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WO2025143742A1 - Polynucléotide à codon optimisé codant pour le facteur ix de coagulation sanguine et plasmide producteur de raav le comprenant - Google Patents

Polynucléotide à codon optimisé codant pour le facteur ix de coagulation sanguine et plasmide producteur de raav le comprenant Download PDF

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WO2025143742A1
WO2025143742A1 PCT/KR2024/021013 KR2024021013W WO2025143742A1 WO 2025143742 A1 WO2025143742 A1 WO 2025143742A1 KR 2024021013 W KR2024021013 W KR 2024021013W WO 2025143742 A1 WO2025143742 A1 WO 2025143742A1
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recombinant aav
treating hemophilia
itr
plasmid
producing
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Korean (ko)
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김한울
강서경
서범석
이다형
이유경
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Samsung Bioepis Co Ltd
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Samsung Bioepis 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue

Definitions

  • the present invention relates to a codon-optimized polynucleotide encoding coagulation factor IX, a plasmid for producing a recombinant AAV comprising the polynucleotide, a recombinant AAV produced by the plasmid, and a use of the recombinant AAV for treating hemophilia B.
  • Hemophilia is a bleeding disease caused by a deficiency of clotting factors in the blood due to an innate, hereditary mutation in a gene located on the X chromosome.
  • Hemophilia A is caused by a deficiency of clotting factor VIII (FVIII)
  • hemophilia B is caused by a deficiency of clotting factor IX (FIX).
  • adeno-associated virus AAV
  • rAAV recombinant AAV
  • the first problem is low efficiency and productivity.
  • a production cell line through triple transfection, a total of three components including Rep/Cap, Helper, and GOI (gene-of-interest) are required. All three components must be introduced into the production cell line at the same time and in an appropriate ratio so that recombinant AAV can be produced with high efficiency.
  • this introduction process works independently for each component, the ratio of all three plasmids being injected simultaneously is low at less than 30%.
  • the ratios of the three plasmids injected into the cells cannot be precisely controlled, which can cause differences in transfection efficiency and GOI (gene-of-interest) depending on the production batch, resulting in a decrease in the productivity of recombinant AAV.
  • the third problem is low therapeutic gene expression and immune response.
  • AAV gene therapy development materials targeting hemophilia B have problems with low therapeutic gene expression and reduced therapeutic efficacy due to immune response induction.
  • the focus is on developing therapeutic gene sequences with CpG removed, promoters with tissue specificity, and capsids.
  • rational engineering is not yet mature, and the complexity of gene regulation of AAV vectors with comprehensive functional improvements in off-target tissues, overexpression toxicity, and innate immune response should be evaluated, and the stability of the translated therapeutic gene should be verified.
  • the present disclosure relates to codon-optimized polynucleotides capable of enhancing the expression and activity of FIX and minimizing the immune response.
  • the present disclosure relates to scAAV for treating hemophilia B, which is produced by the all-in-one vector system and has enhanced expression and activity of FIX.
  • one aspect is to provide a codon-optimized polynucleotide encoding Factor IX (FIX).
  • Another aspect is to provide plasmids for producing recombinant adeno-associated virus (AAV) for treating hemophilia B.
  • AAV adeno-associated virus
  • Another aspect provides a method for producing recombinant AAV for treating hemophilia B.
  • Another aspect is to provide a recombinant AAV produced by a plasmid according to one aspect or by a method according to one aspect.
  • Another aspect is to provide recombinant AAV for treating hemophilia B.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating hemophilia B, comprising a recombinant AAV produced by a plasmid according to one aspect or a recombinant AAV according to one aspect.
  • Another aspect provides a method of delivering FIX to a subject in need thereof, comprising administering to the subject an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.
  • Another aspect provides a method of treating hemophilia B, comprising administering to a subject an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.
  • Another aspect provides a use of the plasmid according to one aspect, the recombinant AAV produced by said plasmid, or the recombinant AAV according to one aspect, for the manufacture of a medicament for the treatment of hemophilia B.
  • FIX Factor IX
  • FIX Factor IX
  • hFIX human FIX
