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WO2018139637A1 - Particules vides de aav encapsulant un acide nucléique - Google Patents

Particules vides de aav encapsulant un acide nucléique Download PDF

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WO2018139637A1
WO2018139637A1 PCT/JP2018/002687 JP2018002687W WO2018139637A1 WO 2018139637 A1 WO2018139637 A1 WO 2018139637A1 JP 2018002687 W JP2018002687 W JP 2018002687W WO 2018139637 A1 WO2018139637 A1 WO 2018139637A1
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region
nucleic acid
sequence
aav
cells
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PCT/JP2018/002687
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English (en)
Japanese (ja)
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尚巳 岡田
浩典 岡田
世志幸 宮川
峰野　純一
蝶野　英人
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学校法人日本医科大学
タカラバイオ株式会社
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Priority to JP2018564684A priority Critical patent/JP7173490B2/ja
Publication of WO2018139637A1 publication Critical patent/WO2018139637A1/fr
Priority to JP2022171573A priority patent/JP7440045B2/ja

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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

Definitions

  • the present invention relates to a method for producing AAV hollow particles encapsulating nucleic acid.
  • Viral vectors are one of them, and various vectors derived from lentiviruses, oncoretroviruses, adenoviruses, adeno-associated viruses and the like are known.
  • adeno-associated virus (AAV) vectors are expected as gene transfer vectors useful in gene therapy in recent years.
  • AAV is a non-pathogenic virus belonging to parvovirus, and is not capable of autonomous propagation because it lacks self-replicating ability, and its propagation is low because co-infection with adenovirus and herpes virus is required for growth. It is said that. It also has a low immunogenicity in the host. Because of such characteristics, it has an advantage of high safety as a gene transfer vector.
  • the host range is wide, it is possible to infect various cells, and vectors derived from each serotype AAV (for example, AAV1 to AAV9) have been developed. Utilizing specificity, it is used for gene transfer into specific cells, tissues and organs such as nerve cells, muscle cells, and hepatocytes.
  • conventional virus vectors including AAV have various problems such as acquisition of self-replication ability and contamination with wild-type virus.
  • AAV hollow particles that are formed of capsids and do not contain AAV viral genome inside have virus initial infection activity such as specific recognition of target cells, adsorption and invasion to cells, and threshing, but are necessary for self-replication. Since it does not have a gene derived from it, there is no virus growth activity. Therefore, hollow particles are capable of specifically delivering drugs such as proteins and nucleic acids to target cells, and are carriers of an ideal drug delivery system (DDS: Drug Delivery System) that has safety for the administered individual. It is.
  • DDS Drug Delivery System
  • the DNA genome can take the form of circular double-stranded DNA (cAAV).
  • This double-stranded DNA has one inverted terminal repeat (ITR) sequence.
  • ITR inverted terminal repeat
  • Musatov et al. Have reported that replication and encapsulation of AAV genomic DNA requires a sequence consisting of the sequence of the A region and the D 'region of ITR (Non-patent Document 1). Musatov et al. Disclose that AAV hollow particles encapsulating DNA are produced by introducing circular double-stranded DNA containing the sequence of the A region-D 'region into cells.
  • the present invention aims to develop and provide a simpler and more efficient method for encapsulating nucleic acids in hollow particles.
  • the present inventor succeeded in producing hollow particles encapsulating nucleic acids while maintaining the initial infection activity of the virus by a simpler and more efficient method. It was also confirmed that the nucleic acid encapsulated in the hollow particles can be effectively introduced into the target cells by mixing the nucleic acid-encapsulated hollow particles with the target cells.
  • the present invention is based on these findings and results, and provides the following.
  • a method for producing adeno-associated virus (AAV) nucleic acid-encapsulated hollow particles comprising the following steps; (1) (i) A region sequence of AAV inverted terminal repeat (ITR), Preparing a linear nucleic acid fragment comprising (ii) the sequence of the A ′ region of the AAV ITR and (iii) the sequence of the D region and / or the sequence of the D ′ region of the AAV ITR; (2) a step of introducing the nucleic acid fragment of (1) into a cell producing AAV hollow particles, and (3) a step of culturing the cell of (2).
  • AAV inverted terminal repeat ITR
  • nucleic acid fragment comprises an A ′ region sequence, an A region sequence, and a D ′ region sequence from the 5 ′ end toward the 3 ′ end.
  • nucleic acid fragment comprises a D region sequence, an A ′ region sequence, and an A region sequence from the 5 ′ end toward the 3 ′ end.
  • nucleic acid fragment comprises a D region sequence, an A ′ region sequence, an A region sequence and a D ′ region sequence from the 5 ′ end toward the 3 ′ end.
