Therapeutic applications of cpf1-based genome editing

Abstract

To provide therapeutic applications of CRISPR / Cpf1-based genome editing.SOLUTION: The invention provides a Cpf1 guide RNA (gRNA) that targets a dystrophin gene and comprises a polynucleotide sequence corresponding to specific sequences, or a complement thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Patent Application No. 62 / 363,888, filed on July 19, 2016, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, the entire content of which is incorporated herein by reference. The above - mentioned ASCII copy was created on July 19, 2017, named 028193 - 9250 - WO00 Sequence Listing.txt, and has a size of 46,056 bytes.

[0003] Statement of Government Rights This invention was made with government support under Federal Grant numbers: AR069085 and MD140071, awarded by NIH and Army / MRMC, respectively. The United States government has certain rights in this invention.

[0004] This disclosure relates to the field of modification of gene expression, genomic manipulation of genes, and genomic modification using systems based on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR / Cpf1) from the genera Prevotella and Francisella 1, and viral delivery systems.

Background Art

[0005] RNA-guided nucleases have been engineered for genome modification in human cells, and as such, there are CRISPR / Cpf1 systems derived from Streptococcus pyogenes and Staphylococcus aureus. A variety of microorganisms have been shown to possess DNA editing or RNA editing systems. Cas9 from S. pyogenes and S. aureus causes blunt-ended double-strand breaks (DSBs) in genomic DNA, which are repaired by non-homologous end joining (NHEJ), leaving small insertions and deletions (indels) at the repair site, or by homologous recombination repair in the presence of a template. These indels can be used to knock out genes, remove splice acceptors, or cleave gene regulatory elements.

[0006] Inherited genetic diseases have a devastating impact on children in the United States. These diseases currently have no cure and can only be managed by attempts to alleviate symptoms. For decades, the field of gene therapy has promised a cure for these diseases. However, this approach has been limited by technical hurdles related to the safe and efficient delivery of therapeutic genes to cells and patients. Duchenne muscular dystrophy (DMD) is a fatal genetic disease characterized clinically by muscle fatigue, loss of walking, and typically death by the age of 30 years, due to the loss of functional dystrophin. DMD is the result of genetic or spontaneous mutations in the dystrophin gene. Most mutations that cause DMD are the result of exon deletions, which shift the translational reading frame out of frame.

[0007] Dystrophin is an important component of a protein complex involved in regulating the integrity and function of muscle cells. Patients with DMD generally lose the ability to support themselves physically in childhood, become increasingly weak in their teens, and die in their twenties. Current experimental gene therapy strategies for DMD require repeated administration of transient gene delivery vehicles or rely on the permanent integration of foreign gene material into genomic DNA. Both of these methods have serious safety concerns. Furthermore, these strategies are limited by their inability to deliver the large and complex dystrophin gene sequence. There is a need for more accurate and efficient gene editing tools to correct or treat patients with mutations in the dystrophin gene.

Summary of the Invention

Means for Solving the Problems

[0008] The present invention targets the dystrophin gene and is directed to a Cpf1 guide RNA (gRNA) comprising a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0009] The present invention is directed to a DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA as described herein.

[0010] The present invention is directed to a DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA, wherein the first Cpf1 gRNA and the second Cpf1 gRNA each comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and wherein the first Cpf1 gRNA and the second Cpf1 gRNA target the dystrophin gene.

[0011] The present invention is directed to an isolated polynucleotide comprising a polynucleotide sequence encoding the Cpf1 gRNA described above or the DNA targeting composition described above.

[0012] The present invention is directed to a vector comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, or the isolated polynucleotide described above.

[0013] The present invention is directed to a vector encoding (a) a first Cpf1 guide RNA (gRNA), (b) a second Cpf1 gRNA, and (c) at least one Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences.

[0014] The present invention is directed to a cell comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, the isolated polynucleotide described above, or the vector described above.

[0015] The present invention is directed to a kit comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, the isolated polynucleotide described above, the vector described above, or the cell described above.

[0016] The present invention is directed to a composition for deleting a segment containing exon 51 in the dystrophin gene, the composition comprising: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36 to 64, 71 to 119, or a complement thereof, the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first vector and the second vector are configured to form first and second double-strand breaks in the first intron and the second intron adjacent to exon 51 of the human DMD gene, whereby a segment containing exon 51 in the dystrophin gene is deleted.

