WO2022114243A1

WO2022114243A1 - Method for treating muscular dystrophy by targeting dmpk gene

Info

Publication number: WO2022114243A1
Application number: PCT/JP2021/044002
Authority: WO
Inventors: Eiji Yoshimi; Tomoya Oe; Mikio Takeuchi; Tetsuya Yamagata; Keith M. CONNOLLY
Original assignee: Astellas Pharma Inc; Modalis Therapeutics Corp
Current assignee: Astellas Pharma Inc; Modalis Therapeutics Corp
Priority date: 2020-11-25
Filing date: 2021-11-24
Publication date: 2022-06-02
Anticipated expiration: 2023-05-25
Also published as: AR124143A1

Abstract

An adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences : (a) a base sequence encodinq a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor, and (b) a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the AAV vector comprises an AAV capsid comprising a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38, are expected to be useful for treating muscular dystrophy.

Description

DESCRIPTION

TITLE OF THE INVENTION

METHOD FOR TREATING MUSCULAR DYSTROPHY BY TARGETING DMPK

GENE

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No.63/I18,345, filed on November 25, 2020, the contents of which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to methods for treating muscular dystrophy by targeting the human myotonin protein kinase (DMPK; dystrophia myotonica protein kinase) gene and the like. More particularly, the present invention relates to methods and pharmaceutical compositions for treating or preventing muscular dystrophy by repressing expression of human DMPK gene by using a guide RNA targeting a particular sequence of human DMPK gene and a fusion protein of a transcriptional repressor and a CRISPR (clustered regularly interspaced short palindromic repeat) effector protein, and the like.

DISCUSSION OF THE BACKGROUND Muscular dystrophy is a generic term for hereditary diseases associated with progressive muscular atrophy and muscle weakness. Even today, a fundamental therapeutic drug effective for muscular dystrophy does not exist, and only symptomatic treatments are performed. Among muscular dystrophies, myotonic dystrophy type 1 (DM1) is caused by mutations in the

DMPK gene.

DM1 is an autosomal dominant genetic disease caused by elongation of CTG repeats in the 3' untranslated region (3' UTR) of the DMPK gene and is one type of triplet repeat disease. It has been reported that, in DM1, RNA containing an expanded CUG repeat sequesters CUG repeat binding proteins such as MBNL (Muscleblind-like) from endogenous RNA targets, thereby causing aberrant splicing patterns, changes in RNA stability/localization, and the like. These findings suggest that silencing of expanded repeat loci has therapeutic value, and various approaches such as antisense oligonucleotide, small RNA, small molecules, and the like are used to silence toxic RNA (see Pinto B et al., Mol Cell. 2017 Nov 2, 68(3):479-490, which is incorporated herein by reference in its entirety).

For example, Jauvin et al. treated DMSXL mice, which is a mouse model of DM1, with an antisense oligonucleotide (ASO) targeting 3 UTR of DMPK gene, and showed that the DMPK mRNA level decreased, nuclear RNA aggregates (RNA foci) decreased and muscle strength increased, whereas no apparent toxicity was detected (see Jauvin D et al., Mol Ther Nucleic Acids. 2017 Jun 16, 7:465-474, which is incorporated herein by reference in its entirety).

W02018/002812 discloses a method for editing a DMPK gene in a cell by genome editing, e.g. using the CRISPR/Cas9 system, which can be used to treat a DMPK related condition or disorder such as DM1 (see W02018/002812, which is incorporated herein by reference in its entirety). Pinto et al. and Batra et al. demonstrated the possibility of the application of deactivated/nuclease- dead Cas9 (dCas9) to the treatment of D 1. To be specific, Pinto et al. combined dCas9 and guide RNA (gRNA) to CTG repeat region and showed that dCas9 effectively blocks transcription of expanded microsatellite repeat, whereby the phenotypes characteristic of DM1, which are due to repeat expansion, can be improved in vitro and in vivo (in HSA^LR mouse which is a mouse model of DM1) (see Pinto B et al., Mol Cell. 2017 Nov 2, 68(3):479-490, which is incorporated herein by reference in its entirety). On the other hand, Batra et al. showed that a combination of dCas9 fused with RNA endonuclease and gRNA for the CUG repeat region of DMPK mRNA can reduce level of CUG repeat expansion RNA and improve splicing abnormality in the cells of DM1 patients (see Batra R et al., Cell.

2017 Aug 24, 170(5):899-912, which is incorporated herein by reference in its entirety).

Adeno-associated virus (AAV) vectors have been known to enable transfer and long-term expression of foreign DNA, including therapeutic genes, in a variety of cell types. The different serotypes of AAV have been shown to have different tropisms in cultured cells and within an organism. For example, AAV8 and AAV9 are known to have relatively strong muscular tropism. Over the last decade many novel synthetic AAV variants have emerged by using a variety of capsid engineering techniques, one of which is the insertion of small, 7 amino acid-long, peptides into an exposed loop of the capsid protein, called variable region VIII (VRVIII).

The implementation of a novel peptide into a wild type capsid has shown to be very promising for permanently changing the tropism of the variant. For example, insertion of a peptide, RGDLGLS (SEQ ID NO: 5), into the capsid of AAV2 was found to increase infection of primary breast cancer cells (see Michelfelder et al., PLoS One. 2009; 4(4):e5122, which is incorporated herein by reference in its entirety), and said insertion into the capsid of AAV9 was found to increase infection of muscle cells (see W02019/207132, which is incorporated herein by reference in its entirety).

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide novel therapeutic methods to muscular dystrophy (particularly DM1).

It is another object of the present invention to provide novel agents which are useful for treating muscular dystrophy.

These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that the expression of human DMPK gene can be strongly suppressed using an AAV vector comprising a polynucleotide encoding a guide RNA targeting a particular sequence of human DMPK gene (Gene ID: 1760) and a fusion protein of a transcriptional repressor and a nuclease-deficient CRISPR effector protein. Based on these findings, the present inventors have completed the present invention.

Thus, the present invention provides the following :

[1] An adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences :

(a) a base sequence encoding a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor, and

(b) a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the AAV vector comprises an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[2] The AAV vector of the above-mentioned [1], wherein the polynucleotide comprises at least two base sequences encoding the guide RNAs, wherein the at least two base sequences are different.

[3] The AAV vector of the above-mentioned [1] or [2], wherein the transcriptional repressor is selected from the group KRAB domain of KOX1 or ZIM3, and the transcription repression domain of MeCP2, SIN3A, HDT1, MBD2B, NIPP1, or HP1A.

[4] The AAV vector of the above-mentioned [3], wherein the transcriptional repressor is KRAB domain of KOX1.

[5] The AAV vector of any of the above-mentioned [1] to [4], wherein the nuclease-deficient CRISPR effector protein is dCas9.

[6] The AAV vector of the above-mentioned [5], wherein the dCas9 is derived from Staphylococcus aureus.

[7] The AAV vector of any of the above-mentioned [1] to [6], wherein the polynucleotide further comprises a promoter sequence for the base sequence .encoding the guide RNA and/or a promoter sequence for the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor.

[8] The AAV vector of the above-mentioned [7], wherein the promoter sequence for the base sequence encoding the guide RNA is selected from the group U6 promoter, SNR6 promoter, SNR52 promoter, SCR1 promoter, RPR1 promoter, U3 promoter, and Hi promoter.

[9] The AAV vector of the above-mentioned [8], wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter.

[10] The AAV vector of any of the above-mentioned [7] to [9], wherein the promoter sequence for the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor is a ubiquitous promoter or a muscle specific promoter.

[11] The AAV vector of the above-mentioned [10], wherein the ubiquitous promoter is selected from the group EFS promoter, CMV promoter and CAG promoter.

[12] The AAV vector of the above-mentioned [10], wherein the muscle specific promoter is selected from the group CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, and Des promoter.

[13] The AAV vector of the above-mentioned [12], wherein the muscle specific promoter is CK8 promoter.

[14] The AAV vector of the above-mentioned [7], wherein the base sequence encoding the guide RNA comprises the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, the transcriptional repressor is KRAB domain of

KOX1, the nuclease-deficient CRISPR effector protein is dCas9 derived from Staphylococcus aureus, the promoter sequence for the base sequence encoding the guide RNA is U6 promoter, and the promoter sequence for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor is CK8 promoter.

[15] The AAV vector of any of the above-mentioned [1] to [14], wherein the AAV vector comprises the AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[16] A pharmaceutical composition comprising an AAV vector of any of the above-mentioned [1] to [15].

[17] The pharmaceutical composition of the above- mentioned [16] for treating or preventing myotonic dystrophy type 1.

[18] A method for treating or preventing myotonic dystrophy type 1, comprising administering an effective amount of an AAV vector of any of the above-mentioned [1] to [15] to a subject in need thereof.

[19] Use of an AAV vector of any of the above-mentioned [1] to [15] for the treatment or prevention of myotonic dystrophy type 1.

[20] Use of an AAV vector of any of the above-mentioned [1] to [15] in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of myotonic dystrophy type 1.