  • step of preparing the nucleic acid fragment includes a step of amplifying the nucleic acid fragment by a nucleic acid amplification reaction.
  • cell producing the hollow particle is a cell into which an AAV Cap gene, Rep gene, and AAV helper function are introduced.
  • a method for producing AAV nucleic acid-encapsulated hollow particles comprising the following steps; (A) (i) at least three sequences of the A region-D ′ region of the AAV ITR, or (ii) a sequence complementary to the A region-D ′ region, Preparing a nucleic acid comprising: (B) introducing the nucleic acid of (a) into a cell producing AAV hollow particles, (C) A step of culturing the cell of (b). [8] The method according to [7], wherein the nucleic acid is a plasmid. [9] The method according to [7], wherein the cell producing the hollow particle is a cell into which an AAV Cap gene, Rep gene, and AAV helper function are introduced.
  • the present invention provides a method for producing hollow particles encapsulating nucleic acids more simply and efficiently.
  • the “AAV hollow particle encapsulating nucleic acid” in the present invention is an AAV-like particle that does not hold a complete AAV genome, and is different from normal AAV.
  • the sequence derived from the virus can be reduced, and the target nucleic acid can be encapsulated in the hollow particles simply and efficiently. Thereby, it is possible to provide hollow particles having high safety and target cell specificity.
  • FIG. 3 is a view showing the structure of a plasmid containing a sequence of A region-D ′ region.
  • FIG. 2 is a view of fluorescence of CHO-K1 cells contacted with AAV hollow particles prepared using a plasmid containing a sequence of A region-D ′ region.
  • FIG. 3 is a view showing the structure of a linear nucleic acid fragment containing the sequence of A region-D ′ region.
  • FIG. 3 is a diagram showing the measured AAV hollow particle titer prepared using a linear nucleic acid fragment containing the sequence of the A region-D ′ region. It is a figure which shows the area
  • inverted terminal repeat refers to cis elements present at both ends of AAV genomic DNA. ITR is essential for replication, amplification and encapsulation of AAV genomic DNA.
  • the ITR includes a Rep binding site (also described as RBS, RBE) and a terminal separation site (TRS) and palindromic sequences that allow hairpin formation.
  • the ITR includes an A region, a B region, a B ′ region, a C region, a C ′ region, and an A ′ region in order from the end.
  • a region and A ′ region, B region and B ′ region, C region and C ′ region are complementary sequences in opposite directions, and each region anneals into a double strand to form a hammerhead structure as shown in FIG. Form.
  • a ′ region there is a D region on the opposite side (3 ′ end side or 5 ′ end side) of the C ′ region.
  • 3 ′ terminal side means that the 3 ′ terminal portion of a certain sequence (region) is positioned further in the 3 ′ terminal direction.
  • 5 ′ terminal side means that the 5 ′ terminal portion of a certain sequence (region) is located further in the 5 ′ terminal direction. Therefore, in the present specification, when “3 ′ terminal side” or “5 ′ terminal side” is described, the target sequence or the like is arranged in contact with the 3 ′ terminal part or 5 ′ terminal part of a certain sequence (region).
  • the target sequence or the like is arranged without contacting the 3 ′ end portion or 5 ′ end portion of a certain sequence (region) (that is, the 3 ′ end portion or 5 ′ end portion of a certain sequence (region) And any sequence between the target sequence and the like).
  • a “capsid” is one of the elements constituting a virus particle (virion), and is a coat or shell consisting of a plurality of unit proteins (capsomere) surrounding a genomic DNA or core. ). Particles composed only of capsid proteins that do not contain any viral nucleic acid, core, or other substance inside the capsid are referred to herein as “empty particles” and are referred to as “empty capsid particles”. ) ".
  • the “target gene” means any gene that is desired to be encapsulated in a hollow particle and introduced into a target cell.
  • the target gene encodes a functional gene such as a structural gene (for example, a functional protein such as an enzyme, a transcription factor, a reporter molecule, or a growth factor) and a regulatory gene (for example, an antisense RNA, siRNA, miRNA, or ribozyme). Gene).
  • a functional gene such as a structural gene (for example, a functional protein such as an enzyme, a transcription factor, a reporter molecule, or a growth factor) and a regulatory gene (for example, an antisense RNA, siRNA, miRNA, or ribozyme). Gene).
  • the gene of interest may contain regulatory elements that control transcription and translation, such as promoter sequences, enhancer sequences, poly A addition signal sequences, terminator sequences, and the like.
  • the present invention is a method for producing AAV nucleic acid-encapsulated hollow particles comprising the following steps; (1) (i) the sequence of the A region of the AAV ITR, Preparing a linear nucleic acid fragment comprising (ii) the sequence of the A ′ region of the AAV ITR and (iii) the sequence of the D region and / or the sequence of the D ′ region of the AAV ITR; (2) a step of introducing the nucleic acid fragment of (1) into a cell producing AAV hollow particles, and (3) a step of culturing the cell of (2).
