cl - transport.
  • the 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 is that the NTD of KCC2a has 23 more amino acids than the NTD of KCC2b.
  • KCC2 proteins have been found in many species, including human KCC2 protein, mouse KCC2 protein, rat KCC2 protein, rabbit, dog, pig, etc.
  • the amino acid sequences and nucleotide sequences of various KCC2 proteins are known and can be found in various bioinformation databases such as NCBI, such as human.
  • NCBI Gene ID of the full-length KCC2a is 57468, its amino acid sequence number is NCBI RefSeq: NP_001128243.1 (SEQ ID NO: 3), and its mRNA sequence number is NCBI RefSeq: NM_001134771.2.
  • the KCC2 protein of the present invention may be derived from different species.
  • the KCC2 protein of the present invention is a naturally occurring mature KCC2 protein.
  • the KCC2 protein of the present invention includes various mutants or derivatives that retain biological activity.
  • a modified or optimized KCC2 protein coding sequence is provided.
  • the KCC2 protein coding sequence is a codon-optimized sequence optimized for its expression in the subject species.
  • Subject as used herein 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.
  • the subject is a human.
  • the sequence is codon-optimized for expression in the human body.
  • Codon optimized coding regions can be designed by various methods. Such optimization can be performed using methods available online (e.g., GeneArt), disclosed methods, or companies providing codon optimization services, such as DNA2.0 (Menlo Park, CA). For example, a codon optimization method is described in International Patent Publication No. WO 2015/012924A2, which is incorporated herein by reference in its entirety. See also, for example, US2014/0032186A1 and US2006/0136184A1. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only fragments of the ORF can be changed. By using one of these methods, the frequency can be applied to any given polypeptide sequence, and a codon optimized coding sequence encoding the polypeptide can be produced.
  • ORF open reading frame
  • 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 approximately 500 base pairs) ligated 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 ligated together to form the final construct. The final construct is then The gene can be 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.
  • the nucleotide sequences encoding KCC2 proteins described herein can be synthesized using techniques well known in the art.
  • 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.
  • kits and protocols for generating DNA via PCR are commercially available. These include the use of polymerases, including but not limited to Taq polymerase, (New England Biolabs), High-Fidelity DNA polymerase (New England Biolabs) and G2 polymerase (Promega).
  • DNA can also be generated from cells transfected with a plasmid containing the KCC2 protein sequence described herein.
  • Kits and protocols are known and commercially available and include, but are not limited to, QIAGEN plasmid kits, Pro Filter plasmid kit (Invitrogen) and GenElute TM plasmid kit (Sigma Aldrich).
  • Other techniques available in this article include sequence-specific isothermal amplification methods, which eliminate the need for thermal cycling. These methods typically use strand 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 reverse transcriptase
  • 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.
  • the nucleic acid molecule encoding the KCC2 protein is DNA or RNA. In some specific embodiments of the present invention, the nucleic acid molecule encoding the KCC2 protein has a polynucleotide sequence selected from the following:
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides of the polynucleotide sequence shown in SEQ ID NO: 2; or
  • Polyadenylation sequence (PolyA)
  • the polyadenylic acid sequence (polyA) is BGH polyA.
  • BGH polyA is a bovine growth hormone polyadenylation signal, which is a transcription termination signal of eukaryotic organisms.
  • sequence of BGH polyA is shown in SEQ ID NO: 8.
  • the recombinant nucleic acid molecule further comprises other expression control elements, such as one or more of a Kozak sequence, a WPRE and a post-transcriptional regulatory element, wherein the Kozak sequence can enhance translation efficiency, and the WPRE sequence can enhance and stabilize mRNA expression.
  • other expression control elements such as one or more of a Kozak sequence, a WPRE and a post-transcriptional regulatory element, wherein the Kozak sequence can enhance translation efficiency, and the WPRE sequence can enhance and stabilize mRNA expression.
  • the recombinant nucleic acid molecule comprises a polynucleotide sequence selected from the group consisting of:
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides of the polynucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; or
  • polynucleotide sequence shown in SEQ ID NO: 12 contains the following elements: hMeCp2 promoter, nucleic acid sequence encoding full-length KCC2, and BGH polyA sequence.
  • polynucleotide sequence shown in SEQ ID NO: 13 contains the following elements: short hMeCp2 promoter, nucleic acid sequence encoding full-length KCC2 protein, and BGH polyA sequence.