[21] A composition or kit for suppressing an expression of human DMPK gene, comprising the following:

(a) a first AAV vector comprising a polynucleotide comprising a base sequence encoding a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor, and

(b) a second AAV vector comprising a polynucleotide comprising a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the first AAV vector and the second AAV vector comprise an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[22] The composition or kit of the above-mentioned [21], wherein the second AAV vector comprises a polynucleotide comprising at least two base sequences encoding the guide RNAs, and wherein the at least two base sequences are different.

[23] The composition or kit of the above-mentioned [21] or [22] further comprising a third AAV vector, wherein the third AAV vector comprises a polynucleotide comprising a base sequence encoding the guide RNA, and wherein the base sequence is different from the base sequence comprised in the second AAV vector.

[24] The composition or kit of any of the above- mentioned [21] to [23], wherein the transcriptional repressor is selected from the group KRAB domain of KOX1 or ZIM3, and the transcription repression domain of MeCP2, SIN3A, HDT1, MBD2B, NIPP1, or HP1A.

[25] The composition or kit of the above-mentioned [24], wherein the transcriptional repressor is KRAB domain of KOX1.

[26] The composition or kit of any of the above- mentioned [21] to [25], wherein the nuclease-deficient CRISPR effector protein is dCas9.

[27] The composition or kit of the above-mentioned [26], wherein the dCas9 is derived from Staphylococcus aureus . [28] The composition or kit of any of the above- mentioned [21] to [27], wherein the first AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the fusion protein, and/or the second AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the guide RNA.

[29] The composition or kit of any of the above- mentioned [23] to [28], wherein the third AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the guide RNA.

[30] The composition or kit of the above-mentioned [28] or [29], wherein the promoter sequence for the base sequence encoding the guide RNA is selected from the group U6 promoter, SNR6 promoter, SNR52 promoter, SCR1 promoter, RPR1 promoter, U3 promoter, and HI promoter.

[31] The composition or kit of the above-mentioned [30], wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter.

[32] The composition or kit of the above-mentioned [28], wherein the promoter sequence for the base sequence encoding the fusion protein is a ubiquitous promoter or a muscle specific promoter.

[33] The composition or kit of the above-mentioned [32], wherein the ubiquitous promoter is selected from the group EFS promoter, CMV promoter and CAG promoter. [34] The composition or kit of the above-mentioned [32], wherein the muscle specific promoter is selected from the group CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, and Des promoter.

[35] The composition or kit of the above-mentioned [34], wherein the muscle specific promoter is CK8 promoter .

[36] The composition or kit of the above-mentioned [28] or [29], wherein the base sequence encoding the guide RNA comprises the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the transcriptional repressor is KRAB domain of KOX1, wherein the nuclease-deficient CRISPR effector protein is dCas9 derived from Staphylococcus aureus, wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter, and wherein the promoter sequence for the base sequence encoding the fusion protein is CK8 promoter.

[37] The composition or kit of any of the above- mentioned [21] to [36], wherein the AAV vector comprises the AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[38] A pharmaceutical composition comprising a composition of any of the above-mentioned [21] to [37]. [39] The pharmaceutical composition of the above- mentioned [38] for treating or preventing myotonic dystrophy type 1.

[40] A method for treating or preventing myotonic dystrophy type 1, comprising an effective amount of administering a composition of any of the above- mentioned [21] to [37] to a subject in need thereof.

[41] Use of a composition of any of the above-mentioned [21] to [37] for the treatment or prevention of myotonic dystrophy type 1.

[42] Use of a composition of any of the above-mentioned [21] to [37] in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of myotonic dystrophy type 1.

[43] An adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences :

(b) a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the AAV vector comprises an AAV capsid comprising a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

[44] The AAV vector of the above-mentioned [43], wherein the polynucleotide comprises at least two base sequences encoding the guide RNAs, wherein the at least two base sequences are different.

[45] The AAV vector of the above-mentioned [43] or [44], wherein the transcriptional repressor is selected from the group KRAB domain of KOXl or ZIM3, and the transcription repression domain of MeCP2, SIN3A, HDTl, MBD2B, NIPP1, or HP1A.

[46] The AAV vector of the above-mentioned [45], wherein the transcriptional repressor is KRAB domain of KOXl .

[47] The AAV vector of any of the above-mentioned [43] to [46], wherein the nuclease-deficient CRISPR effector protein is dCas9.

[48] The AAV vector of the above-mentioned [47], wherein the dCas9 is derived from Staphylococcus aureus.

[49] The AAV vector of any of the above-mentioned [43] to [48], wherein the polynucleotide further comprises a promoter sequence for the base sequence encoding the guide RNA and/or a promoter sequence for the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor.

[50] The AAV vector of the above-mentioned [49], wherein the promoter sequence for the base sequence encoding the guide RNA is selected from the group U6 promoter, SNR6 promoter, SNR52 promoter, SCR1 promoter, RPR1 promoter, U3 promoter, and HI promoter.

[51] The AAV vector of the above-mentioned [50], wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter.

[52] The AAV vector of any of the above-mentioned [49] to [51], wherein the promoter sequence for the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor is a ubiquitous promoter or a muscle specific promoter.

[53] The AAV vector of the above-mentioned [52], wherein the ubiquitous promoter is selected from the group EFS promoter, CMV promoter and CAG promoter.

[54] The AAV vector of the above-mentioned [52], wherein the muscle specific promoter is selected from the group CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, and Des promoter.

[55] The AAV vector of the above-mentioned [54], wherein the muscle specific promoter is CK8 promoter.

[56] The AAV vector of the above-mentioned [49], wherein the base sequence encoding the guide RNA comprises the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, the transcriptional repressor is KRAB domain of

[57] The AAV vector of any of the above-mentioned [43] to [56], wherein the AAV vector comprises the AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 38.

[58] A pharmaceutical composition comprising an AAV vector of any of the above-mentioned [43] to [57].

[59] The pharmaceutical composition of the above- mentioned [58] for treating or preventing myotonic dystrophy type 1.

[60] A method for treating or preventing myotonic dystrophy type 1, comprising administering an effective amount of an AAV vector of any of the above-mentioned [43] to [57] to a subject in need thereof.

[61] Use of an AAV vector of any of the above-mentioned [43] to [57] for the treatment or prevention of myotonic dystrophy type 1. [62] Use of an AAV vector of any of the above-mentioned [43] to [57] in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of myotonic dystrophy type 1.

[63] A composition or kit for suppressing an expression of human DMPK gene, comprising the following:

(a) a first AAV vector comprising a polynucleotide comprising a base seguence encoding a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor, and

(b) a second AAV vector comprising a polynucleotide comprising a base seguence encoding a guide RNA comprising the base seguence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the first AAV vector and the second AAV vector comprise an AAV capsid comprising a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

[64] The composition or kit of the above-mentioned [63], wherein the second AAV vector comprises a polynucleotide comprising at least two base sequences encoding the guide RNAs, and wherein the at least two base sequences are different.

[65] The composition or kit of the above-mentioned [63] or [64] further comprising a third AAV vector, wherein the third AAV vector comprises a polynucleotide comprising a base sequence encoding the guide RNA, and wherein the base sequence is different from the base sequence comprised in the second AAV vector.

[66] The composition or kit of any of the above- mentioned [63] to [65], wherein the transcriptional repressor is selected from the group KRAB domain of KOXl or ZIM3, and the transcription repression domain of MeCP2, SIN3A, HDT1, MBD2B, NIPP1, or HP1A.

[67] The composition or kit of the above-mentioned [66], wherein the transcriptional repressor is KRAB domain of KOXl.

[68] The composition or kit of any of the above- mentioned [63] to [67], wherein the nuclease-deficient CRISPR effector protein is dCas9.

[69] The composition or kit of the above-mentioned [68], wherein the dCas9 is derived from Staphylococcus aureus .

[70] The composition or kit of any of the above- mentioned [63] to [69], wherein the first AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the fusion protein, and/or the second AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the guide RNA.

[71] The composition or kit of any of the above- mentioned [65] to [70], wherein the third AAV vector further comprises a polynucleotide comprising a promoter sequence for the base sequence encoding the guide RNA.

[72] The composition or kit of the above-mentioned [70] or [71], wherein the promoter sequence for the base sequence encoding the guide RNA is selected from the group U6 promoter, SNR6 promoter, SNR52 promoter, SCR1 promoter, RPRl promoter, U3 promoter, and HI promoter.

[73] The composition or kit of the above-mentioned [72], wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter.

[74] The composition or kit of the above-mentioned [70], wherein the promoter sequence for the base sequence encoding the fusion protein is a ubiquitous promoter or a muscle specific promoter.

[75] The composition or kit of the above-mentioned [74], wherein the ubiquitous promoter is selected from the group EFS promoter, CMV promoter and CAG promoter.

[76] The composition or kit of the above-mentioned [74], wherein the muscle specific promoter is selected from the group CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, and Des promoter.

[77] The composition or kit of the above-mentioned [76], wherein the muscle specific promoter is CK8 promoter .

