Virus-like particles and their use

Abstract

To provide virus-like particles encapsulating a target protein and / or a target RNA and methods for producing the virus-like particles.SOLUTION: A virus-like particle encapsulating a target protein is disclosed, where the virus-like particle contains a Gag protein forming a dimer with the target protein, the Gag protein is a fusion protein with an FK506-binding protein (FKBP12) and the target protein is a fusion protein with an FKBP12-rapamycin-associated protein 1, FRAP1 fragment (FRB), and the FKBP12, rapamycin or a rapamycin derivative and the FRB are bound together in the dimer, but except for virus particles in which FKBP12 is fused to N-terminus of the Gag protein).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to virus-like particles encapsulating a target protein and / or a target RNA, and uses thereof. More specifically, the present invention relates to virus-like particles encapsulating a Cas family protein or a Cas family protein / RNA RNP complex, a method for producing the virus-like particles, a kit for producing the virus-like particles, and a method for producing cells in which genomic DNA has been cleaved in a sequence-specific manner.

Background Art

[0002] By introducing a genome editing enzyme such as a CRISPR-Cas9 / gRNA RNP complex into a cell or a living body, DNA cleavage, base substitution, alteration of epigenetic state, etc. can be performed at any location on the genome.

[0003] As methods for introducing a Cas9 protein, a Cas9 gene, a gRNA, etc. into a cell, various methods have been developed, such as a lipofection method using a lipid, a lipid nanoparticle (LNP) method, an electroporation method for physically introducing, a microinjection method, and a virus vector method that utilizes the cell entry mechanism of a virus.

[0004] Among these, the virus vector method is widely used. In particular, adeno-associated virus (AAV) vectors, lentivirus vectors, etc. are known to have high introduction efficiency and low cytotoxicity.

[0005] However, the expression of a foreign gene by a virus vector persists for a long period. For this reason, for example, when a Cas9 gene is introduced into a cell by the virus vector method, the expression of the Cas9 protein persists in the cell for a long period. As a result, the risk of introducing off-target mutations that cleave sequences other than the target sequence increases.

[0006] Therefore, a technique has been developed that utilizes only the viral cell entry mechanism by eliminating the viral replication mechanism and the infection persistence function, and using virus-like particles (hereinafter sometimes referred to as "VLPs") composed only of the viral envelope. Once VLPs enter a cell, they release the foreign gene inside and disappear, so a major feature is that they exhibit a transient expression pattern. Virus-like particles are particles with a diameter of 1,000 nm or less that contain at least one or more virus-derived proteins, and are classified into those with a lipid bilayer membrane and those without.

[0007] For example, Non-Patent Document 1 reports a method of encapsulating the Cas9 protein inside VLPs by using a fusion protein of the Gag structural protein of lentivirus and the Cas9 protein.

[0008] In addition, Clontech sells the Gesicle system, which enhances the encapsulation efficiency of the Cas9 protein inside VLPs. In the Gesicle system, CherryPicker, a membrane-localized fluorescent protein, and the Cas9 protein are associated in a compound A / C Heterodimerizer-dependent manner using the iDimerize Inducible Heterodimer System, thereby enhancing the encapsulation efficiency of the Cas9 protein inside VLPs.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, in the method described in Non-Patent Document 1, the encapsulation efficiency of Cas9 protein into VLPs and the genome cleavage efficiency were low. In addition, in the Gesicle system of Clontech, since the CherryPicker fluorescent protein is not specifically incorporated into VLPs, the encapsulation efficiency of Cas9 may be insufficient in some cases.

[0011] Furthermore, it was not easy to produce VLPs that encapsulated a sufficient amount of the active Cas9 / gRNA RNP complex. To form an active Cas9 / gRNA RNP complex within VLPs, first, a sufficient amount of Cas9 and gRNA need to be encapsulated into VLPs. And for gRNA to be efficiently encapsulated into VLPs, gRNA needs to be transported from the nucleus to the cytoplasm in VLP-producing cells and be localized in the vicinity at the time of VLP budding.

[0012] However, in the conventional general method, there was a problem that gRNA tended to remain in the nucleus of producing cells and was difficult to be encapsulated into VLPs.

[0013] Therefore, an object of the present invention is to provide a technique for efficiently encapsulating a target protein, or a target protein and a target RNA, into virus-like particles (VLPs).

Means for Solving the Problems

[0014] In response to the problems of the above prior art, the inventors expressed Gag protein together with the target protein in VLP-producing cells, and further expressed these proteins as fusion proteins with either FK506-binding protein (FKBP12) or FKBP12-rapamycin associated protein 1, FRAP1 fragment (FRB), respectively, and found that the target protein was efficiently encapsulated into VLPs in a rapamycin compound-dependent manner.

[0015] Furthermore, the inventors sandwiched the sequence of gRNA between the sequences of two RNAs having self-splicing activity (Self-cleaving RNA, hereinafter sometimes referred to as ribozyme), arranged the packaging signal of retrovirus upstream thereof, and further expressed it as an mRNA transcriptionally controlled by an LTR promoter, so that the mRNA was actively encapsulated in VLPs, and the gRNA was automatically excised from the mRNA, and it was found that VLPs with higher Cas9 activity per particle than before could be obtained. Based on these results, the inventors have completed the present invention.

[0016] That is, the present invention includes the following aspects. [1] A virus-like particle encapsulating a target protein, wherein the virus-like particle contains a Gag protein, and the Gag protein forms a dimer with the target protein. [2] One of the Gag protein and the target protein is a fusion protein with FK506-binding protein (FKBP12), and the other is a fusion protein with FKBP12-rapamycin associated protein 1, FRAP1 fragment (FRB), and in the dimer, the FKBP12, rapamycin or a rapamycin derivative, and the FRB are bound. The virus-like particle according to claim 1. [3] The virus-like particle according to [1] or [2], wherein the target protein is a Cas family protein. [4] The virus-like particle according to [3], further encapsulating an mRNA having a gRNA sequence and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence, or a self-cleavage product of the mRNA. [5] A method for producing a genome-edited cell, comprising inoculating the cell with the virus-like particle according to [4]. [6]A method for producing virus-like particles encapsulating a target protein, comprising the following steps (1) and (2): (1) In the presence of rapamycin or a rapamycin derivative, a cell is caused to express a fusion protein of FKBP12 and a Gag protein, and a fusion protein of FRB and the target protein, or a fusion protein of FRB and a Gag protein, and a fusion protein of FKBP12 and the target protein; (2) A step of obtaining a culture medium containing virus-like particles encapsulating the target protein. [7] The production method according to [6], wherein in the step (1), a nucleic acid encoding the fusion protein is introduced into the cell by a lipofection method or an electroporation method. [8] A method for producing virus-like particles encapsulating a Cas family protein and a gRNA, comprising the following steps (1) and (2): (1) In the presence of rapamycin or a rapamycin derivative, a cell is caused to express a fusion protein of FKBP12 and a Gag protein, and a fusion protein of FRB and a Cas family protein, or a fusion protein of FRB and a Gag protein, and a fusion protein of FKBP12 and a Cas family protein, and an mRNA having a gRNA sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence; (2) A step of obtaining a culture medium containing virus-like particles encapsulating the Cas family protein and the gRNA. [9] The production method according to [8], wherein in the step (1), a nucleic acid encoding the fusion protein or the mRNA is introduced into the cell by a lipofection method or an electroporation method.

[10] A kit for producing virus-like particles encapsulating a target protein, the kit comprising an expression vector for a fusion protein of FKBP12 and a Gag protein, or an expression vector for a fusion protein of FRB and a Gag protein.

[11] A kit for producing virus-like particles encapsulating a Cas family protein, comprising an expression vector for a fusion protein of FKBP12 and a Gag protein, and an expression vector for a fusion protein of FRB and a Cas family protein, or an expression vector for a fusion protein of FRB and a Gag protein, and an expression vector for a fusion protein of FKBP12 and a Cas family protein.

[12] The kit according to

[11] , further comprising an expression vector for an mRNA having a base sequence of a target RNA or a multiple cloning site sandwiched between a first ribozyme sequence and a second ribozyme sequence, and a packaging signal sequence.

[13] A virus-like particle encapsulating a target RNA, wherein the virus-like particle contains a Gag protein, and the target RNA is encapsulated in the form of an mRNA having a base sequence of the target RNA sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence or a self-cleavage product of the mRNA.

[14] A method for producing a virus-like particle encapsulating a target RNA, comprising the following steps (1) and (2): (1) a step of expressing in a cell a Gag protein and an mRNA having a base sequence of the target RNA sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence; (2) a step of obtaining, in the culture medium of the cell, the virus-like particle in which the target RNA is encapsulated in the form of the mRNA or a self-cleavage product of the mRNA.

[15] The production method according to

[14] , wherein in the step (1), a nucleic acid encoding the mRNA is introduced into the cell by a lipofection method or an electroporation method.

[16] A kit for producing a virus-like particle encapsulating a target RNA, comprising an expression vector for a Gag protein, and an expression vector for an mRNA having a base sequence of the target RNA or a multiple cloning site sandwiched between a first ribozyme sequence and a second ribozyme sequence, and a packaging signal sequence. A therapeutic agent for a disease, infectious disease or cancer caused by a gene mutation, comprising, as an active ingredient, the virus-like particle according to

[17] [1] to [4] or

[13] .

Advantages of the Invention

[0017] According to the present invention, a technique for efficiently encapsulating a target protein into virus-like particles can be provided.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out the Invention

[0019] [Virus-like particles encapsulating a target protein] In one embodiment, the present invention provides virus-like particles encapsulating a target protein, wherein the virus-like particles contain a Gag protein, and the Gag protein forms a dimer with the target protein.

[0020] As will be described later in the examples, according to the virus-like particles (hereinafter sometimes referred to as "VLPs") of the present embodiment, a target protein can be efficiently encapsulated. By allowing the VLPs of the present embodiment to enter cells, the target protein can be efficiently introduced into the cells.

[0021] VLPs are particles composed only of the viral envelope, excluding the viral replication mechanism and the function of maintaining infection. The VLPs of the present embodiment contain Gag protein. Examples of the Gag protein include Gag proteins derived from retroviruses. As the Gag protein, Gag proteins derived from human immunodeficiency virus (HIV), mouse leukemia virus (MLV), etc. can be preferably used.

[0022] The amino acid sequence of the Gag protein derived from HIV is shown in SEQ ID NO: 1, and the amino acid sequence of the Gag protein derived from MLV is shown in SEQ ID NO: 2. The Gag protein may have mutations as long as the effects of the VLPs of the present embodiment can be obtained.

[0023] In the VLPs of the present embodiment, the Gag protein may be cleaved into matrix (MA), capsid (CA), nucleocapsid (NC), etc., or these may remain bound.

[0024] As will be described later in the examples, it is preferable that the VLPs of the present embodiment do not contain Pol derived from retroviruses. When the VLPs contain Pol, the target protein may be degraded by the protease of Pol.

[0025] By the way, in order for VLPs to enter cells, the process of membrane fusion between the viral membrane and the cell membrane is required. The process of membrane fusion is carried out by a viral protein called an envelope protein. The envelope (Env) protein is present on the surface of the viral envelope virus and has the ability of membrane fusion.

[0026] The outer envelope of the VLP of this embodiment may contain viral envelope proteins well-known in the art. Examples of envelope proteins include Env proteins of viruses in the Retroviridae family (such as human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), human T-lymphotropic virus (HTLV), murine leukemia virus (MLV), feline leukemia virus (FLV), Rous sarcoma virus (RSV), endogenous retrovirus, etc.), Env proteins (G proteins) of viruses in the Rhabdoviridae family (such as vesicular stomatitis virus (VSV), rabies virus, Mokola virus, etc.), Env proteins of viruses in the Arenaviridae family (such as lymphocytic choriomeningitis virus (LCMV), etc.), Env proteins of the Togaviridae family (such as Sindbis virus, etc.), Env proteins (hemagglutinin (HA) protein, fusion (F) protein) of viruses in the Paramyxoviridae family (such as measles virus, etc.), envelope proteins (hemagglutinin (HA) protein, neuraminidase (NA) protein) of viruses in the Orthomyxoviridae family (such as influenza virus, etc.). Among these, VSV-G, a glycoprotein derived from VSV, can be particularly preferably used. The amino acid sequence of VSV-G is shown in SEQ ID NO: 3. Here, examples of host cells into which the VLP is to be introduced include cells derived from humans, cells derived from non-human animals, cells in vivo, etc. Also, examples of organisms include humans or non-human animals.

[0027] In the VLP of the present embodiment, the target protein is not particularly limited. For example, sequence-specific DNA cleavage enzymes such as RNA-guided nucleases and artificial nucleases; proteins that induce cell reprogramming such as Oct3 / 4 protein, Sox2 protein, Klf4 protein, c-Myc protein; proteins that convert cell types such as MyoD protein, GATA4 protein, MEF2C protein, TBX5 protein, FOXA1 protein, FOXA2 protein, FOXA3 protein, HNF4A protein, ASCL1 protein, BRN2 protein, MYT1L protein; fluorescent proteins (such as mCherry); luciferase proteins, etc. can be mentioned.

[0028] When the target protein is a sequence-specific DNA cleavage enzyme, by allowing the VLP of the present embodiment to invade cells, the sequence-specific DNA cleavage enzyme can be efficiently introduced into the cells. As a result, DNA cleavage, base substitution, change in epigenetic state, etc. can be performed at any location on the genome.

[0029] Sequence-specific DNA cleavage enzymes are roughly classified into RNA-guided nucleases and artificial nucleases. The sequence-specific DNA cleavage enzyme may be an RNA-guided nuclease or an artificial nuclease.

[0030] An RNA-guided nuclease is an enzyme that binds a short guide RNA to a target sequence and recruits a nuclease having two DNA cleavage domains (nuclease domains) to induce sequence-specific cleavage. Examples of RNA-guided nucleases include CRISPR-Cas family proteins.

[0031] Examples of CRISPR-Cas family proteins include Cas9, Cpf1 (also known as Cas12a), C2C1 (also known as Cas12b), C2C2 (also known as Cas13a), CasX, CasY, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, etc. The RNA-guided nuclease may be a homolog of a CRISPR-Cas family protein, or may be a modified CRISPR-Cas family protein. For example, it may be a nickase-modified nuclease in which one of the two wild-type nuclease domains is modified to be inactive, or it may be dCas9 in which both are modified to be inactive. Alternatively, it may be Cas9-HF, HiFi-Cas9, eCas9, etc. with improved target specificity. Further, it may be a fusion of those Cas9 with another protein (such as an enzyme).

[0032] Cas9 is derived from, for example, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus, etc. Examples of Cpf1 include those derived from Acidaminococcus, Ruminococcus, Chlamydomonas, Francisella novicida, etc.

[0033] The amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes (hereinafter sometimes referred to as "SpCas9 protein") is shown in SEQ ID NO: 4.

[0034] An artificial nuclease is an artificial restriction enzyme having a DNA-binding domain designed and produced to specifically bind to a target sequence and a nuclease domain (such as the DNA cleavage domain of the restriction enzyme FokI). Examples of artificial nucleases include, but are not limited to, Zinc finger nuclease (ZFN), Transcription activator-like effector nuclease (TALEN), meganuclease, etc.

[0035] In the VLP of this embodiment, the Gag protein forms a dimer with the target protein. Here, forming a dimer means being reversibly bound. Therefore, it is preferable that the Gag protein is not a fusion protein with the target protein.

[0036] Since the Gag protein and the target protein are dissociable, after the VLP of this embodiment invades target cells and the target protein is introduced into the cells, the Gag protein and the target protein can be dissociated, and the target protein can function sufficiently.

[0037] In addition, since the Gag protein and the target protein form a dimer, the target protein can be efficiently encapsulated in the VLP in the VLP-producing cells.

