CN118272373A

CN118272373A - Application of SpG-ABE in preparation of Du's muscular dystrophy treatment composition

Info

Publication number: CN118272373A
Application number: CN202410204314.7A
Authority: CN
Inventors: 金铭; 陈万金; 王柠; 杨辉; 李国玲; 林佳佳
Original assignee: Huida Shanghai Biotechnology Co ltd; First Affiliated Hospital of Fujian Medical University
Current assignee: Huida Shanghai Biotechnology Co ltd; First Affiliated Hospital of Fujian Medical University
Priority date: 2024-02-23
Filing date: 2024-02-23
Publication date: 2024-07-02

Abstract

The application discloses a base editing system targeting nonsense mutations on the human dystrophin (DMD) gene and its use for treating duchenne muscular dystrophy.

Description

Application of SpG-ABE in preparation of Du's muscular dystrophy treatment composition

Sequence listing

The present disclosure includes a sequence Listing XML file submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML file was created by WIPO Sequence software according to WIPO Standard ST.26, 22 nd 2 nd year 2024, named HGP042CN.xml, size 153,103 bytes.

The symbol "T" is used to denote T in DNA and U in RNA according to WIPO Standard ST.26. Thus in the present sequence listing prepared according to st.26, when the sequence is RNA, T in the sequence should be regarded as U.

Technical Field

The present invention is in the field of gene editing and gene therapy technology, and in particular relates to a guide nucleic acid targeting nonsense mutations (nonsense mutation) in the human dystrophin (DMD) gene, a system comprising the guide nucleic acid, and uses thereof, for example for the treatment of duchenne muscular dystrophy.

Background

Dunaliella Muscular Dystrophy (DMD) is an X-linked fatal disease caused by mutation of human dystrophin (dystrophin; uniProt: P11532) gene (DMD), has become the most common neuromuscular disease in childhood, and has a global prevalence of DMD of up to 1 person per 3500 male newborns. Dystrophin is a key scaffold protein that stabilizes the myofiber membrane and directs the organization of signaling molecules, thereby ensuring homeostasis and function of the muscle system. Thus, the lack of functional dystrophin can lead to disturbances in myomembrane integrity and muscle cell regeneration, leading to progressive muscle atrophy and weakness. Thus, DMD patients develop loss of walking ability, pulmonary insufficiency, heart failure, and early death in the age of twenty years. DMD patients still lack curative therapy.

Of the thousands of mutations that lead to DMD, nonsense mutations are estimated to account for about 10% of all DMD cases. Non-homologous end joining (NHEJ) and Homology Directed Repair (HDR) strategies based on CRISPR-Cas systems have been used to treat pathological point mutations and partially restore dystrophin expression in mdx or mdx ^4Cv DMD mouse models carrying nonsense mutations in mouse exons 23 or 53, respectively. However, the above strategy relies on a random and inefficient repair mechanism following a wild-type Cas9 nuclease-induced DNA Double Strand Break (DSB), resulting in limited therapeutic efficacy and a significant and non-negligible risk of introducing erroneous genomic modifications (e.g., large DNA insertions/deletions, inversions, and chromosomal rearrangements). Thus, there is an urgent need to explore safe and effective gene editing methods to correct pathological nonsense mutations affecting DMD patients without inducing genomic DSBs.

Disclosure of Invention

The present invention meets the above-described need by providing products, methods and uses for treating pathological nonsense mutation-related duchenne muscular dystrophy by means of gene editing that utilize base editing without reliance on DNA double strand breaks, achieve efficient pathological DMD nonsense mutation correction and low off-target editing, and can achieve effective and accurate delivery through AAV.

In one aspect, the present disclosure provides a guide RNA comprising (1) a scaffold sequence capable of complexing with an RNA-guided DNA binding protein; and (2) a guide sequence capable of hybridizing to a target sequence on a human dystrophin gene that is 100% reverse complementary to a protospacer sequence on the opposite strand of the human dystrophin gene, the protospacer sequence comprising a nonsense mutation selected from the group consisting of ：E30,c.4174C>T,p.Gln1392*;E23,c.2977C>T,p.Gln993*;E24,c.3189G>A,p.Trp1063*;E44,c.6292C>T,p.Arg2098*;E55,c.8038C>T,p.Arg2680*;E6,c.433C＞T,p.Arg145*;E13,c.1510C>T,p.Gln504*;E19,c.2302C>T,p.Arg1577*;E20,c.2407C>T,p.Gln803*;E20,c.2527G>T,p.Glu843*;E21,c.2695G>T,p.Glu899*;E21,c.2776C>T,p.Gln926*;E25,c.3337C>T,p.Gln1113*;E25,c.3414G>A,p.Trp1138*;E30,c.4117C>T,p.Gln1373*;E34,c.4729C>T,p.Arg768*;E35,c.4996C>T,p.Arg1666*;E36,c.5125A>T,Lys1709*;E37,c.5247C>A,p.Cys1749*;E41,c.5899C>T,p.Arg1967*;E43,c.6188T>A,p.Leu2063*;E54,c.8009A>G,p.Trp2670*;E56,c.8230G>T,p.Glu2744*;E59,c.8713C>T,p.Arg2950*; and E70, c.10141c > T, p.arg 3381.

In another aspect, the present disclosure provides a polynucleotide encoding a guide RNA of the present disclosure.

In yet another aspect, the present disclosure provides a system comprising (1) a guide RNA of the present disclosure or a polynucleotide encoding the guide RNA; and (2) a fusion protein comprising an RNA-guided DNA binding domain and an adenylate deaminase domain or a polynucleotide encoding said fusion protein.

In yet another aspect, the present disclosure provides a carrier system comprising a first carrier and a second carrier,

A) Wherein the first vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding an N-terminal RNA-guided DNA binding domain; and

A polynucleotide encoding an N-terminal intein;

b) Wherein the second vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding a C-terminal intein; and

Polynucleotides encoding a C-terminal RNA-guided DNA binding domain;

wherein the vector system further comprises a polynucleotide encoding an adenylate deaminase domain;

Wherein delivering the vector system into a cell results in production of a fusion protein in the cell, the fusion protein comprising (1) an RNA-guided DNA-binding domain consisting of the N-terminal RNA-guided DNA-binding domain and the C-terminal RNA-guided DNA-binding domain, and (2) the adenylate deaminase domain;

Wherein the vector system further comprises a polynucleotide of the present disclosure.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a guide RNA of the present disclosure, a polynucleotide of the present disclosure, a system of the present disclosure, a vector of the present disclosure, or a vector system of the present disclosure.

In yet another aspect, the present disclosure provides a method of treating duchenne muscular dystrophy comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

In yet another aspect, the present disclosure provides a guide RNA of the present disclosure, a polynucleotide of the present disclosure, a system of the present disclosure, a vector of the present disclosure, or a vector system of the present disclosure for use in the manufacture of a medicament for treating duchenne muscular dystrophy.

In yet another aspect, the present disclosure provides a method of editing a target DNA comprising contacting the target DNA with a guide RNA of the present disclosure, a polynucleotide of the present disclosure, a system of the present disclosure, a vector of the present disclosure, or a vector system of the present disclosure, whereby the target DNA is edited.

In yet another aspect, the present disclosure provides a cell comprising a target DNA edited by a method of the present disclosure.

Drawings

Fig. 1: adenine base editor corrected DMD nonsense mutations in vitro. Fig. 1A: immunohistochemistry of dystrophin in the left biceps of normal donor and DMD patients. Dystrophin appears brown. Scale bar: 50 μm. Fig. 1B: percentage of premature stop codons caused by nonsense mutations in DMD. Fig. 1C: schematic of a fluorescence reporting assay for determining optimal sgrnas. The reporter construct comprises mCherry, human exon variant sequences, and EGFP. Correction of nonsense mutations in the exon variant sequences allows EGFP expression. Fig. 1D: representative flow cytometry plots of EGFP expression in HEK293T cells treated with reporter plasmid alone (control) or in combination with SpG-ABE constructs (E30 sgRNA-4 and E23 sgRNA-1, respectively). Fig. 1E: quantification of percentage of EGFP positive cells after transfection of SpG-ABE and sgrnas for nonsense mutations in human DMD exons 23, 24, 30 and 44 (n=3). Triangles are biological replicates and quantification is shown as mean ± SD.