[78] The composition or kit of the above-mentioned [70], wherein the base sequence encoding the guide RNA comprises the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the transcriptional repressor is KRAB domain of KOX1, wherein the nuclease-deficient CRISPR effector protein is dCas9 derived from Staphylococcus aureus, wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter, and wherein the promoter sequence for the base sequence encoding the fusion protein is CK8 promoter.

[79] The composition or kit of any of the above- mentioned [63] to [78], wherein the AAV vector comprises the AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 38.

[80] A pharmaceutical composition comprising a composition of any of the above-mentioned [63] to [79].

[81] The pharmaceutical composition of the above- mentioned [80] for treating or preventing myotonic dystrophy type 1.

[82] A method for treating or preventing myotonic dystrophy type 1, comprising administering an effective amount of a composition of any of the above-mentioned [63] to [79] to a subject in need thereof.

[83] Use of a composition of any of the above-mentioned [63] to [79] for the treatment or prevention of myotonic dystrophy type 1.

[84] Use of a composition of any of the above-mentioned [63] to [79] in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of myotonic dystrophy type 1.

EFFECT OF THE INVENTION

According to the present invention, the expression of human DMPK gene can be suppressed and, consequently, the present invention is expected to be able to treat and/or prevent DM1.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

Fig. 1 shows DMPK downregulation in human muscular cells. Dark filled circles show the values of each data and bars show the average values of each condition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Adeno-associated virus (AAV) vector

The present invention provides an adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences (hereinafter sometimes to be also referred to as "the AAV vector of the present invention"):

(b) a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the AAV vector comprises an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or wherein the AAV vector comprises an AAV capsid comprising a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

By transduction with the AAV vector of the present invention, the polynucleotide comprised in the AAV vector of the present invention is introduced into a desired cell and transcribed to produce a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor, and a guide RNA targeting a particular region of the expression regulatory region of the human DMPK gene. These fusion protein and guide RNA form a complex (hereinafter the complex is sometimes referred to as "ribonucleoprotein; RNP") and cooperatively act on the aforementioned particular region, thus repressing transcription of the human DMPK gene. In one embodiment of the present invention, the expression of the human DMPK gene can be suppressed by, for example, not less than about 30%, not less than about 40%, not less than about 50%, not less than about 60%, not less than about 70%, not less than about 75%, not less than about 80%, not less than about 85%, not less than about 90%, not less than about 95%, or about 100% .

(1) Definition

In the present specification, "the expression regulatory region of human DMPK gene" means any region in which the expression of human DMPK gene can be repressed by binding RNP to that region. That is, the expression regulatory region of human DMPK gene may exist in any region such as the promoter region, enhancer region, intron, exon of the human DMPK gene, and neighboring genes of human DMPK gene (e.g., human DMWD (DM1 locus, WD repeat containing) gene), as long as the expression of the human DMPK gene is repressed by the binding of RNP. In the present specification, when the expression regulatory region is shown by the particular sequence, the expression regulatory region includes both the sense strand sequence and the antisense strand sequence conceptually.

In the present invention, a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor is recruited by a guide RNA into a particular region in the expression regulatory region of the human DMPK gene. In the present specification, the "guide RNA targeting..." means a "guide RNA recruiting a fusion protein into...".

In the present specification, the "guide RNA (to be also referred to as 'gRNA') " is an RNA comprising a genome specific CRISPR-RNA (to be referred to as "crRNA") . crRNA is an RNA that binds to a complementary sequence of a targeting sequence (described later).

When Cpfl is used as the CRISPR effector protein, the "guide RNA" refers to an RNA comprising an RNA consisting of crRNA and a specific sequence attached to its 5'-terminal (for example, an RNA sequence set forth in SEQ ID NO: 10 in the case of FnCpfl). When Cas9 is used as the CRISPR effector protein, the "guide RNA" refers to a chimera RNA (to be referred to as "single guide RNA(sgRNA)") comprising crRNA and trans-activating crRNA attached to its 3'-terminal (to be referred to as "tracrRNA") (see, for example, Zhang F. et al., Hum Mol Genet. 2014 Sep 15; 23(Rl):R40-6 and Zetsche B. et al., Cell. 2015 Oct 22; 163(3):759-71, which are incorporated herein by reference in their entireties).

In the present specification, a sequence complementary to the sequence to which crRNA binds in the expression regulatory region of the human DMPK gene is referred to as a "targeting sequence". That is, in the present specification, the "targeting sequence" is a DNA sequence present in the expression regulatory region of the human DMPK gene and adjacent to PAM (protospacer adjacent motif). PAM is adjacent to the 5'-side of the targeting sequence when Cpfl is used as the CRISPR effector protein. PAM is adjacent to the 3'-side of the targeting sequence when Cas9 is used as the CRISPR effector protein. The targeting sequence may be present on either the sense strand sequence side or the antisense strand sequence side of the expression regulatory region of the human DMPK gene (see, for example, the aforementioned Zhang F. et al., Hum Mol Genet. 2014 Sep 15; 23(Rl):R40-6 and Zetsche B. et al., Cell. 2015 Oct 22; 163(3):759-71, which are incorporated herein by reference in their entireties).

As used herein, "Adeno-associated virus" or "AAV" relates to the group of viruses containing a short single-stranded DNA and depending on the presence of an Adenovirus for their lytic replication. AAVs are members of the Parvoviridae family of viruses. In the present specification, "Adeno-associated virus vector" or "AAV vector" means vectors derived from Adeno- associated virus, i.e. gene transfer vehicles using the AAV capsid polypeptide to mediate the transfer of recombinant polynucleotides into desired cells.

In the present specification, "AAV capsid" means a proteinaceous shell of the AAV vector and does not comprise a per se replication competent AAV genome. Components of the AAV capsid are known in the art, and the AAV capsid comprises a polypeptide having the activity of self-assembly to produce the proteinaceous shell of the AAV vector, also referred to as AAV capsid polypeptide, coat protein or VP proteins.

(2) Nuclease-deficient CRISPR effector protein

In the present invention, using a nuclease- deficient CRISPR effector protein, a transcriptional repressor fused thereto is recruited to the expression regulatory region of the human DMPK gene. The nuclease- deficient CRISPR effector protein (hereinafter sometimes to be simply referred to as "CRISPR effector protein") to be used in the present invention is not particularly limited as long as it forms a complex with gRNA and is recruited to the expression regulatory region of the human DMPK gene. For example, nuclease-deficient Cas9 (hereinafter sometimes to be also referred to as "dCas9") or nuclease-deficient Cpfl (hereinafter sometimes to be also referred to as "dCpfl") can be included .

Examples of the above-mentioned dCas9 include, but are not limited to, a nuclease-deficient variant of Streptococcus pyogenes-derived Cas9 (SpCas9; PAM sequence: NGG (N is A, G, T or C. hereinafter the same) ), Streptococcus thermophilus-derived Cas9 (StlCas9; PAM sequence: NNAGAAW (W is A or T. hereinafter the same), St3Cas9; PAM sequence: NGGNG), Neisseria meningitidis-derived Cas9 (NmCas9; PAM sequence: NNNNGATT), or Staphylococcus aureus-derived Cas9 (SaCas9; PAM sequence: NNGRRT (R is A or G. hereinafter the same)) and the like (see, for example, Nishimasu et al., Cell. 2014 Feb 27; 15 156(5):935-49, Esvelt KM et al., Nat Methods. 2013 Nov; 10(11):1116-21, Zhang Y. Mol Cell. 2015 Oct 15; 60(2):242-55, and Friedland AE et al., Genome Biol. 2015 Nov 24; 16:257, which are incorporated herein by reference in their entireties) . For example, in the case of SpCas9, a double mutant in which the Asp residue at the 10th position is converted to Ala residue and the His residue at the 840th position is converted to Ala residue (sometimes referred to as "dSpCas9") can be used (see, for example, the aforementioned Nishimasu et al., Cell. 2014, which is incorporated herein by reference in their entireties). Alternatively, in the case of SaCas9, a double mutant in which the Asp residue at the 10th position is converted to Ala residue and the Asn residue at the 580th position is converted to Ala residue (SEQ ID NO: 11), or a double mutant in which the Asp residue at the 10th position is converted to Ala residue and the His residue at the 557th position is converted to Ala residue (SEQ ID NO: 12) (hereinafter any of these double mutants is sometimes to be referred to as "dSaCas9") can be used (see, for example, Nishimasu H, et al., Cell. 2015 Aug 27; 162(5):1113-26, which is incorporated herein by reference in its entirety). In addition, in one embodiment of the present invention, as dCas9, a variant obtained by modifying a part of the amino acid sequence of the aforementioned dCas9, which forms a complex with gRNA and is recruited to the expression regulatory region of the human DMPK gene, may also be used. Examples of such variants include a truncated variant with a partly deleted amino acid sequence. In one embodiment of the present invention, the variant described in WO2019/235627 and W02020/085441, which are incorporated herein by reference in their entireties, can be used as dCas9. Specifically, dSaCas9 obtained by deleting the 721st to 745th amino acids from dSaCas9 that is a double mutant in which the Asp residue at the 10th position is converted to Ala residue and the Asn residue at the 580th position is converted to Ala residue (SEQ ID NO: 13), or dSaCas9 in which the deleted part is substituted by a peptide linker (e.g., one in which the deleted part is substituted by GGSGGS linker (SEQ ID NO: 14) is set forth in SEQ ID NO: 15) (hereinafter any of these double mutants is sometimes to be referred to as "dSaCas9[- 25]"), or dSaCas9 obtained by deleting the 482nd to 648th amino acids of dSaCas9 that is the aforementioned double mutant (SEQ ID NO: 16), or dSaCas9 in which the deleted part is substituted by a peptide linker (one in which the deleted part is substituted by GGSGGS linker is set forth in SEQ ID NO: 17) may also be used.

