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WO2025205770A1 - Plasmide recombiné contenant de l'adn de bourrage - Google Patents

Plasmide recombiné contenant de l'adn de bourrage

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Publication number
WO2025205770A1
WO2025205770A1 PCT/JP2025/011738 JP2025011738W WO2025205770A1 WO 2025205770 A1 WO2025205770 A1 WO 2025205770A1 JP 2025011738 W JP2025011738 W JP 2025011738W WO 2025205770 A1 WO2025205770 A1 WO 2025205770A1
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WO
WIPO (PCT)
Prior art keywords
aav
sequence
recombinant plasmid
cells
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/011738
Other languages
English (en)
Japanese (ja)
Inventor
尚巳 岡田
美加子 和田
英人 蝶野
多佳久 月原
功 岡本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takara Bio Inc
University of Tokyo NUC
Original Assignee
Takara Bio Inc
University of Tokyo NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takara Bio Inc, University of Tokyo NUC filed Critical Takara Bio Inc
Publication of WO2025205770A1 publication Critical patent/WO2025205770A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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

Definitions

  • Recombinant viral vectors are generally produced by separating the elements essential for viral particle formation, those that require cis supply, and those that can be supplied in trans, and then introducing them into host cells. This prevents the production of wild-type virus and the autonomous replication of the recombinant virus in the infected host.
  • the elements essential for viral particle formation are introduced into cells in the form of a nucleic acid construct, creating cells capable of producing viruses (hereafter referred to as virus-producing cells). These cells are then cultured until all of the elements essential for viral particle formation are expressed within the cells, resulting in the production of a viral vector.
  • the virus-producing cells that have achieved virus production are then harvested and disrupted.
  • the viral vector can also be produced from the culture supernatant.
  • the resulting cell lysate, supernatant, or a mixture thereof can be subjected to appropriate processes such as filtration, ultracentrifugation, chromatography, and ultrafiltration to purify the recombinant viral vector, resulting in the final product.
  • stuffer sequences also known as "stuffer DNA” or "filler sequences"
  • Stuffer sequences are sequences that are used to adjust the size of any nucleic acid construct to an appropriate length.
  • Non-Patent Document 1 adjusting the size of the AAV genome to an appropriate length by positioning a stuffer sequence inside the two AAV ITR sequences is useful for producing full AAV particles.
  • positioning a stuffer sequence outside the two AAV ITR sequences is also useful for preventing the mistaken packaging of regions that should not be packaged.
  • Patent Document 1 discloses a stuffer sequence comprising a nucleic acid having a length of 3,300 to 4,200 nucleotides and having at least 90% identity to a specified nucleotide sequence.
  • Patent Document 2 discloses a combination of a first stuffer sequence located inside the two AAV ITR sequences and a second stuffer sequence located outside the two AAV ITR sequences.
  • Patent Document 4 discloses novel inert, non-coding stuffer sequences comprising a predetermined nucleotide sequence or a fragment thereof.
  • the stuffer sequence needs to be further improved in order to efficiently produce full AAV particles.
  • the present inventors discovered that full AAV particles can be efficiently produced by using a recombinant plasmid containing (a) an adeno-associated virus (AAV) genome containing two ITR sequences of AAV and a gene sequence of interest sandwiched between them, and (b) stuffer DNA consisting of a base sequence that does not contain an inverted repeat sequence that can form a stem loop, and thus completed the present invention.
  • AAV adeno-associated virus
  • the present invention is [1] (a) an adeno-associated virus (AAV) genome containing two ITR sequences of AAV and a gene sequence of interest sandwiched between them, and (b) a recombinant plasmid containing stuffer DNA consisting of a nucleotide sequence that does not contain an inverted repeat sequence capable of forming a stem loop; [2] The recombinant plasmid according to [1], wherein the AAV genome of (a) has a chain length of 3.0 to 5.5 kb.
  • AAV adeno-associated virus
  • [3] The recombinant plasmid according to [1] or [2], wherein the chain length of the recombinant plasmid excluding (a) is 5.0 kb or more.
  • [4] The recombinant plasmid according to any one of [1] to [3], wherein the stuffer DNA (b) is a DNA containing a sequence obtained by modifying the base sequence of a naturally occurring nucleic acid.