Fig. 2: adenine base editing restored dystrophin expression in human DMD cardiomyocytes. Fig. 2A: schematic of the application of adenine base editing in the human DMD iPSC model. Human DMD ipscs from patient fibroblasts were edited by SpG-ABE and subsequently differentiated into cardiomyocytes for downstream analysis. Fig. 2B: schematic representation of the binding position of hEx sgRNA-4 in exon 30 mutant. PAM and sgRNA are shown in red and blue, respectively. Adenine (e.g., a 15) in the edit window is numbered from after PAM. Genomic sequencing chromatograms of exon 30 mutants in human ipscs are shown at the bottom. Fig. 2C: percentage of genome editing events in human DMD ipscs treated with SpG-ABE and hEx sgRNA-4 (n=3). The target (mid target) adenine (A15) is shown in blue. Fig. 2D: immunofluorescence analysis of dystrophin and cTnI levels in normal, DMD and corrected cardiomyocytes. Dystrophin and cTnI are shown in red and green, respectively. Nuclei were labeled with blue DAPI staining. Scale bar: 50 μm. Quantitative data are expressed as mean ± SEM.

Fig. 3: local ABE administration restored dystrophin expression in adult humanized DMD ^E30mut mice. Fig. 3A: schematic representation of c.4174c > T nonsense mutations in the human exon 30 sequence of humanized DMD ^E30mut mice. Fig. 3B: an overview of the components of the split ABE system were administered intramuscularly to 8 week old DMD ^E30mut mice. AAV expressing N-and C-terminal ABEs was injected into the tibialis anterior muscle of the right leg of the mouse at a dose of 5X 10 ¹¹ vg/leg/AAV, and saline was injected into the tibialis anterior muscle of the left leg of the mouse as a negative control. All mice were dissected three weeks after injection for analysis. Fig. 3C: percentage of genome editing in tibialis anterior of mice treated with ABE or saline (n=3). Fig. 3D: deep sequencing of the percentage of editing events in transcripts in tibialis anterior of mice treated with ABE or saline (n=3). Fig. 3E: immunofluorescence analysis of dystrophin and ghost expression in tibialis anterior in age-matched wild-type mice and DMD ^E30mut mice treated with ABE or saline. Dystrophin appears green. Scale bar: 200 μm. Fig. 3F: western blot analysis of dystrophin and neogenin (vinculin) in tibialis anterior of mice treated with ABE or saline. Proteins from age-matched wild-type mice were used to normalize dystrophin expression levels. Fig. 3G: quantification of dystrophin + myofibers in cross section of mouse tibialis anterior as in fig. 3E. Fig. 3H: quantification of dystrophin expression levels in tibialis anterior in DMD ^E30mut mice treated as shown in fig. 3F. Quantitative data were calculated after normalization to neogenin expression. Quantification is shown as mean ± SEM (n=3). Each triangle represents a single mouse. The P value was evaluated by the Student's t test. NS: is not significant; * P <0.05.

Fig. 4: systemic ABE delivery improves muscle function by restoring extensive dystrophin in humanized DMD ^E30mut mice. Fig. 4A: overview of systemic administration of components of the split ABE system to neonatal DMD ^E30mut mice. Mice on postnatal day 3 (P3) were intraperitoneally infused with AAV9 expressing split ABE components at a total dose of 2 x 10 ¹¹ vg, with saline treatment as a negative control. Four weeks later, all mice were dissected for downstream analysis. Fig. 4B: percentage of genome editing in myocardium, diaphragm and tibialis anterior of DMD ^E30mut mice treated with ABE or saline (n=3). Fig. 4C: deep sequencing analysis of modification events in transcripts of myocardium, diaphragm and tibialis anterior of DMD ^E30mut mice treated with ABE or saline (n=3). Fig. 4D: immunohistochemistry for dystrophin expression in myocardium, diaphragm muscle and tibialis anterior in age-matched wild-type mice and DMD ^E30mut mice treated with AAV or saline. Dystrophin appears green. Scale bar: 200 μm. Fig. 4E: quantification of dystrophin-positive muscle fibers in a cross section of the mouse tibialis anterior as in fig. 4D. Fig. 4F: western blot analysis of dystrophin expression in myocardium, diaphragm muscle and tibialis anterior of wild type mice and DMD ^E30mut mice treated with ABE or saline. Neurobin was used as a loading control. Fig. 4G: rotating bar running test of wild type mice and age-matched DMD ^E30mut mice treated with ABE or saline (n=9). Fig. 4H: rodent treadmill running test of age-matched wild-type mice and DMD ^E30mut mice treated with ABE or saline (n=9). Quantitative data are shown as mean ± SEM. The P value was evaluated by the Student's test. NS: is not significant; * P <0.01.

Fig. 5: efficiency of correcting nonsense mutations in vitro in SpG-ABE and human sgRNA. Fig. 5A: representative fluorescence microscopy pictures of mCherry and EGFP expression in HEK293T cells after transfection of reporter plasmid alone (control) or in combination with SpG-ABE construct (using E23 sgRNA-1). Scale bar: 200 μm. Fig. 5B: FACS detection of EGFP expression levels in HEK293T cells treated with SpG-ABE and sgrnas targeting human DMD exons 4, 6, 21, 25, 35, 37, 41, 54, 55 and 59 (n=3). Quantification is expressed as mean ± SEM. * P <0.05; * P <0.01; * P <0.001.

Fig. 6: characterization and off-target analysis of human DMD iPSC cells. Fig. 6A: representative morphology of fibroblasts in DMD patients, the image was taken under an optical microscope at 10 x. Fig. 6B: representative pictures of human DMD iPSC colonies taken at 20 x under an optical microscope. Fig. 6C: karyotyping of human DMD iPSC cells. Fig. 6D: RT-PCR analysis of the pluripotency markers OCT4 and NANOG in human DMD iPSC and normal H9 cells (n=3). Fibroblasts from DMD patients served as negative controls. Fig. 6E: left: schematic representation of the binding position of hEx sgRNA-2, sgRNA-3 or sgRNA-5 in exon 30 mutants. PAM and sgRNA sequences are represented in red and blue, respectively. Adenine is numbered from after PAM. Representative chromatograms of genomic sequencing were performed in human DMD ipscs. Right figure: percentage of genome editing performed in human DMD iPSC for full length SpG-ABE and hEx sgrnas (n=3). The middle target edit is shown in blue. Fig. 6F: sanger sequence of corrected myocardial DNA. Fig. 6G: sanger sequence of corrected myocardial cDNA. Fig. 6H: and hEx deep sequencing analysis of genomic modifications of sgRNA-3 at mid-target site and highest predicted off-target site (n=3). The middle target edit is shown in blue. Fig. 6I: schematic representation of intein-mediated cleavage SpG-ABE system. N-and C-terminal ABEs in the ABEv or ABEv system are driven by the CBh promoter or the Spc5.12 promoter, respectively, whereas the human sgRNA cassettes in the ABEv and ABEv systems are both controlled by the U6 promoter. Fig. 6J: percent DNA editing at a15 in human DMD iPSC treated with hEx sgRNA-4 and split SpG-ABE systems (n=3). Quantitative data are shown as mean ± SEM.

Fig. 7: restoration of dystrophin expression in skeletal muscle of ABE-driven adult DMD ^E30mut mice. Immunofluorescence analysis of dystrophin protein expression in the entire tibialis anterior of wild-type mice and DMD ^E30mut mice treated with ABE or saline. Dystrophin is green. Scale bar: 500 μm.