  • the linear nucleic acid fragment of the step (1) comprises (i) the sequence of the A region of ITR, (ii) the sequence of the A ′ region of ITR, and (iii) the sequence of the D region. And / or a sequence of the D ′ region.
  • genomic DNA sequences of known natural AAV serotypes for example, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11
  • an A region, a B region, a B ′ region, a C region, a C ′ region, and an A ′ region are specified.
  • the D region is present on the opposite side (3 ′ end side or 5 ′ end side) of the C ′ region among the ends of the A ′ region of the ITR.
  • the sequence of the D region of each serotype and the sequence of the D ′ region that is complementary to the sequence of the D region and that is the reverse sequence are also specified.
  • the A region, A ′ region, D region, and D ′ region may be derived from different serotypes.
  • a mutant of the sequence of the natural A region can be used as long as it retains the binding ability to Rep and the ability to form a hairpin, and the sequence of the A ′ region in this case is complementary to the sequence of the A region of the mutant. It is.
  • the present invention can also use natural D region sequence variants, in which case the D ′ region sequence is complementary to the variant D region sequence.
  • the linear nucleic acid fragment in the step (1) includes an A ′ region sequence, an A region sequence, and a D ′ region sequence from the 5 ′ end toward the 3 ′ end.
  • the linear nucleic acid fragment of another embodiment includes a D region sequence, an A ′ region sequence and an A region sequence from the 5 ′ end to the 3 ′ end.
  • the linear nucleic acid fragment of another embodiment includes a D region sequence, an A 'region sequence, an A region sequence and a D' region sequence from the 5 'end toward the 3' end.
  • the linear nucleic acid fragment in the step (1) may include a loop sequence (spacer sequence) between the sequence of the A ′ region and the sequence of the A region.
  • the loop sequence may be any sequence that can form a stem loop structure (hairpin structure) with the sequences of the A ′ region and the A region.
  • the chain length of the loop sequence is 3 to 500 bp, preferably 5 to 200 bp, more preferably 7 to 100 bp.
  • the linear nucleic acid fragment in the step (1) can further contain a target gene.
  • the gene of interest can be foreign to AAV.
  • the target gene can be placed on either the 5 ′ end side of the A ′ region sequence, between the A ′ region sequence and the A region sequence, or on the 3 ′ end side of the A region sequence. Or it can arrange
  • the linear nucleic acid fragment in the step (1) includes at least one set of (i), (ii) and (iii) sequences.
  • a nucleic acid fragment containing 1 set of the above sequences a nucleic acid fragment containing 2 sets, a nucleic acid fragment containing 3 sets, a nucleic acid fragment containing 4 sets, or a nucleic acid fragment containing 5 or more sets can be used.
  • the linear nucleic acid fragment in the step (1) is DNA or RNA, preferably DNA. Further, the nucleic acid fragment may be a double-stranded nucleic acid obtained by annealing two complementary nucleic acids or a single-stranded nucleic acid.
  • a linear nucleic acid means that the both ends of a nucleic acid are not couple
  • the linear nucleic acid fragment in the step (1) can be prepared by a known nucleic acid preparation method.
  • the nucleic acid fragment can be prepared in vitro by a nucleic acid amplification reaction such as PCR or chemical synthesis.
  • the nucleic acid can be prepared by extracting from a cell a nucleic acid generated by polymerase reaction, replication and transcription in vivo such as in a eukaryotic cell or a prokaryotic cell.
  • the present invention is a method for producing AAV nucleic acid-encapsulated hollow particles comprising the following steps; (A) (i) at least three AAV ITR A region-D ′ region sequences, or (ii) a sequence complementary to the A region-D ′ region (D region-A ′ region sequence), Preparing a nucleic acid comprising: (B) introducing the nucleic acid of (a) into a cell producing AAV hollow particles, (C) A step of culturing the cell of (b).
  • the nucleic acid of the step (a) includes (i) at least three sequences of AAV ITR A region-D ′ region, or (ii) a sequence complementary to A region-D ′ region.
  • a region-D ′ region sequence means a continuous sequence extending from the A region to the D ′ region of the AAV ITR, or a sequence intermittently including the A region and the D ′ region.
  • D region-A ′ region sequence means a continuous sequence extending from the D region of the AAV ITR to the A ′ region or a sequence intermittently including the D region and the A ′ region.
  • the sequence of the A region-D ′ region, the sequence complementary to this region is a sequence of genomic DNA of a known natural AAV serotype (for example, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) can be used.