  • polynucleotide may refer to any form of nucleic acid, including DNA and RNA, oligonucleotides. Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The sequence or structure of a particular polynucleotide may be described according to the convention of providing the sequence in the 5' to 3' direction.
  • polypeptide may include functional subsequences, modified forms or sequence variants, as long as they retain the functionality of the native protein.
  • modified or variant means that the sequence of the polynucleotide or polypeptide deviates from the reference sequence.
  • the modified sequence or variant sequence may have substantially the same activity or function as the reference sequence, or greater or lesser activity or function than that of the reference sequence, but retains at least a partial activity or function of the reference sequence.
  • Non-limiting examples of modifications include substitutions, insertions, and/or deletions of one or more nucleotides or amino acids.
  • An example of an amino acid substitution is a conservative amino acid substitution. Examples of conservative amino acid substitutions are well known.
  • a "conservative substitution” is the replacement of an amino acid with a biologically, chemically, or structurally similar residue.
  • Biological similarity means that the substitution does not destroy biological activity.
  • Structural similarity means that the amino acids have similar lengths (e.g., alanine, glycine, and serine) or similar sizes.
  • Chemical similarity means that the residues have the same charge or have the same hydrophilic or hydrophobic properties, or both.
  • conservative amino acid substitutions include substitutions within the following groups: glycine/alanine, valine/isoleucine/leucine, aspartic acid/glutamic acid, asparagine/glutamine, serine/threonine, lysine/arginine, phenylalanine/tyrosine.
  • gene and protein variants possessing one or more biological activities may be included.
  • naturally occurring and non-naturally occurring variant genes can have at least 50%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to a reference gene.
  • the naturally occurring and non-naturally occurring variant proteins can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the reference protein.
  • sequence identity refers to the degree of identity of amino acid residues or bases between sequences after the two sequences are aligned to the greatest extent possible over a specific comparison region. Sequence identity can be determined by methods known in the art. The percent of sequence identity can be determined using known sequence comparison programs, such as NCBI's BLAST.
  • the above FIX may be a FIX mutant or a wild-type FIX.
  • FIX can be a FIX variant comprising the R338L mutation (FIX-R338L); a FIX variant comprising the T148A and R338L mutations (FIX-T148A/R338L); or a wild-type FIX.
  • the nucleic acid molecule comprises (b) the Rep gene of AAV.
  • the Rep gene may include one or more (one, two, or three) selected from Rep68, Rep52, and Rep40.
  • Rep78 when overexpressed, may increase cytotoxicity and decrease AAV productivity. Therefore, the Rep gene may include a Rep gene other than Rep78.
  • the Rep gene can comprise a Rep gene of AAV2.
  • the Rep gene can comprise Rep78, Rep68, Rep52, and Rep40.
  • the above variant may be an engineered Rep (ERep) protein.
  • the variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.
  • Cap (Capsid) gene is a gene that encodes the viral capsid protein.
  • the Cap gene may be derived from any AAV serotype.
  • the nucleotide sequence of the Cap gene may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13, but is not limited thereto.
  • each gene may be derived from the same or different AAV serotype.
  • the nucleotide sequence of the Cap gene may be derived from AAV8.
  • the above Cap gene may comprise a gene encoding a known Cap protein or a variant thereof.
  • the above Cap gene may encode one or more selected from capsid protein, VP1 protein, VP2 protein, VP3 protein, and variants thereof, but is not limited thereto.
  • the Cap gene may encode all of VP1, VP2, and VP3 proteins.
  • the above variant may be an engineered Cap (ECap) protein.
  • the variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.
  • the Cap gene may comprise the wild type Cap gene of AAV8.
  • the nucleotide sequences of the Rep gene and the Cap gene may be derived from the same or different AAV serotypes.
  • the Rep gene may be derived from AAV2
  • the Cap gene may be derived from AAV8.
  • the nucleic acid molecule comprises (d) a transgene.
  • the transgene is a gene that is transferred from one organism to another.
  • the transgene may be a heterologous polynucleotide.
  • the transgene may be a gene-of-interest (GOI) to be packaged into a recombinant AAV capsid.
  • the transgene may be a therapeutic gene. Therefore, the plasmid for producing the recombinant AAV can be used for the production of a gene therapy clinical agent for the treatment of a patient.