Fig. 8: local ABE delivery restored dystrophin expression in adult DMD ^E23mut mice. Fig. 8A: schematic representation of nonsense c.2977c > T mutations in the human exon 23 sequence of the humanized DMD ^E23mut mouse. Fig. 8B: graphical representation of intramuscular injection of components of the split ABE system into 8 week old humanized DMD ^E23mut mice, N-and C-terminal ABE delivered by AAV9 particles at a total dose of 5 x 10 ¹¹ vg/leg to the right tibial anterior muscle of the mice, with saline-treated left legs as negative controls. Fig. 8C: schematic representation of the binding position of hEx s.sup.23 sgRNA-1 in exon 23 mutants. PAM and sgRNA are shown in red and blue, respectively. Adenine in the edit window is numbered from after PAM. Fig. 8D: percentage of genome editing events in tibialis anterior of mice treated with saline or ABE (n=3). Fig. 8E: percentage of modification events in transcripts of tibialis anterior in mice treated with saline or ABE (n=3). Fig. 8F: immunohistochemistry for dystrophin expression in tibialis anterior of age-matched wild-type mice and DMD ^E23mut mice treated with ABE or saline. Dystrophin is green. Scale bar: 200 μm. Fig. 8G: quantification of dystrophin-positive muscle fibers in cross section of tibialis anterior of mice treated with ABE or saline (n=3). Fig. 8H: western blot of dystrophin and neomycin in tibialis anterior of age-matched wild-type mice and DMD ^E23mut mice treated with ABE or saline. Proteins from wild-type mice were used to normalize dystrophin expression levels. Fig. 8I: quantification of dystrophin expression levels in ABE-treated DMD ^E23mut mice (n=3) after normalization to neogenin expression. Quantification is shown as mean ± SEM. Each triangle represents a single mouse. NS: is not significant; * P <0.05; * P <0.01.

Fig. 9: restoration of dystrophin expression in DMD ^E23mut mice following local ABE infusion. Immunohistochemistry for dystrophin expression in the entire tibialis anterior of wild-type mice and DMD ^E23mut mice that did not receive or received ABE treatment. Dystrophin appears green. Scale bar: 500 μm.

Fig. 10: systemic dystrophin recovery of systemic administration ABE in neonatal DMD ^E23mut mice. Immunohistochemistry of dystrophin in whole myocardium, diaphragm muscle and tibialis anterior tissue of wild type mice and DMD ^E23mut mice receiving saline or ABE treatment. Dystrophin appears green. Scale bar: 500 μm.

Fig. 11: cardiac function of DMD mice after systemic delivery ABEv a was assessed using echocardiography. Fig. 11A: representative echocardiographic images of DMD ^E30mut mice with or without ABEv2 were monitored for 6 weeks. Age-matched wild type and DMD mice served as controls. Fig. 11B: at week 6 post injection, echocardiographic analysis was performed on WT, DMD simulation, and ABEv 2-treated DMD mice. LVID; d or LVID; s: left ventricular inner diameter during diastole or systole; LVPW; d or LVPW; s: the left ventricular posterior wall thickness during diastole or systole; LVAW; d or LVAW; s: left ventricular anterior wall thickness during diastole or systole; EF: ejection fraction; FS: fractional shortening; CO: cardiac output; LV mass (correction): left ventricular mass corrected based on body surface area. Values are shown as mean ± SEM. Significance was indicated by asterisks and determined by unpaired two-tailed Student's t test. ns indicates no statistical significance.

Fig. 12: characterization of CK activity after intraperitoneal injection (n=6). Significance was indicated by asterisks and determined by unpaired two-tailed Student's t test. ns indicates no statistical significance.

Detailed Description

The present disclosure provides products, methods and uses for treating pathological nonsense mutation related muscular dystrophies by gene editing means. In particular, the present disclosure provides guide RNAs that target nonsense mutations on human dystrophin genes that can complex with RNA-guided DNA binding proteins (e.g., cas9 proteins, such as Cas9 nickases) to guide editing of the nonsense mutations (e.g., base editing by deaminase fused to the RNA-guided DNA binding proteins), restore human dystrophin gene expression, enabling treatment of muscular dystrophy. The present disclosure provides a variety of guide RNAs for a wide variety of nonsense mutations in human dystrophin genes, enabling universal treatment of muscular dystrophy caused by different nonsense mutations, and which can be achieved by the same base editing tool. The present disclosure demonstrates that an adenylate base editing tool consisting of Cas9 (e.g., cas9 nickase) and an adenylate deaminase can achieve efficient editing of a wide variety of nonsense mutations on human dystrophin genes through the guidance of the guide RNAs of the present disclosure. The present disclosure also demonstrates that Cas9 can be split into two parts using an intein system in order for a dual AAV system to deliver the adenylate base editing tools and guide RNAs of the present disclosure and achieve efficient editing.

Thus, in one aspect, the present disclosure provides a guide RNA comprising (1) a scaffold sequence capable of complexing with an RNA-guided DNA binding protein; and (2) a guide sequence capable of hybridizing to a target sequence on a human dystrophin gene that is 100% reverse complementary to a protospacer sequence on the opposite strand of the human dystrophin gene, the protospacer sequence comprising a nonsense mutation selected from the group consisting of ：E30,c.4174C>T,p.Gln1392*;E23,c.2977C>T,p.Gln993*;E24,c.3189G>A,p.Trp1063*;E44,c.6292C>T,p.Arg2098*;E55,c.8038C>T,p.Arg2680*;E6,c.433C＞T,p.Arg145*;E13,c.1510C>T,p.Gln504*;E19,c.2302C>T,p.Arg1577*;E20,c.2407C>T,p.Gln803*;E20,c.2527G>T,p.Glu843*;E21,c.2695G>T,p.Glu899*;E21,c.2776C>T,p.Gln926*;E25,c.3337C>T,p.Gln1113*;E25,c.3414G>A,p.Trp1138*;E30,c.4117C>T,p.Gln1373*;E34,c.4729C>T,p.Arg768*;E35,c.4996C>T,p.Arg1666*;E36,c.5125A>T,Lys1709*;E37,c.5247C>A,p.Cys1749*;E41,c.5899C>T,p.Arg1967*;E43,c.6188T>A,p.Leu2063*;E54,c.8009A>G,p.Trp2670*;E56,c.8230G>T,p.Glu2744*;E59,c.8713C>T,p.Arg2950*; and E70, c.10141c > T, p.arg 3381.

In some embodiments, the guide sequence is as set forth in any one of SEQ ID NOs 14, 15, 13, 3, 8, 23, 24, 29, 1-2, 4-7, 9-12, 16-22, 25-28 and 30-33 or differs from any one of SEQ ID NOs 14, 15, 13, 3, 8, 23, 24, 29, 1-2, 4-7, 9-12, 16-22, 25-28 and 30-33 by NO more than 1, 2, 3, 4, or 5 nucleotides.

In some embodiments, the RNA-guided DNA binding protein comprises a Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 nickase. In one embodiment, the amino acid sequence of the Cas9 nickase is set forth in SEQ ID NO. 84.

In some embodiments, the scaffold sequence (1) has a secondary structure substantially identical to SEQ ID NO. 77; or (2) is shown as SEQ ID NO: 77.

In some embodiments, the guide RNA is as set forth in any one of SEQ ID NOs 47, 48, 46, 36, 41, 56, 57, 62, 34-35, 37-40, 42-45, 49-55, 58-61, and 63-66.

In some embodiments, the RNA-guided DNA-binding domain comprises a Cas9 nickase.

In some embodiments, the amino acid sequence of the Cas9 nickase is set forth in SEQ ID NO. 84.

In some embodiments, the amino acid sequence of the adenylate deaminase domain is shown as SEQ ID NO. 82.

In some embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO. 80.

In yet another aspect, the present disclosure provides a vector of the present disclosure, wherein the vector is selected from the group consisting of a plasmid, an adeno-associated viral vector, and a lentiviral vector.

In some embodiments, the adeno-associated viral vector is AAV9.

C) Wherein the first vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding an N-terminal RNA-guided DNA binding domain; and

A polynucleotide encoding an N-terminal intein;

d) Wherein the second vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding a C-terminal intein; and

Polynucleotides encoding a C-terminal RNA-guided DNA binding domain;

In some embodiments, the amino acid sequence of the N-terminal RNA directed DNA binding domain is shown in SEQ ID NO. 93 and the amino acid sequence of the C-terminal RNA directed DNA binding domain is shown in SEQ ID NO. 101.

In some embodiments, the amino acid sequence of the N-terminal intein is shown as SEQ ID NO. 94 and the amino acid sequence of the C-terminal intein is shown as SEQ ID NO. 100.

In some embodiments, the amino acid sequence of the N-terminal intein is shown as SEQ ID NO. 109 and the amino acid sequence of the C-terminal intein is shown as SEQ ID NO. 115.

In some embodiments, the polynucleotide encoding the N-terminal RNA-guided DNA binding domain and/or the polynucleotide encoding the C-terminal RNA-guided DNA binding domain is operably linked to a ubiquitous promoter or a muscle-specific promoter.

In some embodiments, the ubiquitous promoter is set forth in SEQ ID NO. 90.