Examples of the above-mentioned dCpfl include, but are not limited to, a nuclease-deficient variant of Francisella novicida-derived Cpfl (FnCpfl; PAM sequence: TTN), Acidaminococcus sp.-derived Cpfl (AsCpfl; PAM sequence: TTTN), or Lachnospiraceae bacterium-derived Cpfl (LbCpfl; PAM sequence: TTTA, TCTA, TCCA, or CCCA) and the like (see, for example, Zetsche B. et al., Cell. 2015 Oct 22; 163(3):759-71, Yamano T et al., Cell. 2016 May 5; 165(4):949-62, and Yamano T et al., Mol Cell.

2017 Aug 17; 67(4):633-45, which are incorporated herein by reference in their entireties). For example, in the case of FnCpfl, a double mutant in which the Asp residue at the 917th position is converted to Ala residue and the Glu residue at the 1006th position is converted to Ala residue can be used (see, for example, the aforementioned Zetsche B et al., Cell. 2015, which is incorporated herein by reference in its entirety). In one embodiment of the present invention, as dCpfl, a variant obtained by modifying a part of the amino acid sequence of the aforementioned dCpfl, which forms a complex with gRNA and is recruited to the expression regulatory region of the human DMPK gene, may also be used.

In one embodiment of the present invention, dCas9 is used as the nuclease-deficient CRISPR effector protein. In one embodiment, the dCas9 is dSaCas9, and, in a particular embodiment, dSaCas9 is dSaCas9[-25].

A polynucleotide comprising a base sequence encoding a nuclease-deficient CRISPR effector protein can be cloned by, for example, synthesizing an oligoDNA primer covering a region encoding a desired part of the protein based on the cDNA sequence information thereof, and amplifying the polynucleotide by PCR method using total RNA or mRNA fraction prepared from the cells producing the protein as a template. In addition, a polynucleotide comprising a base sequence encoding a nuclease-deficient CRISPR effector protein can be obtained by introducing a mutation into a nucleotide sequence encoding a cloned CRISPR effector protein by a known site-directed mutagenesis method to convert the amino acid residues (e.g., Asp residue at the 10th position, His residue at the 557th position, and Asn residue at the 580th position in the case of SaCas9; Asp residue at the 917th position and Glu residue at the 1006th position in the case of FnCpfl, and the like can be included, but are not limited to these) at a site important for nuclease activity to other amino acids.

Alternatively, a polynucleotide comprising a base sequence encoding nuclease-deficient CRISPR effector protein can be obtained by chemical synthesis or a combination of chemical synthesis and PCR method or Gibson Assembly method, based on the cDNA sequence information thereof, and can also be further constructed as a base sequence that underwent codon optimization to be codons suitable for expression in human.

(3) Transcriptional repressor

In the present invention, human DMPK gene expression is repressed by the action of the transcriptional repressor fused with the nuclease- deficient CRISPR effector protein. In the present specification, the "transcriptional repressor" means a protein having the ability to repress gene transcription of human DMPK gene or a peptide fragment retaining the function thereof. The transcriptional repressor to be used in the present invention is not particularly limited as long as it can repress expression of human DMPK gene. It includes, for example, Kruppel-associated box (KRAB) domain derived from various genes, such as KRAB domain of KOX1 or ZIM3 (see, for example, Alerasool N et al., Nat Methods. 2020 Nov;17(11):1093-1096, which is incorporated herein by reference in its entirety), transcription repression domain of MBD2B, v-ErbA, SID (including chain state of SID (SID4X)), MBD2, MBD3, DNMT family (e.g., DNMTl, DNMT3A, DNMT3B), Rb, MeCP2, ROM2, LSD1 , AtHD2A, SET1, HDAC11, SETD8, EZH2, SUV39H1, PHFl9, SALI , NUE, SUVR4, KYP, DIM5, HDAC8, SIRT3, SIRT6,

MES0L04 , SET8, HST2, COBB, SET-TAF1B, NCOR, SIN3A, HDT1, NIPP1, or HP1A, and ERF repressor domain (ERD), and variants thereof having transcriptional repression ability, fusions thereof and the like (see, for example, Yeo N C et al., Nat Methods. 2018 Aug; 15(8):611-616, which is incorporated herein by reference in its entirety) . In one embodiment of the present invention, KRAB domain of KOX1 is used as the transcriptional repressor .

A polynucleotide comprising a base sequence encoding a transcriptional repressor can be constructed by chemical synthesis or a combination of chemical synthesis and PCR method or Gibson Assembly method. Furthermore, a polynucleotide comprising a base sequence encoding a transcriptional repressor can also be constructed as a codon-optimized DNA sequence to be codons suitable for expression in human.

A polynucleotide comprising a base sequence encoding a fusion protein of a transcriptional repressor and a nuclease-deficient CRISPR effector protein can be prepared by ligating a base sequence encoding the CRISPR effector protein to a base sequence encoding the transcriptional repressor directly or after adding a base sequence encoding a linker, NLS (nuclear localization signal) (for example, a base sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19), a tag and/or others. In the present invention, the transcriptional repressor may be fused with either N-terminal or C- terminal of the nuclease-deficient CRISPR effector protein. As the linker, a linker with an amino acid number of about 2 to 50 can be used, and specific examples thereof include, but are not limited to, a G-S- G-S linker in which glycine (G) and serine (S) are alternately linked and the like. In one embodiment of the present invention, as the polynucleotide comprising a base sequence encoding a fusion protein of a nuclease- deficient CRISPR effector protein and a transcriptional repressor, the base sequence set forth in SEQ ID NO: 20, which encodes SV40 NLS, dSaCas9, NLS and KRAB as a fused protein, can be used.

(4) Guide RNA

In the present invention, a fusion protein of nuclease-deficient CRISPR effector protein and transcriptional repressor can be recruited to the expression regulatory region of the human DMPK gene by guide RNA. As described in the aforementioned "(1) Definition", guide RNA comprises crRNA, and the crRNA binds to a complementary sequence of the targeting sequence. crRNA may not be completely complementary to the complementary sequence of the targeting sequence as long as the guide RNA can recruit the fusion protein to the target region, and may be a sequence in which at least 1 to 3 bases are deleted, substituted, inserted and/or added.

When dCas9 is used as the nuclease-deficient CRISPR effector protein, for example, the targeting sequence can be determined using a published gRNA design web site (CRISPR Design Tool, CRISPR direct etc.). To be specific, from the sequence of the target gene (i.e., human DMPK gene) and neighboring gene thereof, candidate targeting sequences of about 20 nucleotides in length for which PAM (e.g., NNGRRT in the case of SaCas9) is adjacent to the 3'-side thereof are listed, and one having a small number of off-target sites in human genome among these candidate targeting sequences can be used as the targeting sequence. The base length of the targeting sequence is 18 to 24 nucleotides in length, preferably 18 to 23 nucleotides in length, more preferably 18 to 22 nucleotides in length. As a primary screening for the prediction of the off-target site number, a number of bioinformatic tools are known and publicly available, and can be used to predict the targeting sequence with the lowest off-target effect. Examples thereof include bioinformatics tools such as Benchling (https://benchling.com), and COSMID (CRISPR Off-target Sites with Mismatches, Insertions and Deletions) (Available on https://crispr.bme.gatech.edu on the internet). Using these, the similarity to the base sequence targeted by gRNA can be summarized. When the gRNA design software to be used does not have a function to search for off-target site of the target genome, for example, the off-target site can be searched for by subjecting the target genome to Blast search with respect to 8 to 12 nucleotides on the 3'-side of the candidate targeting sequence (seed sequence with high discrimination ability of targeted nucleotide sequence).

In one embodiment of the present invention, the targeting sequence may be a base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

[Table 1]

In Table 1, "Coordinate" indicates the coordinate of the 5' end of each sequences set forth in SEQ ID NOs: 1-4 .

In one embodiment of the present invention, a base sequence encoding crRNA may be the same base sequence as the targeting sequence. For example, when the targeting sequence set forth in SEQ ID NO: 1

(AGAAACCAGTGACCAGTGAGC) is introduced into the cell as a base sequence encoding crRNA, crRNA transcribed from the sequence is AGAAACCAGUGACCAGUGAGC (SEQ ID NO: 21) and is bound to GCTCACTGGTCACTGGTTTCT (SEQ ID NO: 22), which is a sequence complementary to the base sequence set forth in SEQ ID NO: 1 and is present in the expression regulatory region of the human DMPK gene. In another embodiment, a base sequence which is a targeting sequence in which at least 1 to 3 bases are deleted, substituted, inserted and/or added can be used as the base sequence encoding crRNA as long as guide RNA can recruit a fusion protein to the target region.