  • the A region, A 'region, D region, and D' region may be derived from different serotypes.
  • a variant of the sequence of the natural A region can be used as long as it retains Rep binding activity and hairpin formation, in which case the sequence of the A 'region is complementary to the sequence of the A region of the variant.
  • the present invention can also use natural D region sequence variants, in which case the D 'region sequence is complementary to the variant D region sequence.
  • a nucleic acid comprising at least three A region-D ′ region sequences (hereinafter also referred to as “AD ′ sequences”) is adjacent to one AD ′ sequence and another AD ′ sequence.
  • AD ′ sequences A region-D ′ region sequences
  • a spacer sequence may be included between one AD ′ sequence and another AD ′ sequence.
  • the spacer sequence has a length of 3 to 500 bp, preferably 5 to 200 bp, more preferably 7 to 100 bp.
  • the nucleic acid in the step (a) can further contain a target gene.
  • the gene of interest can be foreign to AAV.
  • the gene of interest can be arranged at one or a plurality of positions on either the 5 'end side or the 3' end side of the sequence of the A region-D 'region.
  • the nucleic acid in the step (a) is DNA or RNA, preferably DNA.
  • the complementary two-molecule nucleic acid may be an annealed double-stranded nucleic acid or a single-molecule single-stranded nucleic acid. Further, it may be a circular nucleic acid or a linear nucleic acid.
  • the nucleic acid in the step (a) is a circular double-stranded DNA, preferably a plasmid.
  • the nucleic acid in the step (a) may further contain an origin of replication that functions in the host cell.
  • an origin of replication that functions in the host cell.
  • the origin of replication of prokaryotic cells ColdE1 ori, f1, ori etc.
  • the origin of replication of eukaryotic cells SV40 ori, EBV ori etc.
  • the nucleic acid having the origin of replication can be maintained episomally in the host cell.
  • the nucleic acid fragment of the step (1) and the nucleic acid of the step (a) are AAV2-derived sequences
  • the sequence of the A ′ region is SEQ ID NO: 8
  • the sequence of the A region is SEQ ID NO: 9 shows.
  • the sequence of the D region is shown in SEQ ID NO: 7
  • the sequence of the D ′ region is shown in SEQ ID NO: 10.
  • the nucleic acid fragment in the step (1) and the nucleic acid in the step (a) include natural nucleic acids, chemically modified nucleic acids, artificial nucleic acids, nucleic acid analogs, and combinations thereof.
  • Natural nucleic acids are DNA and RNA in which only natural nucleotides existing in nature are linked.
  • the chemically modified nucleic acid is a nucleic acid that has been artificially chemically modified. For example, methylphosphonate DNA / RNA, phosphorothioate DNA / RNA, phosphoramidate DNA / RNA, 2′-O-methyl DNA / RNA Examples thereof include RNA.
  • An artificial nucleic acid is a nucleic acid obtained by linking only a non-natural nucleotide or a non-natural nucleotide to a part of a natural nucleic acid.
  • non-natural nucleotide refers to a non-naturally occurring nucleotide that is artificially constructed or artificially chemically modified and has properties and / or structures similar to those of the natural nucleotide.
  • Nucleic acid analogs are artificially constructed polymeric compounds that have similar structures and / or properties to natural nucleic acids.
  • peptide nucleic acids including PNA: Peptide Nucleic Acid
  • peptide nucleic acids having a phosphate group PONA
  • BNA / LNA Bridged Nucleic Acid / Locked Nucleic Acid
  • morpholino nucleic acids morpholino oligos, etc.
  • the nucleic acid may be labeled with a phosphate group, sugar, and / or base as necessary.
  • a labeling substance known in the art can be used for labeling.
  • radioisotopes eg, 32P, 3H, 14C
  • DIG diatomaceous iotide
  • biotin e.g, FITC, Texas, cy3, cy5, cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA
  • luminescent materials eg, acridinium esters.
  • nucleic acid fragment in the step (1) and the nucleic acid in the step (a) a nucleic acid purified and extracted using a known purification method or a commercially available product can be used.
  • known purification methods include purification methods such as extraction with a phenol / chloroform mixture, alcohol precipitation, column purification, filter filtration, and agarose gel electrophoresis.
  • the size of the nucleic acid fragment and the nucleic acid is not limited as long as it can be included in the AAV hollow particles, but is usually 5 kb or less.
  • the nucleic acid fragment of the step (1) and the nucleic acid of the step (a) do not contain the complete natural AAV genome sequence, and lack the sequence of the AAV Rep gene and / or the AAV Cap gene.
  • the nucleic acid fragment and the nucleic acid do not retain the natural complete ITR sequence, and the A region, the A ′ region, and the B region within the ranges described in the steps (1) and (a).