  • the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence; preferably, the AAV inverted terminal repeat sequence is selected from AAVs of different serotypes; preferably, the AAV inverted terminal repeat sequence is selected from AAVs of any serotype in evolutionary branches A-F or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid/chimeric types; more preferably, the AAV inverted terminal repeat sequence is from AAV2.
  • the third aspect of the present invention also provides a recombinant vector, which comprises the aforementioned nucleic acid molecule (or, may also be referred to as an initiator) promoter) or recombinant nucleic acid molecule.
  • the vector is selected from a plasmid vector, a phage vector or a viral vector, wherein the viral vector is selected from an adeno-associated viral vector, an adenoviral vector, a lentiviral vector or a hybrid viral vector.
  • the nucleic acid molecules encoding KCC2 proteins described herein can be engineered into suitable genetic elements (vectors) that can be used to generate viral vectors and/or delivered to host cells, such as naked DNA, bacteriophages, transposons, cosmids, episomes, etc., which transmit the nucleic acid molecules encoding KCC2 proteins carried thereon.
  • the selected 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. Methods for making such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • the fourth aspect of the present invention also provides a recombinant adeno-associated virus, which comprises an AAV capsid and a vector genome, wherein the vector genome comprises the aforementioned nucleic acid molecule (or, also referred to as a promoter) or a recombinant nucleic acid molecule.
  • 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.
  • the AAV capsid is selected from those AAV capsids that can effectively transduce neuronal cells.
  • 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.
  • AAV capsids available herein include AAVrh.39, AAVrh.20, AAVrh.25, AAV10, AAVbb.1 and AAVbb.2 and variants thereof.
  • 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.
  • the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins.
  • the AAV capsid is a chimera of Vpl, Vp2 and Vp3 monomers from two or three different AAVs or recombinant AAVs.
  • 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.
  • 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.
  • the comparison can be made to any variable protein (e.g., vp1, vp2, or vp3).
  • scAAV self-complementary AAV
  • ssAAV single-stranded AAV
  • the AAV capsid is selected from any serotype of AAV in clades A-F or any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or their hybrid/chimeric forms.
  • the capsid of the recombinant adeno-associated virus is AAV9, and its amino acid sequence is shown in SEQ ID NO: 14.
  • the recombinant adeno-associated virus is a single-stranded recombinant adeno-associated virus.
  • the recombinant nucleic acid molecules described herein can be packaged into the capsid of a viral vector (e.g., virion).
  • a viral vector e.g., virion
  • such recombinant nucleic acid molecules used to generate viral vectors contain the coding sequence of the KCC2 protein described herein, the packaging signal of the viral genome on its side, and other expression control sequences, such as those described herein.
  • the packaging signal is 5' reverse terminal repeats (ITR) and 3' ITR.
  • ITR reverse terminal repeats
  • the recombinant nucleic acid molecule and the ITRs associated therewith are referred to herein as "recombinant AAV (rAAV) genome” or "vector genome”.
  • the recombinant adeno-associated virus vector (rAAV) provided by the present invention comprises an AAV capsid and a vector genome as described herein.
  • the vector genome comprises a protein encoding KCC2.
  • Nucleic acid molecules and regulatory sequences such as nucleic acid molecules described herein (or, may also be referred to as promoters) that direct and/or regulate the expression of the above nucleic acid molecules in host cells.
  • the vector genome comprises the recombinant nucleic acid molecules described in the present invention.
  • the vector genome also comprises the ITR sequence of AAV.
  • the ITRs are from an AAV serotype different from the one that provides the capsid.
  • the ITR sequence is from AAV2, or a deletion variant thereof ( ⁇ ITR), which may be used for convenience and accelerate regulatory approval.
  • ITRs from other AAV sources can be selected.
  • the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as a pseudotype.
  • the AAV vector genome comprises an AAV 5'ITR and an AAV 3'ITR.
  • a deletion variant of the 5'ITR in which the D-sequence and the terminal resolution site (trs) are deleted (referred to as ⁇ ITR) has been described.
  • the 5' and 3'ITRs of the full-length AAV are used.
  • the ITR or ⁇ ITR sequence of each serotype is well known to those skilled in the art.
  • the capsid is an AAV9 capsid (whose amino acid sequence is shown in SEQ ID NO: 14) or a variant thereof.
  • single-stranded AAV can be selected.
  • double-stranded AAV, or self-complementary AAV can be selected.