Therefore, in one embodiment of the present invention, as a base sequence encoding crRNA, the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added can be used. In one embodiment of the present invention, as a base sequence encoding crRNA, the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted and/or added can be used.

In one embodiment of the present invention, the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or

SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added can be used as the base sequence encoding crRNA to produce gRNA comprising crRNA set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or crRNA set forth in SEQ ID

NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, respectively. In another embodiment of the present invention, the gRNA can comprise the base sequence set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or the base sequence set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 in which 1 to 3 bases are deleted, substituted, inserted, and/or added. In still another embodiment of the present invention, the gRNA can comprise the base sequence set forth in SEQ ID NO: 24, or the base sequence set forth in SEQ ID NO: 24 in which 1 to 3 bases are deleted, substituted, inserted, and/or added.

When dCpfl is used as the nuclease-deficient CRISPR effector protein, a base sequence encoding gRNA can be designed as a DNA sequence encoding crRNA with particular RNA attached to the 5'-terminal. Such RNA attached to the 5'-terminal of crRNA and a DNA sequence encoding said RNA can be appropriately selected by those of ordinary skill in the art according to the dCpfl to be used. For example, when dFnCpfl is used, a base sequence in which SEQ ID NO: 26; AATTTCTACTGTTGTAGAT is attached to the 5'-side of the targeting sequence can be used as a base sequence encoding gRNA (when transcribed to RNA, the sequences of the underlined parts form base pairs to form a stem-loop structure). The sequence to be added to the 5'-terminal may be a sequence generally used for various Cpfl proteins in which at least 1 to 6 bases are deleted, substituted, inserted and/or added, as long as gRNA can recruit a fusion protein to the expression regulatory region after transcription.

When dCas9 is used as the nuclease-deficient CRISPR effector protein, a base sequence encoding gRNA can be designed as a DNA sequence in which a DNA sequence encoding known tracrRNA is linked to the 3'- terminal of a DNA sequence encoding crRNA. Such tracrRNA and a DNA sequence encoding the tracrRNA can be appropriately selected by those of ordinary skill in the art according to the dCas9 to be used. For example, when dSaCas9 is used, the base sequence set forth in SEQ ID NO: 27 is used as the DNA sequence encoding tracrRNA. The DNA sequence encoding tracrRNA may be a base sequence encoding tracrRNA generally used for various Cas9 proteins in which at least 1 to 6 bases are deleted, substituted, inserted and/or added, as long as gRNA can recruit a fusion protein to the expression regulatory region after transcription.

A polynucleotide comprising a base sequence encoding gRNA designed in this way can be chemically synthesized using a known DNA synthesis method. In another embodiment of the present invention, the polynucleotide comprised in the AAV vector of the present invention may comprise at least two different base sequences respectively encoding a gRNA, wherein the at least two different base sequences are selected from a base sequence comprising a sequence set forth in SEQ

ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID

NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added.

In one embodiment of the present invention, the polynucleotide comprised in the AAV vector of the present invention may comprise the base sequence comprising a sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted and/or added, and at least one base sequence comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4, or a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added.

(5) Promoter sequence

In one embodiment of the present invention, a promoter sequence may be operably linked to the upstream of each of a base sequence encoding fusion protein of nuclease-deficient CRISPR effector protein and transcriptional repressor and/or a base sequence encoding gRNA. The promoter to be possibly linked is not particularly limited as long as it shows a promoter activity in the target cell. Examples of the promoter sequence possibly linked to the upstream of the base sequence encoding gRNA include, but are not limited to, U6 promoter, SNR6 promoter, SNR52 promoter, SCR1 promoter, RPR1 promoter, U3 promoter, HI promoter, and tRNA promoter, which are pol III promoters, and the like. In one embodiment of the present invention, U6 promoter can be used as the promoter sequence for the base sequence encoding the guide RNA. In one embodiment of the present invention, when a polynucleotide comprises two or more base sequences respectively encoding a guide RNA, a single promoter sequence may be operably linked to the upstream of the two or more base sequences. In another embodiment, when a polynucleotide comprises two or more base sequences respectively encoding a guide RNA, a promoter sequence may be operably linked to the upstream of each of the two or more base sequences, wherein the promoter sequence operably linked to each base sequence may be the same or different .

As the aforementioned promoter sequence possibly linked to the upstream of the base sequence encoding fusion protein, a ubiquitous promoter or muscle specific promoter may be used. Examples of the ubiquitous promoter include, but are not limited to, EF-la promoter, EFS promoter, CMV (cytomegalovirus) promoter, hTERT promoter, SRa promoter, SV40 promoter, LTR promoter, CAG promoter, RSV (Rous sarcoma virus) promoter, and the like. In one embodiment of the present invention, EFS promoter, CMV promoter or CAG promoter can be used as the ubiquitous promoter.

Examples of the muscle specific promoter include, but are not limited to, CK8 promoter, CK6 promoter, CK1 promoter, CK7 promoter, CK9 promoter, cardiac muscle troponin C promoter, a-actin promoter, myosin heavy chain kinase (MHCK) promoter (e.g., MHCK7 promoter etc.)_/ MHC promoter, myosin light chain 2A promoter, dystrophin promoter, muscle creatine kinase (MCK) promoter, dMCK promoter, tMCK promoter, enh348 MCK promoter, synthetic C5-12 (Syn) promoter, Myf5 promoter, MLCl/3f promoter, MLC-2 promoter, MYOD promoter, Myog promoter, Pax7 promoter, Des promoter, cTnC promoter and the like (for the detail of the muscle specific promoter, see, US2011/0212529A1, McCarthy JJ et al., Skeletal Muscle. 2012 May; 2(1):8, Wang B. et al., Gene Ther. 2008 Nov; 15(22):1489-99, and the like, which are incorporated herein by reference in their entireties).

In one embodiment of the present invention, CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, or Des promoter can be used as the muscle specific promoter. In one embodiment of the present invention, CK8 promoter can be used as the muscle specific promoter. The aforementioned promoter may have any modification and/or alteration as long as it has promoter activity in the target cell.

In one embodiment of the present invention, U6 promoter is used as a promoter for a base sequence encoding the guide RNA, and CK8 promoter can be used as the promoter sequence for the base sequence encoding the fusion protein. Specifically, as for the U6 promoter, the following base sequences can be used; (i) the base sequence set forth in SEQ ID NO: 28, (ii) a base sequence set forth in SEQ ID NO: 28 wherein 1 or several (e.g., 2, 3, 4, 5 or more) bases are deleted, substituted, inserted and/or added with a promoter activity in the target cell, or (iii) a base sequence not less than 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above) identical with the base sequence set forth in SEQ ID NO: 28 showing a promoter activity in the target cell. As for the CK8 promoter, the following base sequences can be used; (i) the base sequence set forth in SEQ ID NO: 29, (ii) a base sequence set forth in SEQ ID NO: 29 wherein 1 or several

(e.g., 2, 3, 4, 5 or more) bases are deleted, substituted, inserted and/or added with a promoter activity in the target cell, or (iii) a base sequence not less than 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above) identical with the base sequence set forth in SEQ ID NO: 29 showing a promoter activity in the target cell.

(6) Other base sequence

Furthermore, the polynucleotide comprised in the AAV vector of the present invention may further comprise known sequences such as Polyadenylation (polyA) signal, Kozak consensus sequence and the like besides those mentioned above for the purpose of improving the translation efficiency of mRNA produced by transcription of a base sequence encoding a fusion protein of nuclease-deficient CRISPR effector protein and transcriptional repressor. In addition, the polynucleotide comprised in the AAV vector of the present invention may comprise a base sequence encoding a linker sequence, a base sequence encoding NLS and/or a base sequence encoding a tag.

(7) AAV capsid

The AAV vector of the present invention comprises an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

WO2019/207132 discloses that AAV variants having a capsid with a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) showed increase infection of muscle cells. AAV variants having a capsid with the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, which are named as AAVS1_P1 or AAV9S1_P1 and AAVS10_P1 or AAV9S10_P1 respectively, showed reduced infectivity for the liver to about 1/100 and improved infectivity for the muscles by about 10 times, compared with AAV9 vector (see WO2019/207132, which is incorporated herein by reference in its entirety) . The alignment between AAVS1_P1 and AAVS10_P1 is about 96.0% identity according to EMBOSS NEEDLE program below.

In one embodiment of the present invention, the AAV capsid can comprise a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is not less than 95% (e.g. 96%, 97%, 98%, 99% or above) identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, as long as the AAV vector retains of its ability to transduce a cell. In one embodiment of the present invention, the AAV capsid can comprise the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

In one embodiment of the present invention, the AAV capsid may comprise a polypeptide encoded by a base sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a base sequence which is at least 95% identical to the base sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, as long as the polypeptide comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and the AAV vector retains its ability to transduce a cell.