  • B ′ region, C region, C ′ region, and at least one region selected from the group consisting of D region is missing. More preferably, the nucleic acid fragment and the nucleic acid lack the sequence of the B region, B ′ region, C region and C ′ region.
  • Step of introducing nucleic acid into cell The nucleic acid fragment of step (1) or the nucleic acid of step (a) is introduced into a cell producing AAV hollow particles.
  • a cell producing AAV hollow particles As the cell into which the nucleic acid fragment or nucleic acid is introduced, various eukaryotic cells such as mouse cells, mammalian cells including primate cells (for example, human cells), insect cells and the like can be used. In the present specification, cells that produce AAV and nucleic acid-encapsulated hollow particles are sometimes referred to as packaging cells or producer cells.
  • Suitable mammalian cells include, but are not limited to, primary cells and cell lines, and suitable cell lines include HEK293 cells, 293EB cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, Examples thereof include C3H10T1 / 2 fibroblasts, CHO cells, and cells derived from these cells.
  • suitable insect cells include, but are not limited to, primary cells and cell lines, and suitable cell lines include Sf9 cells and cells derived from these cells.
  • the cells that produce the hollow particles are those that express the AAV rep gene product and the cap gene product. These gene products may be encoded by AAV rep gene and cap gene stably integrated into the cell genome, and are introduced into the cell before, simultaneously with, or after the introduction of the nucleic acid fragment or nucleic acid. May be encoded by a vector.
  • the rep gene and the cap gene may be encoded by the same vector, or may be encoded by different vectors.
  • the rep gene and cap gene can be used with any serotype of AAV sequences (eg, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11).
  • the rep gene and the cap gene may be sequences derived from the same serotype or may be sequences derived from different serotypes.
  • expression vectors suitable for the cells to be introduced can be used as long as they can produce hollow particles, and examples thereof include viral vectors, plasmid vectors, cosmid vectors, and artificial chromosomes. .
  • a plasmid vector that can be maintained episomally can be used as necessary.
  • the vector may be placed under the control of a promoter and terminator in an appropriate expression system so that the rep gene and the cap gene can be expressed in cells.
  • the nucleic acid expression system is a system having at least one set of expression regulatory elements necessary for gene expression in a functional state.
  • expression control elements include an enhancer and a poly A addition signal as necessary.
  • a further helper function can be introduced into cells that produce hollow particles.
  • the helper function is also called a helper virus function or an accessory function.
  • Adenovirus is generally used for introduction of the helper function, but viruses such as type 1 or type 2 herpes simplex virus and vaccinia virus can also be used. When a virus is used, the cells are infected with the virus as a helper virus.
  • adenoviruses that do not exhibit late gene expression may be used because only adenovirus early gene expression is required for AAV particle packaging.
  • Adenovirus mutants that lack late gene expression eg, ts100K or ts149 adenovirus mutants) can be used.
  • a nucleic acid construct that provides a helper virus function can be prepared and introduced into a cell using a nucleic acid necessary for the helper virus function isolated from the helper virus.
  • a construct that provides a helper virus function includes a nucleotide sequence that provides one or more types of helper virus functions, including a gene encoding an E1 gene region, an E2A gene region, an E4orf6 gene, and a VA RNA, and includes plasmids, phages, and transposons. Provided to the host cell in the form of a cosmid or other virus.
  • Examples of the method for introducing the nucleic acid fragment of the step (1) and the nucleic acid of the step (a) into a cell include, for example, calcium phosphate method, lipofection method, DEAE dextran method, polyethyleneimine method, electroporation method, direct microinjection method.
  • Fast particle guns can be used.
  • commercially available reagents such as TransIT (registered trademark) -293 Reagent, TransIT (registered trademark) -2020 (manufactured by Miras), Lipofectamine 2000 Reagent, Lipofectamine 2000 CD Reagent (manufactured by Life Technologies), FuGene (registered trademark). ) Transfection Reagent (manufactured by Promega), PEI max (manufactured by Cosmo Bio), or the like may be used.
  • the nucleic acid fragment in the step (1) is introduced into 1 ng to 10 ⁇ g, preferably 5 ng to 1 ⁇ g, more preferably 10 ng to 500 ng per 10 6 cells.
  • the step (a) of the nucleic acid per 10 6 cells 100 ng ⁇ 1000 [mu] g, preferably from 1 [mu] g ⁇ 100 [mu] g, more preferably introduces 5 [mu] g ⁇ 50 [mu] g.
  • Culture of the cells into which the nucleic acid fragment or nucleic acid has been introduced in (B) can be performed under known culture conditions depending on the cells.
  • the culture is performed at a temperature of 30 to 37 ° C., a humidity of 95%, and a CO 2 concentration of 5 to 10%, but the present invention is not limited to such conditions.