  • the transgene may be one or two or more.
  • the above transgene comprises a codon-optimized polynucleotide encoding FIX according to one aspect. Therefore, the plasmid for producing a recombinant AAV can produce a recombinant AAV that can be used as a gene therapy agent for treating hemophilia B.
  • the transgene may be arranged between ITRs (Inverted Terminal Repeats).
  • the transgene may have ITRs arranged on both sides.
  • the transgene may have two ITRs arranged on either side.
  • the transgene may be arranged between L-ITR and R-ITR.
  • the L-ITR (first ITR), the transgene, and the R-ITR (second ITR) may be arranged in sequence in the 5' to 3' direction or in the 3' to 5' direction.
  • the above Inverted Terminal Repeat is involved in the replication of the AAV genome and the packaging of AAV particles.
  • the ITR contains the Rep binding element (RBE), RBE', A, A', B, B', C, C', and D regions.
  • the ITR consists of two arm palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A').
  • A-A' stem palindrome
  • the ITR has a T-shaped stem-loop structure.
  • the ITR can have two configurations, namely, flip and flop.
  • the flip and flop configurations have the B-B' and C-C' palindromes closest to the 3' end, respectively.
  • the two ITRs on either side are referred to as the first ITR and the second ITR, or as the L(left)-ITR and the R(right)-ITR, or as the 5'-ITR and the 3'-ITR.
  • the D region occurs only once at each terminus and thus remains single-stranded.
  • the RBE is the region where the Rep78 and Rep68 proteins of AAV bind.
  • the strand- and site-specific endonuclease catalytic domains of Rep78 and Rep68 introduce nicks into the terminal resolution site (trs).
  • the structure and sequence of ITRs are known.
  • the above ITR may be derived from a virus belonging to the genus Dependovirus in the family Parvoviridae .
  • the above ITR may be derived from AAV.
  • the above ITR may be derived from any AAV serotype.
  • the above ITR may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13, but is not limited thereto.
  • the above AAV serotype may also include other AAV serotypes currently known or to be discovered later.
  • the above AAV serotype may also include an artificial AAV serotype.
  • the two ITRs on both sides may be derived from the same or different AAV serotypes.
  • the ITR may be derived from AAV2.
  • the L-ITR and R-ITR may both be derived from AAV2.
  • the ITR may be a wild-type ITR or a mutant thereof.
  • the ITR may comprise all or a portion of a wild-type ITR sequence.
  • the ITR may have a lower %GC content than the wild-type ITR.
  • the ITR may be a synthetic ITR.
  • the ITR may be an ITR derived from self-complementary AAV (scAAV).
  • the L-ITR and R-ITR may be modified to be capable of producing scAAV.
  • one of the L-ITR and the R-ITR can serve as a primer for DNA replication.
  • the R-ITR can serve as a primer for DNA replication.
  • One of the L-ITR and the R-ITR may not comprise trs.
  • One of the L-ITR and the R-ITR may not comprise trs, and the other may be a wild-type ITR.
  • the L-ITR may not comprise trs, and the R-ITR may be a wild-type ITR.
  • One of the L-ITR and the R-ITR may not comprise trs and may comprise an RBE (Rep binding element).
  • the L-ITR may not comprise trs and may comprise an RBE.
  • One of the L-ITR and the R-ITR may not comprise trs and may have a hairpin structure.
  • the L-ITR may not comprise trs and may have a hairpin structure.
  • One of the above L-ITR and R-ITR may be an ITR mutant having a D region deleted, and the other may be a wild-type ITR.
  • the L-ITR may be an ITR mutant having a D region deleted, and the R-ITR may be a wild-type ITR.
  • the L-ITR may be composed of SEQ ID NO: 3, and the R-ITR may be composed of SEQ ID NO: 8. Accordingly, the recombinant AAV produced by the plasmid may be a self-complementary AAV (scAAV).
  • the sequence added to the ITR may be a nucleotide sequence (46 bp) consisting of SEQ ID NO: 7.
  • the sequence added to the ITR may be a sequence added to the 5'-end of the R-ITR based on the (+) strand.
  • the nucleic acid molecule may further comprise a nucleotide sequence comprising SEQ ID NO: 7 upstream of the R-ITR.
  • the above enhancer may be a liver tissue-specific enhancer.
  • liver tissue-specific enhancers include apolipoprotein (ApoE) HCR-1 and HCR-2 enhancers, mouse transthyretin (mTTR) enhancer, etc.
  • the enhancer may be an mTTR enhancer or a variant thereof.
  • VPC2.0 is a clonal cell line derived from the HEK293F parental cell line and is a host cell suitable for AAV production.
  • the mammalian cell can be a HEK293F cell or a cell derived therefrom.
  • the host cell may be an insect cell.
  • the insect cell may include a cell derived from the tropical armyworm ( Spodoptera frugiperda ) or the cabbage moth ( Trichoplusia ni ).
  • the insect cell may be selected from, but is not limited to, a Sf9 cell, a Sf21 cell, a TN-5B1-4 cell, a High Five cell, or a derivative or functional equivalent thereof.