  • the stuffer DNA (b) is a DNA containing a sequence obtained by modifying the base sequence of a nucleic acid derived from an intron of the human HPRT1 gene.
  • the recombinant plasmid of the present invention By using the recombinant plasmid of the present invention, incorrect packaging is reduced during the process of producing recombinant AAV vectors. Furthermore, because the recombinant plasmid of the present invention does not contain sequences of unknown origin, even if part of the plasmid is inserted into the subject's genome after gene therapy, canceration due to sequences of unknown origin will not occur.
  • FIG. 1 shows the structure of the recombinant plasmid prepared in Example 1.
  • FIG. 10 is a diagram showing the ratio of the copy number of the ampicillin resistance gene to the copy number of the ITR in Example 3.
  • FIG. 10 shows the expression rate of ZsGreen1 (infection efficiency of AAV vector) in Example 4.
  • FIG. 10 is a diagram showing the fluorescence intensity of ZsGreen1 in Example 4.
  • FIG. 10 shows the ratio of AAV full particles in Example 5.
  • FIG. 10 shows the ratio of the copy number of the ampicillin resistance gene to the copy number of the ITR in Example 8 (AAVRKO cells).
  • FIG. 10 shows the ratio of the copy number of the ampicillin resistance gene to the copy number of the ITR in Example 8 (VPC2.0 cells).
  • FIG. 10 shows the ratio of AAV full particles in Example 9 (AAVRKO cells).
  • FIG. 1 shows the ratio of AAV full particles in Example 9 (VPC2.0 cells).
  • vector includes any viral vector, plasmid vector, cosmid vector, phage vector, and binary vector capable of transforming a prokaryotic or eukaryotic host.
  • AAV vector refers to an AAV having vector function.
  • AAV particle refers to a viral particle composed of at least one AAV capsid protein. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide not derived from the wild-type AAV genome), it is referred to as a "recombinant AAV (rAAV)."
  • rAAV recombinant AAV
  • recombinant means produced using genetic recombination technology.
  • recombinant AAV means AAV produced using genetic recombination technology
  • recombinant DNA means DNA produced using genetic recombination technology.
  • AAV genome refers to DNA that can be packaged into AAV particles. It encompasses, for example, genomic DNA derived from various AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, as well as modified versions thereof, and artificially designed DNA based on the sequences of these DNAs.
  • AAV genomes derived from wild-type viruses may be partially deleted, or any nucleic acid sequence may be inserted.
  • AAV genomes contain functional ITR sequences required for viral replication and packaging.
  • capsid refers to the protein outer shell that surrounds the viral genome in a virus particle.
  • the AAV capsid is composed of three types of capsid proteins (VP1, VP2, and VP3), which are encoded by the cap gene possessed by AAV.
  • VP1, VP2, and VP3 are all included in the capsid proteins.
  • AAV full particles refers to AAV particles that contain the full-length AAV genome, i.e., single-stranded DNA flanked by the 5' ITR and 3' ITR. "AAV full particles” are also referred to as “AAV full capsids,” “AAV complete particles,” “AAV vector particles,” or “AAV virions.”
  • AAV partial particles refers to AAV particles that have nucleic acid, but the nucleic acid is not the full-length AAV genome.
  • AAV partial particles are also referred to as “AAV partial capsids.”
  • nucleic acids encapsulated within AAV partial particles including, for example, nucleic acid fragments derived from the AAV genome retained in AAV-producing cells and nucleic acids not derived from the AAV genome.
  • Nucleic acids encapsulated within AAV partial particles that are not derived from the AAV genome may be referred to as "contaminating nucleic acids" herein.
  • contaminating nucleic acids include nucleic acids derived from host cells and nucleic acids derived from recombinant plasmids (e.g., nucleic acids encoding replication origins and selection markers).
  • AAV empty particles refers to AAV particles that do not contain nucleic acid. "AAV empty particles” are also called “AAV empty capsids,” “AAV hollow particles,” or “AAV empty capsid particles.”
  • base sequence refers to a description of the bonding order of nucleotides that make up nucleic acids such as DNA and RNA, focusing on the types of organic bases that make up part of those nucleotides.
  • Base sequence is also called “nucleotide sequence.”