  • transgenic construct encoding side ITR and a construct encoding rep and cap are transiently transfected to produce a cell line.
  • a transgenic construct encoding side ITR is transiently transfected to stably provide a packaging cell line of rep and cap.
  • AAV virions are produced in response to infection with a helper adenovirus or herpes virus, wherein it is necessary to separate rAAV from contaminating viruses.
  • helper functions i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29 and herpesvirus polymerase
  • the helper functions could be provided by transiently transfecting the cells with constructs encoding the required helper functions, or the cells could be engineered to stably contain genes encoding the helper functions, the expression of which could be controlled at the transcriptional or post-transcriptional level.
  • transgenic and rep/cap genes flanking ITRs were introduced into insect cells by infection with a baculovirus-based vector.
  • baculovirus-based vector For an overview of these production systems, see, for example, Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production", Human Gene Therapy 20:922-929, each of which is hereby referenced. Methods of making and using these and other AAV production systems are also described in the following U.S.
  • Patents each of which is incorporated herein by reference in its entirety: 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.
  • the recombinant nucleic acid molecules described herein can also be used to produce other viral vectors.
  • Such other viral vectors can include any virus suitable for gene therapy that can be adopted, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc.
  • adenovirus adenovirus
  • herpes virus lentivirus
  • retrovirus a virus suitable for gene therapy
  • when one of these other vectors is produced it is produced as a replication-defective viral vector.
  • the fifth aspect of the present invention also provides an isolated host cell, which comprises the aforementioned nucleic acid molecule (or, may also be referred to as a promoter) or recombinant nucleic acid molecule or the aforementioned recombinant vector or recombinant adeno-associated virus.
  • the cells are neuronal cells; in some specific embodiments, the cells are cholinergic neuronal cells, noradrenergic neuronal cells, dopaminergic neuronal cells, 5-hydroxytryptaminergic neuronal cells, ⁇ -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.
  • the sixth aspect of the present invention also provides a pharmaceutical composition, which comprises the aforementioned nucleic acid molecule (or, it can also be referred to as a promoter) or a recombinant nucleic acid molecule, a recombinant vector or a recombinant adeno-associated virus and/or the aforementioned host cell and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can be formulated for intravenous injection, intrathecal injection, intraventricular injection, intraspinal injection, thoracic injection, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and/or other parenteral administration.
  • the pharmaceutical composition may also include other drugs that can be used to treat spinal cord injury.
  • the pharmaceutical composition of the present invention can be designed to be delivered to a subject in need thereof by any suitable route or a combination of different routes.
  • the composition is delivered via intravenous injection.
  • the pharmaceutical composition is delivered via intraspinal injection.
  • it can be carried out via intrathecal injection.
  • the composition is delivered via intraventricular injection.
  • the composition is delivered via thoracic injection.
  • the in situ injection of the target tissue can be performed.
  • other routes of administration e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and other parenteral routes
  • routes of administration e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraperitoneal and other parenteral routes
  • routes of administration e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intra
  • the recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses or The isolated host cells can be delivered in a single composition or multiple compositions.
  • two or more different AAVs can be delivered (see, for example, WO 2011/126808A2 and WO 2013/049493A1]).
  • such multiple viruses can contain different replication-deficient viruses (e.g., AAV, adenovirus, and/or lentivirus).
  • delivery can be mediated by non-viral vectors, such as "naked DNA”, “naked plasmid DNA”, RNA and mRNA; in combination with various delivery compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions and other polymers, lipid-based and/or cholesterol-nucleic acid conjugates, and other constructs as described herein, see, for example, X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp.
  • the viral vector or non-viral DNA or RNA transfer part can be formulated with a physiologically acceptable carrier for gene transfer and gene therapy.
  • the delivery vector can select a variety of suitable purification methods, describing examples of purification methods suitable for separating empty capsids from vector particles, such as the methods described in International Patent Application No. PCT/US16/65976 filed on December 9, 2016 and its priority documents, 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 are hereby incorporated by reference into this article.
  • purification comprises a two-step purification scheme that selectively captures and isolates rAAV vector particles containing the genome from the clarified, concentrated supernatant of the rAAV 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.
  • purification comprises affinity chromatography, ultracentrifugation, molecular sieve chromatography, and the like.