  • the method can also be easily scaled up to industrial production because it requires only a single transfection of the host cell with the plasmid for recombinant AAV production with excellent recombinant AAV productivity.
  • the above recombinant AAV may have a high Full capsid ratio (%Full capsid). In addition, the above recombinant AAV may have a low impurity content. Therefore, the above recombinant AAV may have excellent quality and safety.
  • the above recombinant AAV may have both increased expression and activity of the FIX protein.
  • Another aspect provides recombinant AAV for treating hemophilia B.
  • the above genome contains a transgene.
  • the above AAV capsid may be any one AAV capsid selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.
  • the AAV capsid can be a wild-type AAV8 capsid.
  • the above AAV capsid may comprise a VP1 protein, a VP2 protein, and a VP3 protein.
  • the “genome” of the above AAV refers to the sequences that are ultimately packaged or encapsidated to form viral particles.
  • the genome may be linear single-stranded DNA.
  • the above transgene may be arranged between ITRs.
  • the above transgene may be arranged between L-ITR and R-ITR.
  • the above ITR may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.
  • the ITR may be derived from AAV2.
  • the above ITR may be a wild-type ITR or a mutant thereof.
  • the ITR may comprise all or part of a wild-type ITR sequence.
  • one of the L-ITR and the R-ITR may not comprise trs.
  • One of the L-ITR and the R-ITR may not comprise trs and the other may be a wild-type ITR.
  • the L-ITR may not comprise trs and the R-ITR may be a wild-type ITR.
  • One of the L-ITR and the R-ITR may be an ITR mutant having a D region deleted and the other may be a wild-type ITR.
  • the L-ITR may be an ITR mutant having a D region deleted and the R-ITR may be a wild-type ITR.
  • the L-ITR may be composed of SEQ ID NO: 3 and the R-ITR may be composed of SEQ ID NO: 8. Accordingly, the recombinant AAV may be a self-complementary AAV (scAAV).
  • the genome may further comprise a sequence added to the ITR. Details regarding the sequence added to the ITR are as described above.
  • the genome may further comprise a sequence added to the ITR upstream of the R-ITR.
  • the sequence added to the ITR may comprise or consist of SEQ ID NO: 7.
  • the genome may further comprise an expression regulatory element operably linked to the transgene.
  • the expression regulatory element may be a tissue-specific expression regulatory element.
  • the expression regulatory element may comprise at least one selected from an enhancer and a promoter.
  • the above enhancer may be a liver tissue-specific enhancer, and the promoter may be a liver tissue-specific promoter.
  • the enhancer may be an mTTR enhancer or a variant thereof, and the promoter may be an mTTR promoter or a variant thereof.
  • the above enhancer and promoter may be a variant of the mTTR enhancer and promoter in which nucleotides at positions 1 to 22 and 124 to 138 are deleted from the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA is inserted after position 361.
  • the variant of the mTTR enhancer and promoter may comprise or consist of SEQ ID NO: 4.
  • the genome may further comprise an intron.
  • the intron may be a modified SV40 intron.
  • the modified SV40 intron may comprise or consist of SEQ ID NO: 5.
  • the genome may further comprise a polyadenylation (poly A) signal sequence.
  • the polyadenylation signal sequence may be a rabbit globin polyadenylation signal sequence.
  • the rabbit globin polyadenylation signal sequence may comprise or consist of SEQ ID NO: 6.
  • the genome may comprise, in 5' to 3' order, an L-ITR, an enhancer, a promoter, an intron, a codon-optimized polynucleotide encoding FIX according to one aspect, a polyadenylation signal sequence, and an R-ITR.
  • the genome may comprise, in 5' to 3' order, an L-ITR, an enhancer, a promoter, an intron, a codon-optimized polynucleotide encoding FIX according to one aspect, a polyadenylation signal sequence, a sequence appended to the ITR, and an R-ITR.
  • the L-ITR is an ITR variant of AAV2, wherein the variant does not comprise trs; the enhancer and promoter are a variant of an mTTR enhancer and promoter, wherein the variant has deletions of nucleotides at positions 1 to 22 and 124 to 138 in the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA is inserted after position 361; the intron is a modified SV40 intron; the polyadenylation signal sequence is a rabbit globin polyadenylation signal sequence; and the R-ITR can be a wild-type ITR of AAV2.
  • the sequence added to the ITR can be a non-ITR viral DNA sequence derived from the wild-type AAV2 genome.
  • the L-ITR may be composed of SEQ ID NO: 3; the enhancer and promoter may be composed of SEQ ID NO: 4; the intron may be composed of SEQ ID NO: 5; the polyadenylation signal sequence may be composed of SEQ ID NO: 6; and the R-ITR may be composed of SEQ ID NO: 8.
