Vector for nucleic acid introduction, nucleic acid introduction method, polynucleotide, and vector

Abstract

This vector for nucleic acid introduction contains a nucleotide sequence that encodes a Cas protein, a nucleotide sequence that encodes a guide RNA, and a nucleotide sequence of a nucleic acid to be introduced. The guide RNA targets an intron adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon. Also provided is a nucleic acid introduction method comprising a step for introducing a nucleic acid to be introduced through non-homologous end joining by targeting an intron that is adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon.

Description

Nucleic acid transfer vector, nucleic acid transfer method, polynucleotide, and vector

[0001] The present invention relates to a vector for nucleic acid transfer, a nucleic acid transfer method, a polynucleotide, and a vector. This application claims priority based on Japanese Patent Application No. 2024-073679, filed on April 30, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, technologies for applying bacterial and archaeal CRISPR / Cas systems to genome editing have been actively developed. Types I to VI of CRISPR / Cas systems are known. The types commonly used for genome editing include the type II CRISPR / Cas9 system and the type V CRISPR / Cas12 system.

[0003] Cas12f has been reported as a Cas protein with a smaller molecular size than Cas9 and Cas12a. In addition, modified AsCas12f (AsCas12f) derived from Acidibacillus sulfuroxidans has been reported to have enhanced genome editing activity (Patent Document 1, Non-Patent Document 1).

[0004] International Publication No. 2022 / 092317

[0005] Hino T, et al. An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell. 2023;186:4920-4935.e23.

[0006] When gene transfer is performed using genome editing, low expression of the introduced gene is often a problem, so there is a need to develop technologies to improve the expression of the introduced gene.

[0007] Therefore, an object of the present invention is to provide a nucleic acid transfer vector and a nucleic acid transfer method that can improve the expression level of a transferred gene. Another object of the present invention is to provide a polynucleotide that encodes ornithine transcarbamylase and that is expressed at a high level in human cells into which it is transferred, and a vector containing the polynucleotide.

[0008] The present invention includes the following aspects: [1] A nucleic acid transfer vector comprising a nucleotide sequence encoding a Cas protein, a nucleotide sequence encoding a guide RNA, and a nucleotide sequence of a nucleic acid to be introduced, wherein the guide RNA targets an intron adjacent to the 5' side of an exon containing a stop codon, relative to the sense strand. [2] The nucleic acid transfer vector of [1], further comprising the guide RNA recognition sequence. [3] The nucleic acid transfer vector of [1] or [2], further comprising at least a portion of the nucleotide sequence of the exon, wherein at least a portion of the nucleotide sequence of the exon is located 5' to the nucleotide sequence of the nucleic acid to be introduced, relative to the sense strand, and wherein at least a portion of the nucleotide sequence of the exon does not include the stop codon of the exon and the nucleotide sequence 3' to the stop codon, relative to the sense strand. [4] The nucleic acid transfer vector of [3], further comprising a nucleotide sequence encoding a self-cleaving peptide, wherein at least a portion of the nucleotide sequence of the exon, the nucleotide sequence encoding the self-cleaving peptide, and the nucleotide sequence of the nucleic acid to be introduced are located in this order, from the 5' side, relative to the sense strand. [5] The nucleic acid introduction vector according to any one of [1] to [4], wherein the nucleic acid to be introduced is knocked into the intron by non-homologous end joining. [6] The nucleic acid introduction vector according to any one of [1] to [5], wherein the Cas protein is AsCasl2f. [7] The nucleic acid introduction vector according to any one of [1] to [6], wherein a single vector comprises a nucleotide sequence encoding the Cas protein, a nucleotide sequence encoding the guide RNA, and a nucleotide sequence of the nucleic acid to be introduced. [8] A nucleic acid introduction method, comprising a step of introducing the nucleic acid to be introduced by non-homologous end joining, targeting an intron adjacent to the 5' side of an exon containing a stop codon, relative to the sense strand. [9] A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 16 or 20.

[10] A vector comprising the polynucleotide of [9].

[11] The nucleic acid introduction vector according to any one of [1] to [6], wherein the nucleotide sequence of the nucleic acid to be introduced comprises the nucleotide sequence of SEQ ID NO: 16.

[0009] The present invention provides a nucleic acid transfer vector and a nucleic acid transfer method that can improve the expression level of a transgene. Also provided are a polynucleotide that encodes ornithine transcarbamylase and that is expressed at a high level in a human cell into which it is transferred, and a vector containing the polynucleotide.

[0010]

[0023] Figure 1 is a schematic diagram illustrating the structure of a nucleic acid transfer vector and an outline of a nucleic acid transfer method according to one embodiment. It shows the structure of a plasmid used to prepare an AAV vector in Experimental Example 1. Three types of plasmids were prepared: HDR intron 14, NHEJ intron 13, and NHEJ intron 13,14. It is a schematic diagram illustrating knock-in of blood coagulation factor IX (FIX) R338L cDNA via homologous directed repair (HDR) using the HDR intron 14 vector prepared in Experimental Example 1. It is a schematic diagram illustrating knock-in of FIX R338L cDNA via non-homologous end-joining (NHEJ) using the NHEJ intron 13 vector prepared in Experimental Example 1.

[0033] Figure 1 shows the results of a T7 endonuclease assay in Experimental Example 1.

[0034] Figure 1-1 shows the results of measuring FIX activity and FIX antigen levels in newborn hemophilia B mice after AAV vector administration.

[0035] Figure 1-1 shows the results of fluorescent immunostaining of liver tissue sections excised from newborn hemophilia B mice after AAV vector administration.

[0036] Figure 1-1 shows the results of analyzing the FIX-positive cell rate from fluorescent immunostained images.

[0037] Figure 1-2 shows the results of measuring FIX activity in adult hemophilia B mice after AAV vector administration.

[0038] Figure 1-2 shows the results of fluorescent immunostaining of liver tissue sections excised from adult hemophilia B mice after AAV vector administration.

[0039] Figure 1-2 shows the results of analyzing the FIX-positive cell rate from fluorescent immunostained images.

[0039] Figure 1-2 shows the results of measuring plasma human protein C activity in wild-type newborn mice (C57BL / 6J) after AAV vector administration. In Experimental Example 2, neonatal mice were administered with an AAV vector and an anti-FVIII antibody (Proc ＋／＋ ,Proc. ＋／－ ,Proc. －／－ 2 shows the Kaplan-Meier survival curves of neonatal mice (C57BL / 6J, Proc ＋／＋ ,Proc. ＋／－ ,Proc. －／－ ,Proc. －／－F8 －／－ 1 shows the results of measuring plasma human protein C activity in neonatal mice (C57BL / 6J, Proc ＋／＋ ,Proc. ＋／－ ,Proc. －／－ ,Proc. －／－ F8 －／－ In Experimental Example 2, the plasma factor V activity of newborn mice (C57BL / 6J, Proc ＋／＋ ,Proc. ＋／－ ,Proc. －／－ ,Proc. －／－ F8 －／－ 1 shows the results of measuring plasma factor VIII activity in OTC mice administered with an AAV vector. ｓｐｆ－ａｓｈ The graph shows the weight change in male mice. Untreated: untreated mice (mice administered with physiological saline), 3E+11: 3.0 x 10 １１ vg / body AAV vector-administered mice, 1E+12: 1.0 × 10 １２ vg / body AAV vector-administered mice. The same applies below. In Experimental Example 3, OTC mice administered with AAV vectors ｓｐｆ－ａｓｈ 1 shows the change in blood ammonia concentration in male mice. ｓｐｆ－ａｓｈ The figure shows the OTC enzyme activity in the liver of male mice. *: p<0.05. The significance test was performed by one-way ANOVA. In Experimental Example 3, the OTC enzyme activity in the liver of male mice administered with the AAV vector was ｓｐｆ－ａｓｈ This shows the results of Western blotting for hOTC_FLAG in male mice. #1 to #4 indicate the individual mouse numbers. This shows the results of analyzing the amount of hOTC_FLAG protein from Western blotting images in Experimental Example 3. The amount of hOTC_FLAG protein is shown as a relative amount when the protein amount of the positive control is set to 1. ns: p≧0.05. A one-way ANOVA was used to test for significance. In Experimental Example 3, OTC administered with an AAV vector ｓｐｆ－ａｓｈThe expression level of hOTC_FLAG mRNA in male mice is shown. The expression level of hOTC_FLAG mRNA was normalized to the expression level of GAPDH mRNA. *: p<0.05, **: p<0.01. Significance test was performed by one-way ANOVA. In Experimental Example 3, the OTC_FLAG expression level in male mice administered with an AAV vector was normalized to the expression level of GAPDH mRNA. ｓｐｆ－ａｓｈ

[0033] Figure 1 shows the results of fluorescent immunostaining of liver tissue sections excised from male mice. Scale bar: 100 μm. Experimental Example 3 shows the results of analyzing the hOTC_FLAG-positive cell rate from fluorescent immunostaining images. Experimental Example 4 shows the results of comparing the expression levels of human OTC protein in cells transfected with wild-type human OTC cDNA (WT) and three modified cDNAs (CO#1, CO#2, CO#3). Experimental Example 4 shows the results of measuring OTC enzyme activity in cell lysates transfected with wild-type human OTC cDNA (WT) and three modified cDNAs (CO#1, CO#2, CO#3). Experimental Example 5 shows the results of a T7 endonuclease assay.

[0011] Unless otherwise specified, "a," "an," and "the" are inclusive of singular and plural and are understood to mean "one or more."

[0012] The term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that would exert a special function (such as a manner that would completely lose the effects of the invention). In this specification, when "comprise" is used, it includes both "consist of" and "consist essentially of" embodiments.

[0013] When a protein, polypeptide, or peptide is described as having a specific amino acid sequence, this includes embodiments in which it contains other amino acid sequences in addition to the specific amino acid sequence, and embodiments in which it consists of the specific amino acid sequence.When a nucleic acid or polynucleotide is described as having a specific nucleotide sequence, this includes embodiments in which it contains other nucleotide sequences in addition to the specific nucleotide sequence, and embodiments in which it consists of the specific nucleotide sequence.

[0014] A numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the upper and lower limits. When multiple upper and lower limit values ​​are written for a specific parameter, any upper and lower limit values ​​can be combined to form a suitable numerical range.

[0015] Proteins, peptides, nucleic acids (polynucleotides), vectors, and cells may be isolated. "Isolated" means separated from other components. An "isolated" component may be separated from its natural state. An "isolated" component may be substantially free of other components. "Substantially free of other components" means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component may be, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less, or 0.1% by mass or less. The proteins, peptides, polynucleotides (DNA, RNA), vectors, and cells described herein may be isolated proteins, isolated peptides, isolated nucleic acids (isolated polynucleotides), isolated vectors, and isolated cells, respectively.