[0038] The means by which the Gag protein and the target protein form a dimer are not particularly limited. For example, the fact that FK506-binding protein (FKBP12) and FKBP12-rapamycin associated protein 1, FRAP1 fragment (FRB) form a heterodimer in the presence of rapamycin or a rapamycin derivative can be utilized. Alternatively, a system in which GAI (Gibberellin insensitive) and GID1 (Gibberellin insensitive dwarf1) form a heterodimer in the presence of gibberellin or GA3-AM (see, for example, Miyamoto T., et al., Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System, Nat Chem Biol., 8 (5), 465-470, 2012), a system in which PyL (PYR1-like, amino acid numbers 33-209) and ABI1 (amino acid numbers 126-423) can form a heterodimer in the presence of S-(+)-abscisic acid (ABA) (see, for example, Liang F. S., et al., Engineering the ABA plant stress pathway for regulation of induced proximity, Sci Signal., 4 (164), rs2, 2011), etc. can also be utilized.

[0039] In addition, the description of "rapamycin derivative" in this document includes "rapamycin analog", and "rapamycin" and "rapamycin derivative" may be referred to as "rapamycin compound".

[0040] Specifically, VLP-producing cells (cells expressing an outer envelope protein) are cultured in the presence of rapamycin or a rapamycin derivative. In the VLP-producing cells, one of the Gag protein and the target protein is expressed as a fusion protein with FKBP12, and the other is expressed as a fusion protein with FRB. By this, the FKBP12 domain and the FRB domain can be dimerized via rapamycin or a rapamycin derivative.

[0041] Furthermore, the fusion protein (a fusion protein of FKBP12 or FRB and Gag) may have an amino acid sequence that promotes localization to the cell membrane (hereinafter sometimes referred to as a cell membrane localization sequence). When the fusion protein is localized to the cell membrane in the VLP-producing cells, the target protein can be efficiently recruited near the cell membrane through the interaction between the FKBP12 domain and the FRB domain, and the dimer can be formed near the cell membrane. As a result, since the dimer is efficiently encapsulated into the VLP during the budding of the VLP, it becomes possible to produce a VLP containing a larger amount of the target protein than that produced by the conventional method. In the present disclosure, localization to the cell membrane refers to a state in which a target molecule is directly or indirectly bound to a lipid or protein constituting the cell membrane via another factor.

[0042] Examples of the cell membrane localization sequence include sequences that promote post-translational modification of proteins such as farnesylation, palmitoylation, myristoylation, and GPI anchoring, sequences that constitute a transmembrane domain composed of hydrophobic amino acid residues, domains and peptide sequences that have binding properties to proteins present in the cell membrane structure, and the like. These sequences are not particularly limited, and those well-known in the art can be appropriately used. Among these, sequences that promote post-translational modification of proteins (including lipid modification) can be preferably used.

[0043] For example, as will be described later in the examples, FKBP12 may be fused to the Gag protein, and FRB may be fused to the target protein. For example, when the target protein is the Cas9 protein, it is preferable to fuse FKBP12 to the N-terminal side of the Gag protein. Also, it is preferable to fuse FRB to the N-terminal side of the Cas9 protein.

[0044] The amino acid sequence of the FKBP12 protein is shown in SEQ ID NO: 5, and the amino acid sequence of the FRB protein is shown in SEQ ID NO: 6. The FKBP12 protein or the FRB protein may have mutations as long as the effects of the VLPs of the present embodiment can be obtained.

[0045] As the rapamycin derivative, for example, AP21967 (C-16-(S)-7-methylindolerapamycin) can be preferably used.

[0046] [Virus-like particles encapsulating the target protein and the target RNA] The VLP of the present embodiment may further encapsulate a target RNA in addition to the target protein. For example, the target protein may be a Cas family protein and the target RNA may be a gRNA. In this case, by introducing the VLP into a cell, genome editing can be induced at the target sequence site of the genomic DNA of the cell.

[0047] However, gRNA is usually a short-chain RNA with a length of about 100 bases or less. Conventionally, it has not been possible to efficiently encapsulate short-chain RNAs such as gRNA into VLPs.

[0048] In contrast, as will be described later in the examples, the inventors succeeded in efficiently encapsulating the mRNA containing the gRNA sequence and the packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence into VLPs by expressing the mRNA in VLP-producing cells. The above mRNA cuts out the gRNA portion by the self-cleavage activity of the ribozyme within the VLP or within the cell targeted for genome editing, and generates the gRNA as a self-cleavage product.

[0049] That is, the VLP of the present embodiment may further encapsulate an mRNA containing a gRNA sequence and a packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence, or a self-cleavage product of the above mRNA.

[0050] The packaging signal sequence is a base sequence essential for the retroviral genomic RNA to be incorporated into the virus particle. The packaging signal sequence of the retroviral genomic RNA has a characteristic secondary structure and is known to specifically bind to the nucleocapsid (NC) on the Gag protein.

[0051] As the packaging signal sequence, the Ψ sequence of retrovirus can be preferably used. It is also known that when the packaging signal sequence is an Extended packaging signal (Ψ+) including even the head portion of Gag, the encapsulation efficiency into VLPs is further improved. The base sequence of Ψ derived from HIV is shown in SEQ ID NO: 7. The base sequence of Ψ+ derived from HIV is shown in SEQ ID NO: 8.

[0052] In the above mRNA, the position of the packaging signal sequence is not particularly limited. The above mRNA may contain, for example, the packaging signal sequence, the first ribozyme sequence, the gRNA sequence, and the second ribozyme sequence in this order, or, for example, the first ribozyme sequence, the gRNA sequence, the second ribozyme sequence, and the packaging signal sequence in this order.

[0053] Furthermore, the above-mentioned mRNA may further include one or more arbitrary RNA sequences on the 5' side or 3' side of the packaging signal sequence. The arbitrary RNA sequence may, for example, encode a protein.

[0054] When the above-mentioned mRNA contains one arbitrary RNA sequence, the above-mentioned mRNA may, for example, contain an arbitrary RNA sequence, a packaging signal sequence, a first ribozyme sequence, a gRNA sequence, and a second ribozyme sequence in this order, or may contain a packaging signal sequence, an arbitrary RNA sequence, a first ribozyme sequence, a gRNA sequence, and a second ribozyme sequence in this order, or may contain a first ribozyme sequence, a gRNA sequence, a second ribozyme sequence, an arbitrary RNA sequence, and a packaging signal sequence in this order, or may contain a first ribozyme sequence, a gRNA sequence, a second ribozyme sequence, a packaging signal sequence, and an arbitrary RNA sequence in this order.

[0055] In addition, when the above-mentioned mRNA contains a plurality of arbitrary RNA sequences, each RNA sequence may be independently contained at a different position.

[0056] Normally, gRNA is transcribed from a polymerase III promoter such as a U6 promoter or an H1 promoter. Therefore, it is difficult to lengthen gRNA. In contrast, as will be described later in the examples, the inventors used a polymerase II promoter such as an LTR promoter or an EF1α promoter to express a long mRNA of 300 bases or more containing a packaging signal sequence, a first ribozyme sequence, a gRNA sequence, and a second ribozyme sequence in this order, and by excising the gRNA portion by the self-cleavage activity of the ribozyme, it was clarified that the encapsulation efficiency of gRNA into VLP increased dramatically.

[0057] In addition, as will be described later in the examples, by allowing VLP encapsulating Cas9 protein and gRNA to invade cells by this method, a much higher genome editing activity can be obtained compared with conventional VLP.

[0058] As the first ribozyme sequence and the second ribozyme sequence, a hammerhead (HH) ribozyme sequence, a hepatitis delta virus (HDV) ribozyme sequence, a Varkud satellite ribozyme, a hairpin ribozyme, a glmS ribozyme, etc. can be used. The first ribozyme sequence and the second ribozyme sequence may be the same sequence or different sequences from each other.

[0059] An example of the hammerhead (HH) ribozyme sequence is shown in SEQ ID NO: 9. Also, an example of the HDV ribozyme sequence is shown in SEQ ID NO: 10.

[0060] The gRNA may be a complex of a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), or a single gRNA (sgRNA) combining the tracrRNA and the crRNA.

[0061] When the gRNA is a complex of the crRNA and the tracrRNA, two types of mRNAs, an mRNA containing a packaging signal sequence, a first ribozyme sequence, a crRNA sequence, a second ribozyme sequence in this order, and an mRNA containing a packaging signal sequence, a first ribozyme sequence, a tracrRNA sequence, a second ribozyme sequence in this order, may be encapsulated in the VLP.

[0062] Alternatively, for example, one type of mRNA containing a packaging signal sequence, a first ribozyme sequence, a crRNA sequence, a second ribozyme sequence, a third ribozyme sequence, a tracrRNA sequence, a fourth ribozyme sequence in this order can also be encapsulated. Here, the third ribozyme sequence and the fourth ribozyme sequence may be the same sequences as the above-described first ribozyme sequence and second ribozyme sequence. Also, the positions of the crRNA sequence and the tracrRNA sequence can be interchanged.

[0063] As a result, within the VLP or within the cells to be induced for genome editing, crRNA and tracrRNA are generated by the self-cleavage activity of the ribozyme, and a complex of crRNA and tracrRNA is formed.

[0064] The base sequences of crRNA and tracrRNA can be, for example, the following base sequences. First, the base sequence obtained by removing the protospacer adjacent motif (PAM) sequence from the target base sequence is defined as the spacer base sequence. Subsequently, a base sequence in which a scaffold sequence is linked to the 3'-end of the spacer base sequence is designed as the base sequence of crRNA. For example, when the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 11), the base sequence of crRNA can be "5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUG-3'" (SEQ ID NO: 12). Also, the base sequence of tracrRNA can be, for example, "5'-CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'" (SEQ ID NO: 13).

[0065] Also, when the gRNA is sgRNA, the VLP may be encapsulated with an mRNA containing the base sequence of sgRNA sandwiched between the first ribozyme sequence and the second ribozyme sequence and a packaging signal sequence. As a result, within the VLP or within the cells to be induced for genome editing, sgRNA is generated by the self-cleavage activity of the ribozyme.

[0066] The base sequence of the sgRNA can be, for example, the following base sequence. First, the base sequence obtained by removing the PAM sequence from the target base sequence is defined as the spacer base sequence. Subsequently, a base sequence in which a scaffold sequence is linked to the 3'-end of the spacer base sequence is designed. For example, when the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 11), the base sequence of the sgRNA that specifically recognizes the target base sequence can be "5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3'" (SEQ ID NO: 14).

[0067] The VLP of the present embodiment can provide a novel genome editing treatment in the medical field. For example, by encapsulating CRISPR-Cas9 or CRISPR-Cas9 RNP in VLP and injecting it into muscle tissue, it becomes possible to induce the desired genome editing in muscle cells. Also, by injecting it into liver tissue, it becomes possible to induce the desired genome editing in liver cells.

[0068] In the present specification, genome editing means DNA cleavage, single-strand DNA cleavage, homologous recombination induction, base editing, induction or removal of DNA methylation, control of gene expression level, etc.

[0069] Also, genome editing treatments such as inoculating the collected cells, for example, hematopoietic stem cells, with VLP and re-transplanting the cells in which the desired genome editing has been induced are possible. Such a technique can be a novel treatment method for diseases caused by gene mutations. In addition, it is considered applicable to infectious disease treatments that destroy virus receptors and cancer treatments combined with CAR-T cells, etc.

[0070] Also, in the livestock and fisheries industries, by inoculating VLP into the germ cells, fertilized eggs, early embryos or individuals of the target animals, it is possible to induce breed improvement according to the genome editing site.

[0071] The VLP of this embodiment may further contain donor DNA (double-stranded DNA or single-stranded DNA) for inducing gene recombination by the homologous recombination (HR) pathway.

[0072] [Method for producing genome-edited cells] In one embodiment, the present invention provides a method for producing genome-edited cells, which includes inoculating the cells with a VLP encapsulating a Cas family protein and a gRNA. By the production method of this embodiment, genome-edited cells can be produced in a sequence-specific manner.

[0073] Here, inoculating the cells with the VLP means finally allowing the VLP to enter the cells. For example, it may be contacting the cells with the VLP, or adding the VLP to the cell culture medium, etc.

[0074] [Method for producing VLP encapsulating a target protein] In one embodiment, the present invention provides a method for producing a VLP encapsulating a target protein, which includes expressing in cells, in the presence of rapamycin or a rapamycin derivative, a combination of a fusion protein of FKBP12 and a Gag protein and a fusion protein of FRB and the target protein, or a combination of a fusion protein of FRB and a Gag protein and a fusion protein of FKBP12 and the target protein, and as a result, a VLP encapsulating the target protein is released into the culture medium of the cells. By the production method of this embodiment, a VLP encapsulating the target protein can be produced.

[0075] It can also be said that the production method of this embodiment is a method for producing a VLP encapsulating a target protein, including the following steps (1) and (2).

[0076] (1) In the presence of rapamycin or a rapamycin derivative, a step of expressing, in cells, a fusion protein of FKBP12 and a Gag protein, and a fusion protein of FRB and a target protein, or a fusion protein of FRB and a Gag protein, and a fusion protein of FKBP12 and a target protein. (2) A step of obtaining a medium containing VLPs encapsulating the target protein.

[0077] In the step (1), it is preferable to introduce the nucleic acid encoding the fusion protein into the cells using a lipofection method or an electroporation method (including a flow electroporation method). Further, after introduction of the nucleic acid, the cells may be treated with an endonuclease.

[0078] The flow electroporation method is an electroporation method capable of introducing DNA, mRNA, siRNA, proteins, etc. into various cells with high efficiency and high viability on a scale of 50 μL to 1 L. Therefore, by adopting the flow electroporation method, it becomes possible to easily mass-produce VLPs.

[0079] Further, as will be described later in the examples, by treating the cells with an endonuclease after introducing the nucleic acid by flow electroporation, the amount of VLP formation can be increased. This is presumably because the survival rate of the cells can be increased by removing the unintroduced nucleic acid by the endonuclease treatment.

[0080] In addition, for medical applications of VLPs, it is preferable to produce VLPs without using animal-derived components (xeno-free). Therefore, the cell culture medium is preferably a serum-free medium.

[0081] In the production method of this embodiment, the fusion protein of FKBP12 and Gag protein, the fusion protein of FRB and the target protein, the fusion protein of FRB and Gag protein, and the fusion protein of FKBP12 and the target protein may be expressed by an expression vector that functions in VLP-producing cells. In addition, the VLPs released into the medium can be concentrated by, for example, ultracentrifugation, polyethylene glycol (PEG) precipitation, column chromatography, ion exchange chromatography, etc.

[0082] In the production method of this embodiment, the VLPs, the target protein, the rapamycin derivative, the Gag protein, FKBP12, and FRB are the same as those described above. In addition, as the VLP-producing cells, human cells and non-human animal cells can be used. However, when inoculating the living body with VLPs, from the viewpoint of reducing immunogenicity, it is preferable to use cells derived from the same species as the inoculation target species as the VLP-producing cells. Specific VLP-producing cells are not particularly limited, and for example, HEK293T cells, HEK293 cells, etc., which are cell lines derived from human fetal kidneys, can be used.

[0083] According to the production method of this embodiment, the target protein is efficiently encapsulated in VLPs as compared with the prior art. Therefore, more target proteins can be encapsulated (included) by VLPs. The number of target proteins that can be encapsulated per VLP particle is, for example, 3 molecules or more, preferably 4 molecules or more, more preferably 5 molecules or more, still more preferably 6 molecules or more, and most preferably 7 molecules or more.