[0017] The present invention is directed to a cell comprising the composition described above.

[0018] The present invention is directed to a modified adeno-associated virus vector for genome editing that edits a mutant dystrophin gene in a subject, the modified adeno-associated virus vector comprising a first polynucleotide sequence encoding the Cpf1 gRNA described above and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0019] The present invention is directed to a method of modifying a mutant dystrophin gene in a cell, the method comprising administering to the cell the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, the isolated polynucleotide described above, the vector described above, the composition described above, or the modified adeno-associated virus vector described above.

[0020] The present invention is directed to a method of genome editing a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, the isolated polynucleotide described above, the vector described above, the composition described above, or the modified adeno-associated virus vector described above.

[0021] The present invention is directed to a method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, the isolated polynucleotide described above, the vector described above, the composition described above, or the modified adeno-associated virus vector described above.

[0022] The present invention relates to a method for correcting a mutant dystrophin gene in a cell, the method comprising administering to the cell: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the vectors are each configured to form first and second double-strand breaks in the first and second introns adjacent to exon 51 of the human dystrophin gene, whereby a segment containing exon 51 in the dystrophin gene is deleted, thereby correcting the mutant dystrophin gene in the cell.

[0023] The present invention is directed to a method of treating a subject having a mutant dystrophin gene, the method comprising administering to the subject: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first vector and the second vector are configured to form first and second double-strand breaks in the first and second introns adjacent to exon 51 of the human dystrophin gene, whereby a segment containing exon 51 in the dystrophin gene is deleted to treat the subject.

[0024] The present invention is directed to a Cpf1 guide RNA (gRNA) that targets an enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 65-70, or a complement thereof.

[0025] The present invention is directed to a method of disrupting an enhancer of the B-cell lymphoma / leukemia 11A gene in a cell, the method comprising administering to the cell at least one Cpf1 gRNA and a Cpf1 endonuclease as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

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DETAILED DESCRIPTION OF THE INVENTION

[0027] In part, the present disclosure provides a therapeutic application of CRISPR / Cpf1-based genome editing for treating diseases. Cpf1, a type V CRISPR-Cas effector endonuclease, is involved in prokaryotic adaptive immunity, including, among others, the genera Acidaminococcus and Lachnospiraceae, and exhibits gene editing activity in human cells through a single RNA-guided approach. The present disclosure provides methods by which a CRISPR / Cpf1-based system can be used for the treatment of genetic diseases such as Duchenne muscular dystrophy (DMD), sickle cell anemia (SCA), and β-thalassemia.

[0028] According to one aspect of the present disclosure, the first method includes a splice acceptor knockout. Cpf1 generates a relatively large indel footprint, which facilitates the disruption of the splice acceptor and the removal of the target exon from the transcript (see FIG. 1A). As shown in FIG. 1A, Cpf1 causes a 5-base pair staggered double-strand break from DNA (which can be repaired via non-homologous end joining (NHEJ)), resulting in a relatively large insertion or deletion (indel) footprint, and then produces Streptococcus pyogenes or Staphylococcus aureus Cas9. This allows for a more potent disruption of the splice acceptor and even the removal of the target exon, as the repair leaves a relatively large indel footprint and can more efficiently knockout gene elements such as splice acceptors and enhancers. Cpf1 also has an individual protospacer adjacent motif (PAM) that increases the diversity of genomic regions that can be targeted. Cpf1 recognizes TTTN, while Streptococcus pyogenes Cas9 recognizes NGG and Staphylococcus aureus Cas9 recognizes NNGRRT. Additionally, Cpf1 does not require a tracrRNA and thus only requires a crRNA and therefore also uses a small guide RNA.