[0016] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a nucleotide polymer in which nucleotides are linked by phosphodiester bonds. "Nucleic acid" and "polynucleotide" may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of DNA and RNA. "Nucleic acid" and "polynucleotide" may be a polymer of natural nucleotides, a polymer of natural nucleotides and non-natural nucleotides, or a polymer of non-natural nucleotides. "Non-natural nucleotide" refers to a nucleotide in which a portion of the structure of a natural nucleotide has been modified. Any of the base, sugar, and phosphate of a non-natural nucleotide may be modified.

[0017] Nucleotide sequences of "nucleic acids" and "polynucleotides" are written in the commonly accepted single-letter code unless otherwise specified. Nucleotide sequences are written from the 5' to the 3' end unless otherwise specified. Nucleotide residues constituting "nucleic acids" and "polynucleotides" may be written simply as adenine, thymine, cytosine, guanine, or uracil, or as their single-letter codes.

[0018] The terms "protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of amino acids linked by amide bonds. A "protein," "polypeptide," and "peptide" may be a polymer of natural amino acids, a polymer of natural amino acids and unnatural amino acids, or a polymer of unnatural amino acids. An "unnatural amino acid" refers to an amino acid in which a portion of the structure of a natural amino acid has been modified. The amino acid sequences of "proteins," "polypeptides," and "peptides" are written using the commonly accepted one-letter or three-letter code unless otherwise specified. Unless otherwise specified, the amino acid sequences are written from the N-terminus to the C-terminus. The amino acid numbers in the amino acid sequences indicate the position of the amino acid from the N-terminus.

[0019] Unless otherwise specified, the terms "5' side" and "3' side" of DNA refer to the sense strand. That is, the "5' side" refers to the 5' side of the sense strand. The "3' side" refers to the 3' side of the sense strand.

[0020] Amino acid substitutions are sometimes expressed by the single letter code of the original amino acid, followed by the amino acid number, followed by the single letter code of the replacement amino acid.

[0021] [Nucleic Acid Introduction Vector] A first aspect of the present disclosure is a nucleic acid introduction vector. The nucleic acid introduction vector includes a nucleotide sequence encoding a Cas protein, a nucleotide sequence encoding a guide RNA, and a nucleotide sequence of a nucleic acid to be introduced. The guide RNA targets an intron adjacent to the 5' side of an exon containing a stop codon, relative to the sense strand.

[0022] (Nucleotide sequence encoding Cas protein) "Cas protein" refers to a CRISPR-associated protein. The Cas protein forms a complex with a guide RNA and exhibits sequence-specific endonuclease activity when guided by the guide RNA. The Cas protein may be any of types I to VI, but type II or type V is preferred. Examples of Cas proteins include Cas9 such as SpCas9 (Cas9 derived from Streptococcus pyogenes) and SsCas9 (Cas9 derived from Staphylococcus aureus); Cas12a such as AsCas12a (Cas12a derived from Acidaminococcus sp.); and Cas12f such as AsCas12f (Cas12f derived from Acidibacillus sulfuroxidans).

[0023] The Cas protein may be a wild-type protein or a modified version obtained by modifying a wild-type protein. Examples of the modified Cas protein include the following proteins (a) and (b):

[0024] (a) A protein having an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence of a wild-type Cas protein, and having RNA-guided endonuclease activity. (b) A protein having 80% or more sequence identity with the amino acid sequence of a wild-type Cas protein, and having RNA-guided endonuclease activity.

[0025] In the above (a) and (b), the "RNA-guided endonuclease activity" refers to an endonuclease activity that forms a complex with a guide RNA and is guided by the guide RNA to cleave nucleic acids in a sequence-specific manner.

[0026] In (a), "several" includes 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, and 2. In (a), "mutation" may be any of deletion, substitution, addition, and insertion, or a combination thereof. In (b), sequence identity includes 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more.

[0027] In one embodiment, the Cas protein is preferably AsCas12f because it has a small molecular size and can be incorporated into a single vector together with the donor nucleic acid. As used herein, "AsCas12f" encompasses wild-type AsCas12f and modified AsCas12f. Examples of the amino acid sequence of wild-type AsCas12f include the amino acid sequence set forth in SEQ ID NO: 11. Examples of modified AsCas12f include the modified AsCas12f described in International Publication No. 2022 / 092317 and Hino T, et al. Cell. 2023;186:4920-4935.e23.

[0028] Specific examples of modified AsCas12f include the following proteins (1) to (18): (1) A protein comprising at least one of a substitution with histidine at amino acid position 188 and a substitution with lysine at amino acid position 195 in the amino acid sequence represented by SEQ ID NO: 11. (2) A protein comprising at least a substitution with histidine at amino acid position 188 in the amino acid sequence represented by SEQ ID NO: 11. (3) A protein comprising at least a substitution with histidine at amino acid position 188 and a substitution with alanine at amino acid position 232 in the amino acid sequence represented by SEQ ID NO: 11. (4) A protein comprising at least a substitution with histidine at amino acid position 188 and a substitution with alanine at amino acid position 232 in the amino acid sequence represented by SEQ ID NO: 11. (5) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, a substitution with histidine at amino acid position 188 and a substitution with alanine at amino acid position 232, and also comprising one or more substitutions of histidine at amino acid position 123, methionine at amino acid position 246, methionine at amino acid position 316, and leucine at amino acid position 337. (6) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with histidine at amino acid position 188, a substitution with alanine at amino acid position 232, and a substitution with methionine at amino acid position 316. (7) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with histidine at amino acid position 188, a substitution with alanine at amino acid position 232, and a substitution with methionine at amino acid position 316. (8) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with histidine at amino acid position 188, a substitution with alanine at amino acid position 232, a substitution with methionine at amino acid position 316, and a substitution with tyrosine at amino acid position 48. (9) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with lysine at amino acid position 195.(10) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with lysine at amino acid position 195 and a substitution with alanine at amino acid position 232. (11) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with lysine at amino acid position 195 and a substitution with alanine at amino acid position 232. (12) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, a substitution with lysine at amino acid position 195 and a substitution with alanine at amino acid position 232, and one or more of a substitution with tyrosine at amino acid position 70, a substitution with arginine at amino acid position 208, and a substitution with methionine at amino acid position 318. (13) A protein comprising, in the amino acid sequence represented by SEQ ID NO: 11, at least a substitution with lysine at amino acid position 195, a substitution with alanine at amino acid position 232, and a substitution with arginine at amino acid position 208. (14) A protein having the amino acid sequence represented by SEQ ID NO: 11, which contains at least a lysine substitution at position 195, an alanine substitution at position 232, and an arginine substitution at position 208. (15) A protein having the amino acid sequence represented by SEQ ID NO: 11, which contains at least a lysine substitution at position 195, an alanine substitution at position 232, an arginine substitution at position 208, and a histidine substitution at position 123. (16) A protein having the amino acid sequence represented by SEQ ID NO: 11, which does not have a mutation at position 195, and which contains at least a histidine substitution at position 188 and alanine substitution at position 232. (17) A protein having the amino acid sequence represented by SEQ ID NO: 11, which does not have a mutation at position 188, and which contains at least a lysine substitution at position 195 and alanine substitution at position 232.(18) A protein having the amino acid sequence represented by SEQ ID NO: 11, in which the amino acid at position 188 is not mutated, and which contains at least a substitution with lysine at position 195, a substitution with arginine at position 208, and a substitution with alanine at position 232.

[0029] In one embodiment, the modified AsCas12f is a protein having the amino acid sequence set forth in SEQ ID NO: 12 or 13. The amino acid sequence set forth in SEQ ID NO: 12 is an amino acid sequence in which the phenylalanine at amino acid position 48 has been substituted with tyrosine (F48Y), the serine at amino acid position 188 has been substituted with histidine (S188H), the valine at amino acid position 232 has been substituted with alanine (V232A), and the glutamic acid at amino acid position 316 has been substituted with methionine (E316M) in the amino acid sequence of wild-type AsCas12f (SEQ ID NO: 11). The modified AsCas12f having the amino acid sequence set forth in SEQ ID NO: 12 may be referred to as "AsCas12f-YHAM." The amino acid sequence of SEQ ID NO: 13 is an amino acid sequence in which the isoleucine at amino acid position 123 is substituted with histidine (I123H), the aspartic acid at amino acid position 195 is substituted with lysine (D195K), the aspartic acid at amino acid position 208 is substituted with arginine (D208R), and the valine at amino acid position 232 is substituted with alanine (V232A) in the amino acid sequence of wild-type AsCas12f (SEQ ID NO: 11). Modified AsCas12f having the amino acid sequence of SEQ ID NO: 13 may be referred to as "AsCas12f-HKRA."

[0030] The nucleotide sequence encoding the Cas protein may be any nucleotide sequence that encodes a Cas protein, and may be the nucleotide sequence of a wild-type Cas gene, or a modified nucleotide sequence of a wild-type Cas gene. The nucleotide sequence encoding the Cas protein may include all degenerate isomers. "Degenerate" refers to the presence of multiple codons corresponding to one amino acid. "Degenerate isomers" refer to multiple types of nucleotide sequences that encode the same amino acid sequence.

[0031] The nucleotide sequence encoding the Cas protein may be codon-optimized according to the biological species of the cell into which the nucleic acid is to be introduced. Codon optimization refers to replacing at least one codon in the original nucleotide sequence with a codon more frequently used in the target biological species while maintaining the original amino acid sequence. Codon usage tables are readily available, for example, from the "Codon Usage Database" (www.kazusa.or.jp / codon / ) provided by the Kazusa DNA Research Institute, a public interest incorporated foundation. Codons can be optimized according to the biological species using these known codon usage tables. Computer algorithms for codon-optimizing specific sequences for expression in specific biological species are available, for example, from GeneForge (Aptagen; Jacobus, PA).

[0032] In one embodiment, the nucleotide sequence encoding the Cas protein (hereinafter also referred to as the "Cas protein coding sequence") is operably linked to a promoter. "Operatively linked to a promoter" means that the Cas protein coding sequence is linked to a promoter so as to be expressed under the control of the promoter. The promoter is not particularly limited as long as it has the function of expressing the Cas protein. Examples of promoters include Pol II promoters. Examples of Pol II promoters include, but are not limited to, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, and CBh promoter. When expressing a gene to be introduced in an organ-specific manner, a promoter of a gene that is highly expressed in an organ-specific manner may be used. For example, an example of a promoter of a gene that is highly expressed in the liver is the transthyretin (TTR) gene promoter. A poly(A) addition signal may be linked to the 3' end of the Cas protein coding sequence.

[0033] (Nucleotide sequence encoding guide RNA) "Guide RNA" refers to an RNA that can form a complex with a Cas protein and guide the Cas protein to a target sequence. Guide RNA usually includes CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The crRNA is involved in binding to the target region of the genome, and the tracrRNA is involved in binding to the Cas protein. The crRNA includes a spacer sequence (guide sequence). The guide sequence binds complementarily to the target strand (TS) of the DNA to be edited.