[0084] [Method for Producing VLPs Encapsulating Cas Family Protein and gRNA] 1 In one embodiment, the present invention provides a method for producing VLPs encapsulating a Cas family protein and a gRNA, the method comprising expressing, in the presence of rapamycin or a rapamycin derivative, in a cell, a combination of a fusion protein of FKBP12 and a Gag protein and a fusion protein of FRB and a Cas family protein, or a combination of a fusion protein of FRB and a Gag protein and a fusion protein of FKBP12 and a Cas family protein, and an mRNA comprising a gRNA sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence, whereby VLPs encapsulating the Cas family protein and the gRNA are released into the culture medium of the cell.

[0085] The production method of this embodiment can also be said to be a method for producing VLPs encapsulating a Cas family protein and a gRNA, the method including the following steps (1) and (2).

[0086] (1) A step of expressing, in the presence of rapamycin or a rapamycin derivative, in a cell, a combination of a fusion protein of FKBP12 and a Gag protein and a fusion protein of FRB and a Cas family protein, or a combination of a fusion protein of FRB and a Gag protein and a fusion protein of FKBP12 and a Cas family protein, and an mRNA having a gRNA sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence. (2) A step of obtaining a culture medium containing the VLPs encapsulating the Cas family protein and the gRNA.

[0087] In the step (1), it is preferable to introduce the fusion protein or the nucleic acid encoding the mRNA into the cells using a lipofection method or an electroporation method (including a flow electroporation method). Further, after the introduction of the nucleic acid, the cells may be treated with an endonuclease. Also, the cell culture medium is preferably a serum-free medium. The flow electroporation method and the endonuclease treatment are the same as those described above.

[0088] According to the production method of the present embodiment, the Cas family protein and the gRNA are efficiently encapsulated in the VLP as compared with the prior art. Therefore, a larger number of complexes (ribonucleoprotein, RNP) of the Cas family protein and the gRNA can be encapsulated by the VLP. The number of RNPs that can be encapsulated per VLP particle is, for example, 3 molecules or more, preferably 4 molecules or more, more preferably 5 molecules or more, still more preferably 6 molecules or more, and most preferably 7 molecules or more.

[0089] By the production method of the present embodiment, VLPs encapsulated with the Cas family protein and the gRNA can be produced. Also, the VLPs released into the medium can be concentrated by, for example, ultracentrifugation, PEG precipitation, column chromatography, ion exchange chromatography, etc.

[0090] In the production method of the present embodiment, the VLP, the Cas family protein, the gRNA, the rapamycin derivative, the Gag protein, FKBP12, FRB, the packaging signal sequence, the ribozyme sequence, and the VLP-producing cells are the same as those described above.

[0091] In the production method of the present embodiment, the mRNA containing the fusion protein of FKBP12 and Gag protein, the fusion protein of FRB and Cas family protein, the fusion protein of FRB and Gag protein, the fusion protein of FKBP12 and Cas family protein, the gRNA sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence, and the packaging signal sequence may be expressed by an expression vector that functions in VLP-producing cells.

[0092] In particular, the mRNA containing the gRNA sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence and the packaging signal sequence is preferably expressed by a polymerase II promoter. Examples of the polymerase II promoter include an LTR promoter (5'LTR of lentivirus), an EF1α promoter, etc. As the LTR promoter, the 5'LTR of HIV can be particularly preferably used.

[0093] When the mRNA containing the gRNA sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence and the packaging signal sequence is expressed by an LTR promoter, it is preferable to express the Tat protein in VLP-producing cells.

[0094] [Kit for producing VLP encapsulating a target protein] In one embodiment, the present invention provides a kit for producing VLP encapsulating a target protein, which includes an expression vector of a fusion protein of FKBP12 and Gag protein or an expression vector of a fusion protein of FRB and Gag protein. With the kit of the present embodiment, VLP encapsulating a target protein can be produced.

[0095] In the kit of the present embodiment, VLP, target protein, Gag protein, FKBP12, and FRB are the same as those described above.

[0096] The kit of this embodiment may further include a vector for preparing an expression vector of a fusion protein of FKBP12 and a target protein, an expression vector of a fusion protein of FKBP12 and a target protein, a vector for preparing an expression vector of a fusion protein of FRB and a target protein, an expression vector of a fusion protein of FRB and a target protein, and the like.

[0097] Here, the vector for preparing an expression vector of a fusion protein of FKBP12 and a target protein includes, for example, a vector containing a gene encoding FKBP12 and a multiple cloning site downstream of a promoter.

[0098] In this specification, the multiple cloning site is a region in which one or more base sequences recognized by restriction enzymes are arranged. In the multiple cloning site, there may be one restriction enzyme site or a plurality of restriction enzyme sites.

[0099] By incorporating a gene encoding a target protein into the multiple cloning site of the above vector, an expression vector of a fusion protein of FKBP12 and a target protein can be prepared. Here, the multiple cloning site may be located on the 5' side or the 3' side of the gene encoding FKBP12.

[0100] Similarly, the vector for preparing an expression vector of a fusion protein of FRB and a target protein includes, for example, a vector containing a gene encoding FRB and a multiple cloning site downstream of a promoter. By incorporating a gene encoding a target protein into this multiple cloning site, an expression vector of a fusion protein of FRB and a target protein can be prepared. Here, the multiple cloning site may be located on the 5' side or the 3' side of the gene encoding FRB.

[0101] The kit of this embodiment may further include rapamycin or a rapamycin derivative, cells for producing VLP, etc. Rapamycin or a rapamycin derivative and cells for producing VLP are the same as those described above.

[0102] [Kit for Producing VLP Encapsulating Cas Family Protein] In one embodiment, the present invention provides a kit for producing VLP encapsulating a Cas family protein, which includes a combination of an expression vector for a fusion protein of FKBP12 and a Gag protein and an expression vector for a fusion protein of FRB and a Cas family protein, or a combination of an expression vector for a fusion protein of FRB and a Gag protein and an expression vector for a fusion protein of FKBP12 and a Cas family protein. With the kit of this embodiment, VLP encapsulating a Cas family protein can be produced.

[0103] In the kit of this embodiment, VLP, Cas family protein, Gag protein, FKBP12, and FRB are the same as those described above.

[0104] For example, as described later in the examples, FKBP12 may be fused to the Gag protein and FRB may be fused to the Cas family protein. In this case, it is preferable to fuse FKBP12 to the N-terminal side of the Gag protein. Also, it is preferable to fuse FRB to the N-terminal side of the Cas family protein.

[0105] The kit of this embodiment may further include an mRNA expression vector containing a base sequence of a target RNA or a multiple cloning site and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence. In this case, with the kit of this embodiment, VLP encapsulating a Cas family protein and a target RNA can be produced.

[0106] Here, the packaging signal sequence and the ribozyme sequence are the same as those described above. Also, as the target RNA, short-chain RNA can be mentioned, for example, gRNA. In this specification, as the short-chain RNA, for example, RNA having a length of about 200 bases or less, for example, about 150 bases or less, for example, about 100 bases or less can be mentioned.

[0107] By incorporating a DNA fragment encoding the target RNA into the multiple cloning site of an mRNA expression vector containing a multiple cloning site and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence, an mRNA expression vector containing the nucleotide sequence of the target RNA and the packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence can be prepared.

[0108] The kit of this embodiment may further contain rapamycin or a rapamycin derivative, cells for VLP production, etc. Rapamycin or a rapamycin derivative and cells for VLP production are the same as those described above.

[0109] [VLP encapsulating the target RNA] In one embodiment, the present invention provides a VLP in which the target RNA is encapsulated, the VLP contains a Gag protein, and the target RNA is encapsulated in the form of an mRNA containing the nucleotide sequence of the target RNA sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence or a self-cleavage product of the mRNA.

[0110] The VLP of this embodiment is mainly different from the above-described VLP in that the target RNA is encapsulated instead of the target protein.

[0111] As will be described later in the examples, according to the VLP of this embodiment, the target RNA can be efficiently encapsulated. By allowing the VLP of this embodiment to enter cells, the target RNA can be efficiently introduced into the cells.

[0112] Examples of the target RNA include short-chain RNAs. In this specification, examples of short-chain RNAs include RNAs having a length of about 200 bases or less, about 150 bases or less, or about 100 bases or less. Examples of the target RNA include siRNA, shRNA, miRNA, gRNA, and the like.

[0113] In the VLP of this embodiment, the VLP, Gag protein, packaging signal sequence, ribozyme sequence, and gRNA are the same as those described above.

[0114] [Method for Producing VLP Encapsulating Target RNA] In one embodiment, the present invention provides a method for producing a VLP encapsulating a target RNA, the method including expressing in a cell an mRNA containing a Gag protein, a base sequence of a target RNA sandwiched between a first ribozyme sequence and a second ribozyme sequence, and a packaging signal sequence, whereby virus-like particles encapsulating the target RNA are released into the culture medium of the cell, and the target RNA is encapsulated in the virus-like particles in the form of the mRNA or a self-cleavage product of the mRNA.

[0115] The production method of this embodiment can also be said to be a method for producing a VLP encapsulating a target RNA, including the following steps (1) and (2).

[0116] (1) A step of expressing in a cell an mRNA having a Gag protein, a base sequence of a target RNA sandwiched between a first ribozyme sequence and a second ribozyme sequence, and a packaging signal sequence. (2) A step of obtaining a culture medium containing VLP in which the target RNA is encapsulated in the form of the mRNA or a self-cleavage product of the mRNA.

[0117] In the step (1), it is preferable to introduce the nucleic acid encoding the mRNA into the cells using a lipofection method or an electroporation method (including a flow electroporation method). Further, after the introduction of the nucleic acid, the cells may be treated with an endonuclease. Also, the cell culture medium is preferably a serum-free medium. The flow electroporation method and the endonuclease treatment are the same as those described above.

[0118] By the production method of the present embodiment, VLPs encapsulating the target RNA can be produced. In the production method of the present embodiment, the VLPs, Gag protein, packaging signal sequence, first ribozyme sequence, target RNA, second ribozyme sequence, and VLP-producing cells are the same as those described above. Also, the VLPs released into the medium can be concentrated by, for example, ultracentrifugation, PEG precipitation, column chromatography, ion exchange chromatography, etc.

[0119] According to the production method of the present embodiment, the target RNA is efficiently encapsulated in the VLPs as compared with the prior art. Therefore, more target RNA can be encapsulated by the VLPs. The target RNA that can be encapsulated per VLP particle is, for example, 3 molecules or more, preferably 4 molecules or more, more preferably 5 molecules or more, still more preferably 6 molecules or more, particularly preferably 7 molecules or more, and most preferably 8 molecules or more.

[0120] [Kit for producing VLPs encapsulating target RNA] In one embodiment, the present invention provides a kit for producing VLPs encapsulating a target RNA, the kit comprising an expression vector for a Gag protein, and an expression vector for an mRNA comprising the nucleotide sequence of the target RNA or a multiple cloning site and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence. By the kit of the present embodiment, VLPs in which the target RNA is efficiently encapsulated can be produced.

[0121] In the kit of this embodiment, the VLP, target RNA, packaging signal sequence, ribozyme sequence, and multiple cloning site are the same as those described above.

[0122] As described above, by incorporating a DNA fragment encoding the target RNA into the multiple cloning site of the mRNA expression vector containing the multiple cloning site and the packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence, an mRNA expression vector containing the base sequence of the target RNA and the packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence can be prepared.

[0123] Also, as described above, when an mRNA containing the Gag protein, and the base sequence of the target RNA and the packaging signal sequence sandwiched between the first ribozyme sequence and the second ribozyme sequence is expressed in VLP-producing cells, the packaging signal sequence on the mRNA takes a characteristic secondary structure, specifically binds to the nucleocapsid (NC) on the Gag protein, and is efficiently encapsulated into the VLP.

[0124] The kit of this embodiment may further include cells for VLP production and the like. The cells for VLP production are the same as those described above.

[0125] [Therapeutic agent for diseases, infectious diseases or cancers caused by gene mutations] 1. In one embodiment, the present invention provides a therapeutic agent for a disease, an infectious disease, or cancer caused by a gene mutation, the therapeutic agent containing the above-described virus-like particles as an active ingredient. Examples of diseases caused by gene mutations include, but are not limited to, Duchenne muscular dystrophy (DMD), myotonic dystrophy, facioscapulohumeral muscular dystrophy, hemophilia, congenital encephalopathy, phenylketonuria, biopterin metabolism disorder, maple syrup urine disease, homocystinuria, vitiligo, xeroderma pigmentosum, etc. By administering the therapeutic agent of the present embodiment and inducing exon skipping by genome editing, repairing gene mutations, or deleting abnormal genes, diseases caused by gene mutations can be treated.

[0126] Examples of infectious diseases include AIDS, hepatitis B, EBV infection, etc. By administering the therapeutic agent of the present embodiment and destroying the genes of infected viruses or bacteria, or by destroying the receptor genes necessary for virus or bacteria infection, infectious diseases can be treated.

[0127] As used herein, "cancer" means not only cancers arising from epithelial cells but also all malignant tumors. Examples of cancers include congenital pediatric cancers, etc. By administering the therapeutic agent of the present embodiment and destroying genes essential for the survival of cancer cells or cancer genes, or by repairing mutations in tumor suppressor genes, cancer can be treated.

[0128] The administration method and dosage of the therapeutic agent of the present embodiment are not particularly limited and may be appropriately determined according to the patient's symptoms, weight, age, gender, etc. For example, it can be administered intravenously, intraarterially, intramuscularly, etc. in the form of an injection.

[0129] [Other Embodiments] 1. In one embodiment, the present invention provides a method for treating a disease, an infectious disease, or cancer caused by a gene mutation, the method including administering an effective amount of a VLP encapsulating a target protein, a VLP encapsulating a target RNA, or a VLP encapsulating a target protein and a target RNA to a patient in need of treatment.

[0130] In one embodiment, the present invention provides a VLP encapsulating a target protein, a VLP encapsulating a target RNA, or a VLP encapsulating a target protein and a target RNA for the treatment of a disease, an infectious disease, or cancer caused by a gene mutation.

[0131] In one embodiment, the present invention provides the use of a VLP encapsulating a target protein, a VLP encapsulating a target RNA, or a VLP encapsulating a target protein and a target RNA for manufacturing a therapeutic agent for a disease caused by a gene mutation, a therapeutic agent for an infectious disease, or a therapeutic agent for cancer.

[0132] In each of the above embodiments, the VLP encapsulating a target protein, the VLP encapsulating a target RNA, and the VLP encapsulating a target protein and a target RNA are the same as those described above.

[0133] In each of the above embodiments, the VLP is a VLP encapsulating a target protein or a VLP encapsulating a target protein and a target RNA. The target protein may be a Cas family protein, and the target RNA may be a gRNA. Alternatively, the VLP is a VLP encapsulating a target RNA, and the target RNA may be siRNA, shRNA, miRNA, gRNA, etc.

Example

[0134] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0135] [Experimental method] (Cell culture) HEK293T cells, which are human fetal kidney-derived cell lines, and HEK293T cells transfected with the reporter construct EGxxFP (hereinafter sometimes referred to as "HEK293T EGxxFP cells"). HEK293T EGxxFP cells that constitutively express sgRNA DMD#1 targeting the 5'-side (near the splicing acceptor) of exon 45 of the human DMD gene (the target sequence is shown in SEQ ID NO: 15) were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin.

[0136] Healthy donor-derived iPS cells (404C2 strain and 138D2 strain) were cultured in StemFit AK03N medium (Ajinomoto Health Supply Co., Ltd.) on culture dishes coated with iMatix511-E8. Duchenne muscular dystrophy (DMD) patient-derived iPS cells were cultured in StemFit AK03N medium on culture dishes coated with iMatix511-E8.

[0137] C2C12 cells, which are mouse skeletal muscle-derived cell lines, were cultured in DMEM medium containing 15% fetal bovine serum, 0.1 mM essential amino acids, 100 mM sodium pyruvate, 100 mM 2-mercaptoethanol, penicillin, and streptomycin.