  • inverted repeat sequence means that a nucleotide sequence and a nucleotide sequence complementary (or partially complementary) to that sequence exist on the same strand, and these two sequences are referred to as inverted repeat sequences. These two sequences may be adjacent to each other, or may be separated by one or more bases. The inverted repeat sequences may preferably exist adjacent to each other.
  • stem-loop structure refers to a structure formed by single-stranded RNA or DNA having an inverted repeat sequence, consisting of a double-stranded portion (stem) formed by hydrogen bonding between complementary sequences of the inverted repeat sequence, and a single-stranded portion (loop) sandwiched between the stem and the loop.
  • the hydrogen-bonded inverted repeat sequences may be completely complementary or partially complementary.
  • a bulge may also be inserted in the stem region.
  • Stem-loop structures can be predicted and confirmed using nucleic acid secondary structure prediction algorithms. Examples of this algorithm include Vienna RNA Package (Hofacker I et al., Nucleic Acids Research, Vol. 31(13), pp.
  • the "stem-loop structure” is also called a “stem-loop,” “hairpin loop,” or “hairpin loop structure.”
  • the terms “3' end” and “3' side” mean located toward the 3' end of a sequence (region).
  • the terms “5' end” and “5' side” mean located toward the 5' end of a sequence (region).
  • the present invention provides a recombinant plasmid comprising (a) an adeno-associated virus (AAV) genome comprising two AAV ITR sequences and a gene sequence of interest sandwiched between them, and (b) a stuffer DNA consisting of a nucleotide sequence that does not contain an inverted repeat sequence that can form a stem-loop.
  • AAV adeno-associated virus
  • Adeno-associated virus is a non-enveloped virus classified in the genus Dependovirus within the family Parvoviridae.
  • AAV has an icosahedral outer shell and a linear, single-stranded DNA genome of approximately 4.7 kb.
  • AAV can infect primates, including humans, and other vertebrates.
  • AAV includes wild-type viruses and their derivatives, and all serotypes and clades, unless otherwise specified.
  • the serotype of rAAV is determined according to the origin of the cap gene used in rAAV preparation and is not dependent on the AAV genome encapsulated in the rAAV particle.
  • the capsid protein is derived from AAV6 and the ITRs in the AAV genome packaged in the rAAV particle are derived from AAV2, the rAAV particle is serotype 6 as used herein.
  • the present invention can also be applied to AAVs containing mutants of the AAV capsid proteins of each of the above serotypes.
  • the AAV2 genome is 4,680 nucleotides long and contains two open reading frames (ORFs).
  • the first ORF encodes the nonstructural Rep proteins (Rep40, Rep52, Rep68, and Rep78).
  • the Rep proteins are involved in regulating replication and transcription and in the production of the AAV genome.
  • Rep68/78 possess NTP-binding activity, DNA helicase activity, and RNA helicase activity.
  • the Rep proteins possess a nuclear localization signal and several potential phosphorylation sites.
  • the stuffer sequence is inert and harmless, having no function or activity. Also, in certain embodiments, the stuffer sequence is not a sequence that encodes a protein or peptide, and the stuffer sequence is not a sequence that encodes a gene of interest, an AAV ITR sequence, an expression control sequence, an origin of replication, or a selectable marker.
  • the stuffer sequence can be placed at any desired position in the recombinant plasmid of the present invention, as long as it does not impair the function of the recombinant plasmid of the present invention to supply the AAV genome into cells.
  • the stuffer sequence can be placed inside the two ITR sequences of the AAV genome. That is, the AAV genome contains a stuffer sequence. More specifically, the stuffer sequence is placed between the 5' ITR sequence and the gene sequence of interest, or between the gene sequence of interest and the 3' ITR sequence.
  • the stuffer sequence can be placed outside the two ITR sequences of the AAV genome. That is, the AAV genome does not contain a stuffer sequence.
  • the stuffer sequence is placed on the 5'-end side of the 5' ITR or on the 3'-end side of the 3' ITR.
  • the stuffer sequence can be placed within the gene sequence of interest.
  • the AAV genome does not contain a stuffer sequence, more preferably, the stuffer sequence is located outside the two ITR sequences of the AAV genome, and even more preferably, the stuffer sequence is located on the 5' end side of the 5' ITR.
  • the size of the stuffer sequence there are no particular limitations on the size of the stuffer sequence.