In some embodiments, the muscle-specific promoter is set forth in SEQ ID NO. 106.

In some embodiments, the vector system comprises one, two, or three copies of a polynucleotide of the present disclosure.

In some embodiments, the adenylate deaminase domain is shown as SEQ ID NO. 82.

In some embodiments, the first vector comprises a polynucleotide encoding an N-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID NO. 92 and the second vector comprises a polynucleotide encoding a C-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID NO. 99.

In some embodiments, the first vector comprises a polynucleotide encoding an N-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID NO. 108 and the second vector comprises a polynucleotide encoding a C-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID NO. 114.

In some embodiments, the first vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 97 and the second vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 104.

In some embodiments, the first vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 112 and the second vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 116.

In some embodiments, the vector system is selected from the group consisting of a plasmid vector system, an adeno-associated viral vector system, and a lentiviral vector system.

In some embodiments, the adeno-associated viral vector system is an AAV9 vector system.

In yet another aspect, the present disclosure provides a method of treating muscular dystrophy comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

In yet another aspect, the present disclosure provides a guide RNA of the present disclosure, a polynucleotide of the present disclosure, a system of the present disclosure, a vector of the present disclosure, or a use of a vector system of the present disclosure in the manufacture of a medicament for treating muscular dystrophy.

It should be understood that any headings or sub-headings of this disclosure are used for illustration purposes only and not for limitation, and that any embodiment in any section under any heading or sub-heading may be combined with any other embodiment in the same section or in a different section without departing from the scope of this disclosure.

Examples

The technical scheme of the invention is further illustrated and described below by specific embodiments in combination with the accompanying drawings.

The following examples operate following all applicable institutional and/or national guidelines for animal care and use. The experiments of the following examples were approved by the ethical committee of the first affiliated hospital of the university of focalization of medical science. Informed consent was obtained from all individual participants. All mouse experimental procedures were approved by the animal care and use research committee (IACUC) of the biological sciences limited of hui (Shanghai). Humanized muscular dystrophy mice were generated as previously described (Li,G.,et al.,Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy.J Clin Invest,2022:p.e162809). All mice were kept in a room at constant temperature (24-26 ℃) and humidity (40-60%) and light-dark cycled for 12h and fed with standard food.

The experimental methods and data processing methods according to the following examples are as follows:

Constructing a plasmid: the SpG-ABEmax plasmid (# 140002, addgene) was given away by David Liu laboratories and reconstituted to form the pU6-BpiI-EF 1. Alpha. -ABEmax-SpG-CBh-BFP vector (NG-ABEmax vector). The reporter vector contained a CMV driven mCherry cassette, ATG-deleted EGFP, and the human exon mutant sequences identified in DMD patients. sgRNA designed to target pathological nonsense mutations (SEQ ID NO:34-66; table 2) was synthesized as a DNA oligonucleotide, annealed and cloned into the NG-ABEmax vector to form a CRISPR targeting plasmid. The split SpCas9 variant in the Cbh_v5AAV-ABEN terminal (# 137177, addgene) and Cbh_v5AAV-ABEC terminal (# 137178, addgene) plasmids was replaced at the same split point by SpG Cas9-D10A (SEQ ID NO: 84), forming the ABEv system. The ABEv system was further constructed by replacing the CBh promoter with the SPc5-12 promoter and inserting an additional U6-sgRNA into the N-and C-terminal plasmids, respectively.

Cell culture and transfection: HEK293T cells from the American Type Culture Collection (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (# 11965092, gibco) supplemented with 10% fetal bovine serum (# 04-001-1ACS,Biological Industries) and 1% penicillin/streptomycin (# 15140122,Thermo Fisher Scientific). For sgRNA screening, HEK293T cells were seeded in the same amount on 12-well plates (# 3513, corning). The ABE expression plasmid and reporter plasmid were then co-transfected using polyethylenimine reagent (# 101000029, polyplus). After two days, the percentage of EGFP positive cells in the mCherry positive cell population was detected with Beckman CytoFlex flow cytometry.

Establishment and maintenance of human DMD iPSC: the fibroblasts were derived from a male patient carrying the mutation at exon 30 c.4174C > T of the DMD gene. The donor is free of other diseases including infectious diseases and cancers. Written informed consent for cell donation was obtained from the donor and its family. Fibroblasts were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin and passaged with trypsin-EDTA (# 25200056, gibco). Human DMD fibroblasts were reprogrammed to DMD ipscs using CytoTune-IPS SENDAI reprogramming kit (#a16517, thermo FISHER SCIENTIFIC). Human DMD ipscs were plated in matrigel (# 354277, corning) coated cell culture dishes and grown in ncTarget medium (# RP01020, nuwacell Biotechnologies) at 37 ℃, 5% co ₂. Ipscs were passaged to 80% confluence using hPSC dissociation buffer (#rp 01007, nuwacell Biotechnologies).

Human iPSC nuclear transfection and sorting: one hour prior to nuclear transfection, human DMD iPSC was treated with 10. Mu. MROCK inhibitor Y-27632 (# 10005583, cayman). Ipscs were dissociated into single cells using Accutase (# 7920,STEMCELL Technologies). After mixing 3×10 ⁶ cells with 6 μg of plasmid in nuclear transfection buffer (#rp 01005, nuwacell Biotechnologies), a nuclear transfection procedure was performed using Lonza2BNucleofector using procedure B016. After 48 hours, fluorescence positive cells were sorted by BDFACS Aria ^TM III sorter. For sgRNA screening, 5×10 ³ cells were collected and their lysates were amplified using different primer sets. To obtain single iPSC clones, cells were immediately seeded onto matrigel coated 100mm dishes (# 430167, corning) and maintained in ncTarget medium containing 10 μ M Y-27632. After 7 days, single cell colonies were picked and transferred to a 12-well plate. After genomic sequencing, the desired cells were expanded in ncTarget medium.

Cardiomyocyte differentiation of human iPSC: human DMD ipscs, with or without ABE treatment, were digested into single cells and seeded onto matrigel coated 12 well plates at a density of 5 x 10 ⁵ cells/well. When the confluence exceeds 95%, cardiomyocyte differentiation is induced using STEMdiff ^TM cardiomyocyte differentiation kit (# 05010,STEMCELL Technologies). The differentiation medium was replaced in a continuous sequence.

Immunofluorescent staining of human iPSC-derived cardiomyocytes: cardiomyocytes were fixed in 4% paraformaldehyde (#p0099, beyotime) for 10min and immersed in blocking buffer (#p0260, beyotime) for 20 min. After 3 washes, cardiomyocytes were stained with primary antibodies against dystrophin (#d8168, sigma) and cTnI (# 21652-1-AP, thermo FISHER SCIENTIFIC) overnight at 4 ℃. Cells were washed in PBS and then probed with secondary antibody for 2 hours at room temperature. The secondary antibodies are listed below: 488AffiniPure donkey anti-rabbit IgG (# 711-545-152,Jackson ImmunoResearch Labs) and 647AffiniPure donkey anti-mouse IgG (# 715-605-151,Jackson ImmunoResearch Labs). After DAPI staining, images were captured under immunofluorescence microscope (NikonC 2).

AAV administration: AAV9 particles are produced by PackGene Biotech (guangzhou, china). Briefly, pHelper, pRepCap and GOI plasmids were co-transfected into cells at a ratio of 2:1:1 when confluency was between 70-90%. After 72h AAV9 particles were purified using iodixanol density gradient centrifugation. For intramuscular injection, 3 week old DMD ^E30mut and DMD ^E23mut mice were anesthetized and injected with AAV9 formulation or the same volume of saline solution at a dose of 5 x 10 ¹¹ vg/leg/AAV into their tibialis anterior. For intraperitoneal infusion, P3 mice were administered either with AAV9 vector at a dose of 1X 10 ¹¹ vg per virus or with saline solution. After 4 weeks, the myocardium, diaphragm and tibialis anterior of the mice were isolated and then cut into small pieces for the following experiments.