[0138] DMEM medium containing 5% horse serum, 0.1 mM essential amino acids, 100 mM sodium pyruvate, 100 mM 2-mercaptoethanol, 0.5% penicillin, and streptomycin was used as the differentiation induction medium for C2C12 cells.

[0139] (Method for producing VLP) 3×10 6 HEK293T cells were seeded on a 10-cm plate. Subsequently, the next day, pHLS-EF1a-FKBP12-Gag was transfected using lipofectamine 2000 (Thermo Fisher Scientific). HIV(SEQ ID NO: 16) 10 μg, pHLS-EF1a-FRB-SpCas9-A (SEQ ID NO: 17) 10 μg, pL-sin-RGR-AmCyan-A (SEQ ID NO: 18) 10 μg, pcDNA3.1-Tat HIV (SEQ ID NO: 19) 2 μg and pMD-VSVG (SEQ ID NO: 20) 5 μg were transfected. Note that each of these expression vectors was appropriately changed by experiments.

[0140] Subsequently, the medium was replaced with 10 mL of a new medium containing 300 nM of AP21967 (Clontech) the next day. Subsequently, the culture supernatant was collected 36 to 48 hours after transfection, cell debris was removed with a syringe filter with a pore size of 0.45 μm, and centrifuged at 100,000 × g for 3 hours using an Avanti JXN-30 centrifuge (Beckman Coulter) to concentrate the VLP.

[0141] Subsequently, the precipitate containing the VLP was resuspended in 100 μL of HBSS (Thermo Fisher Scientific), dispensed into 1.5 mL tubes, and stored at -80°C.

[0142] (Cell introduction of VLP) 2.5 × 10 4 ~5.0 × 10 4 Individual HEK293T EGxxFP cells were seeded in a 48-well plate. Subsequently, the VLP solution was added the next day, and the fluorescence of EGFP was analyzed using an LSR3 Flow Cytometer 3 days later.

[0143] (T7EI assay) Genomic DNA was recovered from cultured cells according to the protocol using a MonoFas Genomic DNA Extraction kit (GL Sciences Inc.). Subsequently, PCR was performed using 100 ng of genomic DNA, primers for the amplification region, and PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.), and the PCR product was purified using a Wizard SV Gel and PCR Clean-up System (Promega).

[0144] Subsequently, 400 ng of the PCR product was heat-denatured at 95°C for 5 minutes in NEBuffer 2.1 buffer (NEB), and then re-annealed by slowly lowering the temperature. The temperature was decreased from 95°C to 85°C at -2°C / second and from 85°C to 25°C at -0.1°C / second. Subsequently, 10 units of T7 endonuclease I (T7EI) enzyme was added and reacted at 37°C for 15 minutes. Subsequently, EDTA was added to a concentration of 6 mM to stop the reaction. Subsequently, the cleavage products were analyzed using DS1000 High Sensitivity Screen Tape and TapeStation 2200 (Agilent Technologies).

[0145] [Experimental Example 1] (Examination of Cas9 Delivery by VLP) VLP using Gag-Pol or Gag was used to deliver Cas9 protein, and an SSA-EGFP reporter experiment was performed to evaluate the genome editing efficiency.

[0146] Figure 1(a) is a schematic diagram showing the structure of HIV Gag-Pol (hereinafter sometimes referred to as "WT Gag-Pol".). As shown in Figure 1(a), the Gag portion is composed of matrix (MA), capsid (CA, also called p24), nucleocapsid (NC), and p6 (transframe). In addition, Pol is composed of protease (PR), reverse transcriptase (RT), RNase H (RN), and integrase (IN). HIV ). Figure 1(b) is a schematic diagram showing the structure of a fusion protein (hereinafter sometimes referred to as "FKBP12-Gag-Pol".) with an FKBP12 domain added to the N-terminus of HIV Gag-Pol. FKBP12 is an FK506-binding protein.

[0147] Figure 1(c) is a schematic diagram showing the structure of a fusion protein (hereinafter sometimes referred to as "FKBP12-Gag".) with an FKBP12 domain added to the N-terminus of HIV Gag. HIV ).

[0148] Figure 1(c) is a schematic diagram showing the structure of a fusion protein (hereinafter sometimes referred to as "FKBP12-Gag".) with an FKBP12 domain added to the N-terminus of HIV Gag. HIV(There are cases where it is referred to as "」.) It is a schematic diagram showing the structure of

[0149] Protease (PR), which is part of Pol, originally has the activity to cleave specific sites of the Gag-Pol protein (indicated by scissors marks in Fig. 1). On the other hand, in the VLP from which Pol containing PR was removed, although the protease target sequence remains, since protease is not supplied, protein cleavage does not occur.

[0150] Fig. 2 is a schematic diagram explaining the SSA-EGFP (hereinafter may be referred to as "EGxxFP") reporter experiment. As the cells, HEK293T cells that constantly express an sgRNA targeting the human Dystrophin (DMD) gene and into which the EGxxFP reporter construct (SEQ ID NO: 21) has been inserted into the genome were used. The EGxxFP reporter construct is a construct designed such that a sequence containing the target sequence of the sgRNA is inserted into the EGFP cDNA sequence, so that a functional EGFP protein is not expressed.

[0151] As shown in Fig. 2, when VLPs encapsulating Cas9 are introduced into these cells, a complex of the Cas9 protein and the sgRNA (RNP complex) is formed, and the inserted sequence is cleaved. Subsequently, the cleaved EGFP cDNA is repaired by single strand annealing (SSA), and as a result, functional EGFP is expressed and the fluorescence of EGFP can be observed. In this experimental system, by quantifying the fluorescence of EGFP in the cells with a flow cytometer, the cleavage efficiency of the target gene by the Cas9 protein (genome editing efficiency) in the cell population can be evaluated.

[0152] First, in the presence or absence of darunavir (a protease inhibitor of HIV), wild-type Gag-Pol derived from HIV HIV or FKBP12-Gag-Pol HIVIt was composed of, and VLPs (Figs. 1(a) and (b)) encapsulating a Cas9 protein with a fused FRB domain (hereinafter sometimes referred to as "FRB-Cas9 protein") were prepared. Note that the FKBP12 domain and the FRB (FKBP12-rapamycin associated protein1, FRAP1 fragment) domain are known to form a heterodimer in the presence of a rapamycin analog.

[0153] Subsequently, each of the prepared VLPs (15 μL) was introduced into HEK293T EGxxFP reporter cells (5×10 4 cells, with sgRNA expression). Subsequently, three days later, the proportion of GFP-positive cells was analyzed by flow cytometry.

[0154] Figure 3 is a graph showing the analysis results by flow cytometry. In Figure 3, the vertical axis represents the proportion of GFP-positive cells. As a result, it became clear that the genome editing efficiency by FRB-Cas9 delivery was higher in FKBP12-Gag-Pol HIV VLPs than in WT Gag-Pol HIV VLPs (comparison between the groups without darunavir addition). Furthermore, when darunavir, an HIV protease inhibitor, was added to the cells during VLP preparation, it became clear that the genome editing efficiency was further improved in the cells transfected with FKBP12-Gag-Pol HIV VLPs (in Figure 3, the arrow indicates the improvement in genome editing efficiency).

[0155] Subsequently, FKBP12-Gag-Pol HIV in each of the above VLPs and the FRB-Cas9 protein with an HA tag were quantified by Western blotting. Figure 4 is a photograph showing the results of Western blotting. As shown in Figure 4, in the case where darunavir, an HIV protease inhibitor, was added to the cells during VLP preparation, compared with the case without darunavir addition, FKBP12-Gag-Pol HIVThe amounts of the FRB-Cas9 protein with the HA tag (「Full length」 in Fig. 4) and 「p55+12」 (in Fig. 4) were significantly increased. On the other hand, in the absence of darunavir, lower molecular weight molecular species (「p41+12」, 「p24 Capsid」, 「Cleaved 1,2」 in Fig. 4) were increased compared to those with darunavir addition (comparison between + and - darunavir). That is, it was revealed that the cleavage of the Gag protein and the degradation of the Cas9 protein were suppressed by the addition of darunavir.

[0156] Subsequently, VLP encapsulating the FRB-Cas9 protein was prepared using FKBP12-Gag-Pol HIV from which the Pol portion was deleted, FKBP12-Gag HIV ((c) in Fig. 1), and the genome editing efficiency was examined by the SSA-EGFP reporter experiment. Fig. 5 is a graph showing the analysis results by a flow cytometer. In Fig. 5, the vertical axis indicates the ratio of GFP-positive cells.

[0157] As a result, it was revealed that the VLP of FKBP12-Gag HIV has a higher genome editing efficiency by FRB-Cas9 delivery than the VLP of FKBP12-Gag-Pol HIV .

[0158] From these results, it was also revealed that when the Gag protein is expressed in a form containing Pol, not only the Gag protein but also the Cas9 protein is degraded by the action of the protease in Pol. Therefore, in the conventional VLP (for example, Non-Patent Document 1) produced by the method of expressing the Gag protein in a form containing Pol, the amount of the Cas9 protein retained in the VLP is small because the Cas9 protein is degraded by the protease expressed from Pol, and thus it is considered that a sufficient level of genome cleavage efficiency could not be obtained.

[0159] [Experimental Example 2] (Examination 1 of the FKBP12 domain binding site) To deliver FRB-Cas9, we considered where to localize the FKBP12 domain in the VLP.

[0160] Figure 6(a) is a schematic diagram showing the structure of an expression vector (pHLS-EF1a-VSVG-FKBP12) of a VSVG-FKPB12 fusion protein in which the FKBP12 domain is fused to the C-terminus (cytoplasmic domain side) of VSV-G (vesicular stomatitis virus envelope protein), and the structure of a VLP containing the VSVG-FKPB12 fusion protein. VSV-G has a broad tropism and is an envelope protein generally used for producing pseudotyped viruses of lentiviruses and retroviruses, and is the main component of the VLP outer shell according to the present disclosure. Therefore, the FKBP12 domain portion of the fusion protein expressed from this vector exists in a protruding form on the inner membrane of the VLP outer shell (membrane localization).

[0161] Figure 6(b) is a schematic diagram showing the structure of an expression vector of an FKBP12-EGFP fusion protein in which the FKBP12 domain is fused to the N-terminus of EGFP, and the structure of a VLP containing the FKBP12-EGFP fusion protein. The FKBP12 domain portion of the fusion protein expressed from this vector exists inside the VLP (cytoplasmic localization).

[0162] Figure 6(c) shows the FKBP12 domain fused to the N-terminus of Gag HIV FKBP12-Gag with a myristoylation signal at the N-terminus HIV A schematic diagram showing the structure of an expression vector of the fusion protein and the structure of a VLP containing the FKBP12-Gag HIV fusion protein. The FKBP12 domain portion of the fusion protein expressed from this vector exists in a state of being bound to the inside of the VLP outer shell membrane (inner membrane localization).

[0163] First, in the presence or absence of AP21967, a rapamycin analog, VSVG-FKPB12 fusion protein, FKBP12-EGFP fusion protein, FKBP12-Gag HIVVLP containing each of the fusion proteins and encapsulating FRB-Cas9 were prepared.

[0164] Subsequently, the amount of FRB-Cas9 protein contained in each VLP was quantified. Specifically, each concentrated VLP was dissolved in Lysis buffer (120 nM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1% Triton X-100, protease inhibitor), and Western blotting using an anti-Cas9 antibody was performed using the fully automated Western blot device Wes from ProteinSimple. Fig. 7 is an image showing the results of Western blotting.

[0165] As a result, as shown by the arrow in Fig. 7, the amount of Cas9 encapsulated in the VLP was higher in the VLP conjugated with Gag HIV (localized inside the membrane) than in the VLP conjugated with VSV-G (membrane-localized) or EGFP (cytoplasm-localized).

[0166] Subsequently, to assay the amount of Cas9 protein contained in the VLP, the DNA cleavage activity in vitro was measured. Specifically, Lysis buffer was added to each concentrated VLP, and the VLP was dissolved by standing on ice for 10 minutes.

[0167] Subsequently, the VLP lysate containing Cas9 protein was mixed with DNA (700 bp) containing the dystrophin target sequence and sgRNA DMD#1 targeting the 5' side (near the splicing acceptor) of exon 45 of the human DMD gene prepared by in vitro transcription (IVT) reaction (the target sequence is shown in SEQ ID NO: 15) in a buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.5% BSA), and reacted at 37°C for 1 hour. After the reaction, RNase A was added to remove the sgRNA and reacted at 37°C for 30 minutes. Furthermore, proteinase K was added to remove the protein and reacted at 50°C for 20 minutes.

[0168] Subsequently, these samples were electrophoresed using a 2200 TapeStation (high sensitivity D1000 TapeScreen, Agilent Technologies), and the ratio of DNA containing the dystrophin target sequence that was cleaved was measured.

[0169] Figure 8 is an image showing the results of electrophoresis. Figure 9 is a graph quantifying the results of Figure 8. As a result, the VLP containing FKBP12-Gag HIV had the highest cleavage activity, and it was revealed that the encapsulation efficiency of Cas9 into the VLP was improved by the addition of AP21967, which induces dimerization of FKBP12 and FRB.

[0170] [Experimental Example 3] (Examination of the Binding Site of the FKBP12 Domain 2) To deliver FRB-Cas9, it was examined where the FKBP12 domain should be bound to the VLP.

[0171] After introducing each VLP similar to Experimental Example 2 into HEK293T EGxxFP cells that constantly expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15), the cleavage activity of the target DMD gene on the genome was measured by the T7EI assay.

[0172] Specifically, first, each VLP was introduced into HEK293T EGxxFP cells that constantly expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15). Subsequently, genomic DNA was extracted from each cell after 3 days. Subsequently, the target region of the DMD gene on the genome was amplified by PCR. Subsequently, the PCR product was column-purified, and using 400 ng of the obtained DNA, the mutagenesis efficiency was measured by the T7EI assay.

[0173] Figure 10 is an image showing the results of the T7EI assay. Further, Figure 11 is a graph quantifying the results of Figure 10. Further, Figure 12 is a graph showing the results of analyzing the percentage of GFP-positive cells from the EGxxFP reporter construct in each cell of this experimental example using a flow cytometer.

[0174] As a result, the VLP containing FKBP12-Gag HIV had the highest cleavage activity, and it was revealed that the encapsulation efficiency of Cas9 into the VLP was improved by the addition of AP21967 that induces dimerization of FKBP12 and FRB.

[0175] [Experimental Example 4] (Examination 1 of Encapsulation Efficiency of Cas9 Protein into VLP) VLPs encapsulating Cas9 protein using Gag derived from MLV (Murine Leukemia Virus) (hereinafter sometimes referred to as "FKBP12-Gag MLV VLP") and VLPs encapsulating Cas9 protein using Gag derived from HIV (hereinafter sometimes referred to as "FKBP12-Gag HIV VLP") were prepared, and the delivery efficiency of Cas9 protein was examined.

[0176] Specifically, 10 μL of each VLP was inoculated into HEK293T EGxxFP cells (2.5×10 4 cells) that constantly expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15). As a control, cells not inoculated with VLP were prepared. Subsequently, 3 days later, the fluorescence of the EGxxFP reporter construct in each cell sample was analyzed using a flow cytometer.

[0177] Figures 13(a) to (c) are graphs showing the analysis results by a flow cytometer. Figure 13(a) shows the results of control cells, Figure 13(b) shows the results of inoculation with FKBP12-Gag MLV VLP, and Figure 13(c) shows the results of inoculation with FKBP12-Gag HIV VLP. As a result, FKBP12-GagMLV FKBP12-Gag rather than VLP HIV It was revealed that VLP has a higher delivery efficiency of Cas9 protein.