  • the total length of the target gene sequence and stuffer sequence is preferably 3.0 to 5.5 kb, 4.0 to 5.0 kb, or 4.3 to 4.8 kb.
  • the total length of the portion of the plasmid excluding the AAV genome and the stuffer sequence is preferably 5.0 kb or more, 5.0 to 10.0 Kb, or 6.0 to 8.0 Kb.
  • the stuffer sequence used in the present invention does not contain an inverted repeat sequence that can form a stem loop. More specifically, the stuffer sequence does not contain an inverted repeat sequence that can form a secondary structure having a stem of 12 base pairs or more, 11 base pairs or more, 10 base pairs or more, 9 base pairs or more, 8 base pairs or more, or 7 base pairs or more. Preferably, the stuffer sequence does not contain an inverted repeat sequence that can form a secondary structure having a stem of 8 base pairs or more.
  • the number of identical nucleotides is 15 or less.
  • regions other than the stuffer sequence such as the gene of interest, may be modified to reduce the number of inverted repeat sequences present therein.
  • the gene of interest encodes a protein
  • codon degeneracy can be used to change the base sequence without changing the encoded amino acid sequence.
  • the ITRs may also be modified to reduce the number of inverted repeat sequences present therein.
  • inverted repeat sequences important to the function of each region should not be modified in a way that impairs that function.
  • the present invention provides cells transduced with the recombinant plasmid.
  • Helper functions can also be introduced into packaging cells. Helper functions are also called helper virus functions or accessory functions. Adenovirus is commonly used to introduce helper functions, but herpes simplex virus type 1 or 2, vaccinia virus, etc. can also be used. These are also called helper viruses. When a helper virus is used to introduce helper functions, the packaging cells are infected with the helper virus. Because only the early genes of adenovirus are required for helper function, an adenovirus deficient in late gene expression can be used as the helper virus. Examples of adenoviruses deficient in late gene expression include ts100K and ts149 adenovirus mutants.
  • nucleic acids providing helper functions isolated from helper viruses or artificially produced nucleic acids providing helper functions can be introduced into packaging cells.
  • nucleic acids providing helper functions include nucleic acids encoding adenovirus-derived E1A, E1B, E2A, VA, and E4orf6.
  • the nucleic acid providing the helper function is introduced into the packaging cell in the form of, for example, a plasmid, phage, transposon, cosmid, episomal DNA, a viral genome, or an artificial chromosome.
  • Methods for introducing recombinant plasmids into packaging cells include, for example, the calcium phosphate method, lipofection, DEAE-dextran method, polyethyleneimine method, electroporation, direct microinjection, and high-velocity particle bombardment.
  • Commercially available reagents such as TransIT®-293 Reagent, TransIT®-2020 (all manufactured by Mirus), Lipofectamine 2000 Reagent, Lipofectamine 2000CD Reagent (all manufactured by Life Technologies), FuGene® Transfection Reagent (manufactured by Promega), and PEI max (manufactured by Cosmo Bio) may also be used.
  • the present invention is not particularly limited, in producing the cells of the present invention, 1 ng to 10 ⁇ g, preferably 5 ng to 1 ⁇ g, more preferably 10 ng to 500 ng of recombinant plasmid is introduced per 10 6 packaging cells.
  • the present invention provides a method for producing a recombinant AAV vector by culturing cells into which the recombinant plasmid has been introduced.
  • Packaging cells into which the recombinant plasmid of the present invention has been introduced can be cultured under known culture conditions depending on the type of cell. For example, culture at a temperature of 30 to 37°C, humidity of 95%, and CO2 concentration of 5 to 10% is exemplified, but the present invention is not limited to such conditions. As long as the desired cell growth and/or recombinant AAV vector production can be achieved, culture may be carried out at any temperature, humidity, or CO2 concentration outside the above ranges.
  • cultureware examples include cell culture equipment (containers) such as petri dishes, flasks, bags, shakers, large culture tanks, and bioreactors.
  • a CO2 gas-permeable bag for cell culture is preferred as the bag.
  • a large culture tank may be used.
  • the culture period is not particularly limited, but is, for example, 12 hours to 10 days, preferably 24 hours to 7 days.
  • the recombinant AAV vector is produced within the cells and/or in the culture supernatant.