  • quantification of viral genomes 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 as viral genomes (vg)
  • the units are expressed (i.e., 1 ⁇ 10 7 vg, 1 ⁇ 10 8 vg, 1 ⁇ 10 9 vg, 1 ⁇ 10 10 vg, 1 ⁇ 10 11 vg, 1 ⁇ 10 12 vg, 1 ⁇ 10 13 vg, 1 ⁇ 10 14 vg, 1 ⁇ 10 15 vg, 1 ⁇ 10 16 vg, respectively).
  • 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, 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, respectively).
  • Methods for titrating AAV are described in Clark et al., Hum. Gene Ther 1999; 10: 1031-1039.
  • the volume of the carrier, excipient or buffer is at least about 25 ⁇ L. In one embodiment, the volume is about 50 ⁇ L. In another embodiment, the volume is about 100 ⁇ L. In another embodiment, the volume is about 200 ⁇ L. In another embodiment, the volume is about 300 ⁇ L. In another embodiment, the volume is about 400 ⁇ L. In another embodiment, the volume is about 500 ⁇ L.
  • the volume is about 600 ⁇ L. In another embodiment, the volume is about 700 ⁇ L. In another embodiment, the volume is about 800 ⁇ L. In another embodiment, the volume is about 900 ⁇ L. 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.
  • the rAAV is preferably suspended in a physiologically compatible carrier and can be administered to a human or non-human mammal patient.
  • 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.
  • 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.
  • 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.
  • 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 Chemical stabilizers include gelatin and albumin.
  • compositions of the invention may comprise a pharmaceutically acceptable carrier as defined above.
  • 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.
  • the viral vectors described herein can be used to prepare drugs so as to deliver nucleic acid molecules encoding KCC2 proteins to subjects (e.g., human patients) in need thereof, and to provide KCC2 proteins 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.
  • the seventh aspect of the present invention also provides the use of the aforementioned nucleic acid molecule (or, also referred to as a promoter), recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing or treating spinal cord injury.
  • the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or pharmaceutical composition can be administered in combination with another therapy.
  • the eighth aspect of the present invention also provides a method for treating spinal cord injury in a subject, the method comprising administering the aforementioned recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or the aforementioned pharmaceutical composition to a subject in need thereof; in certain preferred embodiments, the composition is administered intravenously; in certain preferred embodiments, the composition is administered intrathecally; in certain preferred embodiments, the composition is administered intraspinal; in other preferred embodiments, the subject is a mammal; in a more preferred embodiment, the subject is a human. In some embodiments, the treatment method can also be used in combination with other therapies.
  • the above-mentioned recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and/or the aforementioned pharmaceutical compositions can be administered repeatedly.
  • 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 protein and a secondary treatment is required, rAAV10 carrying the above-mentioned nucleic acid molecule can be subsequently administered, and vice versa.
  • the pharmaceutical composition of the present invention may be used in combination with any one of substances including but not limited to CLP290, osteopontin, growth factors or 4-aminopyridine.
  • immunosuppressants are used for transient co-treatment before, during and/or after treatment with the composition of the present invention.
  • Immunosuppressants used for such co-therapy include but are not limited to steroids, antimetabolites, T cell inhibitors and alkylating agents.
  • transient treatment may include taking steroids (e.g., once daily in decreasing doses) at a dose of 1:1.
  • prednisole for 7 days, starting at about 60 mg and decreasing by 10 mg per day (no dose on day 7).
  • Other doses and immunosuppressants may be selected.
  • Example 1 Design of expression cassette and construction of shuttle plasmid carrying the expression cassette
  • this embodiment uses multiple promoters (Syn, mMeCp2, short mMeCp2, hMeCp2 or short hMeCp2) to design multiple expression cassettes containing multiple different promoters.
  • adeno-associated virus vectors were used as delivery vectors to verify the effects of different promoters on the expression of human wild-type full-length KCC2 protein in the nervous system.
  • the expression cassette containing the combination and its nucleic acid sequence are as follows (the schematic diagram of its structure and composition can be seen in Figure 1, which also shows the ITRs located on both sides of the expression cassette, and the ITRs are derived from AAV2):
  • Syn-KCC2-BGH poly A (SEQ ID NO: 9) is an expression cassette that has been disclosed in existing literature (refer to See WO2019226643A1), which is used as a positive control in this example.
  • a shuttle plasmid carrying the above expression cassette is constructed for packaging the corresponding recombinant adeno-associated virus vector.