[0178] [Experimental Example 5] (Examination of encapsulation efficiency of Cas9 protein into VLP 2) In the presence or absence of AP21967, VLPs containing Cas9 with an FRB domain bound to the N-terminus, Cas9 with an FRB domain bound to the C-terminus, and Cas9 with an FRB domain bound to both the N-terminus and the C-terminus were prepared, and the encapsulation efficiency of Cas9 protein into the VLPs was examined.

[0179] AP21967 is known to specifically bind to FRB(T2098L) in which the 2098th residue threonine of the FRB domain is replaced with leucine. Therefore, FRB(T2098L) was used as the FRB.

[0180] Specifically, HEK293T EGxxFP cells (2.5×10 4 cells) that constantly expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) were inoculated with 150 ng of each VLP equivalent to the amount of p24. Subsequently, three days later, the fluorescence of the EGxxFP reporter construct of each cell sample was analyzed by a flow cytometer.

[0181] Figure 14 is a graph showing the analysis results by a flow cytometer. In Figure 14, the vertical axis represents the percentage of GFP-positive cells. As a result, AP21967-dependent delivery of Cas9 protein was observed in all VLPs. Also, it was revealed that the delivery efficiency of Cas9 protein was the highest when the FRB domain was bound to the N-terminus of the Cas9 protein.

[0182] [Experimental Example 6] (Examination of encapsulation efficiency of Cas9 protein into VLP 3) The encapsulation efficiency of Cas9 protein into VLPs was examined in the case where Cas9 protein was encapsulated into VLPs through the binding of FKBP12 domain and FRB domain, and in the case where Cas9 protein was directly fused to Gag and encapsulated into VLPs.

[0183] Figure 15(a) is a schematic diagram showing the structure of the fusion protein of FRB domain and Cas9 protein, and the structure of the fusion protein of FKBP12 domain and Gag. HIV In this case, Cas9 protein is encapsulated into VLPs by dimerization via AP21967.

[0184] Also, Figure 15(b) is a schematic diagram showing the structure of the fusion protein of Gag and Cas9 protein, and the structure of GAG-Pol. MLV In this case, GAG-Pol was added to promote VLP particle formation. MLV And Figure 15(c) is a schematic diagram showing the structure of the fusion protein of Cas9 protein and Gag-Pol, and the structure of GAG-Pol. MLV In this case, GAG-Pol was added to promote VLP particle formation.

[0185] Also, Figure 15(d) is a graph showing the analysis results by flow cytometer. In Figure 15(d), the vertical axis represents the percentage of GFP-positive cells. As a result, it was revealed that the delivery efficiency of Cas9 protein was higher in the VLPs prepared using the binding of FKBP12 domain and FRB domain than in the VLPs prepared by directly fusing Cas9 protein to Gag or Gag-Pol. HIV And Figure 15(c) is a schematic diagram showing the structure of the fusion protein of Cas9 protein and Gag-Pol, and the structure of GAG-Pol. HIV In this case, GAG-Pol was added to promote VLP particle formation. HIV In this case, GAG-Pol was added to promote VLP particle formation.

[0186] Specifically, each VLP was inoculated into HEK293T EGxxFP cells that constitutively expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15). Subsequently, three days later, the fluorescence of the EGxxFP reporter construct in each cell sample was analyzed by a flow cytometer.

[0187] Figure 15(d) is a graph showing the analysis results by flow cytometer. In Figure 15(d), the vertical axis represents the percentage of GFP-positive cells. As a result, it was revealed that the delivery efficiency of Cas9 protein was higher in the VLPs prepared using the binding of FKBP12 domain and FRB domain than in the VLPs prepared by directly fusing Cas9 protein to Gag or Gag-Pol.

[0188] [Experimental Example 7] (Examination of the Concentration of AP21967) VLP was prepared by changing the concentration of AP21967, a hetero-bivalent ligand, to 0, 3, 30, and 300 nM. Subsequently, HEK293T EGxxFP cells (5×10 4 cells) that constantly expressed sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) were inoculated with each VLP. Subsequently, three days later, the fluorescence of the EGxxFP reporter construct in each cell sample was analyzed by a flow cytometer.

[0189] Figures 16 and 17 are graphs showing the analysis results by a flow cytometer. In Figures 16 and 17, the vertical axis represents the percentage of GFP-positive cells. As a result, fluorescence indicating an improvement in genome editing efficiency was observed in a concentration-dependent manner of AP21967 during VLP preparation. In addition, it was revealed that the VLP prepared in the presence of AP21967 can deliver Cas9 in a VLP inoculation amount-dependent manner to induce genome editing.

[0190] [Experimental Example 8] (Examination of the FRB Domain) AP21967 is known to specifically bind to the FRB domain (T2098L) in which the 2098th threonine is replaced with leucine. On the other hand, the FRB domain (T2098A) in which the 2098th threonine is replaced with alanine is known to lose its binding ability to AP21967.

[0191] Therefore, VLP was prepared using Cas9 containing each FRB domain, and the genome editing efficiency was measured. Specifically, HEK293T EGxxFP cells that constantly expressed an sgRNA targeting sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) were inoculated with each VLP. Subsequently, three days later, the fluorescence of the EGxxFP reporter construct in each cell sample was analyzed by a flow cytometer.

[0192] Figures 18(a) to (c) are graphs showing the analysis results by flow cytometry. In Figures 18(a) to (c), the vertical axis indicates the ratio of GFP-positive cells. Also, “+” indicates the result of producing VLPs in the presence of AP21967, and “-” indicates the result of producing VLPs in the absence of AP21967.

[0193] As a result, it became clear that in VLPs containing Cas9 protein bound to the FRB domain (T2098A) mutant, AP21967-dependent Cas9 delivery was inhibited. This result further supports that dimerization of Cas9 protein and Gag via the FRB domain (T2098L) is necessary for efficient delivery of Cas9 protein.

[0194] [Experimental Example 9] (Examination of the localization of FRB-Cas9 protein) The intracellular localization of the FRB-Cas9 protein was examined. Specifically, VLPs containing a fusion protein in which mCherry, a fluorescent protein, was bound to FRB-SpCas9 (with a nuclear localization signal) were produced. Subsequently, concentrated VLPs (8 μL) were inoculated onto HEK293T cells (5×10 4 cells) on a 48-well plate, and the fluorescence of mCherry was analyzed using a 20× objective lens of a Keyence BX-700 microscope 20 hours after inoculation.

[0195] Figure 19 is a fluorescence micrograph showing the analysis results. In Figure 19, “DAPI” indicates the result of staining the nucleus with 4′,6-diamidino-2-phenylindole, “mCherry” indicates the result of detecting the fluorescence of mCherry, and “Merge” indicates the result of merging the fluorescence of DAPI and the fluorescence of mCherry. As a result, it was confirmed that the FRB-Cas9 protein localizes to the nucleus. In Figure 19, the arrow indicates the fluorescence of mCherry localized to the nucleus.

[0196] [Experimental Example 10] (Examination 1 of the method for encapsulating sgRNA into VLPs) Figures 20(a) to (c) are schematic diagrams for explaining the behavior of sgRNA in VLP-producing cells (HEK293T) depending on the method of expressing sgRNA.

[0197] Figure 20(a) is a schematic diagram showing the case where sgRNA is transcribed using a polymerase III promoter. Since sgRNA is a short RNA of about 100 bases, in many cases, it is transcribed using a polymerase III promoter such as the U6 or H1 promoter. However, in this case, as shown in Figure 20(a), it has been reported that sgRNA mainly accumulates in the nucleus.

[0198] Figure 20(b) is a schematic diagram showing the case where mRNA is transcribed using a polymerase II promoter. By adding ribozymes (hammerhead (HH) ribozyme and hepatitis delta virus (HDV) ribozyme), which are self-cleaving sequences, before and after sgRNA, it is possible to transcribe it as an mRNA longer than the original sgRNA from a polymerase II promoter (LTR promoter, EF1α promoter, etc.). In this case, as shown in Figure 20(b), the mRNA is distributed in the cytoplasm.

[0199] Figure 20(c) is a schematic diagram for explaining the method of encapsulating sgRNA into VLPs examined in this experimental example. In this experimental example, in order to more efficiently encapsulate sgRNA transcribed as mRNA into VLPs, it was examined to add a packaging signal (Ψ) derived from HIV to the mRNA. The packaging signal (Ψ) is a sequence possessed by retroviruses and lentiviruses such as MLV and HIV to incorporate their genomic mRNA into virus particles.

[0200] It is known that the viral genomic mRNA is incorporated into virus particles by the binding of the Gag protein and the packaging signal. The inventors examined applying this mechanism to the encapsulation of sgRNA into VLPs.

[0201] Note that it is known that the Extended packaging signal (Ψ+) including the head part of Gag has a higher uptake efficiency into virus particles than the packaging signal array. In this experimental example, the Ψ+ sequence was also used. Note that a mutation was introduced into the start codon in Gag in this part so that the protein is not translated.

[0202] In this experimental example, first, when sgRNA was transcribed from the U6 promoter (indicated as "U6" in FIGS. 21(a) and (b)), sgRNA was transcribed as mRNA containing Ψ+ from the LTR promoter, but when not containing the ribozyme sequence (indicated as "LTR-Ψ n.r." in FIGS. 21(a) and (b)), sgRNA having Ψ+ and the ribozyme sequence was transcribed from the EF1α promoter (indicated as "EF1a" in FIGS. 21(a) and (b)), in the presence of the Tat protein that activates the HIV LTR promoter (indicated as "LTR-Ψ +Tat" in FIGS. 21(a) and (b)) or in the absence (indicated as "LTR-Ψ -Tat" in FIGS. 21(a) and (b)), when sgRNA having Ψ+ and the ribozyme sequence was transcribed, VLPs were prepared respectively in the presence of FRB-Cas9. Subsequently, genomic DNA of VLP-producing cells was recovered, and the target base sequence of the DMD gene was analyzed by the T7EI assay to verify the genome editing efficiency.

[0203] FIG. 21(a) is a graph showing the results of the T7EI assay in VLP-producing cells. As a result, it became clear that the U6 promoter can transcribe the most active sgRNAs. Also, when transcribed as mRNA from the polymerase II promoter (LTR or EF1α), it was found that if it has a ribozyme sequence, it is excised as sgRNA and genome editing can be induced.

[0204] Subsequently, 1, 3, and 10 μL of each prepared VLP were introduced into HEK293T EGxxFP reporter cells (5×10 4 cells), respectively. Subsequently, three days later, the proportion of GFP-positive cells was analyzed by flow cytometry.

[0205] Figure 21(b) is a graph showing the analysis results by flow cytometry. In Figure 21(b), the vertical axis indicates the ratio of GFP-positive cells. As a result, in the U6 promoter that showed high genome editing activity in VLP-producing cells, the encapsulation efficiency of sgRNA into VLP was low, resulting in low genome editing activity.

[0206] On the other hand, in the presence of Tat protein, when sgRNA having Ψ+ and ribozyme sequences was transcribed from the LTR promoter (shown as "LTR-Ψ+Tat" in Figure 21(b)), it was revealed that the delivery efficiency of sgRNA via VLP was the highest.

[0207] [Experimental Example 11] (Examination of the method for encapsulating sgRNA into VLP 2) The HIV Rev protein is known to bind to the RRE (Rev responsive element) sequence on RNA and transport viral RNA with PRE to the cytoplasm. And most lentiviral vectors incorporate the RRE sequence. Since the expression vector of sgRNA used in Experimental Example 10 also contained the RRE sequence, the necessity of the Rev protein was examined.

[0208] Specifically, VLPs were produced in the presence or absence of Rev protein and in the presence or absence of RRE sequence, and the genome editing efficiency was examined. Figure 22(a) is a schematic diagram showing the structure of an expression vector (SEQ ID NO: 22) of sgRNA containing the RRE sequence. Figure 22(b) is a schematic diagram showing the structure of an expression vector (SEQ ID NO: 23) obtained by removing the PRE sequence from the expression vector shown in Figure 22(a).

[0209] The sgRNA is transcribed from the HIV LTR promoter, and the HIV Tat protein is required for the activation of the HIV LTR promoter. Therefore, in the presence or absence of the Tat protein and in the presence or absence of the Rev protein, sgRNA was transcribed from the expression vector shown in Fig. 22(a) to produce VLPs, and the genome editing efficiency by each produced VLP was analyzed.

[0210] Specifically, each VLP was introduced into HEK293T EGxxFP reporter cells, and the percentage of GFP-positive cells was analyzed by flow cytometry 3 days later. Figs. 22(c)-(f) are graphs showing the analysis results by flow cytometry. In Figs. 22(c)-(f), "-Tat" indicates the result of producing VLPs in the absence of the Tat protein, "+Tat" indicates the result of producing VLPs in the presence of the Tat protein, "-Rev" indicates the result of producing VLPs in the absence of the Rev protein, and "+Rev" indicates the result of producing VLPs in the presence of the Rev protein. As a result, a high genome editing efficiency was observed in the presence of the Tat protein. Also, the genome editing efficiency was similar in the presence and absence of the Rev protein.

[0211] Also, in the presence or absence of the Tat protein, sgRNA was transcribed from the expression vector shown in Fig. 22(b) to produce VLPs, and the genome editing efficiency by each produced VLP was analyzed. Specifically, each VLP was introduced into HEK293T EGxxFP reporter cells, and the percentage of GFP-positive cells was analyzed by flow cytometry 3 days later.

[0212] Figures 22(g) and (h) are graphs showing the analysis results by flow cytometry. In Figures 22(g) and (h), "-Tat" indicates the result of producing VLPs in the absence of Tat protein, "+Tat" indicates the result of producing VLPs in the presence of Tat protein, and "-PRE" indicates the result of producing VLPs with an expression vector from which the PRE sequence has been removed. As a result, it became clear that high genome editing efficiency can be obtained even in the absence of the PRE sequence.

[0213] From the above results, it became clear that the RRE sequence is not required for the sgRNA expression vector, nor is the Rev protein.

[0214] [Experimental Example 12] (Examination of the method for encapsulating sgRNA into VLPs 3) As described above, sgRNA is transcribed from the HIV LTR promoter, and the HIV Tat protein is required for the activation of the HIV LTR promoter. Therefore, the expression level of sgRNA in VLP-producing cells was quantified by real-time PCR in the presence and absence of Tat protein in VLP-producing cells. Also, the amount of sgRNA in VLPs was quantified in the same manner.

[0215] Figure 23 is a graph showing the results of real-time PCR. In Figure 23, the vertical axis indicates the expression level (relative value) of sgRNA, "+" indicates the presence, "-" indicates the absence, and "ND" indicates that it was below the detection limit.

[0216] As a result, it became clear that by co-expressing the Tat protein in VLP-producing cells, the expression level of sgRNA and the encapsulation amount of sgRNA into VLPs can be increased.

[0217] [Experimental Example 13] (Examination of the method for encapsulating sgRNA into VLPs 4) The effect of the packaging signal (Ψ+) in the sgRNA expression vector was examined. Specifically, VLPs were prepared using an sgRNA expression vector with Ψ+ or an sgRNA expression vector from which Ψ+ had been deleted.

[0218] Subsequently, each VLP was introduced into HEK293T EGxxFP reporter cells, and 3 days later, the percentage of GFP-positive cells was analyzed using a flow cytometer. Figures 24(a) to (c) are graphs showing the analysis results by flow cytometry. Figure 24(a) shows the results of the control without inoculation of VLP, Figure 24(b) shows the results of VLP prepared using an sgRNA expression vector from which Ψ+ had been deleted, and Figure 24(c) shows the results of VLP prepared using an sgRNA expression vector with Ψ+.

[0219] As a result, it was revealed that deletion of Ψ+ from the sgRNA expression vector decreased the delivery efficiency of sgRNA via VLPs. This result indicates that the packaging signal is important for enhancing the encapsulation efficiency of sgRNA.