[0034] A "guide sequence" refers to a sequence contained in a guide RNA that can bind to a complementary sequence of a target sequence. A "target sequence" is a sequence adjacent to a protospacer adjacent motif (PAM). The target sequence is typically selected as a nucleotide sequence of 15 to 50 nucleotide residues (e.g., 17 to 50 nucleotide residues, preferably 20 to 40 nucleotide residues, more preferably 20 to 30 nucleotide residues, and even more preferably 20 nucleotide residues) adjacent to the 5' or 3' side of the PAM. In type II Cas proteins such as Cas9, the target sequence is the sequence adjacent to the 5' side of the PAM. In type V Cas proteins such as Cas12a and Cas12f, the target sequence is the sequence adjacent to the 3' side of the PAM. The PAM varies depending on the type of Cas protein. For example, the PAM corresponding to SpCas9 is "NGG" (where N is A, T, G, or C). The PAM corresponding to AsCas12f is "TTTG."

[0035] When intron 13 of the mouse albumin gene locus is targeted, examples of the target sequence include the nucleotide sequences set forth in SEQ ID NOs: 1 to 7. Of these, the nucleotide sequence set forth in SEQ ID NO: 5 is preferred as the target sequence for intron 13 of the mouse albumin gene locus.

[0036] When intron 13 of the human albumin locus is targeted, examples of the target sequence include the nucleotide sequences set forth in SEQ ID NOs: 29 to 30. Of these, the nucleotide sequence set forth in SEQ ID NO: 33 is preferred as the target sequence for intron 13 of the human albumin locus.

[0037] The guide RNA may be composed of wild-type crRNA and wild-type tracrRNA, or may be composed of crRNA and tracrRNA with portions of the structure modified or removed. The guide RNA includes a guide sequence and may include one or more stem structures derived from either or both of the crRNA and tracrRNA.

[0038] The guide RNA may be a dual guide RNA or a single guide RNA (sgRNA). Dual guide RNA is a guide RNA in which one molecule of crRNA and one molecule of tracrRNA are complementary linked. sgRNA is a guide RNA in which crRNA and tracrRNA are linked to form a single RNA molecule. In one embodiment, the guide RNA is an sgRNA.

[0039] Various designs of guide RNA have been proposed, and those skilled in the art can design guide RNAs based on known techniques. For example, wild-type guide RNA for AsCas12f has a stem structure of Stem1 to Stem5. Guide RNA for AsCas12f may be a guide RNA from which Stem3 to Stem5 have been removed. Guide RNA for AsCas12f may have a guide sequence and Stem1 and Stem2. Examples of guide RNAs that can be used for AsCas12f include those described in Hino T, et al. Cell. 2023;186:4920-4935.e23.

[0040] The guide RNA of this embodiment targets an intron adjacent to the 5' side of an exon containing a stop codon. An "exon containing a stop codon" is an exon containing a stop codon among exons in a target gene locus into which a nucleic acid to be introduced is to be introduced. A "stop codon" is a codon that has no corresponding amino acid and terminates protein biosynthesis.

[0041] An "intron adjacent to the 5' side of an exon containing a stop codon" is an intron adjacent to the upstream side of an exon containing a stop codon. For example, in the target locus shown in Figure 1, Exon(stop) represents the exon containing the stop codon. Exon(X) is the exon located immediately upstream of Exon(stop). Intron(prestop) is an intron adjacent to the 5' side of an exon containing a stop codon. Intron(prestop) is located between Exon(stop) and Exon(X).

[0042] The guide RNA of this embodiment contains, as a guide sequence, a target sequence adjacent to the 5' or 3' side of a PAM present in an intron (hereinafter also referred to as "intron (prestop)") adjacent to the 5' side of an exon (hereinafter also referred to as "exon (stop)") containing a stop codon. The "target sequence" is a nucleotide sequence of the non-target strand (NTS) corresponding to the nucleotide sequence to which the guide sequence binds in the TS.

[0043] A target locus into which a nucleic acid to be introduced is selected that contains PAM in an intron (prestop). For example, the locus of a constitutively expressed gene can be selected as the target locus. For example, the locus of a highly expressed gene can be selected as the target locus. Examples of target loci include, but are not limited to, the albumin locus, α1-antitrypsin locus, selenoprotein P locus, fibrinogen locus, and transthyretin locus.

[0044] In one embodiment, the target locus is the albumin locus. In one embodiment, the guide RNA targets the 13th intron (intron 13) of the albumin locus. The human and mouse albumin loci contain a stop codon in the 14th exon (exon 14). Thus, in the human and mouse albumin loci, the exon (stop) is exon 14 and the intron (prestop) is intron 13.

[0045] In one embodiment, a nucleotide sequence encoding a guide RNA (hereinafter also referred to as a "guide RNA coding sequence") is operably linked to a promoter. "Operatively linked to a promoter" means that the guide RNA coding sequence is linked to a promoter so as to be expressed under the control of the promoter. The promoter is not particularly limited as long as it has the function of expressing the guide RNA. Examples of promoters include pol III promoters. Examples of pol III promoters include mouse U6-snRNA promoter, human U6-snRNA promoter, human H1-RNase P RNA promoter, and human valine-tRNA promoter.

[0046] (Nucleotide sequence of nucleic acid to be introduced) A "nucleic acid to be introduced" is a nucleic acid intended to be introduced into a target gene locus by a nucleic acid introduction vector. The nucleic acid to be introduced is, for example, a gene intended to be expressed in a target cell (hereinafter also referred to as a "gene to be introduced").

[0047] The gene to be introduced is not particularly limited. For example, the gene to be introduced may be a gene for improving abnormal gene expression in a disease involving abnormal gene expression. For example, the gene to be introduced may be a gene whose gene expression has been reduced or eliminated due to abnormal gene expression. For example, the gene to be introduced may be a gene for an expression control molecule that suppresses the expression of a gene whose gene expression has become excessive due to abnormal gene expression. Examples of the expression control molecule include small interfering nucleic acids (siRNA, shRNA, etc.), transcription inhibitors, etc.

[0048] Examples of genes to be introduced include, but are not limited to, the blood coagulation factor IX (FIX) gene, blood coagulation factor IX variant genes (factor IX Padua; factor IX R338L), protein C gene, ornithine transcarbamylase gene, phenylalanine hydroxylase gene, blood coagulation factor VIII gene, α-L-iduronidase gene, iduronate-2-sulfatase gene, heparan N-sulfatase gene, α-N-acetylglucosaminidase gene, acetyl-CoA:α-glucosaminide N-acetyltransferase gene, N-acetylglucosamine-6-sulfatase gene, N-acetylgalactosamine-6-sulfatase gene, β-galactosidase gene, N-acetylgalactosamine-4-sulfatase gene, β-glucuronidase gene, and hyaluronidase gene.

[0049] From the viewpoint of reducing the molecular size, it is preferable that the gene to be introduced does not contain introns. The gene to be introduced may be, for example, a cDNA of a protein intended to be expressed in the target cell (hereinafter also referred to as a "protein to be expressed"). The gene to be introduced contains a coding sequence (CDS) of the protein to be expressed.

[0050] The nucleotide sequence of the gene to be introduced does not need to contain a start codon (ATG). When the nucleic acid introduction vector contains an exon (stop) described below, the start codon of the gene present in the target locus is used as the start codon of the gene to be introduced. Therefore, the nucleotide sequence of the gene to be introduced contained in the nucleic acid introduction vector does not need to contain a start codon. The nucleotide sequence of the gene to be introduced contained in the nucleic acid introduction vector may be a nucleotide sequence in which the start codon is removed from the CDS of the protein to be expressed.

[0051] The gene to be introduced may be a wild-type gene, or a modified gene in which the wild-type nucleotide sequence has been modified. The modified gene may have a reduced proportion of guanine residues and cytosine residues to improve expression in the introduced cells. The modified gene may be codon-optimized depending on the type of cell to be introduced. The modified gene preferably has a nucleotide sequence that encodes the same amino acid sequence as the wild-type gene. When the gene to be introduced is an ornithine transcarbamylase gene, the nucleotide sequence of the gene to be introduced is preferably the nucleotide sequence set forth in SEQ ID NO: 16. The nucleotide sequence set forth in SEQ ID NO: 16 is the nucleotide sequence obtained by removing the start codon from the nucleotide sequence set forth in SEQ ID NO: 20 and adding a stop codon.

[0052] A poly A addition signal may be linked to the 3' end of the gene to be introduced.

[0053] The nucleic acid introduction vector may further include at least a portion of the nucleotide sequence of an exon (stop). In one embodiment, the exon (stop) is located, for example, on the 5' side (upstream) of the nucleic acid to be introduced. In one embodiment, the nucleotide sequence of the exon (stop) located upstream of the nucleic acid to be introduced does not include the stop codon of the exon (stop) and the nucleotide sequence 3' side (downstream of) the stop codon. In one embodiment, the nucleotide sequence of the exon (stop) located upstream of the nucleic acid to be introduced is a nucleotide sequence up to one nucleotide upstream of the stop codon in the exon (stop). For example, the nucleotide sequence of the exon (stop) located upstream of the nucleic acid to be introduced is a nucleotide sequence from the first codon of the exon (stop) to the codon immediately preceding the stop codon.

[0054] When the target locus is the human or mouse albumin locus, the nucleic acid transfer vector may comprise the nucleotide sequence of exon 14 of the albumin locus on the 5' side (upstream) of the nucleic acid to be transferred. For example, the nucleic acid transfer vector may comprise the nucleotide sequence from the first codon of exon 14 of the albumin locus to the codon immediately preceding the stop codon. An example of the nucleotide sequence of exon 14 of the mouse albumin locus contained in the nucleic acid transfer vector is the nucleotide sequence set forth in SEQ ID NO: 24. The nucleotide sequence of exon 14 of the human albumin locus is set forth in SEQ ID NO: 27. An example of the nucleotide sequence of exon 14 of the human albumin locus contained in the nucleic acid transfer vector is the nucleotide sequence set forth in SEQ ID NO: 28.

[0055] The nucleic acid introduction vector may further include a nucleotide sequence encoding a self-cleaving peptide (hereinafter also referred to as a "self-cleaving peptide coding sequence"). In one embodiment, the exon (stop), the self-cleaving peptide coding sequence, and the nucleic acid to be introduced are arranged in this order from the 5' side. In one embodiment, the exon (stop) is arranged on the 5' side (upstream) of the nucleic acid to be introduced, via the self-cleaving peptide coding sequence. With this configuration, the nucleic acid to be introduced can be expressed using the promoter of the target gene locus without inhibiting expression of the gene at the target gene locus. Furthermore, because the nucleic acid is cleaved by the self-cleaving peptide, the protein encoded by the target gene locus and the protein encoded by the nucleic acid to be introduced can be expressed as separate proteins.