  • the L-ITR may be composed of SEQ ID NO: 3; the enhancer and promoter may be composed of SEQ ID NO: 4; the intron may be composed of SEQ ID NO: 5; the polyadenylation signal sequence may be composed of SEQ ID NO: 6; the sequence added to the ITR may be composed of SEQ ID NO: 7, and the R-ITR may be composed of SEQ ID NO: 8.
  • Another aspect provides a pharmaceutical composition for preventing or treating hemophilia B, comprising a recombinant AAV produced by a plasmid according to one aspect or a recombinant AAV according to one aspect.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable carrier.
  • the pharmaceutical composition above may be for gene therapy for hemophilia B.
  • the pharmaceutical composition may be a composition for delivering FIX protein for gene therapy for hemophilia B.
  • Another aspect provides a method of treating hemophilia B, comprising administering to a subject an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.
  • the All-in-One scAAV-FIX vector of Figure 1 contains each component in the following order: helper gene, plasmid backbone, Rep gene, Cap gene, L-ITR, liver tissue-specific enhancer, liver tissue-specific promoter, intron, GOI, polyadenylation signal sequence, ITR+, R-ITR.
  • Codon-optimized sequences of Factor IX mutants or wild-type (WT) Factor IX were generated using GenSmart Codon Optimization Tool (Genescript) or GeneArt Codon Optimization Tool (GeneArt).
  • Factor IX mutants include the R338L mutation or the T148A/R338L mutation in the wild-type sequence. The generated codon-optimized sequences were used as GOIs.
  • the optimized GOI was designed to optimize transcription of the expressed gene, protein refolding, translation, gene synthesis, and minimize immunotoxicity.
  • codon optimization included the following parameters:
  • a portion corresponding to the 5' ITR to the 3' ITR of the pAAV-ApoE_hAAT-FIX39 vector (SPK9001) of US 10799566 B2 was synthesized. Specifically, the portion includes the 5' AAV2 ITR, ApoE HCR-1/2 enhancer, hAAT promoter, 5' UTR, FIX39-Padua CDS, intron A, 3' UTR, bGH poly A, and 3' AAV2 ITR.
  • FIX39-Padua has CpG dinucleotides completely removed from the FIX coding and intron sequences.
  • the region corresponding to the 5' ITR to the 3' ITR of the AMT-061 vector (AAV5-hFIXco-Padua) of Nathwani et al., Blood (2006), 107(7):2653-61 was synthesized. Specifically, the region includes the 5' AAV2 ITR, HCR enhancer, hAAT promoter, modified SV40 intron, hFIX-Padua, SV40 poly A, and 3' AAV2 ITR.
  • a triple vector was created in the same manner as in Comparative Example 1, except that the portion corresponding to the 5' ITR to the 3' ITR of Comparative Example 2 was inserted into the vector containing GOI (including the portion corresponding to L-ITR to R-ITR), and this was named ttSPK9001 vector.
  • the portion corresponding to 5' ITR to 3' ITR of BAX335 (TAK-748) vector of BA konkle et al., Blood. 2021;137(6):763-774 was synthesized. Specifically, the portion includes 5' AAV2 ITR, 3X CRM8, mTTR enhancer, mTTR promoter, MVM intron, hFIX-Padua, bGH poly A, and 3' AAV2 ITR.
  • An all-in-one vector was produced in the same manner as in Example 1, except that the transgene (GOI) portion of the All-in-One scAAV-FIX vector of Example 1 was replaced with the FIX39-Padua CDS and intron A used in the AIO-SPK9001 vector of Comparative Example 2.
  • the 5’ ITR to 3’ ITR portion of the vector included an AAV2 L-ITR (SEQ ID NO: 3) having the D region deleted from the wild-type sequence; a mutant of the mTTR enhancer/promoter (SEQ ID NO: 4); a modified SV40 intron (SEQ ID NO: 5); the FIX39-Padua CDS of Comparative Example 2; the intron A of Comparative Example 2; the rabbit globin pA polyadenylation signal sequence (SEQ ID NO: 6); It contains a non-ITR viral DNA sequence corresponding to wtAAV2 nt 4489-4534 derived from the upstream of the 3' ITR in the wild-type AAV2 genome (SEQ ID NO: 7) and the wild-type R-ITR of AAV2 (SEQ ID NO: 8).
  • the constructed vector was named scAIO-FIX (SPK9001).
  • the FIX expression level of the All-in-One scAAV-FIX vector was improved by 1.9 to 2.6 times, and the FIX coagulation ability was increased by 1.3 to 1.4 times.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne : un polynucléotide à codon optimisé codant pour le facteur IX (FIX) ; un plasmide de production d'AAV recombinant comprenant le polynucléotide ; un AAV recombinant produit par le plasmide ; et l'utilisation de l'AAV recombinant pour traiter l'hémophilie B. Le plasmide comprenant le polynucléotide peut produire un AVV recombinant qui présente une expression et une activité de FIX élevées et présente une excellente productivité et une qualité élevée.
PCT/KR2024/021013 2023-12-26 2024-12-24 Polynucléotide à codon optimisé codant pour le facteur ix de coagulation sanguine et plasmide producteur de raav le comprenant Pending WO2025143742A1 (fr)