  • the human Syn promoter (human synapsin I promoter, human synapsin I promoter, SEQ ID NO: 1) and its seamless primers (forward primer: 5'-TGGTGGCGGCGATATCCTGCGCTCTCAGGCACGACAC-3', reverse primer: 5'-GGGTTCCTCTGCTCGAGCAGTGCAAGTGGGTTTTAGG-3') and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is shown in SEQ ID NO: 3) and its seamless primers (forward primer: 5'-TCGAGGCAGATCTGTCGAC-3', reverse primer: 5'-CAGGATATCGCCGCCACCATGAGCCGCAGGTTCACGGTC-3') were synthesized respectively (synthesized by Beijing Nosai Genome Research Center Co., Ltd.).
  • the plasmid pSNAV2.0-Syn-KCC2 was double-digested with SwaI and NruI to recover a vector fragment of about 6.2 kb.
  • primer pair 2 forward primer:
  • 5'-AGGTTAAGTCCTCATTTAAATTAGGCAA-3' was used to amplify fragment 1 (about 1.27 kb) and fragment 2 (about 270 bp) on the original vector by PCR using the above plasmid pSNAV2.0-Syn-KCC2 as a template.
  • the kana resistance encoding gene was amplified by PCR using the seamless primer pair 3 (forward primer: 5′-TTAGAAAAACTCATCGAGCATCAAATG-3′, reverse primer: 5′-ATGAGCCATATTCAACGGGA-3′) and the synthetic 1902-Kan (containing the kana resistance encoding gene) as a template, and was named fragment 3.
  • PCR was used to add homology arms between the vector and fragment 1, fragment 1 and fragment 3, fragment 3 and fragment 2, and fragment 2 and the vector. Fragment 1, fragment 2, fragment 3 and the vector fragment of pSNAV2.0-Syn-KCC2 were connected by seamless connection.
  • the resulting shuttle plasmid was named pSNAV2.0-K-Syn-KCC2-BGH ( FIG. 2C , the main structure of its vector genome is shown in FIG. 1A ).
  • the mMeCp2 promoter and KCC2 protein coding sequence were inserted between the 5'ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR method.
  • the constructed shuttle plasmid was named pSNAV2.0-K-mMeCp2-KCC2-BGH ( Figure 3B, the main structure of the vector genome is shown in Figure 1B).
  • the hMeCp2 promoter (SEQ ID NO: 6) and its seamless primer (forward primer: 5'-CATGGTGGCGGCGATATCCGCGGCGGCGGCGGCGGC-3', reverse primer: 5'-CTAGGGGTTCCTCTGCTCGAGCAAGCCAGGGTTGCGATTTGTTG-3') and the KCC2 coding sequence (Genebank NM_001134771.2) (SEQ ID NO: 2, its amino acid sequence is as shown in SEQ ID NO: 3) and its seamless primer (forward primer: 5'-GTCGAGGCAGATCTGTCGACTCAGGAGTAGATGGTGATGACCTC-3', reverse primer: 5'-GATATCGCCGCCACCATGAGCC-3') were synthesized by Beijing Nosai Genome Research Center Co., Ltd.
  • the hMeCp2 promoter and KCC2 protein coding sequence were inserted between the backbone vector 5'ITR sequence and the BGH polyA sequence by seamless cloning PCR method.
  • the constructed shuttle plasmid was named pSNAV2.0-K-hMeCp2-KCC2-BGH (as shown in Figure 3D, the main structure of the vector genome is shown in Figure 1D).
  • the pSNAV2.0-K-Syn-KCC2-BGH vector (as shown in FIG. 3A ) was digested with restriction endonucleases Sal I + Xho I, and the large fragment (about 4.5 kb) was recovered.
  • the short hMeCp2 promoter and KCC2 protein coding sequence were inserted between the 5'ITR sequence and the BGH polyA sequence of the backbone vector by seamless cloning PCR method.
  • the constructed shuttle plasmid was named pSNAV2.0-K-short hMeCp2-KCC2-BGH (as shown in Figure 3E, the main structure of the vector genome is shown in Figure 1E).
  • Example 2 Virus packaging and purification
  • the present invention adopts HEK293T cells as production cell lines and conventional three-plasmid packaging system to produce AAV virus vectors.
  • the experimental methods used are conventional methods in the art. (For example, see Xiao Xiao, Juan Li, and Richard Jude Samulski. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 1998, 72 (3): 2224).