[0220] [Experimental Example 14] (Examination of the copy number of sgRNA in the sgRNA expression vector) In a vector expressing an sgRNA (hereinafter sometimes referred to as "RGR") flanked by ribozymes under the control of the EF1α promoter, the sgRNA cassette was increased from 1 copy to 4 copies, and the genome editing efficiency was examined.

[0221] Specifically, each VLP was introduced into HEK293T EGxxFP reporter cells, and the ratio of GFP-positive cells was analyzed using a flow cytometer 3 days later. Figure 25 is a graph showing the analysis results by the flow cytometer. In Figure 25, under the control of the EF1α promoter, (i) is a schematic diagram showing the structure of an sgRNA expression vector having 1 copy of the sgRNA cassette, (ii) is a schematic diagram showing the structure of an sgRNA expression vector having 2 copies of the sgRNA cassette, (iii) is a schematic diagram showing the structure of an sgRNA expression vector having 3 copies of the sgRNA cassette, and (iv) is a schematic diagram showing the structure of an sgRNA expression vector having 4 copies of the sgRNA cassette.

[0222] As a result, it was revealed that under the control of the EF1α promoter, VLPs can be produced even when up to 4 copies of sgRNA are included in the sgRNA expression vector, and genomic editing can be induced by delivering sgRNA.

[0223] Subsequently, in a vector expressing an sgRNA sandwiched by ribozymes (hereinafter sometimes referred to as "RGR") under the control of the LTR promoter, the sgRNA cassette was increased from 1 copy to 4 copies, and the genomic editing efficiency was examined.

[0224] Specifically, each VLP was introduced into HEK293T EGxxFP reporter cells, and the ratio of GFP-positive cells was analyzed using a flow cytometer 3 days later. Figure 26 is a graph showing the analysis results by the flow cytometer. In Figure 26, (i) is a schematic diagram showing the structure of an sgRNA expression vector having 1 copy of the sgRNA cassette, (ii) is a schematic diagram showing the structure of an sgRNA expression vector having 2 copies of the sgRNA cassette, (iii) is a schematic diagram showing the structure of an sgRNA expression vector having 3 copies of the sgRNA cassette, and (iv) is a schematic diagram showing the structure of an sgRNA expression vector having 4 copies of the sgRNA cassette.

[0225] As a result, it was revealed that even when up to four copies of sgRNA were included in the sgRNA expression vector under the control of the LTR promoter, VLPs could be produced and the sgRNA could be delivered to induce genome editing.

[0226] [Experimental Example 15] (Simultaneous delivery of Cas9 protein and sgRNA by VLP 1) VLPs encapsulating FRB-Cas9 protein and sgRNA (RGR) simultaneously were prepared. Subsequently, the prepared VLPs were introduced into HEK293T EGxxFP reporter cells, and the percentage of GFP-positive cells was analyzed by flow cytometry 3 days later. Figure 27 is a graph showing the analysis results by flow cytometry. As a result, genome editing activity was observed with very high efficiency in a dose-dependent manner of VLPs.

[0227] [Experimental Example 16] (Simultaneous delivery of Cas9 protein and sgRNA by VLP 2) VLPs encapsulating FRB-Cas9 protein and sgRNA (RGR) simultaneously were prepared. Subsequently, 1.25, 2.5, 5, 10, and 20 μL of the prepared VLPs were introduced into human iPS cells (404C2 strain). Subsequently, genomic DNA was recovered 3 days later, the target base sequence DMD gene region was amplified by PCR, and the genome editing efficiency was examined by T7EI assay. Figure 28 is an image showing the results of the T7EI assay. As a result, genome editing activity was observed with very high efficiency in a dose-dependent manner of VLPs.

[0228] [Experimental Example 17] (Comparison with existing systems) A comparison was made between a system (Gesicle system, Clontech) that delivers the Cas9 protein / sgRNA RNP complex using exosome-like vesicles (Gesicle) and a system using VLPs.

[0229] The Gesicle system is a system in which the Cas9 protein / sgRNA RNP complex and the red fluorescent protein CherryPicker associate within the Gesicle and are efficiently encapsulated therein. The red fluorescent protein CherryPicker is a fusion protein of the red fluorescent protein mCherry and the transferrin receptor membrane anchor domain.

[0230] In this experimental example, first, VLPs were prepared only with VSV-G, and VLPs encapsulating Cas9 protein and sgRNA by free diffusion (shown as "VSVG Only VLP" in Fig. 29), the Gesicle system (Clontech, shown as "Clontech Gesicle System" in Fig. 29), and FKBP12-Gag encapsulating sgRNA expressed from an sgRNA expression vector having FRB-Cas9 protein and Ψ+ HIV VLP (shown as "FKBP12-Gag HIV VLP" in Fig. 29) were each prepared.

[0231] Subsequently, 5 μL of each VLP or Gesicle was inoculated into HEK293T EGxxFP reporter cells (2.5×10 4 cells), and after 72 hours, analysis was performed by fluorescence microscopy and flow cytometry.

[0232] Figs. 29(a) to (c) are fluorescence micrographs observing the fluorescence of GFP. Also, Fig. 29(d) is a graph showing the analysis result by flow cytometry. In Fig. 29(d), the vertical axis represents the ratio of GFP-positive cells. As a result, it became clear that FKBP12-Gag HIV VLP showed significantly higher genome editing efficiency compared to other VLPs or Gesicles.

[0233] [Experimental Example 18] (Simultaneous delivery of Cas9 protein and sgRNA by VLP 3) Cas9 protein and sgRNA were simultaneously delivered with various VLPs, and the genome editing efficiency was examined.

[0234] Specifically, first, the following VLPs (a) to (e) were prepared. (a) A VLP was prepared only with VSV-G, and Cas9 protein and sgRNA were encapsulated by free diffusion (shown as "VSVG Only VLP" in Fig. 30). (b) A VLP in which Gag- MLV was directly fused to the C-terminus of the Cas9 protein and encapsulated (shown as "Cas9-Gag-Pol HIV Fusion VLP" in Fig. 30). To promote VLP particle formation, Gag-Pol HIV was added. (c) A VLP in which Gag MLV was directly fused to the N-terminus of the Cas9 protein and encapsulated (shown as "Gag MLV -Cas9 Fusion VLP" in Fig. 30). To promote VLP particle formation, Gag-Pol MLV was added. (d) Cas9 was fused with FRB, and FKBP12 was fused to Gag MLV so that Cas9 was encapsulated by dimerization via AP21967 (shown as "FKBP12-Gag MLV Fusion VLP" in Fig. 30). (e) Cas9 was fused with FRB, and FKBP12 was fused to Gag HIV so that Cas9 was encapsulated by dimerization via AP21967 (shown as "FKBP12-Gag HIV Fusion VLP" in Fig. 30).

[0235] Subsequently, each prepared VLP was introduced into HEK293T EGxxFP reporter cells, and 3 days later, the ratio of GFP-positive cells was analyzed by a flow cytometer. Figs. 30(a) to (e) are graphs showing the analysis results by the flow cytometer. Figs. 30(a) to (e) are the results of introducing the above-mentioned VLPs (a) to (e) into cells, respectively. Fig. 31 is a graph quantifying the results of Figs. 30(a) to (e). In Fig. 31, the vertical axis represents the ratio of GFP-positive cells. As a result, very high efficiency of genome editing activity was observed in FKBP12-Gag HIV Fusion VLP and FKBP12-Gag MLV Fusion VLP.

[0236] [Experimental Example 19] (Examination of VLP delivery of two types of sgRNAs 1) When delivering two types of sgRNAs into cells using VLPs, two methods are envisioned: co-introducing two types of VLPs encapsulating separate sgRNAs, and introducing one type of VLP encapsulating two types of sgRNAs. Figures 32(a) and (b) are schematic diagrams explaining these methods. Figure 32(a) is a schematic diagram explaining the method of co-introducing two types of VLPs encapsulating separate sgRNAs. Further, Figure 32(b) is a schematic diagram explaining the method of introducing one type of VLP encapsulating two types of sgRNAs. In this experimental example, an examination was conducted to deliver two types of sgRNAs into cells using these two methods.

[0237] First, VLPs encapsulating sgRNA DMD#1 targeting Cas9 and the 5'-side (near the splicing acceptor) of exon 45 of the human DMD gene (the target sequence is shown in SEQ ID NO: 15) (hereinafter sometimes referred to as "DMD1 VLPs") and VLPs encapsulating sgRNA DMD#23 targeting Cas9 and the 3'-side (near the splicing donor) (the target sequence is shown in SEQ ID NO: 24) (hereinafter sometimes referred to as "DMD23 VLPs") were prepared.

[0238] Subsequently, 10 μL of each of these VLPs were introduced alone or mixed into four types of target iPS cells seeded at 2.5×10 4 cells per well in a 12-well plate. As the target iPS cells, the 404C2 strain and the 1383D2 strain, which are iPS cell lines derived from healthy individuals, iPS cells derived from a DMD patient with exon 44 deletion (hereinafter sometimes referred to as "ΔEx44 iPS Cells"), and iPS cells derived from a DMD patient with exon 46-47 deletion (hereinafter sometimes referred to as "ΔEx46-47 iPS Cells") were used. Subsequently, genomic DNA was extracted from each iPS cell, and the genomic editing efficiency at each target site was measured by the T7EI assay.

[0239] Figures 33(a) and (b) are graphs showing the results of the T7EI assay. Figure 33(a) is a graph showing the genome editing efficiency at the target site of sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15), and Figure 33(b) is a graph showing the genome editing efficiency at the target site of sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24).

[0240] As a result, differences in genome editing efficiency were observed for each iPS cell line. However, even when co-introducing the VLP encapsulating sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) and the VLP encapsulating sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24), genome editing efficiency equivalent to that of the single VLP case was confirmed.

[0241] [Experimental Example 20] (Examination of VLP delivery of two types of sgRNAs 2) 10 μL each of the VLP encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) and the VLP encapsulating Cas9 and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24), prepared in the same manner as in Experimental Example 19, were introduced alone or mixed into two types of target iPS cells seeded at 2.5×10 4 cells each in a 12-well plate. As the target iPS cells, the 404C2 cell line and 1383D2 cell line, which are iPS cell lines derived from healthy individuals, were used. Subsequently, genomic DNA was extracted from each iPS cell, a 300-base region containing the target sites of both sgRNAs was amplified by PCR, and analysis was performed using an electrophoresis system (2200 TapeStation, Agilent Technologies).

[0242] Figure 34 is an image showing the results of electrophoresis. As a result, it was confirmed that co-introducing the two types of VLPs made it possible to induce a large deletion of approximately 150 bases that excises the genomic sequence between the target sites of both.

[0243] This method can also be applied to induce exon skipping by deleting specific single or multiple exons, for example, when normal dystrophin protein cannot be expressed due to out-of-frame mutations, and to restore the protein reading frame.

[0244] [Experimental Example 21] (Examination of VLP delivery of two types of sgRNAs 3) sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24) were tandemly loaded into one expression vector, and VLPs (hereinafter sometimes referred to as "Tandem1 / 23") encapsulating Cas9 and two types of sgRNAs simultaneously in one type of VLP were prepared.

[0245] Subsequently, the prepared VLPs were introduced into iPS cells derived from DMD patients. As iPS cells derived from DMD patients, iPS cells derived from exon 44-deficient DMD patients (hereinafter sometimes referred to as "ΔEx44 iPS Cells") and iPS cells derived from exon 46-47-deficient DMD patients (hereinafter sometimes referred to as "ΔEx46-47 iPS Cells") were used.

[0246] Also, for comparison, cells were prepared by mixing and introducing VLPs encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) and VLPs encapsulating Cas9 and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24), which were prepared in the same manner as in Experimental Example 19.

[0247] Subsequently, genomic DNA was extracted from each iPS cell, a 300-base region containing the target sites of both sgRNAs was amplified by PCR, and analysis was performed using an electrophoresis system (2200 TapeStation, Agilent Technologies).

[0248] Figure 35 is an image showing the results of electrophoresis. As a result, it was confirmed that even when using VLPs encapsulating two types of sgRNAs simultaneously, it was possible to induce a large deletion of approximately 150 bases that excises the genomic sequence between the two target sites.

[0249] [Experimental Example 22] (Comparison 1 between the method using VLP and other methods) Cas9 and sgRNA were introduced into HEK293T EGxxFP cells by the method using VLP and other methods. Subsequently, three days later, the fluorescence of the EGxxFP reporter construct of each cell sample was analyzed by a flow cytometer.

[0250] Specifically, Cas9 and sgRNA were introduced by the following methods (a) to (d). (a) As a control, cells into which neither Cas9 nor sgRNA was introduced were prepared. (b) 0.5 μg of a Cas9 expression plasmid and 0.5 μg of an sgRNA expression plasmid were introduced into HEK293T EGxxFP cells using a lipofection reagent (FuGENE 6 or FuGENE HD, Promega) (shown as "Plasmid DNA Transfection" in Fig. 36). (c) Purified Cas9 protein (1 μg) and sgRNA (0.25 μg) were mixed to form a complex (ribonucleoprotein, RNP), and the complex was introduced into HEK293T EGxxFP cells using a lipofection reagent (CRISPR-MAX, Thermo Fisher Scientific) (shown as "RNP transfection" in Fig. 36). (d) SpCas9 and sgRNA were introduced into HEK293T EGxxFP cells using VLP (50 μL) (shown as "VLP" in Fig. 36).

[0251] Figures 36(a) to (d) are graphs showing the analysis results by a flow cytometer. Figures 36(a) to (d) are the results of analyzing the cells (a) to (d) described above, respectively. HEK293T cells are originally cells with high gene transfer efficiency, and genome editing can be achieved with sufficient high efficiency by plasmid DNA introduction or RNP introduction. However, it has been revealed that VLP shows genome editing activity equal to or higher than these existing methods.

[0252] [Experimental Example 23] (Comparison between the method using VLP and other methods 2) Cas9 and sgRNA were introduced into 404C2 cells, which are a human iPS cell line, by the method using VLP and other methods. Subsequently, two days later, the genome editing efficiency of the region on the 5' side (near the splicing acceptor) of exon 45 of the human DMD gene, which is the target site of sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15), was examined by the T7EI assay.

[0253] Specifically, Cas9 and sgRNA were introduced by the following methods (a) to (f). (a) Neither Cas9 nor sgRNA was introduced, and T7 endonuclease I (T7EI) was not added in the T7EI assay (shown as "No Transfection, -T7EI" in Fig. 37). (b) Neither Cas9 nor sgRNA was introduced, and T7EI was added in the T7EI assay (shown as "No Transfection, +T7EI" in Fig. 37). (c) 0.5 μg of the Cas9 expression plasmid and 0.5 μg of the sgRNA expression plasmid were introduced into 404C2 cells using a lipofection reagent (FuGENE 6 or FuGENE HD, Promega) (shown as "DNA Transfection, +T7EI" in Fig. 37). (d) Purified Cas9 protein (1 μg) and sgRNA (0.125 μg) were mixed to form a complex (ribonucleoprotein, RNP), and the RNP was introduced into 404C2 cells using a lipofection reagent (CRISPR-MAX, Thermo Fisher Scientific) (shown as "RNP Transfection, +T7EI" in Fig. 37). (e) SpCas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) were introduced into 404C2 cells using VLP (80 μL) (shown as "DMD1 VLP, +T7EI" in Fig. 37). (f) SpCas9 and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24) were introduced into 404C2 cells using VLP (80 μL) (shown as "DMD23 VLP, +T7EI" in Fig. 37).

[0254] Fig. 37 is an image showing the results of electrophoresis. It was revealed that it is difficult to introduce plasmid DNA into human iPS cells by lipofection using the FuGENE reagent, genome editing induction is possible by RNP introduction, and VLP shows genome editing activity equivalent to that of RNP introduction.