Genomic DNA extraction and deep sequencing: human cells or mouse tissues were digested in lysis buffer containing proteinase K and their genomic DNA was extracted according to the manufacturer's instructions. Prior to Sanger or deep sequencing, DNA was amplified using Phantamax super fidelity DNA polymerase (#p505—d1, vazyme) and specific primer sets. To construct deep sequencing products, illumina flow cell binding sequences and barcodes were added to the 5 'and 3' ends of the primer sequences. The DNA product was purified with gel extraction kit (Omega) and analyzed by 150-bp paired-end reading Illumina NovaSeq platform (Genewiz co.ltd.). Depth sequencing data was first demultiplexed by Cutadapt (v.2.8) based on sample barcodes. The demultiplexed reads were then processed through CRISPResso to quantify editing efficiency, including indels, a-to-G conversions, or C-to-T conversions at each target site.

RNA isolation, cDNA synthesis, and sequencing: total mRNA was isolated from mouse tissue or human iPSC-derived cardiomyocytes using TRIzol reagent (# 15596-018, ambion) according to the manufacturer's instructions. mRNA was reverse transcribed into cDNA using HISCRIPT II One Step RT-PCR Kit (#P611-01, vazyme). Cdnas were amplified using Phanta max super-fidelity DNA polymerase and analyzed for conversion efficiency of a to G by Sanger and deep sequencing.

Western blot: mouse tissues were pulverized into powder and homogenized in RIPA lysis buffer (#p0013B, beyotime) containing protease inhibitor cocktail. Protein concentration was measured using BCA protein detection kit (# 23225,Thermo Fisher Scientific) and then adjusted to the same concentration. 10 μg of total protein was loaded into each lane of 3% -8% tris-acetate gel (#EA 03752BOX, invitrogen) and electrophoresed for 1h. The proteins were transferred to PVDF membranes under humid conditions. Subsequently, the membranes were blocked in 5% skim milk/TBST buffer and then incubated with primary antibodies overnight at 4 ℃. The primary antibodies were as follows: dystrophin (# D8168, sigma) and focal adhesion protein (# 13901S,Cell Signaling Technology). After 3 washes, the membrane was probed with HRP conjugated secondary antibody for 1h at room temperature. After incubation with chemiluminescent substrate (#wp 20005, invitrogen), the membrane was observed by Image Lab ^TM.2 software.

Immunohistochemical analysis: for immunofluorescent staining, mouse tissues were embedded in o.c.t compounds and then snap frozen in liquid nitrogen cooled isopentane. They were cut into 10 μm sections with a microtome and transferred to a slide. Sections were fixed in 4% paraformaldehyde for 2h and permeabilized with 0.4% Triton-X/PBS for 30 min at room temperature. After blocking with 10% goat serum/PBS, sections were probed overnight at 4 ℃ with a primary antibody directed against dystrophin (#ab 15277, abcam). After 3 washes, sections were stained with secondary antibody and DAPI for 3h at room temperature. After washing in PBS, the slides were sealed with permanent synthetic mounting medium. All pictures were observed and captured under an Olympus FV3000 inverted microscope.

Rotating bar and rodent treadmill test: mice were trained daily one week prior to the experiment. Three mice were placed simultaneously on a rotating rod (Ugo basic inc.) with an acceleration rate of from 4rpm to 40rpm in 30 seconds. When the mice drop onto the lever, the test is stopped and the time is recorded. Each mouse was tested five times and the average of these five times was taken for further comparison. Mice were trained daily one week prior to the experiment. Three mice were placed simultaneously on a rodent treadmill (Shanghai TOW Intelligent Technology co., ltd, AT-5 MR) with an acceleration rate of from 0 to 15m/s in 30 seconds, recording the running time before the first fall.

Echocardiographic analysis

Echocardiographic analysis evaluation of WT and DMD ^E30mut mice was performed at week 6 post AAV dosing using a transthoracic echocardiograph (Vevo 3100,Visual Sonics) with a 25MHz sensor following the procedure outlined in reference Pesce.et al(2014).Characterization of a murine model of cardiorenal syndrome type 1by high-resolution Doppler sonography.Journal of ultrasound,18(3),229–235. Briefly, mice were anesthetized with isoflurane, placed on a platform with electrocardiogram leads attached, and their fur removed. The pre-warmed ultrasound gel is applied to the chest area and the ultrasound probe is placed in a position that ensures clear visualization of the left ventricle and atrium in B-mode, with the outflow tract being horizontal on the screen. Measurements of the Left Ventricular Posterior Wall (LVPW) and the Left Ventricular Internal Dimension (LVID) were taken in M mode, and the average of three cardiac cycles was taken. Echocardiographic data collection and analysis were performed without knowledge of the mouse genotype or treatment.

Statistical analysis: quantitative data were from at least three independent experiments. Statistical significance of group differences was calculated by unpaired Student's t test between two or more groups. P values less than 0.05 were considered significant.

Example 1: nonsense mutations were identified from DMD patient populations for in vitro adenine base editing

To identify DMD-associated nonsense mutations as potential targets for adenine base editing, the present example performed whole exome sequencing of human peripheral leukocytes obtained from DMD patient populations affiliated with the first hospital of the university of medical sciences, fowly. These patients develop clinical muscular dystrophy symptoms such as abnormal gait and muscle hypertrophy. Histopathological analysis of biceps samples confirmed the absence of dystrophin in these patients (fig. 1B), indicating the pathological cause of the muscular dystrophy phenotype. Subsequent whole exome sequencing and bioinformatic analysis identified nonsense mutations listed in table 1 in the patient population. These nonsense mutations are randomly distributed in exons of the dystrophin gene and can induce premature translation termination of the dystrophin transcript. In these nonsense mutations, 52% (n=14) resulted in TGA stop codons, 30% (n=8) resulted in TAG stop codons, 18% (n=5) resulted in TAA stop codons (fig. 1A).

TABLE 1 identification of DMD-related nonsense mutations from DMD patients

To test the possibility of editing adenine in these stop codons by ABE, 33 guide sequences (guide sequences) were screened, which were individually inserted into reporter plasmids for 12 different nonsense mutation sites in the dystrophin gene (see table 2) to block expression of fluorescent proteins (fig. 1C). The reporter plasmid containing the nonsense mutation was co-transfected into HEK293T cells with a base editing plasmid (expression plasmid) expressing SpG-ABE (having a broader PAM recognition capacity than wild-type SpCas 9) and the corresponding sgrnas to be screened, and then the efficiency of EGFP fluorescence restored by each sgRNA-guided SpG-ABE was quantified relative to upstream mCherry reporter lacking the premature stop codon. Expression of SpG-ABE with non-specific sgRNA (negative control) was found to fail to restore the detectable EGFP signal (FIGS. 1D and 5A), indicating the stringency of the reporting system. In contrast, it was found that the sgrnas of the present invention can guide correction of nonsense mutations to activate powerful EGFP expression (fig. 1D-1E; fig. 5A-5B), even up to 80% restoration of EGFP expression. These results indicate that a wide variety of nonsense mutations in the human dystrophin gene can be corrected by adenine base editing.

The architecture of the expression plasmid is shown in FIG. 1C, which contains, from 5 'to 3': u6 promoter (SEQ ID NO: 76), sgRNA coding sequence, EF 1. Alpha. Promoter (SEQ ID NO: 78), kozak sequence (gccacc), spG ABE coding sequence (SEQ ID NO: 79), SV40 poly A signal coding sequence (SEQ ID NO: 86), CBh promoter (SEQ ID NO: 97), BFP coding sequence (SEQ ID NO: 88), bGH poly A signal coding sequence (SEQ ID NO: 75). Wherein the sgRNA coding sequence encodes the sgRNA shown in one of SEQ ID NOS: 34-66, comprising a scaffold sequence capable of complexing with a Cas9 protein (SEQ ID NO: 77), and a guide sequence capable of hybridizing with a human DMD target sequence (one of SEQ ID NOS: 1-33) comprising an insertion sequence on a reporter plasmid (SEQ ID NO: 72) designed for each nonsense mutation of the sgRNA to be screened. Wherein the ABE coding sequence (SEQ ID NO: 68) encodes the SpG-ABE editor (SEQ ID NO: 80). The SpG-ABE editor (SEQ ID NO: 80) comprises, from N-terminal to C-terminal: the initiation codon ATG encodes methionine (Met/M), bpNLS1 (SEQ ID NO: 81), adenine deaminase TadA e (SEQ ID NO: 82), linker (SEQ ID NO: 83), spG Cas9-D10A nickase (SEQ ID NO: 84), linker (SGGS), bpNLS2 (SEQ ID NO: 85).