  • the recombinant AAV vector may be obtained from the cell culture supernatant, or from the supernatant obtained by centrifuging cells that have been resuspended in an appropriate buffer and disrupted.
  • the recombinant AAV vector may be obtained from a mixture of the cell culture supernatant and the supernatant obtained by centrifuging cells after disruption.
  • the recombinant AAV vector can be concentrated and purified by known methods such as filter filtration, CsCl density gradient centrifugation, chromatography, and ultrafiltration.
  • the recombinant AAV vector obtained in this manner can be stored by an appropriate method, such as freezing, until it is used for the desired purpose.
  • the recombinant AAV vector produced by the method of the present invention has a high ratio of "AAV full particles” and a low ratio of "AAV partial particles” and "AAV empty particles.” Furthermore, the recombinant AAV vector produced by the method of the present invention has a low content of contaminating nucleic acids. That is, the ratio of contaminating nucleic acids to the AAV genome contained in the recombinant AAV vector, excluding empty particles, is 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.03% or less.
  • the present invention provides a recombinant AAV vector obtained by the above-described method for producing a recombinant AAV vector.
  • Subjects include human and non-human (e.g., primate) mammals.
  • the subject will benefit from or require expression of a gene sequence of interest.
  • the recombinant AAV vector of the present invention may be contained in a composition. Such a composition is useful for administering the recombinant AAV vector to a subject.
  • the composition may also contain a carrier and/or other medicinal agents.
  • the composition may also contain two or more different recombinant AAV vectors.
  • the capsid proteins constituting the capsids of the two or more different recombinant AAV vectors may be different from each other, and/or the encapsulated nucleic acids may be different from each other.
  • the composition may contain multiple recombinant AAV vectors targeting different cells.
  • a carrier is a substance that facilitates the formulation of a composition and its application to a living body, and is added to the extent that it does not inhibit or suppress its action. Examples of carriers include, but are not limited to, excipients, binders, disintegrants, fillers, emulsifiers, flow additives, and lubricants. In the present invention, it is preferable to use a pharmaceutically acceptable carrier.
  • the amount of recombinant AAV vector contained in the composition is not particularly limited.
  • the amount of recombinant AAV vector contained is determined taking into consideration the type and/or effective amount of nucleic acid encapsulated in the recombinant AAV vector, the subject or cells to which the recombinant AAV vector is delivered, the administration method, the form of the composition, the carrier, etc.
  • a gene of interest can be introduced into a subject or cells.
  • a gene introduction method is also an embodiment of the present invention.
  • Gene introduction into an individual animal, including a human is carried out by administering the composition of the present invention via a route such as intratissue (e.g., intramuscular), intravenous, subcutaneous, or intraperitoneal administration.
  • Gene introduction into cells can also be carried out by contacting the composition with the cells in vitro.
  • Example 1 Preparation of recombinant plasmid A modified plasmid was prepared based on pAAV-ZsGreen1 (Takara Bio, #6231) by the following procedure.
  • pAAV-ZsGreen1 contains an ampicillin resistance gene, two ITR sequences, a CMV promoter sequence, a sequence encoding ZsGreen1, and a poly(A) addition sequence ( Figure 1).
  • SfiI recognition sites were introduced into the multiple cloning sites at both ends of the sequence encoding ZsGreen1 to allow for easy modification of the gene of interest (GOI).
  • a sequence encoding the degradation domain of mouse ornithine decarboxylase (SEQ ID NO: 1) was added downstream of ZsGreen1. This domain contains a PEST sequence that reduces protein stability, thereby mitigating the effect of intracellular fluorescent protein accumulation on measuring AAV vector infection efficiency.
  • the resulting plasmid was named pAAV-ZsGreen1-DR.
  • the 145-bp ITRs at both ends of the AAV genome have a hairpin structure.
  • repeat sequences nucleotide sequences that could form stem-loop structures in regions (target sequences) other than the hairpin structure within the ITR sequence of pAAV-ZsGreen1-DR, and these repeat sequences were modified to ones that would not form stem-loops. More specifically, the following steps (1) to (4) were carried out in sequence.
  • All consecutive eight-nucleotide sequences in the target sequence are compared, and if there is another eight-nucleotide sequence that is a perfect match, a base substitution is introduced into one of the sequences so that the two sequences are different. This process is repeated until there are no more eight-nucleotide sequences that are a perfect match.