[0255] [Experimental Example 24] (Efficient Genome Editing by VLP Introduction into Muscle Cells 1) We investigated the induction of genome editing in mouse myoblast cell line C2C12 using VLPs. Specifically, first, mouse myoblast cell line C2C12 transfected with an EGxxFP reporter construct was seeded at 2.5×10 4 cells / well in a collagen I-coated 12-well plate.

[0256] Subsequently, the cells were inoculated with 0, 1, 3, 10, 30 μL of VLPs encapsulating FRB-Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15). Subsequently, three days later, the percentage of GFP-positive cells was analyzed by flow cytometry.

[0257] Figure 38 is a graph showing the analysis results by flow cytometry. In Figure 38, the vertical axis represents the cell number, and the horizontal axis represents the fluorescence intensity of GFP. Also, the percentage of GFP-positive cells is shown on the right side of the graph. As a result, it became clear that genome editing was induced with very high efficiency in a VLP inoculation amount-dependent manner.

[0258] [Experimental Example 25] (Efficient Genome Editing by Introduction of VLPs into Muscle Cells 2) After inducing the differentiation of mouse myoblast cell line C2C12 into muscle fibers, we investigated the induction of genome editing using VLPs. Specifically, first, mouse myoblast cell line C2C12 transfected with an EGxxFP reporter construct was seeded at 2.5×10 4 cells / well in a collagen I-coated 12-well plate.

[0259] Subsequently, the medium was replaced with a differentiation induction medium (DMEM, 5% horse serum, 100 mM sodium pyruvate, 100 mM 2-mercaptoethanol, penicillin & streptomycin), and differentiation into muscle fibers was induced over 4 days.

[0260] Subsequently, 20 μL of VLP was inoculated. As the VLP, VLP encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) (hereinafter sometimes referred to as "DMD1 VLP") or VLP encapsulating Cas9 and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24) (hereinafter sometimes referred to as "DMD23 VLP") was inoculated. Subsequently, four days after inoculation, the fluorescence of the EGxxFP reporter construct was analyzed by a fluorescence microscope (Keyence Corporation) or flow cytometry.

[0261] FIG. 39(a) is a schematic diagram explaining the structure of the EGxxFP reporter construct used in this experimental example. As shown in FIG. 39(a), the EGxxFP reporter construct has only the target sequence of sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) and does not have the target sequence of sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24). Therefore, cleavage of the EGxxFP reporter construct is induced only when DMD1 VLP is inoculated.

[0262] FIG. 39(b) is a photograph showing the observation results by a fluorescence microscope. In FIG. 39(b), "Bright Field" indicates that it is a bright-field (phase contrast) observation image, and "GFP Field" indicates that it is a fluorescence detection image of GFP. As a result, it became clear that genome editing in the EGxxFP reporter construct was induced and fluorescence of GFP was observed only when DMD1 VLP was introduced.

[0263] FIG. 39(c) is a graph showing the analysis results by a flow cytometer. In FIG. 39(c), the vertical axis indicates the ratio of GFP-positive cells. As a result, it became clear that genome editing in the EGxxFP reporter construct was induced and fluorescence of GFP was observed only when DMD1 VLP was introduced.

[0264] Generally, differentiated cells stop cell proliferation and it becomes difficult to introduce genes or perform genome editing. In contrast, it has been revealed that genome editing can be efficiently induced even in differentiated cells by using VLPs.

[0265] [Experimental Example 26] (Time-course change of genome editing activity by VLP introduction) The time-course change of genome editing activity by VLP introduction was examined. Specifically, first, 404C2 iPS cells were seeded at a cell density of 1.0×10 5 cells / 24-well plate. Subsequently, VLPs encapsulating Cas9 and sgRNA were inoculated. Subsequently, genomic DNA was collected over time, and the genome editing efficiency was measured by the T7EI assay.

[0266] Figure 40(a) is a diagram showing the experimental schedule. Figure 40(b) is a representative image showing the results of the T7EI assay. Figure 40(c) is a graph showing the time-course change of genome editing activity. As a result, it was revealed that no genome editing activity was observed 8 hours after introducing Cas9 and sgRNA with VLPs. Also, it was revealed that genome editing gradually accumulated after 12 hours from the introduction of VLPs and reached almost a plateau 36 hours later.

[0267] [Experimental Example 27] (Examination of the safety of the VLP system 1) It has been reported that sgRNA targeting the VEGFA gene (the target sequence is shown in SEQ ID NO: 25) is likely to introduce mutations at off-target sequences. Therefore, in the introduction of VLPs showing transient expression, the genome editing efficiency at the target site and off-target sites was examined.

[0268] First, VLPs (shown as "VEGFA VLP" in Figure 41) encapsulating Cas9 and sgRNA targeting the VEGFA gene (the target sequence is shown in SEQ ID NO: 25) were prepared. Subsequently, 100, 200, 300 ng (equivalent to the p24 amount) of the prepared VLPs were inoculated into 5×10 4 HEK293T cells.

[0269] Also, for comparison, cells transfected with plasmid DNA expressing Cas9 and sgRNA were also prepared. Subsequently, genomic DNA was recovered from each cell after 3 days, and the genome editing efficiency at the target site region (On-Target) of VEGFA was examined by the T7EI assay. Also, the genome editing efficiency at the off-target site region (Off-Target) similar to the target sequence was examined by the T7EI assay.

[0270] Table 1 below shows the target sequence, On-Target sequence, and Off-Target sequence of the sgRNA.

[0271]

Table 1

[0272] Figure 41(a) is a graph showing the results of the T7EI assay. In Figure 41(a), "DNA Plasmid" indicates the result of transfection with plasmid DNA expressing Cas9 and sgRNA, and "Empty Plasmid" indicates the result of transfection with empty plasmid DNA.

[0273] Figure 41(b) is a graph showing the ratio of on-target cleavage to off-target cleavage. In Figure 41(b), "VEGFA DNA Plasmid" indicates the result of transfection with plasmid DNA expressing Cas9 and sgRNA, and "VEGFA VLP" indicates the result of transfection with VLP.

[0274] As a result, in the target site region of the cells transfected with 300 ng (equivalent to the amount of p24) of VLP, a genome editing efficiency equal to or higher than that of the cells transfected with plasmid DNA was observed.

[0275] In addition, in the introduction of plasmid DNA, unintentional mutation introduction into off-target sites was confirmed. In contrast, in the introduction of VLP, no mutation introduction into off-target sites was confirmed. In the introduction of VLP, the existence time of Cas9 is short and it disappears before mutations are introduced into off-target sites. Therefore, it was confirmed that the target base sequence specificity is higher compared to the introduction of plasmid DNA.

[0276] [Experimental Example 28] (Examination of the safety of the VLP system 2) HEK293T EGxxFP cells (1×10 5 cells) were transfected with 6 μL of VLP encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) to induce genome editing. Also, for comparison, cells transfected with the same amount of empty VLP were prepared.

[0277] Subsequently, 48 hours later, the genome editing efficiency was measured with a flow cytometer. Also, the number of surviving cells was stained with trypan blue and counted using an automatic cell counter (name: "Countess II", Thermo Fisher Scientific).

[0278] Figure 42 is a graph showing the results. In Figure 42, the upper part is a graph showing the analysis results by flow cytometer. The vertical axis of the graph indicates the percentage of GFP-positive cells. Also, "DMD1 VLP" indicates the result of introducing VLP encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15), "Empty VLP" indicates the result of introducing empty VLP, and "Not treated" indicates the result of HEK293T EGxxFP cells without any treatment. Also, the lower part of Figure 42 is a graph showing the result of counting the number of surviving cells. The vertical axis of the graph indicates the number of cells. As a result, even under the VLP introduction conditions showing sufficient genome editing activity, the number of surviving cells hardly decreased, and it was confirmed that the cytotoxicity of VLP is low.

[0279] [Experimental Example 29] (Induction of exon skipping in human skeletal muscle cells) It was examined whether the VLP according to the present invention can induce exon skipping also in human skeletal muscle cells. First, by adding doxycycline to the medium of iPS cells derived from a DMD patient lacking exon 44 that express the MYOD1 gene in a doxycycline-inducible manner and overexpressing the MYOD1 gene, differentiation was induced into skeletal muscle cells.

[0280] Subsequently, 10 μL each of a VLP encapsulating Cas9 and sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) (hereinafter sometimes referred to as "DMD1 VLP"), which was prepared in the same manner as in Experimental Example 19, and a VLP encapsulating Cas9 and sgRNA DMD#23 (the target sequence is shown in SEQ ID NO: 24) (hereinafter sometimes referred to as "DMD23 VLP") were introduced alone or in combination into the obtained human skeletal muscle cells.

[0281] Subsequently, total RNA was extracted from each human skeletal muscle cell, and a region containing exon 45 of the cDNA of the DMD gene was amplified by PCR by RT-PCR, and analysis was performed using an electrophoresis system (2200 TapeStation, Agilent Technologies).

[0282] Figure 43(a) is an image showing the results of electrophoresis. The band indicated by the upper arrow in Figure 43(a) is a band derived from cDNA containing exon 45. Also, the band indicated by the lower arrow in Figure 43(a) is a band derived from cDNA lacking exon 45 due to exon skipping.

[0283] Figure 43(b) is a graph obtained by digitizing the results of Figure 43(a). In Figure 43(b), the ratio of the intensity of the lower band to the total intensity of the upper and lower bands in Figure 43(a) is shown as the exon skipping efficiency (%) of exon 45. In Figures 43(a) and (b), "DMD1" indicates DMD1 VLP, "DMD23" indicates DMD23 VLP, "+" indicates introduction into human skeletal muscle cells, and "-" indicates no introduction into human skeletal muscle cells. In addition, in Figure 43(b), "****" indicates that there is a significant difference with P < 0.0001 as a result of analysis by one-way ANOVA.

[0284] As a result, it was revealed that by introducing DMD1 VLP alone, exon skipping of exon 45 could be induced in human skeletal muscle cells with an efficiency of about 36%. On the other hand, when DMD23 VLP was introduced alone, it was revealed that the exon skipping efficiency was low. In contrast, by co-introducing two types of VLPs, DMD1 VLP and DMD23 VLP, it was revealed that exon skipping could be induced with a very high efficiency of about 92%.

[0285] In addition, the expression of dystrophin protein in each human skeletal muscle cell was examined. Specifically, Western blotting was performed using an anti-dystrophin antibody using the fully automated Western blot device Wes from ProteinSimple. In addition, myosin heavy chain protein was detected as a loading control. In addition, as a control, HEK293T cells overexpressing dystrophin cDNA were analyzed in the same manner.

[0286] Figure 43(c) is an image showing the results of Western blotting. In Figure 43(c), "DYS Ctrl." indicates the result of the control, "DMD1" indicates the result of introducing DMD1 VLP alone, "DMD23" indicates the result of introducing DMD23 VLP alone, and "DMD1+23" indicates the result of co-introducing DMD1 VLP and DMD23 VLP.

[0287] As a result, it was revealed that the expression level of dystrophin protein in human skeletal muscle cells was correlated with the exon skipping efficiency, and high expression of dystrophin protein could be induced by co-introducing two types of VLPs, DMD1 VLP and DMD23 VLP.

[0288] This result indicates that when normal dystrophin protein cannot be expressed due to an out-of-frame mutation, exon skipping can be induced by deleting specific single or multiple exons, and the reading frame of the protein can be restored.

[0289] [Experimental Example 30] [Preparation of VLP Encapsulating Luciferase] VLPs encapsulating luciferase protein were prepared. Figures 44(a) to (c) are schematic diagrams explaining the structure of the VLPs prepared in this experimental example. Figure 44(a) is a schematic diagram showing the structure of an expression construct of a fusion protein (hereinafter sometimes referred to as "FKBP12-Gag") in which an FKBP12 domain is added to the N-terminus of HIV Gag. HIV (Sometimes referred to as such.) Figure 44(b) is a schematic diagram showing the structure of an expression construct of a luciferase protein (hereinafter sometimes referred to as "FRB-Luc") fused with an FRB domain. Figure 44(c) is a schematic diagram showing the state of encapsulating FKBP12-Gag HIV and FRB-Luc into VLPs.

[0290] In the presence or absence of AP21967, a rapamycin analog, FRB-Luc, FKBP12-Gag HIV , and VSV-G were expressed in HEK293T cells in the combinations shown in Figure 45 to prepare VLPs. In Figure 45, "+" indicates introduction, and "-" indicates no introduction.

[0291] Subsequently, FRB-Luc, FKBP12-Gag, and VSV-G in each of the above VLPs were quantified by Western blotting. Figure 45 is a photograph showing the results of Western blotting. As a result, it was confirmed that more luciferase protein was encapsulated in the VLPs prepared in the presence of AP21967 compared to the absence.

[0292] Subsequently, HEK293T cells were seeded in 96-well plates, and each of the above VLPs was introduced. Subsequently, after 16 hours, the cells were disrupted and luciferase activity was measured. Figure 46 is a graph showing the measurement results of luciferase activity. In Figure 46, "control" shows the results of VLPs not containing FRB-Luc. Also, "Luc VLP" shows the results of VLPs prepared by expressing HIV FRB-Luc, FKBP12-Gag and VSV-G. Also, "-" indicates the results of VLPs prepared in the absence of AP21967, and "+" indicates the results of VLPs prepared in the presence of AP21967.

[0293] As a result, it became clear that the VLPs prepared in the presence of AP21967 delivered 12 times more luciferase protein to HEK293T cells compared to the VLPs prepared in the absence of AP21967.

[0294] [Experimental Example 31] (Delivery of Protein in Vivo Using VLP) Examination was carried out on delivering protein into the body using VLP. 30 μL or 60 μL of the Luc VLP prepared in Experimental Example 30 was administered intramuscularly to the gastrocnemius muscle of C57BL / 6 mice. Also, a group administered with PBS was prepared as a control. Subsequently, after 16 hours, 2 days, and 3 days, the luminescence of luciferase was detected.

[0295] Specifically, 5 to 15 minutes before the analysis, the mice were anesthetized with isoflurane, and 3 mg / mouse of luciferin was administered intravenously. Subsequently, the luminescence of luciferase was detected by in vivo luminescence and fluorescence imaging using an IVIS imaging device (PerkinElmer).

[0296] Figure 47 is a photograph showing the results of IVIS analysis. Figure 48 is a graph obtained by quantifying the results of Figure 47. As a result, luminescence of luciferase was observed in a dose-dependent manner near the muscle of the mice injected with Luc VLP. In addition, almost no leakage into the liver or other organs was observed. In addition, complete disappearance of the luciferase protein was observed within 3 days after the introduction of Luc VLP. This result indicates that the delivery of the protein by VLP is transient.

[0297] [Experimental Example 32] (Induction of exon skipping in vivo) Examination was conducted to induce exon skipping in vivo using VLP. Figure 49 is a diagram explaining the luciferase reporter knock-in mouse model used in this experimental example. As shown in Figure 49, a luciferase reporter gene was knocked in by genome editing at the Gt(ROSA)26Sor locus of C57BL / 6 mice. The human DMD gene exon 45 and its 5'- and 3'-flanking introns were introduced into this luciferase reporter gene.

[0298] As shown in Figure 49, when this exon 45 is incorporated into luciferase mRNA during the splicing process, it becomes out-of-frame, so that a normal luciferase protein cannot be expressed. However, when the same DMD1 VLP and DMD23 VLP as in Experimental Example 19 are introduced into this luciferase reporter knock-in mouse model, exon skipping of exon 45 can be induced, and it becomes possible to express a normal luciferase protein.

[0299] 50 μL of DMD1 VLP and 50 μL of DMD23 VLP, which were the same as those in Experimental Example 19, were administered intramuscularly to the gastrocnemius muscle of the above luciferase reporter knock-in mouse model. Subsequently, in the same manner as in Experimental Example 31, luminescence of luciferase was detected over time by in vivo luminescence / fluorescence imaging using an IVIS imaging device (PerkinElmer).