The architecture of the reporter plasmid is shown in FIG. 1C, which contains, from 5 'to 3': CMV enhancer (SEQ ID NO: 67), CMV promoter (SEQ ID NO: 68), kozak sequence (gccacc), mCherry coding sequence (SEQ ID NO: 70), T2A coding sequence (SEQ ID NO: 71), insert sequence, P2A coding sequence (SEQ ID NO: 73), EGFP coding sequence (SEQ ID NO: 74), bGH poly A signal coding sequence (SEQ ID NO: 75). Wherein the insertion sequence (e.g., SEQ ID NO: 72) comprises a human DMD target sequence. The human DMD target sequences may be exchanged according to the DMD exon nonsense mutations targeted to screen for corresponding sgrnas for each DMD exon nonsense mutation.

It should be noted that although SEQ ID NOS.1-33 are labeled as RNA in the electronic sequence Listing, they represent both the protospacer sequence as a DNA sequence and the guide sequence as an RNA sequence, where t represents u when it represents the guide sequence.

Example 2: spG-ABE can restore dystrophin expression in human DMD cardiomyocytes by correcting nonsense mutations

To further verify the results obtained in the fluorescence reporting model in example 1, patient-derived induced pluripotent stem cells (ipscs) are important in vitro models to test the efficacy of SpG-ABE in correcting DMD mutations and restoring dystrophin expression. To investigate whether SpG-ABE could restore dystrophin expression in human DMD cells, iPSC was generated from DMD patients with the c.4174C > T mutation (FIG. 2; FIGS. 6A-6D). The ipscs were corrected using the SpG-ABE system and monoclonal cell lines with corrected mutation sites were collected. The selected clones were then differentiated into cardiomyocytes and evaluated for dystrophin expression. This example uses SpG-ABE and E30-sgRNA-1 to E30-sgRNA-5 (guide sequences SEQ ID NO:11-15, respectively) to nuclear transfect DMD iPSC carrying the c.4174C > T mutation to induce base editing. Deep sequencing results showed that SpG-ABE and hEx sgRNA-4 (guide sequence SEQ ID NO: 14) can provide an average mid-target (A15 site) editing rate of 58.5+0.707% with NO detectable bystanders (e.g., A7, A8, A18, A19 sites) (off-target) editing (FIGS. 2B and 2C). hEx30 SgRNA-2 (guide sequence SEQ ID NO: 12), sgRNA-3 (guide sequence SEQ ID NO: 13) or SgRNA-5 (guide sequence SEQ ID NO: 15) induced SpG-ABE with an on-target editing efficiency of about 20% -30% (FIG. 6E). Corrected individual iPSC colonies were selected for expansion.

Immunofluorescent staining for dystrophin showed high expression of dystrophin in these corrected cardiomyocytes, at levels comparable to normal cardiomyocytes derived from H9 cells (fig. 2D). Both genomic and cDNA sequencing confirmed that the c.4174C > T mutation in cardiomyocytes from ABE-edited DMD iPSC was corrected (FIGS. 6F and 6G). Furthermore, no significant genomic modifications above background were detected in any of the first 9 predicted off-target sites (fig. 6H), supporting that the SpG-ABE system of the invention exhibits a high degree of specificity in editing the c.4174c > T nonsense mutation in the dystrophin gene.

For in vivo administration, the SpG-ABE was split into two separate fragments using the trans-spliced intein from Nostoc punctiforme (Npu), each separately packaged in a single AAV vector (fig. 6I). Due to the preferential tendency of AAV9 to skeletal and cardiac muscle, the double adeno-associated virus 9 (AAV 9) system was selected to deliver the SpG-ABE component. For ABEv1 construct, the framework established by Levy et al (Levy,J.M.,et al(2020).Cytosine and adenine base editing of the brain,liver,retina,heart and skeletal muscle of mice via adeno-associated viruses.Nature biomedical engineering,4(1),97–110) was used, but SpG Cas9-D10A was used instead of nCas (D10A). ABEv1 (FIG. 6I) carrying the ubiquitous promoter Cbh was used to test the feasibility of mutation editing in patient-derived iPSCs after cleavage of the ABE into two parts with the intein system, while ABEv (FIG. 6I) carrying the muscle-specific promoter Spc5.12 was designed primarily for in vivo ABE muscle-specific expression to avoid ABE expression in non-muscle cells. In addition, additional sgRNA expression units were added to the ABEv design (3 total; FIG. 6I). ABEv1 and ABEv were each nuclear transfected into human DMD iPSC, yielding a mid-target edit frequency of 75.7% ± 3.8% and 30.3% ± 13.0% at target adenine a15, respectively (fig. 6J), indicating that both split SpG-ABE systems could correct the c.4174c > T mutation, whereas the ubiquitous (ubiquitous) CBh promoter provided higher expression efficiency in undifferentiated patient ipscs.

ABEv1 are delivered by the dual AAV vector system shown in fig. 2E, expressing N-terminal ABEv1 and C-terminal ABEv1, respectively.

AAV vectors expressing N-terminus ABEv1 comprise, from 5 'to 3': 5'ITR (SEQ ID NO: 89), CBh promoter (SEQ ID NO: 90), kozak sequence (gccacc), N-terminal ABEv coding sequence (SEQ ID NO: 91), bGH poly A signal coding sequence (SEQ ID NO: 95), 3' ITR (SEQ ID NO: 96). The AAV vector comprises 5'ITR to 3' ITR sequences as shown in SEQ ID NO. 97. Wherein the N-terminal ABEv coding sequence (SEQ ID NO: 91) encodes N-terminal ABEv1 (SEQ ID NO: 92) comprising, from N-terminal to C-terminal: the initiation codon ATG encodes the amino acids Met (M), bpNLS1 (SEQ ID NO: 81), tadA e (SEQ ID NO: 82), linker (SEQ ID NO: 83), N-terminal SpG Cas9-D10A (SEQ ID NO: 93), N-terminal Npu intein (SEQ ID NO: 94), linker (SGGS), bpNLS2 (SEQ ID NO: 85).

AAV vectors expressing C-terminal ABEv1 comprise, from 5 'to 3': 5'ITR (SEQ ID NO: 89), CBh promoter (SEQ ID NO: 90), kozak sequence (gccacc), C-terminal ABEv coding sequence (SEQ ID NO: 98), bGH poly A signal coding sequence (SEQ ID NO: 75), reverse complement of sgRNA coding sequence (SEQ ID NO: 102), reverse complement of U6 promoter (SEQ ID NO: 103), 3' ITR (SEQ ID NO: 96). The AAV vector comprises 5'ITR to 3' ITR sequences as shown in SEQ ID NO. 104. Wherein the C-terminal ABEv coding sequence (SEQ ID NO: 98) encodes C-terminal ABEv1 (SEQ ID NO: 99) comprising, from N-terminal to C-terminal: the initiation codon ATG encodes the amino acids Met (M), bpNLS1 (SEQ ID NO: 81), the C-terminal Npu intein (SEQ ID NO: 100), the C-terminal SpG Cas9-D10A (SEQ ID NO: 101), the linker (SGGS), bpNLS2 (SEQ ID NO: 85). Wherein the sgRNA coding sequence encodes E30 sgRNA-4 (SEQ ID NO: 47) and can be replaced with other sgRNAs as desired.

ABEv2 are delivered by the dual AAV vector system shown in fig. 2E, expressing N-terminal ABEv2 and C-terminal ABEv2, respectively.

AAV vectors expressing N-terminus ABEv2 comprise, from 5 'to 3': 5'ITR (SEQ ID NO: 105), U6 promoter (SEQ ID NO: 76), sgRNA coding sequence, spc5.12 promoter (SEQ ID NO: 106), kozak sequence (gccacc), N-terminal ABEv coding sequence (SEQ ID NO: 107), CW3SL (SEQ ID NO: 110), 3' ITR (SEQ ID NO: 111). The AAV vector comprises 5'ITR to 3' ITR sequences as shown in SEQ ID NO. 112. Wherein the N-terminal ABEv coding sequence (SEQ ID NO: 107) encodes N-terminal ABEv2 (SEQ ID NO: 108) comprising, from N-terminal to C-terminal: the initiation codon ATG encodes amino acids Met (M), bpNLS1 (SEQ ID NO: 81), tadA e (SEQ ID NO: 82), a linker (SEQ ID NO: 83), N-terminal SpG Cas9-D10A (SEQ ID NO: 93), N-terminal Rma intein (SEQ ID NO: 109).