[0056] A "self-cleaving peptide" is a peptide that has the function of splitting a protein translated from one mRNA into two. When two CDSs are linked in-frame via a self-cleaving peptide coding sequence, proteins are produced from the upstream CDS and the downstream CDS of the self-cleaving peptide coding sequence, respectively. The self-cleaving peptide induces the production of two proteins from one mRNA by ribosomal skipping or the like. Any known self-cleaving peptide can be used without particular limitation. Examples of self-cleaving peptides include, but are not limited to, 2A peptides. Examples of 2A peptides include, but are not limited to, P2A peptide, F2A peptide, E2A peptide, T2A peptide, etc.

[0057] (Other Components) The nucleic acid introduction vector of this embodiment may include other components in addition to the above components. Examples of other components include a guide RNA recognition sequence and an intron (prestop) nucleotide sequence.

[0058] <Guide RNA Recognition Sequence> The "guide RNA recognition sequence" is a sequence consisting of a PAM and a target sequence of a guide RNA adjacent to the PAM. The guide RNA recognition sequence is recognized by a complex of a Cas protein and a guide RNA and cleaved by the Cas protein. The guide RNA recognition sequence is preferably positioned between the set of the Cas protein coding sequence and the guide RNA coding sequence and the set of nucleic acids to be introduced. As a result, upon cleavage of the guide RNA recognition sequence, the set of the Cas protein coding sequence and the guide RNA coding sequence is separated from the set of nucleic acids to be introduced. This allows only the set of nucleic acids to be introduced to be knocked into the target gene locus.

[0059] In addition, by cleaving the nucleic acid introduction vector at the guide RNA recognition sequence, it is possible to suppress the sustained expression from the nucleic acid introduction vector.This is thought to be due to the decrease in the stability of the nucleic acid introduction vector.For example, AAV vectors are stable in the form of circular concatemers or the like.It is presumed that the AAV vector contains a guide RNA recognition sequence, and by being cleaved at the guide RNA recognition sequence, the circular structure of the AAV vector is inhibited.This is thought to impair the stability of the AAV vector, making it impossible to sustain expression.

[0060] The region containing the guide RNA recognition sequence may be a region in which the 5' and 3' sides of a region containing a target sequence present in an intron (prestop) of the target gene locus are reversed. The length of the region containing the target sequence can be, for example, 25 to 50 bp.

[0061] <Nucleotide sequence of intron (prestop)> When the nucleic acid introduction vector contains the nucleotide sequence of an exon (stop), the nucleic acid introduction vector may further contain at least a portion of the nucleotide sequence of the intron (prestop). In one embodiment, the intron (prestop) is positioned on the 5' side (upstream) of the exon (stop), similar to its position in the target gene locus. In one embodiment, the nucleotide sequence of the intron (prestop) is positioned adjacent to the 5' side of the nucleotide sequence of the exon (stop).

[0062] The nucleotide sequence of the intron (prestop) contained in the nucleic acid introduction vector may include at least a portion of the nucleotide sequence of the intron (prestop). The nucleotide sequence of the intron (prestop) preferably includes the nucleotide sequence of the 3' splice site of the intron (prestop). The nucleotide sequence of the intron (prestop) preferably does not include the nucleotide sequence of the 5' splice site of the intron (prestop). Examples of the nucleotide sequence of the intron (prestop) contained in the nucleic acid introduction vector include nucleotide sequences located 3' (downstream) of the 5' splice site of the intron (prestop). Examples of the nucleotide sequence of the intron (prestop) include nucleotide sequences that include the 3' splice site of the intron (prestop) but do not include the 5' splice site.

[0063] It is preferable that the nucleotide sequence of the intron (prestop) contained in the nucleic acid introduction vector does not include the guide RNA recognition sequence present in the intron (prestop), and that a nucleotide sequence located 3' (downstream) of the guide RNA recognition sequence is used.

[0064] When the target locus is the human or mouse albumin locus, the nucleic acid transfer vector may comprise, on the 5' side (upstream) of the nucleic acid to be transferred, the nucleotide sequence of intron 13 of the albumin locus and the nucleotide sequence of exon 14 of the albumin locus. In one embodiment, the nucleotide sequence of intron 13 of the albumin locus is a nucleotide sequence that includes the 3' splice site of intron 13 but does not include the 5' splice site. The nucleotide sequence of intron 13 of the mouse albumin locus is shown in SEQ ID NO: 22. An example of a nucleotide sequence of intron 13 of the mouse albumin locus contained in the nucleic acid transfer vector is the nucleotide sequence shown in SEQ ID NO: 23. The nucleotide sequence of intron 13 of the human albumin locus is shown in SEQ ID NO: 26. An example of a nucleotide sequence of intron 13 of the human albumin locus contained in the nucleic acid transfer vector is the nucleotide sequence from any nucleotide residue located 3' (downstream) from the target sequence to the 569th nucleotide residue in the nucleotide sequence shown in SEQ ID NO: 26. An example of the nucleotide sequence of intron 13 of the human albumin gene locus contained in the nucleic acid transfer vector is the nucleotide sequence set forth in SEQ ID NO:37.

[0065] <Homology Arm> A "homology arm" refers to a region subjected to homologous recombination in knock-in of a nucleic acid to be introduced via HDR. A homology arm includes the nucleotide sequence of the knock-in target region and its surrounding region. In HDR-mediated knock-in using the CRISPR / Cas system, a homology arm including a region adjacent to the 5' side of the target sequence is called a 5' homology arm. A homology arm including a region adjacent to the 3' side of the target sequence is called a 3' homology arm.

[0066] In one embodiment, the nucleic acid to be introduced is knocked into an intron (prestop) by NHEJ. In this case, the nucleic acid introduction vector of this embodiment does not contain a homology arm (particularly a 3' homology arm). However, even in this case, the nucleic acid introduction vector of this embodiment may contain at least a portion of the nucleotide sequence of the intron (prestop) and exon (stop).

[0067] (Vector) In the nucleic acid introduction vector of this embodiment, the type of vector is not particularly limited, and known vectors can be used. Examples of vectors include plasmid vectors and viral vectors. The plasmid vector can be selected appropriately depending on the type of target cell. Examples of plasmid vectors for expression in animal cells include pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors), Sendai viral vectors, herpes viral vectors, vaccinia viral vectors, pox viral vectors, polio viral vectors, Simbis viral vectors, rhabdoviral vectors, paramyxoviral vectors, orthomyxoviral vectors, EB viral vectors, papilloma viral vectors, and foamy viral vectors.

[0068] In one embodiment, the nucleic acid transfer vector is a viral vector. In one embodiment, the nucleic acid transfer vector is an AAV vector. By using AsCasl2f as the Cas protein, the size of the Cas protein coding sequence can be reduced. This allows a single viral vector, such as an AAV vector, to carry the Cas protein coding sequence, the guide RNA coding sequence, and the nucleic acid to be transferred.

[0069] (Example of the structure of a vector for nucleic acid introduction) Figure 1 shows an example of the structure of a vector for nucleic acid introduction. In the example of Figure 1, a genome editing cassette and a donor DNA cassette are mounted between ITRs (inverted terminal repeats). In the example of Figure 1, an AsCas12f coding sequence is used as the Cas protein coding sequence. The AsCas12f coding sequence may be a wild-type AsCas12f coding sequence or a modified AsCas12f coding sequence. Examples of modified AsCas12f coding sequences include an AsCas12f-YHAM coding sequence and an AsCas12f-HKRA coding sequence. An sgRNA coding sequence is used as the guide RNA coding sequence. Examples of sgRNA coding sequences include, for example, the coding sequence of an sgRNA that targets intron 13 of the human or mouse albumin locus. The P2A peptide coding sequence is used as the self-cleaving peptide coding sequence. The sgRNA recognition sequence is used as the guide RNA recognition sequence. In FIG. 1, p1 represents a promoter operably linked to AsCas12f. p2 represents a promoter operably linked to sgRNA. pA represents a polyA addition signal.

[0070] In the example of FIG. 1, the genome editing cassette includes an AsCas12f expression cassette (p1 and AsCas12f coding sequence) and an sgRNA expression cassette (p2 and sgRNA coding sequence). The donor DNA includes an intron (prestop), an exon (stop), a P2A peptide coding sequence, and a nucleic acid to be introduced. The intron (prestop), exon (stop), a P2A peptide coding sequence, and a nucleic acid to be introduced are linked in frame in this order from the 5' side (upstream). An sgRNA recognition sequence is located between the genome editing cassette and the donor DNA. Examples of the nucleotide sequence of the exon (stop) include the nucleotide sequence from the 5' end (first codon) of exon 14 of the human or mouse albumin locus to the codon immediately preceding the stop codon. Examples of the nucleotide sequence of the intron (prestop) include a nucleotide sequence that includes the 3' splice site but not the 5' splice site of intron 13 of the human or mouse albumin locus. Examples of the nucleotide sequence of the intron (prestop) include a nucleotide sequence from a nucleotide residue located 3' (downstream) from the target sequence of intron 13 of the human or mouse albumin locus to the 3' end of intron 13. The nucleic acid to be transferred is not particularly limited. When the nucleic acid to be transferred contains an ornithine transcarbamylase gene, the nucleic acid to be transferred may contain the nucleotide sequence set forth in SEQ ID NO: 16.

[0071] When the nucleic acid transfer vector shown in FIG. 1 is introduced into a cell, AsCas12f and sgRNA are expressed from the genome editing cassette. AsCas12f and sgRNA form a complex and cleave the sgRNA recognition sequence in the vector. In addition, the sgRNA recognition sequence in the intron (prestop) at the target locus of the cell genome (e.g., human or mouse albumin locus) is cleaved. As a result, the donor DNA is knocked into the intron (prestop) (e.g., intron 13 of the human or mouse albumin locus). Because the nucleic acid transfer vector shown in FIG. 1 does not contain homology arms, the donor DNA is knocked in by NHEJ.

[0072] The nucleic acid to be introduced knocked into the intron (prestop) is transcribed together with a gene present in the target locus (hereinafter also referred to as the "target locus gene") (e.g., a human or mouse albumin gene) by a promoter located upstream of the target locus. In the mRNA produced in this way, the transcription sequence from the target locus gene and the transcription sequence from the nucleic acid to be introduced are linked via the transcription sequence from the P2A peptide coding sequence. When this mRNA is translated, the protein encoded by the target locus gene and the protein encoded by the nucleic acid to be introduced are produced separately.

[0073] In the example of Figure 1, the genome editing cassette is placed upstream of the donor DNA via the sgRNA recognition sequence, but the genome editing cassette may also be placed downstream of the donor DNA via the sgRNA recognition sequence. In the example of Figure 1, the AsCas12f expression cassette is placed upstream of the sgRNA expression cassette, but the AsCas12f expression cassette may also be placed downstream of the sgRNA expression cassette. In the example of Figure 1, AsCas12f is used as the Cas protein, but other Cas proteins may also be used. In the example of Figure 1, the P2A peptide is used as the self-cleaving peptide, but other self-cleaving peptides may also be used.