In one embodiment of the present invention, the AAV capsid may comprise a polypeptide encoded by a base sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a base sequence which is not less than 95% (e.g. 96%, 97%,

98%, 99% or above) identical to the base sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, as long as the polypeptide comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and the AAV vector retains its ability to transduce a cell.

In one embodiment of the present invention, the AAV capsid can comprise a polypeptide which is not less than 95% (e.g. 96%, 97%, 98%, 99% or above) identical to the amino acid sequence set forth in SEQ ID NO: 38, as long as the AAV vector retains its ability to transduce a cell. In one embodiment of the present invention, the AAV capsid can comprise the amino acid sequence set forth in SEQ ID NO: 38.

In one embodiment of the present invention, the AAV capsid may comprise a polypeptide encoded by a base sequence set forth in SEQ ID NO: 37 or a base sequence which is at least 95% identical to the base sequence set forth in SEQ ID NO: 37, as long as the AAV vector retains its ability to transduce a cell.

In one embodiment of the present invention, the AAV capsid may comprise a polypeptide encoded by a base sequence set forth in SEQ ID NO: 37 or a base sequence which is not less than 95% (e.g. 96%, 97%, 98%, 99% or above) identical to the base sequence set forth in SEQ ID NO: 37, as long as the AAV vector retains its ability to transduce a cell.

In the present specification, the "identity" means Identity which is a value obtained from EMBOSS NEEDLE program (J Mol Biol 1970; 48:443-453) search using default parameters. The parameters are as follows.

Gap penalty = 10 Extend penalty = 0.5 Matrix = EBLOSUM62

When the AAV vector of the present invention is prepared, a known method such as (1) a method using a plasmid, (2) a method using a baculovirus, (3) a method using a herpes simplex virus, (4) a method using an adenovirus, or (5) a method using yeast can be used (e.g., Appl Microbiol Biotechnol. 2018; 102(3):1045- 1054, etc., which is incorporated herein by reference in its entirety). For example, when the AAV vector of the present invention is prepared by a method using a plasmid, first, a vector plasmid comprising inverted terminal repeat (ITR) at both ends of wild-type AAV genomic sequence and the polynucleotide encoding a guide RNA targeting a particular sequence of human DMPK gene and a fusion protein of a transcriptional repressor and a nuclease-deficient CRISPR effector protein inserted in place of the DNA encoding Rep protein and capsid protein is prepared. On the other hand, the DNA encoding Rep protein and capsid protein which are necessary for forming virus particles are inserted into other plasmids. Furthermore, a plasmid comprising genes (E1A, E1B, E2A, VA and E4orf6) responsible for the helper action of adenovirus necessary for proliferation of AAV is prepared as an adenovirus helper plasmid. The co transfection of these three kinds of plasmids into the host cell causes the production of recombinant AAV (i.e., AAV vector) in the cell. As the host cell, a cell capable of supplying a part of the gene products (proteins) of the genes responsible for the aforementioned helper action (e.g., 293 cell etc.) is preferably used. When such cell is used, it is not necessary to carry the gene encoding a protein that can be supplied from the host cell in the aforementioned adenoviral helper plasmid. The produced AAV vector is present in the culture medium and/or cell. Thus, a desired AAV vector is prepared by collection of the virus from the culture medium after destroying the host cell with freeze-thawing or the like and then subjecting the virus fraction to separation and purification by density gradient ultracentrifugation method using cesium chloride, column method or the like.

AAV vector has great advantages in terms of safety, gene transduction efficiency and the like, and is used for gene therapy. However, it is known that the size of polynucleotide that can be packaged is limited. For example, in one embodiment of the present invention, the full-length including the base length of the polynucleotide comprising (i) a base sequence encoding a fusion protein of dSaCas9 and KRAB domain, (ii) a base sequence encoding gRNA targeting an expression regulatory region of human DMPK gene, (iii) CK8 promoter sequence and U6 promoter sequence as the promoter sequences, and (iv) the ITR region is about 4.9kb, and the polynucleotide can be carried in a single AAV vector.

(8) Exemplified embodiments of the present invention

In one embodiment of the present invention, an AAV vector comprising a polynucleotide is provided comprising the following base sequences: a base sequence encoding a fusion protein of a nuclease-deficient CRISPR effector protein and a transcriptional repressor,

CK8 promoter for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor, one or two base sequences respectively encoding a guide RNA, wherein the one or two base sequences are selected from a base sequence comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a base sequence comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, and

U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of KOX1 or ZIM3, and wherein the AAV vector comprises an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

CK8 promoter for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor, one or two base sequences respectively encoding a guide RNA, wherein the one or two base sequences are selected from a base sequence comprising a sequence set forth in SEQ ID NO: 3, or a base sequence comprising a sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, and U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of KOX1, and wherein the AAV vector comprises an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

CK8 promoter for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor, one or two base sequences respectively encoding a guide RNA, wherein the one or two base sequences are selected from a base sequence comprising a sequence set forth in SEQ ID NO: 3, or a base sequence comprising a sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, and U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of KOX1, and wherein the AAV vector comprises an AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of KOX1 or ZIM3, and wherein the AAV vector comprises an AAV capsid comprising a polypeptide is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

CK8 promoter for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor, one or two base sequences respectively encoding a guide RNA, wherein the one or two base sequences are selected from a base sequence comprising a sequence set forth in SEQ ID NO: 3, or a base sequence comprising a sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, and U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of K0X1, and wherein the AAV vector comprises an AAV capsid comprising a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

CK8 promoter for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor, one or two base sequences respectively encoding a guide RNA, wherein the one or two base sequences are selected from a base sequence comprising a sequence set forth in SEQ ID NO: 3, or a base sequence comprising a sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, and

U6 promoter for the base sequence encoding the guide RNA, wherein the nuclease-deficient CRISPR effector protein is dSaCas9, wherein the transcriptional repressor is KRAB domain of KOX1, and wherein the AAV vector comprises an AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 38.

In an embodiment of the present invention, the polynucleotide comprised in the AAV vector of the present invention comprises in order from the 5' end (i) the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor and (ii) the base sequence encoding the gRNA. In another embodiment, the polynucleotide comprises in order from the 5' end (ii) the base sequence encoding the gRNA and (i) the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor.

2. Pharmaceutical composition for treating or preventing DM1

The present invention also provides a pharmaceutical composition comprising the AAV vector of the present invention (hereinafter sometimes referred to as "the pharmaceutical composition of the present invention"). The pharmaceutical composition of the present invention can be used for treating or preventing DM1 .

The pharmaceutical composition of the present invention comprises the AAV vector of the present invention as an active ingredient, and may be prepared as a formulation comprising such active ingredient (i.e., the AAV vector of the present invention) and, generally, a pharmaceutically acceptable carrier.

In an embodiment of the present invention, the pharmaceutical composition of the present invention is administered parenterally , and may be administered topically or systemically. The pharmaceutical composition of the present invention can be administered by, but are not limited to, for example, intravenous administration, intraarterial administration, subcutaneous administration, intraperitoneal administration, or intramuscular administration.

The dose of the pharmaceutical composition of the present invention to a subject is not particularly limited as long as it is an effective amount for the treatment and/or prevention. It may be appropriately optimized according to the active ingredient, dosage form, age and body weight of the subject, administration schedule, administration method and the like.

In one embodiment of the present invention, the pharmaceutical composition of the present invention can be not only_· administered to the subject affected with DM1 but also prophylactically administered to subjects who may develop DM1 in the future based on the genetic background analysis and the like. The term "treatment" in the present specification also includes remission of disease, in addition to the cure of diseases. In addition, the term "prevention" may also include delaying the onset of disease, in addition to prophylaxis of the onset of disease. The pharmaceutical composition of the present invention can also be referred to as "the agent of the present invention" or the like.

3. Method for treatment or prevention of DM1

The present invention also provides a method for treating or preventing DM1, comprising administering an effective amount of the AAV vector of the present invention to a subject in need thereof (hereinafter sometimes referred to as "the method of the present invention") . In addition, the present invention includes the AAV vector of the present invention for use in the treatment or prevention of DM1. Furthermore, the present invention includes use of the AAV vector of the present invention in the manufacture of a pharmaceutical composition for the treatment or prevention of DM1.

The method of the present invention can be practiced by administering the aforementioned pharmaceutical composition of the present invention to a subject affected with DM1, and the dose, administration route, subject and the like are the same as those mentioned above.

Measurement of the symptoms may be performed before the start of the treatment using the method of the present invention and at any timing after the treatment to determine the response of the subject to the treatment.

The method of the present invention can improve, but are not limited to, any symptom of DM1 such as the function of skeletal muscle and/or cardiac muscle. Muscles or tissue to be improved in the function thereof are not particularly limited, and any muscles and tissue, and muscle groups can be mentioned.