AAV vectors expressing C-terminus ABEv2 comprise, from 5 'to 3': 5'ITR (SEQ ID NO: 105), U6 promoter (SEQ ID NO: 76), sgRNA coding sequence, spc5.12 promoter (SEQ ID NO: 106), kozak sequence (gccacc), C-terminal ABEv2 coding sequence (SEQ ID NO: 113), CW3SL (SEQ ID NO: 110), 3' ITR (SEQ ID NO: 111). The AAV vector comprises 5'ITR to 3' ITR sequences as shown in SEQ ID NO. 116. Wherein the C-terminal ABEv coding sequence (SEQ ID NO: 113) encodes C-terminal ABEv2 (SEQ ID NO: 114) comprising, from N-terminal to C-terminal: the initiation codon ATG encodes the amino acid Met (M), the C-terminal Rma intein (SEQ ID NO: 115), the C-terminal SpG Cas9-D10A (SEQ ID NO: 101), the linker (SGGS), bpNLS2 (SEQ ID NO: 85). Wherein the sgRNA coding sequences each encode E30 sgRNA-4 (SEQ ID NO: 47) and can be replaced with other sgRNAs as desired.

The experimental results of this example collectively demonstrate that genome editing using adenine base editing can restore dystrophin expression by correcting nonsense mutations in human DMD cells.

Example 3: local delivery of SpG-ABE restores dystrophin expression in humanized DMD mice

Based on the in vitro results of the previous examples, this example next investigated whether SpG-ABE can directly correct in vivo nonsense mutations using a humanized DMD model mouse (DMD ^E30mut mouse) generated by introducing exon 30 of the human dystrophin gene containing a c.4174c > T nonsense mutation into a mouse dystrophin homolog.

For in vivo evaluation of the split SpG-ABE system, this example intramuscular injected 5x 10 ¹¹ viral genome (vg) doses of each AAV9 vector into the right Tibialis Anterior (TA) of 3 week old humanized dystrophy mice, while the left tibialis anterior injected with saline as a control (fig. 3A and 3B). Four weeks after local AAV injection, skeletal muscle of the SpG-ABE or saline treated leg was collected for analysis. In the humanized DMD ^E30mut mice given a single administration of SpG-ABE and hEx sgRNA-4, a clear a to G transition was observed at a15, but not in mice treated with saline (fig. 3C). Average efficiencies of mid-target genome editing for ABEv1 and ABEv2 treatment groups were 9.7±0.9% and 12.7±1.9%, respectively (fig. 3C). Dmd cDNA sequencing showed that transcript A to G editing rate of ABEv1 was 45.7.+ -. 1.2% and transcript A to G editing rate of ABEv was 68.3.+ -. 10.9% (FIG. 3D). The difference in a to G editing rates at the genomic and cDNA levels may be due to the high abundance of dystrophin transcripts in skeletal muscle. Thus, tibialis anterior with ABEv1 infusion showed recovery of dystrophin expression in 61.0.+ -. 4.1% of skeletal fibres, whereas ABEv treatment resulted in 72.4.+ -. 5.4% recovery (FIGS. 3E and 3G; FIG. 7). Western blot analysis showed that dystrophin levels in ABEv1 or ABEv treated DMD ^E30mut mice were restored to 22.2±1.269% and 59.3±10.1% of wild type controls, respectively (fig. 3F and 3H). Compared to in vitro results showing ABEv1 to be better than ABEv2 in patient ipscs, DMD ^E30mut mice treated with ABEv2 showed almost twice higher dystrophin recovery compared to DMD ^E30mut mice treated with ABEv1 (fig. 3F and 3H).

This example next tests whether the split SpG-ABE system can also correct other DMD nonsense mutations identified from the DMD population. For this, this example constructed another humanized DMD mouse model carrying the c.2977c > T, p.gln993 mutation in human dystrophin exon 23, designated DMD ^E23mut mouse (fig. 8A). Similar to topical AAV treatment of DMD ^E30mut mice, AAV9 vectors carrying ABEv or ABEv and E23 sgRNA-1 targeting the a13 site in exon 23 were injected into skeletal muscle of humanized DMD ^E23mut mice. Three weeks after intramuscular AAV delivery in adult DMD ^E23mut mice, ABEv and ABEv2 were observed with about 1.5% and about 1% of mid-target genomic modifications at a13, respectively (fig. 8B-8D), which also resulted in about 0.5% and about 1% of bystander (off-target) editing events at a18 (fig. 8D). ABEv1 and ABEv had average mid-target a to G substitution rates of 13% and 17%, respectively, at a13 in the dystrophin transcript and bystander editing rates of 3% and 14%, respectively, at a18 (fig. 8E). Immunofluorescent staining showed that dystrophin was present in 26.4±1.5% of the myofibers in the ABEv treated tibialis anterior, while dystrophin was present in 53.6±5.8% of the myofibers in the ABEv2 treated tibialis anterior (fig. 8F-8G and fig. 9). Western blots showed that ABEv and ABEv restored dystrophin to 18.2±5.7% and 55.9±2.8% of wild type mice, respectively (fig. 8H-8I). Overall, these data indicate that the SpG-ABE system with specific sgrnas for pathological nonsense mutations in the human dystrophin gene can strongly restore dystrophin expression in humanized DMD mouse models.

Example 4: systemic administration of SpG-ABE improves muscle function in humanized DMD mice by recovering dystrophin

Given that SpG-ABE and hEx sgRNA-4 restored dystrophin efficiently in tibialis anterior (FIGS. 3G and 3H), this example evaluates the effect of systemic SpG-ABE administration in neonatal DMD ^E30mut mice. After ABEv or ABEv 24 weeks of intraperitoneal AAV delivery, the myocardium (HE), diaphragm (DI) and Tibialis Anterior (TA) tissues of AAV-infused or saline-infused mice were harvested (fig. 4A). Depth sequence analysis showed that systemic ABEv1 treatment resulted in a conversion of a to G in the myocardial, diaphragmatic and tibialis anterior tissues of 37.2±1.4%, 13.2±4.8% and 7.7±1.9%, respectively (fig. 4B). In contrast, ABEv2 treatment resulted in 20.3±4.3%, 4.7±1.9% and 12.0±2.6% of a to G conversion in the myocardium, diaphragm and tibialis anterior tissues, respectively, of DMD ^E30mut mice (fig. 4B). Notably, the frequency of genome editing was significantly higher in the myocardium than in the diaphragm and tibialis anterior (fig. 4B). RT-PCR and cDNA sequencing showed that ABEv1 induced 89.1.+ -. 1.7%, 23.1.+ -. 12.3% and 19.3.+ -. 3.2% of the base substitutions at A15 in endogenous dystrophin transcripts in myocardium, diaphragm and tibialis anterior respectively, while ABEv2 induced 84.3.+ -. 1.8%, 9.1.+ -. 5.5% and 24.7.+ -. 3.3%, respectively (FIG. 4C). Consistent with genomic analysis (fig. 4B), ABEv1 and ABEv2 corrected about 90% of the a15 sites in the myocardial transcripts, about 4-fold higher than in the diaphragm and tibialis anterior (fig. 4C).

Immunofluorescent staining for dystrophin further showed that systemic ABEv1 and ABEv2 delivery could restore dystrophin expression in 95.9±0.5% or 94.0±0.9% of the myofibers in cardiac samples, and dystrophin expression in 44.3±1.0% or 44.6++11.9% of skeletal muscle fibers in tibialis anterior muscle, detected in 71.6±21.3% or about 26.3±19.6% of tibialis anterior muscle fibers in ABEv1 and ABEv groups (fig. 4D-4E and fig. 10). Western blot analysis further demonstrated that effective recovery of full-length dystrophin in myocardium and tibialis anterior after systemic administration ABEv and ABEv2, ABEv treatment can restore high dystrophin expression in tibialis anterior (fig. 4F), consistent with immunofluorescent staining assays (fig. 4D-4E and fig. 10). In addition, H & E histological staining analysis and Creatine Kinase (CK) analysis showed that ABE treatment reduced muscle fiber and blood creatine kinase activity (fig. 11).