[0074] The nucleic acid introduction vector of this embodiment may be a single vector or a combination of multiple types of vectors. When the nucleic acid introduction vector is a single vector, the single vector contains all of the Cas protein coding sequence, guide RNA coding sequence, and nucleic acid to be introduced. In this case, it is preferable that a guide RNA recognition sequence is located between the set of Cas protein coding sequence and guide RNA coding sequence and the nucleic acid to be introduced. When the nucleic acid introduction vector of this embodiment is a combination of multiple types of vectors, it may be a combination of a vector containing a set of Cas protein coding sequence and guide RNA coding sequence and a vector containing the nucleic acid to be introduced. Since this increases the knock-in efficiency of the nucleic acid to be introduced, it is preferable that the nucleic acid introduction vector of this embodiment is a single vector.

[0075] As shown in the examples below, the nucleic acid transfer vector of this embodiment can express the target nucleic acid (target gene) with high efficiency by targeting an intron (prestop) for knock-in. Furthermore, by performing knock-in via NHEJ, the target nucleic acid can be expressed with higher efficiency. This is thought to be because knock-in via NHEJ improves the knock-in efficiency of the target nucleic acid.

[0076] The nucleic acid transfer vector of this embodiment can express the target gene to be transferred in target cells with high efficiency, and is therefore effective in treating diseases associated with abnormal gene expression. For example, in the case of a disease caused by reduced or lost expression of a specific gene, the disease can be treated by using the specific gene as the target gene to be transferred. For example, in the case of a disease caused by excessive expression of a specific gene, the disease can be treated by using a gene that suppresses the expression of the specific gene (e.g., a transcription inhibitor gene, a small interfering nucleic acid gene such as siRNA or shRNA, etc.) as the target gene to be transferred.

[0077] The nucleic acid transfer vector of this embodiment can be used as a pharmaceutical composition for treating or preventing diseases associated with abnormal gene expression. The pharmaceutical composition can contain the nucleic acid transfer vector and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the function of the active ingredient and is not toxic to the recipient. The pharmaceutical composition contains the nucleic acid transfer vector of this embodiment as an active ingredient. Therefore, a pharmaceutically acceptable carrier can be a carrier that does not inhibit the function of the nucleic acid transfer vector and is not toxic to the recipient. Examples of pharmaceutically acceptable carriers include water, saline, phosphate-buffered saline, phosphate buffer, various buffers, and drug carriers such as liposomes. One pharmaceutically acceptable carrier may be used alone, or two or more may be used in combination.

[0078] The dosage form of the pharmaceutical composition is not particularly limited. Examples of dosage forms include liquids, capsules, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. The pharmaceutical composition is preferably administered parenterally in the form of an injection, suppository, topical skin preparation, etc.

[0079] The administration route of the pharmaceutical composition is not particularly limited, and it can be administered orally or parenterally. Examples of parenteral administration routes include intravenous injection, intravenous drip infusion, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular injection, and local injection. The dosage of the pharmaceutical composition can be appropriately determined depending on the type of disease, symptoms of the disease, the subject's age, sex, weight, sensitivity, administration method, administration timing, administration interval, administration period, properties of the formulation, and the type of gene to be introduced. The dosage of the pharmaceutical composition can be a therapeutically effective amount of the nucleic acid transfer vector. The "therapeutically effective amount" refers to the amount of an active ingredient that can exert a therapeutic effect. The therapeutically effective amount of a nucleic acid transfer vector refers to the amount of a nucleic acid transfer vector that can exert a therapeutic effect of the nucleic acid transfer vector.

[0080] The pharmaceutical composition may be administered once or multiple times. In the case of multiple administration, the administration interval of the pharmaceutical composition can be appropriately set depending on the type of disease, symptoms of the disease, age, sex, weight, susceptibility difference of the subject, administration method, administration timing, administration interval, administration period, properties of the formulation, type of gene to be introduced, etc. Examples of administration intervals include daily, once every 1 to 3 days, once a week, once a month, once every 1 to 10 months, once every 1 to several years, etc.

[0081] [Nucleic acid introduction method] A second aspect of the present disclosure is a nucleic acid introduction method. In one embodiment, the nucleic acid introduction method includes a step of introducing a nucleic acid to be introduced by non-homologous end joining (hereinafter also referred to as an "introduction step"), targeting an intron adjacent to the 5' side of an exon containing a stop codon relative to the sense strand.

[0082] (Introduction step) In the introduction step, the nucleic acid to be introduced is introduced by NHEJ, targeting an intron (prestop) in the target gene locus. The introduction step can be performed using a Cas protein, a guide RNA targeting the intron (prestop), and a donor DNA containing the nucleic acid to be introduced. The introduction step may be performed in vitro, ex vivo, or in vivo.

[0083] The Cas protein may be any of those listed above in [Vector for Nucleic Acid Introduction]. In one embodiment, the Cas protein may be AsCas12f. AsCas12 may be wild-type AsCas12f or modified AsCas12f. Examples of modified AsCas12f include AsCas12f-YHAM and AsCas12f-HKRA. The Cas protein may be expressed from a vector containing a Cas protein coding sequence.

[0084] The guide RNA can be appropriately selected and used according to the type of Cas protein. The guide RNA includes a target sequence in an intron (prestop) as a guide sequence. The guide RNA may include, for example, a guide sequence that targets a target sequence in intron 13 of the human or mouse albumin locus. The guide RNA may be expressed from a vector including a guide RNA coding sequence.

[0085] The donor DNA contains a nucleic acid to be introduced. In the nucleic acid introduction method of this embodiment, the nucleic acid to be introduced is introduced via NHEJ, so it does not need to contain a homology arm. The donor DNA preferably has an exon (stop) linked to the 5' side (upstream) of the nucleic acid to be introduced via a self-cleaving peptide coding sequence. The donor DNA preferably has an intron (prestop) linked to the 5' side (upstream) of the exon (stop). Examples of nucleotide sequences of the exon (stop) and intron (prestop) contained in the donor DNA include those listed above in [Vector for nucleic acid introduction]. The donor DNA may have a poly A addition signal linked to the 3' end of the nucleic acid to be introduced. The donor DNA can have a configuration similar to that listed above in [Vector for nucleic acid introduction].

[0086] The introduction step can be carried out by introducing a Cas protein or its expression vector, a guide RNA or its expression vector, and donor DNA into a target cell. These introductions into cells can be carried out by known methods.

[0087] Methods for introducing a vector into cells include, for example, lipofection, microinjection, DEAE-dextran method, gene gun method, electroporation, calcium phosphate method, etc. When the expression vector is a viral vector, methods for infecting cells with the viral vector (for example, polybrene method) can be used.

[0088] The method for introducing RNA into cells is not particularly limited, and any known method can be appropriately selected and used. For example, a commercially available RNA transfection reagent such as Lipofectamine (registered trademark) MessengerMAX (manufactured by Life Technologies) can be used for introducing RNA.

[0089] The method for introducing a protein into a cell is not particularly limited, and any known method can be appropriately selected and used, such as a method using a protein introduction reagent, a method using a protein introduction domain (PTD) fusion protein, or a microinjection method.

[0090] The Cas protein and guide RNA are preferably encoded in the same vector as the donor DNA. That is, the Cas protein coding sequence, the guide RNA coding sequence, and the nucleic acid to be introduced are preferably carried on a single vector. The introduction step may be performed using the nucleic acid introduction vector according to the first aspect.

[0091] The target cells are not particularly limited. The organisms from which the cells are derived are not particularly limited, and examples thereof include mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, cows, horses, pigs, goats, and sheep; birds such as chickens; reptiles such as snakes and lizards; amphibians such as African clawed frogs; fish such as zebrafish, medaka, and tiger pufferfish; chordates such as sea squirts; arthropods such as fruit flies and silkworms; plants such as Arabidopsis thaliana, rice, wheat, and tobacco; fungi such as yeast and Neurospora crassa; and bacteria such as Escherichia coli, Bacillus subtilis, and blue-green algae. The type of cell is not particularly limited, and examples include cells derived from various tissues or having various properties, such as blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, eggs), fertilized eggs, fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, tissue stem cells, iPS cells, ES cells, cancer cells, and various disease cells.

[0092] (Other Steps) The nucleic acid transfer method of this embodiment may include other steps in addition to the transfer step, such as a step of culturing cells (a culture step) and a step of measuring the expression level of the nucleic acid to be transferred (an expression level measurement step).

[0093] <Culturing step> The nucleic acid transfer method of this embodiment may include a step of culturing the target cells after the transfer step. The medium and culture conditions can be selected appropriately depending on the type of cell. When the vector used in the transfer step contains a drug resistance marker, cells into which the vector has been transferred can be efficiently selected by culturing the cells in the presence of the drug. Cells may be cloned by diluting the cell culture medium, plating, or the like.

[0094] <Expression level measurement step> The nucleic acid introduction method of this embodiment may include a step of measuring the expression level of the nucleic acid to be introduced (gene to be introduced) after the introduction step. The expression level of the nucleic acid to be introduced can be measured by a known method. Examples of methods for measuring the expression level of the nucleic acid to be introduced include ELISA, Western blotting, fluorescent immunostaining, and flow cytometry analysis. Alternatively, the activity of a protein expressed from the nucleic acid to be introduced may be measured. The activity measurement can be performed by selecting an appropriate known method depending on the type of protein.

[0095] [Polynucleotide] A third aspect of the present disclosure is a polynucleotide. The polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 16 or 20. The polynucleotide is preferably a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 16 or 20, or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 with an initiation codon (ATG) added to the 5' end.

[0096] The nucleotide sequence set forth in SEQ ID NO: 16 is a nucleotide sequence obtained by removing the start codon from the nucleotide sequence encoding human ornithine transcarbamylase (hOTC). The nucleotide sequence set forth in SEQ ID NO: 16 has a reduced proportion of guanine residues and cytosine residues (hereinafter also referred to as "GC content") compared to the nucleotide sequence of a wild-type cDNA encoding hOTC. The nucleotide sequence set forth in SEQ ID NO: 20 is a nucleotide sequence obtained by adding a start codon to the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 16 and removing a stop codon from the 3'-end. As shown in the Examples below, the nucleotide sequence set forth in SEQ ID NO: 16 or 20 can achieve high expression levels of hOTC when introduced into human cells in an expressible form. The polynucleotide of this embodiment may have a nucleotide sequence obtained by adding a stop codon (TGA) to the 3'-end of the nucleotide sequence set forth in SEQ ID NO: 20.