[0300] Figure 50(a) is a photograph showing the results of IVIS analysis from 1 to 160 days after administration of VLP (n = 5). Figure 50(b) is a representative photograph showing the results of IVIS analysis on the 126th day after administration of VLP. As a result, expression of luciferase due to exon skipping was specifically observed at the site where VLP was injected.

[0301] Unlike the case of injecting Luc VLP, upon a single administration of DMD1 VLP and DMD23 VLP, luciferase activity was observed 3 days later, reached a plateau 7 days later, and luciferase activity was detected for at least 160 days thereafter. This result indicates that exon skipping of exon 45 was stably maintained in the muscles of mice.

[0302] [Experimental Example 33] (Genome Editing in Various Cells Using VLP) VLPs encapsulating Cas9 and sgRNAs against various target sequences were introduced into various cells, and the genome editing efficiency was examined.

[0303] [T Lymphocytes] Figure 51(a) is a photograph showing the results of measuring the genome editing efficiency by T7EI assay after introducing VLPs encapsulating an sgRNA (the target sequence is shown in SEQ ID NO: 28) against the CCR5 gene, which is a co - receptor of HIV, and Cas9 into Jurkat cells, a human T lymphocyte cell line. Figure 51(b) is a graph quantifying the results of Figure 51(a). As a result, it became clear that indel (insertion - deletion mutation) was introduced in a dose - dependent manner by the introduction of VLP, reaching a maximum of 48%.

[0304] [Monocytes] VLP encapsulating sgRNA against the EGFP gene (the target sequence is shown in SEQ ID NO: 29) and Cas9 was introduced into cells (EGFP-U937) in which EFGP was stably expressed in U937 cells, a human monocyte cell line. Also, as a control, VLP encapsulating Cas9 and sgRNA against the EGFP gene (the target sequence is shown in SEQ ID NO: 29) was introduced into cells (SAMHD1-U937) in which the SAMHD1 gene was stably expressed in U937 cells. Subsequently, three days after the introduction of VLP, the fluorescence of EGFP was analyzed by flow cytometry.

[0305] Figure 52 is a graph showing the results of flow cytometry analysis. In Figure 52, "-" indicates that VLP was not introduced, and "+" indicates that VLP was introduced. As a result, it was revealed that in the group into which VLP was introduced, the average value of the fluorescence intensity of EGFP decreased by about 50% compared to the group into which VLP was not introduced.

[0306] 《Neurons》 By overexpressing the Neurogenin2 gene (NGN2-IRES-mCherry) in human iPS cells, differentiation into cortical neuron-like neurons was induced. Subsequently, VLP encapsulating sgRNA against the SAMHD1 gene and Cas9 was introduced into the obtained neurons. As the sgRNA against the SAMHD1 gene, two types, sgRNA#1 (the target sequence is shown in SEQ ID NO: 30) and sgRNA#2 (the target sequence is shown in SEQ ID NO: 31), were each used.

[0307] Figure 53(a) is a schematic diagram showing the positions of sgRNA#1 and sgRNA#2 with respect to the SAMHD1 gene. Figure 53(b) is a photograph of neurons differentiated from human iPS cells. Figure 53(c) is an image showing the results of measuring the genome editing efficiency by the T7EI assay. In Figure 53(c), "anti-SAMHD1 VLP" indicates a VLP encapsulating Cas9 and sgRNA against the SAMHD1 gene, "-" indicates that no VLP was introduced, "#1" indicates that a VLP encapsulating sgRNA#1 was introduced, and "#2" indicates that a VLP encapsulating sgRNA#2 was introduced.

[0308] As a result, it was revealed that the SAMHD1 gene, which is a gene related to congenital encephalopathy, can be efficiently genome-edited by the introduction of VLP. The introduction efficiency of insertion / deletion mutations (Indel) was approximately 36% in both cases using either sgRNA#1 or sgRNA#2.

[0309] [Experimental Example 34] (Large-scale production of VLP) A method for large-scale production of VLP without using animal-derived components (xeno-free) was investigated for medical applications. First, a HEK293 cell line that expresses SV40 large T antigen and can be cultured in suspension in serum-free medium was established.

[0310] 《Examination of electroporation conditions》 Using a flow electroporation device ("MaxCyte STX", MaxCyte), an expression vector for forming VLP was introduced into the above HEK293 cells under low electroporation energy conditions "E4" or high electroporation energy conditions "E9".

[0311] As the expression vector, an expression vector of FKBP12-Gag HIV an expression vector of FRB-SpCas9, an expression vector of sgRNA sandwiched between ribozymes (hereinafter sometimes referred to as "RGR"), Tat HIVAn expression vector of [vector name] and an expression vector of VSVG were used.

[0312] After electroporation, the cells were incubated for 40 minutes in the presence or absence of an endonuclease ("benzonase", Merck). Subsequently, AP21967 was added to the cell culture medium, and the cells were cultured in suspension with stirring at 100 rpm.

[0313] Subsequently, 36 - 48 hours after electroporation, the culture supernatant was collected, cell debris was removed using a syringe filter with a pore size of 0.45 μm, and centrifugation was performed at 100,000 × g for 3 hours using an Avanti JXN - 30 centrifuge (Beckman Coulter) to recover the VLPs.

[0314] Subsequently, the VLPs prepared under each condition were introduced into HEK293T cells (hereinafter sometimes referred to as "HEK293T EGxxFP cells") into which the reporter construct EGxxFP was introduced. Subsequently, 3 days later, the fluorescence of EGFP in each cell was measured using a flow cytometer, and the genome editing efficiency was measured.

[0315] Figure 54(a) is a graph showing the results of measuring the fluorescence of EGFP. As a result, it was revealed that the genome editing efficiency was the highest when electroporation was performed under the electroporation condition of "E9" and the cells were treated with endonuclease after electroporation. This result indicates that the amount of VLP formation was the largest under the above conditions.

[0316] 《Examination of Tat and AP21967》 Using a flow electroporation device ("MaxCyte STX", MaxCyte), VLPs were prepared under the condition of E9. Here, the conditions in the presence or absence of the expression vector of Tat HIV and in the presence or absence of AP21967 were examined.

[0317] Subsequently, the VLPs prepared under each condition were introduced into HEK293T EGxxFP cells, respectively. Subsequently, three days later, the fluorescence of EGFP in each cell was measured using a flow cytometer, and the genome editing efficiency was measured.

[0318] Figure 54(b) is a graph showing the results of measuring the fluorescence of EGFP. As a result, it was revealed that the genome editing efficiency was the highest when VLPs were prepared in the presence of Tat HIV and AP21967. This result further supports the importance of the presence of Tat HIV and AP21967 for the encapsulation of sgRNA and Cas9 into VLPs.

[0319] 《Comparison of VLP production by adherent culture and suspension culture》 Forty-eight 10-cm dishes were used, and HEK293T cells were adherently cultured in a medium containing 10% fetal bovine serum (FBS). Each expression vector was introduced using a lipofection reagent to produce VLPs on a total scale of 480 mL.

[0320] In addition, two 1-L flasks containing 240 mL of medium were used, and HEK293 cells were suspension-cultured in a serum-free medium without animal-derived components. Each expression vector was introduced by flow electroporation to produce VLPs on a 480-mL scale.

[0321] Subsequently, each VLP was concentrated by centrifugation overnight. Subsequently, the VLPs were lysed using Triton-X and reacted with a substrate (DNA containing the dystrophin target sequence, 700 bp) to quantify the abundance of the active Cas9 / sgRNA RNP complex. For quantification, a standard curve prepared using recombinant spCas9 protein and chemically synthesized sgRNA was used in the same manner as in Experimental Example 2.

[0322] Figure 55(a) is a graph showing the results of quantifying the active Cas9 / sgRNA RNP complex. In Figure 55(a), "48×10 cm" shows the results of VLPs prepared by adherent culture, and "2×1 L" shows the results of VLPs prepared by suspension culture.

[0323] As a result, it was revealed that a total of 8.1 μg of Cas9 / sgRNA RNP complex could be produced by suspension culture using serum-free medium. This was approximately 30% less than the VLP produced by adherent culture using serum-containing medium on the same scale. However, for medical applications, animal-derived components should not be used. The above results indicate that the production of VLP by suspension culture can be scaled up and industrially produced for medical applications.

[0324] Also, Fig. 55(b) is a graph showing the results of measuring the induction of insertion / deletion mutations (Indels) by introducing a VLP containing 0.26 μg of an active RNP complex containing sgRNA DMD#1 (the target sequence is shown in SEQ ID NO: 15) into iPS cells derived from DMD patients. In Fig. 55(b), "No RNP" indicates that the Cas9 / sgRNA RNP complex was not introduced, and "RNP Electrop." indicates that the Cas9 / sgRNA RNP complex was introduced by electroporation.

[0325] As a result, it was revealed that VLP induced insertion / deletion mutations with higher efficiency compared to the case of electroporating 10 μg of recombinant RNP into iPS cells. This result indicates that VLP efficiently delivers CRISPR-Cas9 RNP to target cells and exhibits high cleavage activity.

[0326] [Experimental Example 35] (Analysis of VLP particles) The size of VLP particles and the number of molecules of the active Cas9 / sgRNA RNP complex in one VLP particle were analyzed. First, VLP was adsorbed onto a monolithic silica column. Subsequently, it was extracted and purified with buffers having the NaCl concentration adjusted to 0.1 M, 0.2 M, 0.65 M, and 1 M, respectively. Subsequently, the VLP contained in the elution fractions eluted at each salt concentration was subjected to Western blotting to detect SpCas9 protein and capsid (also called CA, p24).

[0327] Figure 56(a) is a photograph showing the result of Western blotting of SpCas9 protein. Also, Figure 56(b) is a photograph showing the result of Western blotting of p24. As a result, it was considered that VLPs were included in the elution fractions with a salt concentration of 0.65 M and a salt concentration of 1 M.

[0328] 《Measurement of particle size》 Subsequently, the purified VLPs were observed with an electron microscope and the particle size was measured. Figure 57(a) is a representative electron micrograph of VLPs contained in the elution fraction with a salt concentration of 0.65 M. Also, Figure 57(b) is a representative electron micrograph of VLPs contained in the elution fraction with a salt concentration of 1 M. Also, Figure 57(c) is a graph showing the results of measuring the particle size (major axis and minor axis) of VLPs contained in the elution fraction with a salt concentration of 0.65 M based on the electron micrograph. Also, Figure 57(d) is a graph showing the results of measuring the particle size (major axis and minor axis) of VLPs contained in the elution fraction with a salt concentration of 1 M based on the electron micrograph.

[0329] Figure 58(a) is a graph showing the results of measuring the particle size of VLPs contained in the elution fraction with a salt concentration of 0.65 M using a commercially available device (“NanoSight”, Malvern Panalytical). Also, Figure 58(b) is a graph showing the results of measuring the particle size of VLPs contained in the elution fraction with a salt concentration of 1 M using a commercially available device (“NanoSight”, Malvern Panalytical). In Figures 58(a) and (b), the vertical axis indicates the concentration of VLPs (10 6 particles / mL), and the horizontal axis indicates the particle size (nm).

[0330] 《Analysis of the number of Cas9 molecules》 The number of Cas9 proteins (the number of active Cas9 / sgRNA RNP complexes) inside VLP1 particles was analyzed. First, using Triton-X, the VLPs contained in the elution fraction with a salt concentration of 0.65 M and the VLPs contained in the elution fraction with a salt concentration of 1 M were dissolved and reacted with a substrate (DNA containing a dystrophin target sequence, 700 bp) to quantify the abundance of active Cas9 / sgRNA RNP complexes. For the quantification, a standard curve prepared using recombinant spCas9 protein and chemically synthesized sgRNA was used in the same manner as in Experimental Example 2.

[0331] Figure 59(a) is an image showing the standard curve. Also, Figure 59(b) is an image showing the results of cleaving a substrate (DNA containing a dystrophin target sequence, 700 bp) using Cas9 / sgRNA RNP complexes contained in VLPs in the elution fractions with salt concentrations of 0.65 M and 1 M, respectively. In Figures 59(a) and (b), “% Cleavage Activity” indicates the ratio of the cleaved substrate (cleavage activity).

[0332] As a result, the concentration of Cas9 protein in the elution fraction with a salt concentration of 0.65 M was calculated to be 3.4 μg / mL. Also, the concentration of Cas9 protein in the elution fraction with a salt concentration of 1 M was calculated to be 13.3 μg / mL. Table 2 below shows the calculated values of the particle diameter of the VLPs, the concentration of the VLPs, the concentration of the Cas9 protein, and the number of Cas9 protein molecules per VLP1 particle.

[0333]

Table 2

Industrial Applicability

[0334] According to the present invention, it is possible to provide a technique for efficiently encapsulating a target protein into virus-like particles.

Claims

1. A virus-like particle encapsulating a target protein, wherein the virus-like particle contains a Gag protein, the Gag protein forms a dimer with the target protein, the Gag protein is a fusion protein with FKBP12-rapamycin associated protein 1, FRAP1 fragment (FRB), the FRB is fused to the N-terminus of the Gag protein, and the target protein is a fusion protein with FK506-binding protein (FKBP12), A virus-like particle in which, in the dimer, the FRB, rapamycin or a rapamycin derivative, and the FKBP12 are bound.

2. The virus-like particle according to claim 1, wherein the target protein is a Cas family protein.

3. The virus-like particle according to claim 2, further encapsulating an mRNA having a gRNA sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence and a packaging signal sequence, or a self-cleaving product of the mRNA.

4. A method for producing genome-edited cells, comprising inoculating the cells with the virus-like particle according to claim 3 in vitro.

5. A method for producing a virus-like particle encapsulating a target protein, comprising the following steps (1) and (2): (1) In the presence of rapamycin or a rapamycin derivative, a step of expressing, in cells, a fusion protein of FRB and a Gag protein, and a fusion protein of FKBP12 and a target protein, wherein the FRB is fused to the N-terminus of the Gag protein, (2) A step of obtaining a culture medium containing the virus-like particle encapsulating the target protein.

6. The production method according to claim 5, wherein, in the step (1), the nucleic acid encoding the fusion protein is introduced into the cells by a lipofection method or an electroporation method.

7. A method for producing a virus-like particle encapsulating a Cas family protein and a gRNA, comprising the following steps (1) and (2): (1) In the presence of rapamycin or a rapamycin derivative, in cells, a fusion protein of FRB and a Gag protein, a fusion protein of FKBP12 and a Cas family protein, and An mRNA having a gRNA sequence and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence, A step of expressing, wherein the FRB is fused to the N-terminus of the Gag protein, (2) A step of obtaining a medium containing virus-like particles encapsulating the Cas family protein and gRNA.

8. The production method according to claim 7, wherein in the step (1), the fusion protein or the nucleic acid encoding the mRNA is introduced into the cell by a lipofection method or an electroporation method.

9. A kit for producing virus-like particles encapsulating a target protein, comprising an expression vector for a fusion protein of FRB and Gag protein, wherein the FRB is fused to the N-terminus of the Gag protein.

10. A kit for producing virus-like particles encapsulating a Cas family protein, comprising an expression vector for a fusion protein of FRB and Gag protein, and an expression vector for a fusion protein of FKBP12 and Cas family protein, Including, the FRB is fused to the N-terminus of the Gag protein, the kit.

11. The kit according to claim 10, further comprising an expression vector for an mRNA having a base sequence or a multiple cloning site of a target RNA and a packaging signal sequence sandwiched between a first ribozyme sequence and a second ribozyme sequence.

12. A therapeutic agent for a disease, an infectious disease or cancer caused by a gene mutation, comprising the virus-like particle according to claim 3 as an active ingredient.

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