4. Compositions or kit

The present invention also provides a composition or kit comprising the following for suppression of the expression of the human DMPK gene (hereinafter sometimes referred to as "the composition or kit of the present invention", or "the composition of the present invention" or "the kit of the present invention" respectively) :

(b) a second AAV vector comprising a polynucleotide comprising a base sequence encoding a guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 , or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the first AAV vector and the second AAV vector comprise an AAV capsid comprising a polypeptide which comprises the amino acid sequence RGDLGLS (SEQ ID NO: 5) and is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a polypeptide which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 38.

In one embodiment of the present invention, the second AAV vector can comprise a polynucleotide comprising at least two base sequences encoding the guide RNAs comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the at least two base sequences are different.

In one embodiment of the present invention, the second AAV vector may comprise the base sequence comprising a sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted and/or added, and at least one base sequence comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4, or a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or

SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added.

In one embodiment of the present invention, the composition or kit of the present invention can further comprise a third AAV vector, wherein the third AAV vector comprises a polynucleotide comprising a base sequence encoding the guide RNA comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID

NO: 4 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, wherein the base sequence comprised in the third AAV vector is different from the base sequence comprised in the second AAV vector.

In one embodiment of the present invention, the second AAV vector may comprise the base sequence comprising a sequence set forth in SEQ ID NO: 3 or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted and/or added, and the third AAV vector may comprise at least one base sequence comprising a sequence set forth in SEQ ID NO:

1, SEQ ID NO: 2, or SEQ ID NO: 4, or a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4 in which 1 to 3 bases are deleted, substituted, inserted and/or added.

In one embodiment of the present invention, the first AAV vector can further comprise a polynucleotide comprising a promoter sequence for the base sequence encoding the fusion protein, and/or the second AAV vector and/or the third AAV vector can further comprise a polynucleotide comprising a promoter sequence for the base sequence encoding the guide RNA.

The present invention also provides a pharmaceutical composition comprising the composition of the present invention. The pharmaceutical composition can be used for treating or preventing DM1. The pharmaceutical composition comprises the first AAV vector and the second AAV vector as active ingredients, and may be prepared as a formulation comprising such active ingredients and, generally, a pharmaceutically acceptable carrier. In one embodiment of the present invention, the pharmaceutical composition can further comprise the third AAV vector as one of active ingredients.

The present invention further provides a method for treating or preventing DM1, comprising administering the composition of the present invention to a subject in need thereof. In addition, the present invention includes the composition of the present invention for use in the treatment or prevention of DM1. Furthermore, the present invention includes use of the composition of the present invention in the manufacture of a pharmaceutical composition for the treatment or prevention of DM1.

As the nuclease-deficient CRISPR effector protein, transcriptional repressor, guide RNA, as well as polynucleotides encoding them and AAV vectors in which they are carried, those explained in detail in the above-mentioned section of "1. Adeno-associated virus (AAV) vector" can be used. The dose, administration route, subject, formulation and the like of the above- mentioned pharmaceutical composition are the same as those explained in the section of "2. Pharmaceutical composition for treating or preventing DM1".

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES

Example 1. Adeno-associated virus (AAV) production (1) Experimental Methods

Construction of plasmids for delivery and expression of dSaCas 9-KRAB:gRNA and generation of AAV

The GOI (gene of interest) plasmid of AAV which contains in order from the 5' end CK8 promoter(SEQ ID NO^~2-9-)^~SV-4-0—NTrS—dS-a-O-a-s9-NLS-KRAB (SEQ ID NO: 20) with an additional terminal stop codon [SEQ ID NO: 30 (DNA) and SEQ ID NO: 31 (protein)], bovine growth hormone (bGH) polyA sequence (SEQ ID NO: 32), U6 promoter sequence (SEQ ID No: 33), SaCas9 gRNA scaffold sequence (SEQ ID NO: 34), and targeting sequence set forth in SEQ ID NO:

3 was cloned to pAAV-CMV (Takara # 6230) as described in Example 2 of PCT/JP2020/021851 between two ITRs and the generated plasmid was named pED148-h695 (comprising the targeting sequence set forth in SEQ ID NO: 3).

Adeno-associated virus (AAV) production

Adeno-associated virus serotype 8 (AAV8), 9

(AAV9), S1_P1 (AAVS1_P1) or S10_P1 (AAVS10_P1) particles were generated using 293T cells (ATCC # CRL-3216) from five Collagen-coated 225 cm² Cell Culture Flasks (Corning # NCO431082) for each serotype. A RepCap plasmid for AAV9 was constructed as follows: AAV9 capsid sequence (see AY530579.1) [SEQ ID NO: 35 (DNA) and SEQ ID NO: 36 (protein)] was subcloned into a pRC2-mi342 vector (Takara # 6230) replacing the AAV2 capsid sequence. A RepCap plasmid for AAV8 was constructed as follows: AAV8 capsid sequence (see NC_006261.1) [SEQ ID NO: 37 (DNA) and SEQ ID NO: 38 (protein)] was subcloned into a pRC5 vector (Takara # 6664) replacing the AAV5 capsid sequence. RepCap plasmids for AAVS1_P1 and AAVS10_P1 comprising AAVS1_P1 capsid sequence [SEQ ID NO: 8 (DNA) and SEQ ID NO: 6 (protein)] and AAVS10_P1 capsid sequence [SEQ ID NO: 9 (DNA) and SEQ ID NO: 7 (protein)] disclosed in WO2019/207132 respectively were used. Cells were seeded at a density of 1.8 ^c 10⁷ cells per Collagen-coated 225 cm² Cell Culture Flask (Corning # NCO431082) and cultured in 60 mL of DMEM (Invitrogen # 11995-065) supplemented with 10% FBS (GE Healthcare # SH30070.03) . Next day, transfection mix was prepared for each capsid. 100 pg of one of the RepCap plasmids, pHelper plasmid included in AAVpro® Helper Free System (Takara # 6230) and pEDl48-h695 were mixed with 30 mL of Opti-MEM (GIBCO # 31985-062) and 900 pL of TransIT-293 Transfection Reagent (MIR # MIR2700) was added and mixed gently. After incubated at room temperature for 15 minutes, 6.1 L of the mix per flask was added and the culture vessel was gently rocked to evenly distribute the complexes of TransIT-293 Transfection Reagent and DNA . Next day, medium was removed and 60 mL of DMEM supplemented with 2% FBS was added to each flask.

After four days from medium change, 750 pL of 0.5M EDTA (pH8.0) (Invitrogen # 15575020) was added to each flask and mixed thoroughly. Supernatant and cells were harvested after incubation at room temperature for 10 minutes. Cells were aliguoted into six 50 mL conical tubes (Falcon # 352070), and centrifuged 2,000 ^c g for 10 minutes at 4°C. AAV particles from supernatant and cell pellet were collected separately with AAVpro® Concentrator (TaKaRa # 6674) or AAVpro® Purification Kit Maxi (All Serotypes) (TaKaRa #6666), respectively.

Supernatant was collected in a storage bottle (Corning # 430282) and 16 mL of Concentrating Solution A included in AAVpro® Concentrator was added and mixed with vortex mixer, followed by the addition of 80 mL of Concentrating Solution B included in AAVpro®

Concentrator and mixed with vortex. The mixture was aliquoted to eight 50 mL conical tubes. The supernatants were incubated overnight at 4°C.

After centrifugation at 2,000 * g at 4°C for 50 minutes, supernatant was removed. 2 mL of dissolving solution included in AAVpro® Concentrator was added to each tube and pipetted for suspension, followed by putting them together to one tube. The suspension was mixed with vortex for 15 seconds before and after incubation at room temperature for 15 minutes. Suspension was centrifuged at 2,000 ^c g at 4°C for 15 minutes and the supernatant was transferred to a new tube and re-centrifuged under the same condition. Supernatant was filtrated with a Millex-HV 0.45 pm filter included in AAVpro® Concentrator (Millipore # SLHV033RB) and added to the Amicon Ultra-15, lOOkDa filter included in AAVpro® Concentrator (Millipore # UFC910008). The supernatant was centrifuged at 2,000 * g at 15°C for 10 minutes and the volume was confirmed to be 1.5 mL or less. 10.5 mL of washing solution was added and pipetted, followed by centrifugation at 2,000 x g at 15°C for 10 minutes and the volume was confirmed to be 1.5 mL or less. The washing process was repeated two more times, followed by concentrating the volume of the supernatant to 500 pL. After resuspension, the AAV solution was aliquoted into 100 pL per tube and stored at -80°C.