[0097] The polynucleotide of this embodiment may be DNA or RNA. When the polynucleotide of this embodiment is DNA, it can be used as a DNA vector. When the polynucleotide of this embodiment is RNA, it can be used as mRNA. When the polynucleotide of this embodiment is RNA, the thymine residues in the nucleotide sequence set forth in SEQ ID NO: 16 or 20 are replaced with uracil residues.

[0098] (mRNA) When the polynucleotide of this embodiment is mRNA, it may contain other components in addition to the nucleotide sequence set forth in SEQ ID NO: 16. Examples of other components include a 5' Cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a Kozak sequence, and a poly(A) tail.

[0099] An mRNA may contain a 5' Cap at its 5' end. Examples of 5' Caps include Cap0, Cap1, and Cap2 structures. The cap structure is typically a 7-methylguanine ribonucleotide, attached via its 5'-triphosphate to the 5' position of the first nucleotide in the 5'-3' direction of the mRNA, i.e., the first cap-adjacent nucleotide. In a Cap0 structure, the ribose of the first cap-adjacent nucleotide and the ribose of the second nucleotide of the mRNA both contain 2'-hydroxyl. In a Cap1 structure, the ribose of the first cap-adjacent nucleotide and the ribose of the second nucleotide of the mRNA both contain 2'-methoxy. In a Cap2 structure, the ribose of the first cap-adjacent nucleotide and the ribose of the second nucleotide of the mRNA both contain 2'-methoxy.

[0100] The cap structure can be incorporated into the 5' end of mRNA during transcription by known methods. For example, the cap structure can be incorporated into mRNA by co-transcription using a commercially available capping kit. The cap structure may also be added to RNA after transcription or chemical synthesis of mRNA using a capping enzyme.

[0101] The mRNA may include either or both of a 5'UTR and a 3'UTR. The 5'UTR and 3'UTR may be those of the wild-type hOTC mRNA or those of a different mRNA.

[0102] The mRNA may contain a Kozak sequence. The Kozak sequence can affect translation initiation and the total amount of protein produced from the mRNA. The Kozak sequence contains a methionine codon that can function as an initiation codon. A minimal Kozak sequence is NNNRUGN (N is any nucleotide residue, and R is a purine residue (A or G)). In the formula, the first N is preferably A or G, and the second N is preferably G. In one embodiment, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG.

[0103] The mRNA may include a poly(A) tail at its 3' end. The poly(A) tail may include a sequence of at least 8 consecutive adenine nucleotides, but may also include one or more non-adenine nucleotide residues (e.g., G, C, U). The length of the poly(A) tail may be, for example, 10 to 500 nucleotides, 30 to 300 nucleotides, or 60 to 250 nucleotides.

[0104] [Vector] A fourth aspect of the present disclosure is a vector. The vector comprises the polynucleotide according to the third aspect. That is, the vector comprises the nucleotide sequence set forth in SEQ ID NO: 16 or 20.

[0105] The vector is preferably an expression vector. An expression vector refers to a vector equipped with a system that enables the expression of a protein from a protein-coding sequence contained in the vector in a cell into which the expression vector has been introduced. The expression vector of this embodiment is equipped with a system that enables the expression of hOTC from an hOTC coding sequence containing the nucleotide sequence set forth in SEQ ID NO: 16 or 20 in a cell into which the expression vector has been introduced. The expression vector may include other components in addition to the nucleotide sequence set forth in SEQ ID NO: 16 or 20. The expression vector includes, for example, a promoter that can function in a target cell and the nucleotide sequence set forth in SEQ ID NO: 16 or 20 operably linked to the promoter. "Operable in a target cell" means that the nucleotide sequence set forth in SEQ ID NO: 16 or 20 operably linked to the promoter can be expressed in the target cell. "Operatively linked to a promoter" means that the nucleotide sequence set forth in SEQ ID NO: 16 or 20 is linked to the promoter so that hOTC is expressed from the nucleotide sequence set forth in SEQ ID NO: 16 or 20 under the control of the promoter in the target cell.

[0106] The promoter is not particularly limited as long as it has the function of expressing hOTC from the nucleotide sequence set forth in SEQ ID NO: 16 or 20 in the target cell. Examples of the promoter include Pol II promoters. Examples of Pol II promoters include those similar to those listed in the above section "Vector for nucleic acid introduction."

[0107] The expression vector may contain other components in addition to the nucleotide sequence and promoter set forth in SEQ ID NO: 16 or 20. Examples of other components include a terminator, an enhancer, a polyA addition signal, a marker gene, a replication origin, and a gene encoding a protein that binds to the replication origin and controls replication. The terminator is linked to the 3' side of the nucleotide sequence set forth in SEQ ID NO: 16 or 20. Terminators commonly used in the biological species from which the target cells are derived can be used. A marker gene refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Examples of drug resistance genes include puromycin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Examples of fluorescent protein genes include the green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, and red fluorescent protein (RFP) gene. Examples of luminescent enzyme genes include the luciferase gene. Examples of chromogenic enzyme genes include the β-galactosidase gene, β-glucuronidase gene, and alkaline phosphatase gene.

[0108] The type of expression vector is not particularly limited, and known expression vectors can be used. Examples of expression vectors include plasmid vectors and viral vectors. Examples of expression vectors include those listed in the above section "Vector for nucleic acid introduction."

[0109] The vector may be the nucleic acid introduction vector according to the first aspect. When the vector is the nucleic acid introduction vector according to the first aspect, it may contain a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 16 or 20 as the nucleic acid to be introduced.

[0110] The polynucleotide according to the third aspect or the vector according to the fourth aspect can be used as a pharmaceutical composition for treating or preventing ornithine transcarbamylase deficiency (OTC deficiency).

[0111] The pharmaceutical composition can contain at least one selected from the group consisting of the polynucleotide and the vector (hereinafter also referred to as "polynucleotide / vector"), as well as a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the function of the active ingredient and is not toxic to the recipient. The pharmaceutical composition contains a polynucleotide / vector as the active ingredient. Therefore, a pharmaceutically acceptable carrier can be any carrier that does not inhibit the function of the polynucleotide / vector and is not toxic to the recipient. Examples of pharmaceutically acceptable carriers include water, saline, phosphate-buffered saline, phosphate buffer, various buffers, and drug carriers such as liposomes. One pharmaceutically acceptable carrier may be used alone, or two or more may be used in combination.

[0112] The dosage form of the pharmaceutical composition is not particularly limited. Examples of dosage forms include liquids, capsules, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. The pharmaceutical composition is preferably administered parenterally in the form of an injection, suppository, topical skin preparation, etc.

[0113] The administration route of the pharmaceutical composition is not particularly limited, and it can be administered orally or parenterally. Examples of parenteral administration routes include intravenous injection, intravenous drip infusion, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular injection, and local injection. The dosage of the pharmaceutical composition can be appropriately determined depending on the type of disease, symptoms of the disease, the subject's age, sex, weight, sensitivity, administration method, administration timing, administration interval, administration period, properties of the formulation, and the type of gene to be introduced. The dosage of the pharmaceutical composition can be a therapeutically effective amount of the vector for nucleic acid introduction. The "therapeutically effective amount" refers to the amount of an active ingredient that can exert a therapeutic effect. The therapeutically effective amount of a polynucleotide / vector refers to the amount of a polynucleotide / vector that can exert a therapeutic effect.

[0114] The pharmaceutical composition may be administered once or multiple times. In the case of multiple administration, the administration interval of the pharmaceutical composition can be appropriately set depending on the type of disease, symptoms of the disease, age, sex, weight, susceptibility difference of the subject, administration method, administration timing, administration interval, administration period, properties of the formulation, type of gene to be introduced, etc. Examples of administration intervals include daily, once every 1 to 3 days, once a week, once a month, once every 1 to 10 months, once every 1 to several years, etc.

[0115] In one embodiment, the present disclosure provides a method for treating ornithine transcarbamylase deficiency, comprising administering to a subject in need of treatment an effective amount of at least one selected from the group consisting of a polynucleotide set forth in SEQ ID NO: 16 or 20 and a vector comprising the polynucleotide.

[0116] In one embodiment, the present disclosure provides use of at least one selected from the group consisting of a polynucleotide set forth in SEQ ID NO: 16 or 20 and a vector comprising the polynucleotide in the manufacture of a pharmaceutical composition for treating ornithine transcarbamylase deficiency.

[0117] In one embodiment, the present disclosure provides at least one polynucleotide selected from the group consisting of a polynucleotide set forth in SEQ ID NO: 16 or 20 and a vector comprising the polynucleotide, for use in treating ornithine transcarbamylase deficiency.

[0118] In one embodiment, the present disclosure provides use of at least one selected from the group consisting of a polynucleotide set forth in SEQ ID NO: 16 or 20 and a vector comprising the polynucleotide, for treating ornithine transcarbamylase deficiency.

[0119] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0120] [Experimental Example 1] <AAV Vector Preparation> For genome editing, enAsCas12f-HKRA (SEQ ID NO: 13), a modified AsCas12f containing four amino acid substitutions (I123H / D195K / D208R / V232A) to enhance genome editing activity, was used (Hino T, et al. Cell. 2023;186:4920-4935.e23.). A promoter, enAsCas12f-HKRA cDNA, SV40 polyA signal, and a single guide RNA (sgRNA) sequence expressed by the U6 promoter were inserted between the inverted terminal repeats (ITR) of the pAAV plasmid. When introduced into cells, a cytomegalovirus (CMV) promoter was used. For in vivo targeting of mouse liver, the mouse transthyretin (mTTR) promoter, a liver-specific promoter, was used. To insert human blood coagulation factor IX (FIX) R338L cDNA (SEQ ID NO: 14; sequence excluding the start codon (ATG)) into the mouse albumin locus (mAlb), an sgRNA target sequence was selected that efficiently cleaves the mAlb intron. The target sequences of the sgRNAs targeting introns 13 and 14 of mAlb are listed in Table 1. The enAsCas12f-HKRA-sgRNA expression cassette and the FIX R338L cDNA were linked via an sgRNA coding sequence containing the target sequence (see Figure 2). To compare knock-in efficiencies via homologous recombination (HDR) and non-homologous end joining (NHEJ), donor sequences were designed to insert FIX via HDR when targeting immediately after the stop codon (HDR intron 14; see Figures 2 and 3). When targeting intron 13 upstream of the stop codon, donor sequences were designed to insert FIX via NHEJ (NHEJ intron 13; see Figures 2 and 4). Plasmids targeting intron 13 and intron 14 were also constructed (NHEJ introns 13 and 14; see Figure 2). To produce AAV vectors in a helper-free system, AAV vectors were packaged by triple-plasmid transfection into AAVpro293T cells.The viral titer of the recombinant AAV vector was determined by quantitative PCR.