Applications Claiming Priority (2)

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KR10-2023-0191861 2023-12-26
KR20230191861 2023-12-26

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WO2025143742A1 true WO2025143742A1 (fr) 2025-07-03

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PCT/KR2024/021013 Pending WO2025143742A1 (fr) 2023-12-26 2024-12-24 Polynucléotide à codon optimisé codant pour le facteur ix de coagulation sanguine et plasmide producteur de raav le comprenant

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WO (1) WO2025143742A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130132833A (ko) * 2010-11-03 2013-12-05 카탈리스트 바이오사이언시즈, 인코포레이티드 변형된 제 ix 인자 폴리펩티드 및 이의 용도
KR20180048580A (ko) * 2015-06-23 2018-05-10 더 칠드런스 호스피탈 오브 필라델피아 변형된 인자 ix, 및 세포, 기관 및 조직으로 유전자를 전달하기 위한 조성물, 방법 및 용도
KR20200010443A (ko) * 2017-05-22 2020-01-30 박스알타 인코퍼레이티드 B형 혈우병 유전자 요법을 위한 증가된 발현을 가지는 재조합 fix 변이체를 암호화하는 바이러스 벡터
KR20200014318A (ko) * 2017-05-31 2020-02-10 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 최적화된 인간 응고 인자 ix 유전자 발현 카세트 및 이의 사용
KR20200057051A (ko) * 2017-09-27 2020-05-25 시질론 테라퓨틱스, 인크. 활성 세포를 포함하는 방법, 조성물 및 이식 가능한 요소

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130132833A (ko) * 2010-11-03 2013-12-05 카탈리스트 바이오사이언시즈, 인코포레이티드 변형된 제 ix 인자 폴리펩티드 및 이의 용도
KR20180048580A (ko) * 2015-06-23 2018-05-10 더 칠드런스 호스피탈 오브 필라델피아 변형된 인자 ix, 및 세포, 기관 및 조직으로 유전자를 전달하기 위한 조성물, 방법 및 용도
KR20200010443A (ko) * 2017-05-22 2020-01-30 박스알타 인코퍼레이티드 B형 혈우병 유전자 요법을 위한 증가된 발현을 가지는 재조합 fix 변이체를 암호화하는 바이러스 벡터
KR20200014318A (ko) * 2017-05-31 2020-02-10 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 최적화된 인간 응고 인자 ix 유전자 발현 카세트 및 이의 사용
KR20200057051A (ko) * 2017-09-27 2020-05-25 시질론 테라퓨틱스, 인크. 활성 세포를 포함하는 방법, 조성물 및 이식 가능한 요소

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