  • it may be carried out at a temperature, humidity, or CO 2 concentration other than the above ranges.
  • the culture medium a known medium can be used.
  • DMEM, IMDM, Ham F12, RPMI-1640 and the like may be used, which are commercially available from Lonza, Life Technologies, Sigma-Aldrich, etc. Can be obtained as
  • the medium may be a serum-free medium or a medium supplemented with fetal bovine serum (FBS) or human serum-derived albumin.
  • FBS fetal bovine serum
  • Cell culture equipment such as petri dishes, flasks, bags, large culture tanks or bioreactors can be used as the culture equipment.
  • a CO 2 gas permeable bag for cell culture is suitable.
  • a large culture tank may be used.
  • the culture period is not particularly limited, and for example, 12 hours to 10 days, preferably 24 hours to 7 days is preferable.
  • nucleic acid-encapsulated hollow particles that is, AAV-like particles that retain nucleic acid therein are produced in the cells and / or in the culture supernatant.
  • the step of obtaining hollow particles encapsulating nucleic acid from the supernatant of the cell culture and the centrifuged supernatant of the collected cells resuspended in an appropriate buffer solution (cell disruption solution) can be implemented.
  • the culture supernatant and cell lysate supernatant thus obtained are used as they are, or further, for example, by concentrating and purifying hollow particles by a known method such as filter filtration or CsCl density gradient centrifugation or a commercially available product. Once performed, it can be stored in a suitable manner, such as frozen and used for the desired application.
  • the present invention provides a hollow particle encapsulating the nucleic acid fragment of the step (1) or the nucleic acid of the step (a).
  • the hollow particles of the present invention do not retain the complete natural AAV genome, nor do they retain nucleic acids containing sequences of the AAV Rep gene and / or the AAV Cap gene.
  • the hollow particles of the present invention further do not retain a natural complete ITR and comprise the group consisting of A region, A ′ region, B region, B ′ region, C region, C ′ region, D region. It does not retain the sequence of at least one region that is more selected.
  • the present invention provides a composition comprising the nucleic acid-encapsulated hollow particles.
  • the composition of the present invention comprises at least one of nucleic acid-encapsulated hollow particles obtained by the method of the present invention.
  • the composition of the present invention can contain a nucleic acid-encapsulated hollow particle as an active ingredient, a carrier, and / or another drug.
  • Two or more different nucleic acid-enclosed hollow particles may be contained in the composition.
  • the two or more different nucleic acid-encapsulated hollow particles may be derived from viruses having different capsids or the like, and / or encapsulated nucleic acids may be of different types.
  • a plurality of nucleic acid-encapsulated hollow particles with different target cells may be included.
  • the carrier is a substance that is added within a range that facilitates formulation of the composition and application to a living body and does not inhibit or suppress its action.
  • an excipient for example, an excipient, a binder, a disintegrant, a filler, an emulsifier, examples include, but are not limited to, flow addition modifiers or lubricants.
  • a pharmaceutically acceptable carrier is used in the composition of the present invention.
  • the content of the nucleic acid-encapsulated hollow particles in the composition of the present invention is not particularly limited.
  • Kind of nucleic acid contained in the particle and / or effective amount thereof, applied cell or individual, application method / route, purpose of application, form of composition (including form and size), and the carrier It is determined as appropriate in consideration of the type of
  • a gene By using the nucleic acid-encapsulated hollow particles of the present invention and the composition containing the hollow particles, a gene can be introduced into a cell or animal individual. Such a gene introduction method is also an embodiment of the present invention. Gene transfer into cells can be carried out by contacting hollow particles with cells in vitro. In addition, gene introduction into individual animals including humans can be achieved by administering the nucleic acid-encapsulated hollow particles of the present invention, preferably an appropriate composition containing the hollow particles, into tissues (for example, intramuscularly), intravenously, subcutaneously, and intraperitoneally. It is carried out by administering by this route.
  • Example 1 Packaging of Plasmid DNA into AAV Hollow Particles (1) Construction of AD′-Mounted Plasmid The AD ′ sequence present in the ITR of AAV2 genomic DNA (61 bp across A region-D ′ region: SEQ ID NO: 1) A double-stranded DNA having a restriction enzyme AflIII recognition sequence at both ends was prepared by annealing both chemically synthesized strands.
  • This double-stranded DNA was inserted into the AflIII site upstream of the CMV promoter of pAcGFP1-N1 Vector (Clontech, manufactured by Takara Bio USA), a plasmid AD-F in which the AD ′ sequence was inserted in a 5 ′ to 3 ′ direction, Plasmid AD-R inserted in the reverse direction was prepared. Furthermore, plasmid AD-3F in which three AD ′ sequences were inserted in the forward direction was prepared. As a negative control, a plasmid AD ( ⁇ ) in which no artificial gene was inserted was prepared. Furthermore, as positive controls, plasmid ss-AAV and plasmid ds-AAV that generate an AAV genome carrying the AcGFP gene were prepared.