[0121]

[0122] <T7 endonuclease assay> On the day before plasmid transfection, 5 × 10 mouse liver-derived TLR3 cells (JCRB Cell Bank) were transfected. ４ Cells / well were seeded onto 48-well plates coated with type I collagen (Cellmatrix type IC, Nitta Gelatin, Osaka, Japan). TLR3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 2% bovine serum albumin, 5 ng / mL human EGF, and ITS-X supplement (Thermo Fisher Scientific, Waltham, MA, USA). Plasmid (200 ng) was added to TLR3 cells with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. After 72 hours, cells were lysed with SimplePrep reagent (Takara, Shiga, Japan) for DNA extraction, and the supernatant was directly used for polymerase chain reaction (PCR). DNA fragments were amplified with ExTaq DNA polymerase (Takara). The purified PCR products were denatured and annealed in a thermal cycler and then treated with T7 endonuclease (NIPPON GENE, Tokyo, Japan). DNA fragments were analyzed using a microchip electrophoresis device (MCE-202 MultiNA; Shimadzu, Kyoto, Japan).

[0123] The results of the T7 endonuclease assay are shown in Figure 5. Of the sgRNAs targeting intron 13, sgRNA5 showed the highest genome editing activity. Of the sgRNAs targeting intron 14, sgRNA1 showed the highest genome editing activity. Based on these results, in animal experiments, sgRNA5 was used as the sgRNA targeting intron 13. sgRNA1 was used as the sgRNA targeting intron 14.

[0124] <Animal Experiments> Blood coagulation factor IX (FIX) knockout mice (B6.129P2-F9tm1Dws) were obtained from The Jackson Laboratory (Sacramento, CA, USA). To obtain plasma samples, mice were anesthetized with isoflurane (1-3%), and blood was collected from the jugular vein using a 29G microsyringe (TERUMO, Tokyo, Japan) containing 1 / 10 (v / v) sodium citrate. Plasma was collected by centrifugation at 5,000 rpm for 10 minutes and frozen at -80°C until analysis. Neonatal mice were intraperitoneally injected with 3 x 10 AAV vectors. １１ Adult mice were administered 20 μL of 1×10 AAV vector via the jugular vein. １２ 200 μL of vg was administered.

[0125] <Measurement of blood coagulation factor IX activity and antigen amount> Human FIX activity (FIX:C) was measured by a one-stage coagulation assay using an automated coagulation analyzer (Sysmex CS-1600, Sysmex, Kobe, Japan). FIX antigen amount (FIX:Ag) was measured as follows. First, a microtiter plate was coated with anti-human FIX antibody (CEDARLANE, Burlington, ON, Canada). After blocking with 5% casein, diluted plasma samples were incubated at 37°C for 1 hour. FIX antigen binding was detected with horseradish peroxidase (Affinity Biologicals, Ancaster, ON, Canada) and anti-human FIX antibody conjugated with ABTS Microwell Peroxidase Substrate (Seracare, Milford, MA, USA).

[0126] <Fluorescent Immunostaining> Hemophilia B mice were sacrificed and perfused with phosphate-buffered saline (PBS). The livers were removed and fixed with 4% paraformaldehyde, substituted with sucrose, and embedded in OTC compound (Sakura Fintek Japan, Tokyo, Japan). The livers were then frozen in liquid nitrogen. Frozen tissue sections were blocked with 5% donkey serum and reacted with anti-human FIX antibody (Affinity Biologicals) and anti-mouse CD146 antibody (BioLegend, San Diego, CA, USA). Streptavidin Alexa Fluor was then used. ＴＭ 594 Conjugate (Thermo Fisher Scientific) and AlexaFluor 488-conjugated anti-rat IgG (Thermo Fisher Scientific) were added to the frozen tissue sections and incubated at 4°C for 2 hours. The frozen tissue sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Immunofluorescently stained sections were observed and photographed using an all-in-one microscope (BZ-X700, Keyence, Tokyo, Japan). FIX-positive cells were quantified with BZ-X 700 imaging software (Keyence).

[0127] Experimental Example 1-1 (Knock-in therapy of newborn mice with hemophilia B) The AAV vectors used were AAV vectors packaged with the plasmid HDR intron14, the plasmid NHEJ intron13, and the plasmid NHEJ intron13,14 (hereinafter referred to as the "HDR intron14 vector," the "NHEJ intron13 vector," and the "NHEJ intron13,14 vector," respectively). The AAV vectors were administered to newborn blood coagulation factor IX (FIX) knockout mice (B6.129P2-F9tm1Dws) using the method described above. Blood samples were then collected over time, and FIX activity and FIX antigen levels were measured. Twelve weeks after AAV vector administration, the mice were sacrificed, the livers were removed, and liver tissue sections were subjected to fluorescent immunostaining.

[0128] The results of measuring FIX activity and FIX antigen levels are shown in Figure 6. Both FIX activity and FIX antigen levels were higher in mice administered with the NHEJ intron13 vector and mice administered with the NHEJ intron13,14 vector compared to mice administered with the HDR intron14 vector. There was no significant difference in FIX activity or FIX antigen levels between mice administered with the NHEJ intron13 vector and mice administered with the NHEJ intron13,14 vector.

[0129] The results of fluorescent immunostaining are shown in Figure 7. More FIX-positive cells were detected in mice administered with the NHEJ intron 13 vector and the NHEJ intron 13,14 vector than in mice administered with the HDR intron 14 vector. Almost no FIX-positive cells were detected in hemophilia B mice (negative control) that had not received the AAV vector.

[0130] Figure 8 shows the results of quantifying the FIX-positive cell rate. The FIX-positive cell rate was higher in mice administered with the NHEJ intron 13 vector and mice administered with the NHEJ intron 13,14 vector compared to mice administered with the HDR intron 14 vector. There was no significant difference in the FIX-positive cell rate between mice administered with the NHEJ intron 13 vector and mice administered with the NHEJ intron 13,14 vector.

[0131] [Experimental Example 1-2] (Knock-in therapy of adult mice with hemophilia B) HDR intron 14 vector and NHEJ intron 13 vector were used as AAV vectors. Adult blood coagulation factor IX (FIX) knockout mice (B6.129P2-F9tm1Dws) were administered the AAV vectors using the method described above. Blood samples were then collected over time, and FIX activity was measured. Eight weeks after administration of the AAV vectors, the mice were sacrificed, the livers were removed, and liver tissue sections were subjected to immunofluorescence staining.

[0132] The results of measuring FIX activity are shown in Figure 9. Mice administered with the NHEJ intron13 vector had higher FIX activity than mice administered with the HDR intron14 vector. On the other hand, FIX activity was barely detectable in mice administered with the HDR intron14 vector.

[0133] The results of the fluorescent immunostaining are shown in Figure 10. In mice administered with the NHEJ intron13 vector, more FIX-positive cells were detected than in mice administered with the HDR intron14 vector. In mice administered with the HDR intron14 vector, almost no FIX-positive cells were detected.

[0134] Figure 11 shows the results of quantifying the FIX-positive cell rate. The FIX-positive cell rate was higher in mice administered with the NHEJ intron13 vector than in mice administered with the HDR intron14 vector. The FIX-positive cell rate in mice administered with the HDR intron14 vector was almost the same as that in hemophilia B mice (negative control) not administered with the AAV vector.

[0135] From the above results, it was confirmed that expression of a target gene can be improved by introducing the target gene by NHEJ, targeting the intron immediately before the stop codon.

[0136] Experimental Example 2 (Knock-in therapy of protein C-deficient mice) An AAV vector was prepared by packaging a plasmid containing the mouse transthyretin (mTTRq) promoter and modified AsCas12f (I123H / D195K / D208R / V232A; enAsCas12f-HKRA), a human U6 promoter and an sgRNA (sgRNA5) targeting mouse albumin locus (mAlb) intron 13, and human PROC (hPROC) cDNA (SEQ ID NO: 15) as a donor sequence. More specifically, an AAV vector was prepared containing a structure in which the FIX R338L cDNA was replaced with hPROC cDNA in the NHEJ intron 13 of Figure 2. The AAV vector was administered to newborn mice.

[0137] Various doses of the AAV vector were intraperitoneally administered to newborn wild-type mice (C57BL / 6J). Six weeks after administration, plasma human protein C activity was measured (n=6-7).

[0138] The results are shown in Figure 12. The higher the dose of AAV vector, the higher the plasma human protein C activity. P values ​​were evaluated by one-way ANOVA with Dunnett's multiple comparison test.

[0139] Next, Proc ＋／－ Female mice were administered anti-FVIII antibodies, and Proc ＋／－ The mice were mated with male mice. Anti-FVIII antibody administration was repeated every 7 days until birth. All newborn mice were administered with the AAV vector (1.0 × 10 １１ The wild-type mice (C57BL / 6J) and Proc mice (Proc) were intraperitoneally administered with anti-FVIII antibody (1 μg). Six weeks after administration, plasma prolein C activity, plasma factor V activity, and plasma factor VIII activity were measured. －／－ F8 －／－ Neonatal mice were also measured for plasma prolein C activity, plasma factor V activity, and plasma factor VIII activity at 6 weeks after birth. ＋／－ F8 －／－ The mice were not administered with AAV vectors or anti-FVIII antibodies. Wild-type mice (C57BL / 6J): n=2, Proc ＋／＋ : n = 4, Proc ＋／－ : n = 9, Proc －／－ : n = 2, Proc －／－ F8 －／－ :n=3.

[0140] Figure 13 shows the Kaplan-Meier survival curves of newborn mice administered with AAV vectors. －／－ In mice, administration of AAV vectors has been confirmed to result in survival for more than 40 days.

[0141] Figure 14 shows plasma human protein C activity. Figure 15 shows plasma factor V activity. Figure 16 shows plasma factor VIII activity. ＋／＋ Mouse, Proc ＋／－ Mouse, Proc －／－ In mice, plasma human protein C activity was detected (Figure 14). ＋／＋ Mouse, Proc ＋／－ Mouse, and Proc －／－ In mice, there were no significant differences in plasma factor V activity and plasma factor VIII activity (FIGS. 15 and 16).

[0142] These results suggest that protein C deficiency may be cured by administering an AAV vector that introduces hPROC cDNA by targeting intron 13 of the mouse albumin locus (mAlb).

[0143] Experimental Example 3 (Knock-in therapy of ornithine transcarbamylase (OTC) low-expressing mouse model) An AAV vector was prepared by packaging a plasmid containing a mouse transthyretin (mTTRq) promoter and modified AsCas12f (I123H / D195K / D208R / V232A; enAsCas12f-HKRA), a human U6 promoter and an sgRNA (sgRNA5) targeting mouse albumin locus (mAlb) intron 13, and a donor sequence containing a cDNA (hOTC_FLAG) in which a FLAG tag coding sequence was added to the cDNA (SEQ ID NO: 16) of human OTC (hOTC). More specifically, an AAV vector was prepared containing a structure in which the FIX R338L cDNA in the NHEG intron 13 of FIG. 2 was replaced with the hOTC_FLAG cDNA. The AAV vector was ｓｐｆ－ａｓｈ Male mice were administered the drug.