  • the shuttle plasmids obtained in Example 1 (pSNAV2.0-K-Syn-KCC2-BGH, pSNAV2.0-K-mMeCp2-KCC2-BGH, pSNAV2.0-K-short mMeCp2-KCC2-BGH, pSNAV2.0-K-hMeCp2-KCC2-BGH and pSNAV2.0-K-short hMeCp2-KCC2-BGH) were used to package recombinant adenovirus-derived vectors carrying the above-mentioned different promoters and expression cassettes.
  • the related viral vectors were named ssAAV 2/9-Syn-KCC2-BGH polyA, ssAAV 2/9-mMeCp2-KCC2-BGH polyA, ssAAV 2/9-short mMeCp2-KCC2-BGH polyA, ssAAV 2/9-short hMeCp2-KCC2-BGH polyA and ssAAV 2/9-short hMeCp2-KCC2-BGH polyA.
  • the AAV virus vector produced by packaging was purified by AAV9 affinity chromatography (Thermo Scientific POROS CaptureSelect AAV9 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 2, and the results of the titer detection of each recombinant AAV virus genome are shown in Table 3.
  • Recombinant viral vectors ssAAV 2/9-mMeCp2-KCC2-BGH polyA, ssAAV 2/9-shortmMeCp2-KCC2-BGHpolyA, ssAAV 2/9-hMeCp2-KCC2-BGHpolyA, and ssAAV 2/9-short hMeCp2-KCC2-BGH polyA carrying different promoter expression cassettes were used to infect neurons at the same MOI (1E+6), and the negative control group (NC) was added with the same volume of virus solvent. Immunofluorescence detection was performed after 7 days of culture in a 37°C, 5% CO 2 incubator.
  • Neurons were fixed with 4% PFA for 10 minutes, then blocked with a blocking solution containing 1% BSA and 0.1% Triton X-100 at room temperature for 1 hour, and the primary antibody anti-rabbit KCC2 (Millipore, 07-432) was incubated at 1:500 at 4°C overnight.
  • the secondary antibody Goat anti-Rabbit IgG (H+L) Alexa FluorTM Plus 555 (Invitrogen, A32732) was added at 1:1000, incubated at room temperature for 1 h, and sealed with anti-fading mounting medium containing DAPI, and observed and photographed under a fluorescence microscope (Olympus, IX71).
  • the average optical density was calculated using Fiji ImageJ software to calculate the changes in the levels of KCC2 protein in neurons of different groups relative to the NC group.
  • the in vitro activity of the above-mentioned different recombinant viral vectors was evaluated by detecting the level of intracellular chloride ions (Cl - ).
  • the biological function of KCC2 protein is to transport Cl- from the neuron to the extracellular space and maintain a low Cl- concentration in the neuron. Therefore, the Cl- efflux activity is an important indicator for evaluating the biological activity of KCC2 protein.
  • SuperClomeleon see Grimley, JS; Li, L.; Wang, W.; Wen, L.; Beese, LS; Hellinga, HW; Augustine, GJ (2013). Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation.
  • SuperClomeleon is a CFP mutant and a YFP mutant fluorescent protein fused and expressed in the cell, which can be used as a fluorescent reporter molecule for changes in intracellular chloride ion ( Cl- ) concentration.
  • the binding of Cl- to SuperClomeleon will cause its conformational changes, and the fluorescence resonance energy transfer (FRET) efficiency between the two fluorescent protein variants will change.
  • FRET fluorescence resonance energy transfer
  • the excitation wavelength of the detection is 440nm, and the emission wavelengths are 485nm and 535nm.
  • the efficiency of FRET is characterized by the ratio value of 535nm/485nm, which is used as an indicator of intracellular chloride ion levels.
  • the final result graph sets the result of the negative control (NC) as 1, and compares the multiples of the results of other recombinant adeno-associated viruses relative to the negative control.
  • lentivirus expressing SuperClomeleon was packaged, and then primary cortical neurons of P0 rats were isolated in vitro.
  • the recombinant adeno-associated viruses ssAAV 2/9-mMeCp2-KCC2-BGH polyA, ssAAV 2/9-short mMeCp2-KCC2-BGH polyA, ssAAV 2/9-hMeCp2-KCC2-BGH polyA, and ssAAV 2/9-short hMeCp2-KCC2-BGH polyA obtained in Example 2 were infected with neurons at the same MOI (1E+6), and the negative control group (NC) was prepared by adding the same volume of virus solvent to the neuronal cells at 37°C and 5% CO.