  • the AcGFP gene is inserted into the multiple cloning site of the pAAV-MSC vector of AAV Helper-Free System (manufactured by Agilent Technologies).
  • the plasmid ds-AAV replaces the eGFP gene of the plasmid pdsAAV-CB-eGFP provided by Dr. Arun Srivastava with AcGFP.
  • FIG. 1 A schematic diagram of each plasmid is shown in FIG. In FIG. 1, the AD ′ sequence is described as “AD”.
  • Example 2- Introduction of plasmid into 293EB cells
  • pAAV2 / 9 Vector SEQ ID NO: 11
  • pAd5N expressing AAV9 Cap and AAV2 Rep.
  • 293EB cells were cultured in DMEM medium containing 1/100 volume of GlutaMax (Gibco) for 3 days at 37 ° C. and 5% CO 2 .
  • Nucleic acid-encapsulated AAV hollow particles were purified from the culture supernatant of 293EB cells cultured in Example 1- (2) by cesium chloride density gradient centrifugation.
  • the purified AAV-like particle solution 2 ⁇ L, dH 2 O 116 ⁇ L, 50 mM MgCl 2 15 ⁇ L, 0.1% Triton X-100 15 ⁇ L, 250 U / ⁇ L Benzonase 2 ⁇ L were mixed, incubated at 37 ° C. for 30 minutes, and released. Genomic DNA and plasmid DNA were degraded. Next, 100 ⁇ L of AL buffer (manufactured by Qiagen) was added to dissolve the AAV capsid, and the DNA packaged in the hollow particles was extracted.
  • AL buffer manufactured by Qiagen
  • an AcGFP-f primer SEQ ID NO: 2
  • an AcGFP-r primer SEQ ID NO: 3
  • SYBR registered trademark
  • Premix DimerEraser trademark
  • Example 1- (3) Each AAV-like particle obtained from the culture supernatant in Example 1- (3) was infected with CHO-K1 cells as follows. The day before infection, 2.8 ⁇ 10 4 cells of CHO-K1 cells were seeded on 48-well plates. The next day, each AAV-like particle obtained in Example 1- (3) was added to 4.0 ⁇ 10 5 v. g. Infected with / cell. After infection, CHO-K1 cells were incubated at 37 ° C., 5% CO 2 for 3 days, and then the expression of AcGFP was observed with a fluorescence microscope (FIG. 2). As shown in FIG.
  • the plasmid in which the AD ′ sequence is loaded in the reverse direction and the plasmid in which the three AD ′ sequences are loaded in the forward direction are more effectively hollow than the plasmid in which the forward AD ′ sequence is loaded.
  • the expression of AcGFP in the infected cells was confirmed. It was shown that AAV-like particles containing the AcGFP gene were produced from cells introduced with a plasmid containing the AD ′ sequence.
  • Example 2 Packaging of AAV Genome by Introducing DNA Fragment (1) Preparation of DNA Fragment Containing AD Sequence Oligonucleotide Full described in SEQ ID NO: 4, Oligonucleotide Plus described in SEQ ID NO: 5, described in SEQ ID NO: 6 Oligonucleotide Minus was chemically synthesized and each complementary strand was also chemically synthesized and annealed to prepare double-stranded DNA.
  • FIG. 3 shows the structure of each double-stranded DNA.
  • AAV ITR A region sequence, D 'region sequence, A' region sequence (complementary to A region), D region sequence (complementary to D 'region), A sequence, D These are referred to as' sequence, A 'sequence and D sequence, and are shown as A, D (+), A' and D (-) in Fig. 3, respectively.
  • the oligonucleotide Full consists of a D sequence, an A ′ sequence, a loop sequence, an A sequence, and a D ′ sequence in this order from the 5 ′ end.
  • Oligonucleotide Plus consists of an A ′ sequence, a loop sequence, an A sequence, and a D ′ sequence in this order from the 5 ′ end.
  • the oligonucleotide Minus is composed of a D sequence, an A ′ sequence, a loop sequence, and an A sequence in this order from the 5 ′ end.
  • the D sequence is shown in SEQ ID NO: 7
  • the A ′ sequence is shown in SEQ ID NO: 8
  • the A sequence is shown in SEQ ID NO: 9
  • the D ′ sequence is shown in SEQ ID NO: 10, respectively.