[0144] The nucleotide sequence of the plasmid used to prepare the AAV vector is shown in SEQ ID NO: 21. Features of the nucleotide sequence shown in SEQ ID NO: 21 are shown in Table 2.

[0145]

[0146] The NHEJ intron 13 vector used in Experimental Example 1-1 and the plasmid used to prepare the AAV vector used in Experimental Example 2 have the same structure as the plasmid used in Example 4, except that hOTC_FLAG_GS-Delta-CG in Table 2 was changed to hF9 (wild) R338L cDNA (SEQ ID NO: 14) and hPROC_cDNA (SEQ ID NO: 15), respectively. The nucleotide sequence of mAlb intron 13 contained in the plasmid is shown in SEQ ID NO: 23. The nucleotide sequence of mAlb exon 14 contained in the plasmid is shown in SEQ ID NO: 24. The P2A coding sequence contained in the plasmid is shown in SEQ ID NO: 25.

[0147] An outline of the test method is shown in Figure 17. The AAV vector was administered to 8-9 week old OTC mice. ｓｐｆ－ａｓｈ To a male mouse, 3.0 × 10 １１ vg / body (3E+11 vg / body AAV) or 1.0×10 １２ The mice were intravenously administered with a dose of 1E+12 vg / body AAV (n=4). The day of AAV vector administration was set as day 0, and a high-protein diet (40% kcal) was administered starting 5 weeks after administration. Untreated mice (intravenously administered saline) were used as controls.

[0148] Figure 18 shows changes in mouse body weight. The changes in body weight (Changes of BW (%)) are shown as relative values ​​when the body weight on day 0 of high-protein diet administration is set as 100%. Weight loss was improved in AAV vector-administered mice (3E+11, 1E+12) compared to untreated mice (Untreated). １２ In mice administered with AAV vectors (1E+12) of vg / body, the １１ Compared with the vg / body AAV vector-administered mice (3E+11), weight loss was significantly improved.

[0149] Changes in blood ammonia levels are shown in Figure 19. Blood ammonia levels were reduced in AAV vector-administered mice (3E+11, 1E+12) compared to untreated mice (Untreated).

[0150] Figure 20 shows the OTC enzyme activity in the liver. 7 to 8 weeks after AAV vector administration, the liver was excised from the mice and the OTC enzyme activity was measured. Compared to untreated mice (Untreated), the OTC enzyme activity was elevated in the AAV vector-administered mice (3E+11, 1E+12). １２ In mice administered with AAV vectors (1E+12) of vg / body, the １１ Compared with the vg / body AAV vector-administered mice (3E+11), OTC enzyme activity was increased.

[0151] Figure 21 shows the results of Western blotting for hOTC_FLAG. Livers were excised from the mice 7 to 8 weeks after AAV vector administration, and cell lysates were prepared. Western blotting was performed on the cell lysates using an anti-FLAG antibody. Untreated mice (Untreated), 3.0 x 10 １１ vg / body AAV vector-administered mice (3E+11), and 1.0 × 10 １２ Livers were collected from four individual AAV vector-administered mice (1E+12) of vg / body, and Western blotting was performed. A cell lysate of Huh7 cells expressing hOTC_FLAG was used as a positive control. A cell lysate of Huh7 cells expressing hOTC_FLAG was used as a negative control. A hOTC_FLAG band was detected in AAV vector-administered mice (3E+11, 1E+12). 1.0 x 10 １２ vg / body AAV vector-administered mice (1E+12) were 3.0 × 10 １１ The amount of hOTC_FLAG protein was higher than that in the vg / body AAV vector-administered mice (3E+11).

[0152] Figure 22 shows the relative amount of hOTC_FLAG protein analyzed from Western blotting images. １２ vg / body AAV vector-administered mice (1E+12) were 3.0 × 10 １１ The amount of hOTC_FLAG protein was higher than that in the vg / body AAV vector-administered mice (3E+11).

[0153] Figure 23 shows the expression level of hOTC_FLAG mRNA in mouse liver. Livers were collected from mice 7 to 8 weeks after AAV vector administration, and RNA was extracted. hOTC mRNA was quantified by RT-qPCR using the RNA. The expression level of hOTC_FLAG mRNA was normalized to the expression level of GAPDH mRNA. 1.0 x 10 １２ vg / body AAV vector-administered mice (1E+12) were 3.0 × 10 １１ The expression level of hOTC_FLAG mRNA was higher compared to the vg / body AAV vector-administered mice (3E+11).

[0154] Figure 24 shows fluorescent immunostained images of mouse liver tissue sections. Livers were excised from the mice 7 to 8 weeks after AAV vector administration, and the liver tissue sections were subjected to fluorescent immunostaining. From the left, the images show a fluorescent immunostained image using an anti-FLAG antibody, a DAPI stained image, a fluorescent immunostained image using an anti-CD146 antibody, and a merged image of these. CD146 was used as a marker for endothelial cells. 1.0 x 10 １２ vg / body AAV vector-administered mice (1E+12) were 3.0 × 10 １１ Compared with the vg / body AAV vector-administered mice (3E+11), the number of hOTC_FLAG-positive cells was higher.

[0155] Figure 25 shows the results of analyzing the hOTC_FLAG positive cell rate from the fluorescent immunostaining images. １２ vg / body AAV vector-administered mice (1E+12) were 3.0 × 10 １１ The rate of hOTC_FLAG-positive cells was higher than that in the vg / body AAV vector-administered mice (3E+11).

[0156] These results suggest that OTC deficiency may be cured by administering an AAV vector that targets intron 13 of the mouse albumin locus (mAlb) and introduces hOTC cDNA.

[0157] [Experimental Example 4] (Study of human OTC gene sequence) A FLAG tag coding sequence was added to the wild-type cDNA (WT) (SEQ ID NO: 17) of the human OTC gene (hOTC) and three modified cDNAs with reduced GC content (CO#1: SEQ ID NO: 18, CO#2: SEQ ID NO: 19, CO#3: SEQ ID NO: 20), and each was inserted into the pCDNA3 plasmid. Each of the plasmids was transfected into HEK293 cells using Lipoefectamin 3000. After transfection, each cell was cultured in DMEM medium containing 10% FBS. After 2 days of culture, the cells were harvested and treated with a cell lysis solution (50 mM Tris-HCl [pH 7.5], 0.1% SDS, 0.15 M NaCl, 1% Triton-X100, 1% sodium deoxycholate, 1% protease inhibitor cocktail) to obtain cell lysates. For OTC activity measurement, cells were treated with a lysis solution (10 mM HEPES, 0.5% Triton-X100, 2 mM EDTA, 0.5 mM DTT [pH 7.2]) to obtain various lysates.

[0158] Each of the cell lysates obtained above was subjected to immunoblotting using an anti-FLAG antibody. The immunoblotting signal was detected, and the expression level of the OTC protein in each cell was evaluated.

[0159] The results are shown in Figure 26. The OTC protein expression ratio is shown as relative activity when the OTC protein amount in cells transfected with wild-type cDNA is set to 1. In cells transfected with the modified hOTC cDNA CO#3, OTC protein expression was improved compared to cells transfected with other cDNAs. On the other hand, in cells transfected with the modified hOTC cDNA CO#1, OTC protein expression was improved compared to cells transfected with wild-type cDNA (WT), but OTC protein expression was lower than in cells transfected with CO#3. In cells transfected with the modified hOTC cDNA CO#2, OTC protein expression was not improved compared to cells transfected with wild-type cDNA (WT).

[0160] Next, the OTC enzyme activity of each of the cell lysates obtained above was measured.

[0161] The results are shown in Figure 27. The relative OTC activity is shown relative to the OTC activity of cells transfected with the wild-type cDNA, which was set to 1. In cells transfected with the modified hOTC cDNA CO#3, the OTC activity was significantly improved compared to cells transfected with the wild-type cDNA (WT).

[0162] These results confirmed that the modified hOTC cDNA CO#3 highly expressed hOTC when introduced into human cells.

[0163] Experimental Example 5 (Study of gRNA targeting human albumin locus (hAlb)) A target sequence for sgRNA targeting intron 13 of the human albumin locus (hAlb) was designed. The designed target sequences are shown in Table 3. For sgRNAs 1 to 7, the cleavage efficiency of target DNA was evaluated by T7 endonuclease assay using HEK293 cells. AsCas12f was used as the Cas protein.

[0164]

[0165] The results are shown in Figure 26. gRNA5 had the highest cleavage efficiency. Sequence analysis of gRNA5 confirmed that it also had low off-target effects. From these results, gRNA5 was considered to be the most preferred target sequence among gRNA1 to 7.

[0166] According to the present invention, a nucleic acid transfer vector and a nucleic acid transfer method are provided that can improve the expression level of a transgene.

[0167] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A nucleic acid transfer vector comprising: a nucleotide sequence encoding a Cas protein; a nucleotide sequence encoding a guide RNA; and a nucleotide sequence of a nucleic acid to be transferred, wherein the guide RNA targets an intron adjacent to the 5' side of an exon containing a stop codon, based on the sense strand.

2. The nucleic acid introduction vector according to claim 1, further comprising the guide RNA recognition sequence.

3. A nucleic acid introduction vector according to claim 1 or 2, further comprising at least a portion of the nucleotide sequence of the exon, wherein at least a portion of the nucleotide sequence of the exon is located on the 5' side of the nucleotide sequence of the nucleic acid to be introduced relative to the sense strand, and wherein at least a portion of the nucleotide sequence of the exon does not include the stop codon of the exon or the nucleotide sequence 3' from the stop codon relative to the sense strand.

4. A nucleic acid transfer vector according to claim 3, further comprising a nucleotide sequence encoding a self-cleaving peptide, wherein at least a portion of the nucleotide sequence of the exon, the nucleotide sequence encoding the self-cleaving peptide, and the nucleotide sequence of the nucleic acid to be transferred are arranged in this order from the 5' end based on the sense strand.

5. A nucleic acid transfer vector according to claim 1 or 2, wherein the nucleic acid to be transferred is knocked into the intron by non-homologous end joining.

6. A nucleic acid introduction vector according to claim 1 or 2, wherein the Cas protein is AsCas12f.

7. The nucleic acid introduction vector according to claim 6, wherein a single vector comprises a nucleotide sequence encoding the Cas protein, a nucleotide sequence encoding the guide RNA, and a nucleotide sequence of the nucleic acid to be introduced.

8. A method for introducing nucleic acid, comprising the step of introducing a nucleic acid to be introduced by non-homologous end joining, targeting an intron adjacent to the 5' side of an exon containing a stop codon, based on the sense strand.

9. A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 16 or 20.

10. A vector comprising the polynucleotide of claim 9.

11. A nucleic acid transfer vector according to claim 1 or 2, wherein the nucleotide sequence of the nucleic acid to be transferred comprises the nucleotide sequence set forth in SEQ ID NO: 16.

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