  • NC negative control group
  • mice C57BL/6 mice, 8 weeks old, female, 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., (2016), Cell, 174 (6) 1599. doi: 10.1016). In short, two transverse hemisections were performed simultaneously at the thoracic vertebrae T7 and T10 of the mouse, 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.
  • the virus solvent (as the model control group) or the recombinant adeno-associated virus of the present application (taking ssAAV 2/9-short hMeCp2-KCC2-BGH polyA as an example) and the positive control ssAAV2/9-Syn-KCC2-BGH polyA were injected into the tail vein at 200 ⁇ L each, with the 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 score (see Table 4 for scoring criteria) every week, and the observation period was 5 weeks.
  • the mouse model and drug administration process were the same as in 4.1.
  • the survival rate the number of animals surviving per week/the number of animals that were initially modeled and dosed (expressed as a percentage).
  • ssAAV 2/9-short hMeCp2-KCC2-BGH polyA was also used as an example.
  • the ssAAV 2/9-short hMeCp2-KCC2-BGH polyA recombinant virus infection group achieved an effect that was basically equivalent to or even better than the prior art positive control ss-AAV 2/9-Syn-KCC2-BGH polyA; in the survival rate detection experiment, the survival rate of mice in the ssAAV 2/9-short hMeCp2-KCC2-BGH polyA recombinant virus infection group was higher than the survival rate of mice in the prior art positive control group (ss-AAV 2/9-Syn-KCC2-BGH polyA). It further confirms that the human hMeCp2 or short hMeCp2 promoter used in the technical solution of this application is consistent with its in vitro effect, and also has surprising and better in vivo activity, and can also bring higher safety.

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Abstract

La présente invention concerne une molécule d'acide nucléique et une molécule d'acide nucléique recombinée. La molécule d'acide nucléique recombinée comporte la molécule d'acide nucléique, une séquence d'acide nucléique codant pour une protéine étrangère et une séquence d'acide polyadénylique (c'est-à-dire poly A). La présente invention concerne également un virus adéno-associé recombiné, comportant une capside AAV et un génome de vecteur, le génome de vecteur comprenant la molécule d'acide nucléique recombinée. La présente invention concerne également l'utilisation du virus adéno-associé recombiné pour traiter plus efficacement les lésions de la moelle épinière.
PCT/CN2024/098967 2023-06-16 2024-06-13 Promoteur mecp2 humain et son utilisation Pending WO2024255792A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110869385A (zh) * 2017-03-24 2020-03-06 爱丁堡大学董事会 MeCP2表达盒
US20200093937A1 (en) * 2017-05-31 2020-03-26 The Trustees Of The University Of Pennsylvania Gene therapy for treating peroxisomal disorders
CN111936163A (zh) * 2017-10-23 2020-11-13 斯托克制药公司 用于治疗基于无义介导的rna衰变的病况和疾病的反义寡聚体
US20210198692A1 (en) * 2018-05-31 2021-07-01 The Governing Council Of The University Of Toronto Adeno-associated viral vector-mediated gene therapy for treating fragile x-associated disorders
CN116113441A (zh) * 2020-08-12 2023-05-12 Ucb生物制药有限责任公司 使用包含甲基cpg结合蛋白2（mecp2）启动子序列的核酸构建体的基因疗法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110869385A (zh) * 2017-03-24 2020-03-06 爱丁堡大学董事会 MeCP2表达盒
US20200093937A1 (en) * 2017-05-31 2020-03-26 The Trustees Of The University Of Pennsylvania Gene therapy for treating peroxisomal disorders
CN111936163A (zh) * 2017-10-23 2020-11-13 斯托克制药公司 用于治疗基于无义介导的rna衰变的病况和疾病的反义寡聚体
US20210198692A1 (en) * 2018-05-31 2021-07-01 The Governing Council Of The University Of Toronto Adeno-associated viral vector-mediated gene therapy for treating fragile x-associated disorders
CN116113441A (zh) * 2020-08-12 2023-05-12 Ucb生物制药有限责任公司 使用包含甲基cpg结合蛋白2（mecp2）启动子序列的核酸构建体的基因疗法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 19 June 2006 (2006-06-19), ANONYMOUS: "Homo sapiens methyl CpG-binding protein 2 (MECP2) gene, promoter reg exon 1", XP093248647, Database accession no. AY523575.1 *

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