To examine muscle function after systemic SpG-ABE treatment, rotary bar running and rodent treadmill running tests were performed on DMD ^E30mut mice receiving saline or SpG-ABE treatment. DMD ^E30mut mice infused with saline alone showed significantly reduced running activity compared to age-matched wild-type mice (fig. 4G-4H), but DMD ^E30mut mice treated with both ABEv1 and ABEv2 showed significant improvement in locomotor performance (fig. 4G-4H), further supporting systemic recovery of dystrophin and demonstrating that systemic ABE treatment can alleviate muscle loss due to nonsense mutations in dystrophin. Together, these findings indicate that systemic administration of ABE can broadly restore dystrophin levels and improve muscle function in humanized dystrophy mice. Next, cardiac function was assessed by echocardiography on untreated and treated DMD mice at week 6 post AAV dosing. No differences in multiple electrocardiographic indices were detected between untreated and treated DMD mice (fig. 12).

Claims

1. A guide RNA comprising (1) a scaffold sequence capable of complexing with an RNA-guided DNA binding protein (e.g., a Cas9 protein, such as Cas9 nickase); and (2) a guide sequence capable of hybridizing to a target sequence on a human dystrophin gene that is 100% reverse complementary to a protospacer sequence on the opposite strand of the human dystrophin gene, the protospacer sequence comprising a nonsense mutation selected from the group consisting of:

E30,c.4174C>T,p.Gln1392*；E23,c.2977C>T,p.Gln993*；E24,c.3189G>A,p.Trp1063*；

E44,c.6292C>T,p.Arg2098*；E55,c.8038C>T,p.Arg2680*；E6,c.433C＞T,p.Arg145*；

E13,c.1510C>T,p.Gln504*；E19,c.2302C>T,p.Arg1577*；E20,c.2407C>T,p.Gln803*；

E20,c.2527G>T,p.Glu843*；E21,c.2695G>T,p.Glu899*；E21,c.2776C>T,p.Gln926*；

E25,c.3337C>T,p.Gln1113*；E25,c.3414G>A,p.Trp1138*；E30,c.4117C>T,p.Gln1373*；

E34,c.4729C>T，p.Arg768*；E35,c.4996C>T,p.Arg1666*；E36,c.5125A>T,Lys1709*；

E37,c.5247C>A,p.Cys1749*；E41,c.5899C>T,p.Arg1967*；E43,c.6188T>A,p.Leu2063*；

E54, c.8009A > G, p.Trp2670; e56, c.8230G > T, p.Glu2744; e59, c.8713C > T, p.Arg2950; and E70, c.10141c > T, p.arg3381.

2. The guide RNA of claim 1, wherein the guide sequence is as set forth in any one of SEQ ID NOs 14, 15, 13, 3, 8, 23, 24, 29, 1-2, 4-7, 9-12, 16-22, 25-28 and 30-33 or is identical to SEQ ID NOs 14, 14,

15. Any of 13, 3, 8, 23, 24, 29, 1-2, 4-7, 9-12, 16-22, 25-28, and 30-33 differ by no more than 1,2,3, 4, or 5 nucleotides.

3. The guide RNA of claim 1 or 2, wherein the scaffold sequence (1) has substantially the same secondary structure as SEQ ID No. 77; or (2) is shown as SEQ ID NO: 77.

4. The guide RNA of claim 1 or 2, wherein the guide RNA is as set forth in SEQ ID NOs 47, 48, 46,

36. 41, 56, 57, 62, 34-35, 37-40, 42-45, 49-55, 58-61, And 63-66.

5. A polynucleotide encoding the guide RNA of any one of claims 1-4.

6. A system comprising (1) the guide RNA of any one of claims 1-4 or a polynucleotide encoding the guide RNA; and (2) a fusion protein comprising an RNA-guided DNA binding domain and an adenylate deaminase domain or a polynucleotide encoding said fusion protein.

7. The system of claim 6, wherein the RNA-guided DNA-binding domain comprises a Cas9 nickase.

8. The system of claim 7, wherein the amino acid sequence of Cas9 nickase is set forth as SEQ ID No. 84.

9. The system of claim 6, wherein the amino acid sequence of the adenylate deaminase domain is depicted as SEQ ID No. 82.

10. The system of any one of claims 6-9, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID No. 80.

11. A vector comprising the polynucleotide of claim 5.

12. The vector of claim 11, wherein the vector is selected from the group consisting of a plasmid, an adeno-associated viral vector, and a lentiviral vector.

13. The vector of claim 12, wherein the adeno-associated viral vector is AAV9.

14. A carrier system comprising a first carrier and a second carrier,

A) Wherein the first vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding an N-terminal RNA-guided DNA binding domain; and

A polynucleotide encoding an N-terminal intein;

b) Wherein the second vector comprises in the 5 'to 3' direction:

i. a polynucleotide encoding a C-terminal intein; and

Polynucleotides encoding a C-terminal RNA-guided DNA binding domain;

Wherein the vector system further comprises the polynucleotide of claim 5.

15. The vector system of claim 14, wherein the RNA-guided DNA-binding domain comprises a Cas9 nickase.

16. The vector system of claim 15, wherein the amino acid sequence of Cas9 nickase is set forth as SEQ ID No. 84.

17. The vector system of claim 14, wherein the amino acid sequence of the N-terminal RNA-guided DNA binding domain is shown in SEQ ID No. 93 and the amino acid sequence of the C-terminal RNA-guided DNA binding domain is shown in SEQ ID No. 101.

18. The vector system of claim 14, wherein (1) the amino acid sequence of the N-terminal intein is shown as SEQ ID No. 94 and the amino acid sequence of the C-terminal intein is shown as SEQ ID No. 100; or (2) the amino acid sequence of the N-terminal intein is shown as SEQ ID NO. 109, and the amino acid sequence of the C-terminal intein is shown as SEQ ID NO. 115.

19. The vector system of any one of claims 14-18, wherein the polynucleotide encoding an N-terminal RNA-guided DNA binding domain and/or the polynucleotide encoding a C-terminal RNA-guided DNA binding domain is operably linked to a ubiquitous promoter or a muscle-specific promoter.

20. The vector system of claim 19, wherein the ubiquitous promoter is set forth in SEQ ID No. 90.

21. The vector system of claim 19, wherein the muscle-specific promoter is set forth in SEQ ID No. 106.

22. The vector system of any one of claims 14-18, wherein the vector system comprises one, two, or three copies of the polynucleotide of claim 5.

23. The vector system of any one of claims 14-18, wherein the adenylate deaminase domain is depicted as SEQ ID No. 82.

24. The vector system of any one of claims 14-18, wherein (1) the first vector comprises a polynucleotide encoding an N-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID No. 92 and the second vector comprises a polynucleotide encoding a C-terminal adenylate base editor having an amino acid sequence as set forth in SEQ ID No. 99; or (2) the first vector comprises a polynucleotide encoding an N-terminal adenylate base editor having an amino acid sequence as shown in SEQ ID NO. 108, and the second vector comprises a polynucleotide encoding a C-terminal adenylate base editor having an amino acid sequence as shown in SEQ ID NO. 114.

25. The vector system of any one of claims 14-18, wherein (1) the first vector comprises a 5'itr to 3' itr sequence as set forth in SEQ ID No. 97 and the second vector comprises a 5'itr to 3' itr sequence as set forth in SEQ ID No. 104; or (2) the first vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 112 and the second vector comprises a 5'ITR to 3' ITR sequence as set forth in SEQ ID NO. 116.

26. The vector system of any one of claims 14-18, wherein the vector system is selected from the group consisting of a plasmid vector system, an adeno-associated viral vector system, and a lentiviral vector system.

27. The vector system of claim 26, wherein the adeno-associated viral vector system is an AAV9 vector system.

28. A pharmaceutical composition comprising the guide RNA of any one of claims 1-4, the polynucleotide of claim 5, the system of any one of claims 6-10, the vector of any one of claims 11-13, or the vector system of any one of claims 14-27.

29. Use of the guide RNA of any one of claims 1-4, the polynucleotide of claim 5, the system of any one of claims 6-10, the vector of any one of claims 11-13, or the vector system of any one of claims 14-27 in the manufacture of a medicament for the treatment of duchenne muscular dystrophy.

30. A method of editing target DNA comprising contacting the target DNA with the guide RNA of any one of claims 1-4, the polynucleotide of claim 5, the system of any one of claims 6-10, the vector of any one of claims 11-13, or the vector system of any one of claims 14-27, whereby the target DNA is edited.

31. A cell comprising target DNA edited by the method of claim 31.