Cell pellet was loosened enough and 1.66 mL of AAV Extraction Solution A plus included in AAVpro® Purification Kit (All Serotypes) was added per 50 mL tube. The mixture was mixed with vortex for 15 seconds and incubated for 5 minutes at room temperature. After vortexing for 15 seconds, the mixture was centrifuged at 5,697 x g for 10 minutes at 4°C and the supernatant from six tubes were transferred to a new 50 mL conical tube and 1 mL of AAV Extraction solution B (1/10 vol) included in AAVpro® Purification Kit (All Serotypes) was added and pipetted. 110 pL of Cryonase Cold-active Nuclease (1/100 vol) was added and incubated at 37°C for 1 hour. 1.11 mL of Precipitator A (1/10 vol) included in AAVpro® Purification Kit (All Serotypes) was added and after vortexing for 10 seconds, incubated at 37°C for 30 minutes and vortexed for 10 seconds. 0.555 mL of Precipitator B (1/20 vol) included in AAVpro® Purification Kit (All Serotypes) was added and vortexed for 10 seconds. After centrifugation at 5,697 ^c g at 4°C for 5 minutes, supernatant was filtered with Millex- HV 0.45 pm. The supernatant was concentrated to 1.5 mL using Amicon Ultra-15 lOOkDa included in AAVpro® Purification Kit (All Serotypes), which was centrifuged at 2,000 x g at 15°C for 5 minutes. To change buffer, after removal of flow-through, 5 mL of suspension buffer was added and pipetted and centrifuged at 2,000 ^c g at 15°C for 5 minutes. After repeating the process for a total of 5 times, AAV solution was concentrated to 500 pL .

After suspending by pipetting, AAV solution was collected and aliquoted to 100 pL per tube and stored at -80 °C.

The titer of purified AAV genome was measured using AAVpro® Titration Kit (for Real Time PCR) (Takara # 6233).

(2) Results

AAV8, AAV9, AAVS1_P1, and AAVS10_P1, which carried transgenes of dSaCas9, KRAB, and sgRNA comprising crRNA encoded by the targeting sequence set forth in SEQ ID NO: 3, were obtained. Genome titer of the AAVs is shown in Table 2. [Table 2]

Example 2. In-vitro pharmacological evaluation of recombinant AAVs carrying the base sequence encoding dSaCas9, transcriptional repressor and sgRNA on DMPK gene repression (1) Experimental Methods Cell culture and AAV infection iCM cells (Immortalized non-DM control myoblast cell line) obtained from Institut de Myologie as human muscular cells were suspended in skeletal muscle cell growth medium kit (Promocell # C23060) (note: media was supplemented with 20% FBS, rather than 5% as directed by the kit, and 50 pg/ml Gentamicin S) and seeded onto a Collagen type I-Coated 24 well plate (IWAKI #4820-010) at a density of 24,000 cells in 1 ml of medium per well. Next day, the media was replaced with differentiation media (DMEM media (Thermo Fisher # 10566-016) supplemented with 10 pg/ l insulin (Sigma # 19278)) and the cells were cultured for 6 days at 37°C with 5% CO2 to differentiate the myoblast cells into myotubes. At day 8, 750 pL of the media was removed and 500 pL of the new differentiation media was added. For AAV infection, 350 pL PBS containing 5.7 ^c 10¹¹, 1.9 * 10¹¹, 6.3 ^c lO¹⁰, or 2.1 x 10¹⁰ vg/ml of each AAV8 (supernatant), AAV9 (Cell pellet), AAVS1_P1 (Cell pellet), or AAVS10_P1 (supernatant) were added to the medium. We used supernatant-derived or pellet-derived AAVs respectively because there are no apparent differences of infectivity reported between them. Regarding AAVS10_P1, 7.1 ^c 10⁹ and 2.4 ^c 10⁹ vg/ml were also added. AAV titers of 2.0 x 10¹¹, 6.7 x 10¹⁰, 2.2 ^c 10¹⁰, 7.4 ^c 10⁹, 2.5 ^c 10⁹ or 8.2 x 10⁸ vg/well were described as H, M, L, L2, L3 or L4 in the graphs of Figure 1, respectively. For control wells, 350 pL PBS was added to the medium. Cells were cultured and incubated at 37°C/5% CO2 for 4 days. After washing with 500 pL PBS, total RNA was extracted using RNeasy Plus Mini Kit (Qiagen # 74134) and QIAshredder (Qiagen # 79656) according to the manufacturer's instruction. RNA from cells without AAV infection was set as control and shown as Ctrl in Figure 1.

Gene expression analysis

For Taqman qPCR, 80 ng of total RNA was converted to cDNA using Superscript™ VILO™ cDNA Synthesis Kit (Thermo Fisher # 11754250) in 20 pL reaction volume.

The cDNA was diluted 160 fold with water and 2 pL was used for the qPCR. The qPCR was run in 5 pL final volume containing Taqman probes for DMPK (Thermo Fisher # Hs01094329_ml, FAM) or GAPDH (Thermo Fisher # Hs99999905_ml , FAM), and Taqman™ Gene Expression Master Mix (Thermo Fisher # 4369016) with ViiA 7 Real-Time PCR System (Thermo Fisher). The qPCR cycling condition was as follows: 95°C for 10 minutes after 50°C for 2 minutes followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The data were analyzed with QuantStudio™ Real-Time PCR software (Thermo Fisher). The expression values were analyzed for each gene using cDNA samples from Ctrl wells as standard. Calibration curves were generated by four-fold serial dilutions of cDNA for 8 times and linear ranges were adopted. The expression levels of DMPK gene were normalized to those of GAPDH gene and relative values to control wells were shown in percentage .

(2) Results

By applying AAV8, AAV9, AAVS1_P1, or AAVS10_P1 into iCM cells, DMPK mRNA downregulation was found, which suggests AAV8, AAV9, AAVS1_P1, or AAVS10_P1 carrying transgenes of dSaCas9, KRAB, and sgRNA comprising crRNA encoded by the targeting sequence set forth in SEQ ID NO: 3 has a pharmacological effect on DMPK downregulation in human muscular cells. Also, AAVS1_P1 and AAVS10_P1 showed comparable or superior downregulat ion of DMPK gene expression to differentiated muscular cells compared to the AAV8 or AAV9 (Figure 1).

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

As used herein the words "a" and "an" and the like carry the meaning of "one or more".

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

All patents and other references mentioned above are incorporated in full herein by this reference, the same as if set forth at length.

INDUSTRIAL APPLICABILITY

According to the present invention, expression of DMPK gene can be suppressed in the human muscular cells Therefore, the present invention is expected to be extremely useful for the treatment and/or prevention of DM1.

Claims

1. An adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences:

2. The AAV vector according to claim 1, wherein the polynucleotide comprises at least two base sequences encoding the guide RNAs, wherein the at least two base sequences are different.

3. The AAV vector according to claim 1 or 2, wherein the transcriptional repressor is selected from the group KRAB domain of KOX1 or ZIM3, and the transcription repression domain of MeCP2, SIN3A, HDT1, MBD2B, NIPPl, or HP1A.

4. The AAV vector according to claim 3, wherein the transcriptional repressor is KRAB domain of KOX1.

5. The AAV vector according to any one of claims 1 to 4, wherein the nuclease-deficient CRISPR effector protein is dCas9.

6. The AAV vector according to claim 5, wherein the dCas9 is derived from Staphylococcus aureus.

7 . The AAV vector according to any one of claims 1 to 6, wherein the polynucleotide further comprises a promoter sequence for the base sequence encoding the guide RNA and/or a promoter sequence for the base sequence encoding the fusion protein of the nuclease- deficient CRISPR effector protein and the transcriptional repressor.

8. The AAV vector according to claim 7, wherein the promoter sequence for the base sequence encoding the guide RNA is selected from the group U6 promoter, SNR6 promoter, SNR52 promoter, SCRl promoter, RPRl promoter, U3 promoter, and HI promoter.

9. The AAV vector according to claim 8, wherein the promoter sequence for the base sequence encoding the guide RNA is U6 promoter.

10. The AAV vector according to any one of claims 7 to 9, wherein the promoter sequence for the base sequence encoding the fusion protein of the nuclease-deficient CRISPR effector protein and the transcriptional repressor is a ubiquitous promoter or a muscle specific promoter .

11. The AAV vector according to claim 10, wherein the ubiquitous promoter is selected from the group EFS promoter, CMV promoter and CAG promoter.

12. The AAV vector according to claim 10, wherein the muscle specific promoter is selected from the group CK8 promoter, myosin heavy chain kinase (MHCK) promoter, muscle creatine kinase (MCK) promoter, synthetic C5-12 (Syn) promoter, and Des promoter.

13. The AAV vector according to claim 12, wherein the muscle specific promoter is CK8 promoter.

14. The AAV vector according to claim 7, wherein the base sequence encoding the guide RNA comprises the base sequence set forth in SEQ ID NO: 3, or the base sequence set forth in SEQ ID NO: 3 in which 1 to 3 bases are deleted, substituted, inserted, and/or added, the transcriptional repressor is KRAB domain of

15. The AAV vector according to any one of claims 1 to 14, wherein the AAV vector comprises the AAV capsid comprising the amino acid sequence set forth in SEQ ID NO: 38.

16. A pharmaceutical composition comprising an AAV vector according to any one of claims 1 to 15.

17. The pharmaceutical composition according to claim 16 for treating or preventing myotonic dystrophy type 1.

18. A method for treating or preventing myotonic dystrophy type 1, comprising administering an effective amount of an AAV vector of any one of claims 1 to 15 to a subject in need thereof.

19. An adeno-associated virus (AAV) vector comprising a polynucleotide comprising the following base sequences :