  • the DNA fragments were ligated to the pCR-Blunt vector of Zero Blunt cloning kit (manufactured by ThermoFisher), and plasmid DNA was prepared according to the instructions. Using these plasmid DNAs as templates, PCR was performed using M13 Forward ( ⁇ 20) primer and M13 Reverse primer (ThermoFisher) to amplify DNA fragment Full, DNA fragment Plus, and DNA fragment Minus. As a negative control, PCR was performed using a pCR-Blunt vector containing an intervening sequence as a template to amplify a DNA fragment Mock.
  • the PCR reaction solution was subjected to 1.5% agarose gel electrophoresis, and the DNA fragment was extracted and purified from the gel after electrophoresis. Two bands were generated from the DNA fragment Full, and were extracted separately as a DNA fragment Full-H and a DNA fragment Full-M.
  • Example 2- (1) Introduction into 293EB cells 75 ng of each DNA fragment obtained in Example 2- (1) was transfected into 293EB cells together with pAAV2 / 9 Vector and pAd5N (pHelper Vector) using Polyethyleneimine “Max”. Two days after transfection, 293EB cells were replaced with DMEM / F12 (ThermoFisher) medium containing glucose, sodium bicarbonate, and 1/100 volume of GlutaMax. Thereafter, the cells were cultured for 5 days at 37 ° C. and 5% CO 2 .
  • DMEM / F12 ThermoFisher
  • DNA fragment containing a sequence derived from the IAV ITR resulted in packaging of the DNA fragment into AAV hollow particles.
  • DNA fragments can be prepared in large quantities by PCR, and AAV-like particles can be prepared more easily.
  • the present invention provides a simpler and more efficient method for encapsulating nucleic acids in hollow particles.
  • the nucleic acid-encapsulated hollow particles produced using the method of the present invention and the composition containing the nucleic acid-encapsulated hollow particles as an active ingredient are useful as gene transfer methods in gene therapy research or clinical fields.
  • SEQ ID NO: 1 AD 'region sequence
  • SEQ ID NO: 2 AcGFP-f
  • SEQ ID NO: 3 AcGFP-r
  • SEQ ID NO: 4 Oligonucleotide Full
  • SEQ ID NO: 5 Oligonucleotide Plus
  • SEQ ID NO: 6 Oligonucleotide Minus
  • SEQ ID NO: 7 D region sequence
  • SEQ ID NO: 8 A 'region sequence
  • SEQ ID NO: 9 A region sequence SEQ ID NO: 10: D 'region sequence SEQ ID NO: 11: pAAV2 / 9
  • SEQ ID NO: 13 3 ′ antisense primer

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Abstract

La présente invention concerne un procédé de fabrication de particules vides encapsulant un acide nucléique du virus adéno-associé (AAV), le procédé comprenant les étapes suivantes : (1) une étape de préparation de fragments d'acide nucléique linéaires, les fragments d'acide nucléique comprenant (i) la séquence de la région A d'une séquence terminale inversée répétée (ITR) de AAV, (ii) la séquence de la région A' de l'ITR du AAV, et (iii) la séquence de la région D et/ou la séquence de la région D' de l'ITR du AAV; (2) une étape d'introduction des fragments d'acide nucléique préparés en (1) dans des cellules qui génèrent des particules de AAV vides; et (3) une étape de culture des cellules obtenues en (2).
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JPWO2020158792A1 (ja) * 2019-01-30 2021-12-02 国立研究開発法人国立精神・神経医療研究センター 核酸送達複合体
JP7481694B2 (ja) 2019-01-30 2024-05-13 国立研究開発法人国立精神・神経医療研究センター 核酸送達複合体
WO2021002412A1 (fr) 2019-07-03 2021-01-07 学校法人日本医科大学 Procédé de production d'une particule creuse de vaa encapsulant un acide nucléique
JPWO2021002412A1 (fr) * 2019-07-03 2021-01-07
KR20220031000A (ko) 2019-07-03 2022-03-11 각꼬호우징 닛뽄 이까다이가꾸 핵산 봉입aav 중공입자의 제조방법
CN114269940A (zh) * 2019-07-03 2022-04-01 学校法人日本医科大学 用于产生包封核酸的aav中空颗粒的方法
EP3995571A4 (fr) * 2019-07-03 2023-08-09 Nippon Medical School Foundation Procédé de production d'une particule creuse de vaa encapsulant un acide nucléique
JP7575761B2 (ja) 2019-07-03 2024-10-30 学校法人日本医科大学 核酸封入ａａｖ中空粒子の製造方法
CN114269940B (zh) * 2019-07-03 2024-12-06 学校法人日本医科大学 用于产生包封核酸的aav中空颗粒的方法
CN115851837A (zh) * 2022-01-25 2023-03-28 广州派真生物技术有限公司 一种提高杆状病毒系统生产腺相关病毒的方法及应用

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