  • All 18 consecutive nucleotide sequences in the target sequence are compared, and if there is a sequence that matches 16 or more nucleotides, base substitutions are introduced into one of them so that the number of matching nucleotides is 15 or less. This procedure is repeated until there are no 18-nucleotide sequences that match 16 or more nucleotides.
  • the resulting plasmid was named pAAV-opti-ZsGreen1-DR.
  • the repeat sequence within the sequence encoding ZsGreen1 in pAAV-opti-ZsGreen1-DR was also modified to prevent the formation of a stem-loop, while maintaining the amino acid sequence.
  • the resulting plasmid was named pAAV-opti-ZsGreen1-DR(opti).
  • a kanamycin resistance gene was added to pAAV-opti-ZsGreen1-DR(opti), and a sequence derived from the intron of the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene (SEQ ID NO: 2) was added as a stuffer sequence to the outer ITR region of the plasmid to prevent the mis-inclusion of contaminating nucleic acids from the plasmid during AAV production.
  • the resulting plasmid was named pAAV-opti-ZsGreen1-DR(opti)backstuff(Amp/Km).
  • the repeat sequence within the stuffer sequence of pAAV-opti-ZsGreen1-DR(opti)backstuff(Amp/Km) was modified to eliminate the formation of a stem-loop using the same procedures as in (1) to (4) above.
  • the resulting plasmid was named pAAV-opti-ZsGreen1-DR(opti)backopti-stuff(Amp/Km).
  • the structure of the constructed plasmid is shown in Figure 1.
  • the optimized nucleotide sequence derived from the intron of the human HPRT1 gene after the repeat sequence was modified is shown in SEQ ID NO: 3.
  • SEQ ID NO: 1 Nucleotide sequence encoding a degradation domain of mouse ornithine decarboxylase aagcttccgcggagccatggcttcccgccggcggtggcggcgcaggatgatggcacgctgcccatgtcttgtgcccaggagagcgggatggaccgtcaccctgcagcctgtgcttctgctaggatcaatgtg
  • Example 2 Production of AAV Vectors Either of the plasmids obtained in Example 1, pAAV2/9 Vector (Addgene, #112865) expressing AAV9 Cap and AAV2 Rep, and pAd5N (Agilent Technologies) were transfected into 293 EB cells (WO 2012/144446) using Polyethyleneimine "Max" (Cosmobio). 293EB cells were cultured in D-MEM medium (High Glucose) (Fujifilm Wako Pure Chemical Industries, Ltd., #048-29763) containing 1/100 volume of Gibco GlutaMax (Thermo Fisher Scientific, #35050061) at 37°C in 5% CO for 3 days.
  • D-MEM medium High Glucose
  • Gibco GlutaMax Gibco GlutaMax
  • Example 7 Production of AAV vectors (VPC2.0 cells) Any of the plasmids obtained in Example 1, pAAV2/9 Vector (Addgene, #112865) expressing AAV9 Cap and AAV2 Rep, and pAd5N (Agilent Technologies) were transfected into VPC2.0 cells (Thermo Fisher Scientific, #A49784) using an AAV-MAX Transfection Kit (Thermo Fisher Scientific, #A50515). VPC2.0 cells were cultured in Viral Production Medium (Thermo Fisher Scientific, #4817901) containing 1/50 volume of GlutaMAX (Thermo Fisher Scientific, #35050061) at 37°C in 8% CO2 with shaking (120 rpm) for 10 days.
  • Viral Production Medium Thermo Fisher Scientific, #4817901
  • GlutaMAX Thermo Fisher Scientific, #35050061

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Abstract

La présente invention concerne un plasmide recombiné comportant : (a) un génome de virus adéno-associé (AAV) comportant deux séquences ITR AAV et une séquence de gène cible flanquée par les séquences ITR de l'AAV ; et (b) un ADN de bourrage contenant une séquence nucléotidique qui ne comporte pas de séquence répétée inversée pouvant former une tige-boucle.
PCT/JP2025/011738 2024-03-26 2025-03-25 Plasmide recombiné contenant de l'adn de bourrage Pending WO2025205770A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
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JP2005512549A (ja) * 2001-12-14 2005-05-12 ケイヘーネ・エヌ・ブイ 環状プローブを用いた複数の標的配列の分析と検出
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