[0029] Another aspect of the disclosure provides a method that includes a compatible overhang deletion. Cpf1 can promote gene deletion by adapting the overhang to remove gene elements (see FIG. 1B). As shown in FIG. 1B, Cpf1 produces a 5-base pair overhang that can be compatible with a second double-strand break. Providing multiple Cpf1 guide RNAs together with the compatible overhang can promote seamless gene deletion. Previous studies using Staphylococcus aureus (S. aureus) Cas9 have demonstrated that approximately 67% of gene deletions are seamless for one guide RNA pair. For example, the compatible overhangs produced by multiplexing Cpf1 around a gene region of interest (e.g., exon 51 in dystrophin) can promote seamless deletion. After NHEJ, gene deletions are achieved that can restore the reading frame of the mutant gene. By adapting the overhang, very accurate ligation can be promoted.

[0030] Yet another aspect of the disclosure provides a method that includes enhancer disruption. Cpf1 can produce a relatively large indel footprint that more reliably disrupts enhancers and other gene regulatory elements (see FIG. 1C). As shown in FIG. 1C, the relatively large indel footprint produced by Cpf1 can be used to disrupt enhancers for the purpose of examining enhancer function or as a candidate for a treatment method for diseases such as SCA.

[0031] For example, the present disclosure describes modifications of Cpf1 for targeted gene excision of single and multiple exons of the dystrophin gene for the treatment of Duchenne muscular dystrophy (DMD). This is achieved by targeted mutagenesis of splice receptors at multiple mutagenic sites for single exon excision, or gene deletion of single or multiple exons. Targeted exon excision can restore the reading frame of dystrophin, leading to improvement of muscle function and patient phenotype. Also, gene enhancers can be targeted as a therapeutic approach for the treatment of diseases, in particular, the BCL11a enhancer region or the γ-globin promoter can be targeted as a treatment for sickle cell anemia (SCA) or β-thalassemia. To restore the expression of functional dystrophin in cells derived from DMD patients, the disclosed Cpf1 gRNAs are used together with a CRISPR / Cpf1-based system to target gene regions such as the intron region around exon 51 of the human dystrophin gene, and gene deletions can be induced in this region.

[0032] Also described herein are gene editing systems based on CRISPR / Cpf1 and gene constructs, compositions and methods for delivering multiple gRNAs to target the dystrophin gene. The subject matter of the present disclosure also provides a method for delivering a gene construct (e.g., a vector) or a composition comprising this gene construct to skeletal muscle. This vector can be an AAV (e.g., a modified AAV vector). The subject matter of the present disclosure describes a method for delivering the active form of this class of therapeutic agents to skeletal muscle that is effective, efficient and facilitates the success of genome modification, and further provides means for rewriting the human genome for therapeutic use and target model species for basic science use.

[0033] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0034] 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0035] The terms "comprising," "comprised of," "having," "has," "can," "containing," and variations thereof, as used herein, are intended to be open-ended phrases, terms, or words that do not exclude additional acts or structural possibilities. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements provided herein, whether explicitly described or not.

[0036] Regarding the recitation of numerical ranges herein, each number therebetween is clearly intended with the same degree of precision. For example, for the range of 6 - 9, in addition to 6 and 9, the numbers 7 and 8 are intended, and for the range 6.0 - 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are clearly intended.

[0037] As used herein, the terms "about" or "approximately" mean within an acceptable error range with respect to a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within three or more standard deviations in accordance with the conventions in the art. Alternatively, "about" can mean within a range of up to 20% of a given value, preferably within a range of up to 10%, more preferably within a range of up to 5%, and even more preferably within a range of up to 1%. Alternatively, especially with respect to biological systems or biological processes, the term can mean within an order of magnitude of a value, preferably within a factor of five, and more preferably within a factor of two.

[0038] As used interchangeably herein, "adeno-associated virus" or "AAV" refers to a small virus belonging to the genus Dependovirus of the family Parvoviridae that infects humans and several other primate species. AAV is not currently known to cause disease and thus elicits a very mild immune response.

[0039] As used herein, "binding region" refers to a region within a nuclease target region that is recognized and bound by a nuclease.

[0040] As used interchangeably herein, "cardiac muscle" or "heart muscle" means a type of involuntary striated muscle (myocardium) found in the walls and histological basis of the heart. Cardiac muscle is made up of cardiomyocytes or myocardiocytes. Cardiomyocytes exhibit striations similar to those on skeletal muscle cells but contain only a single, unique nucleus, unlike multinucleated skeletal muscle. In certain embodiments, "cardiac condition" refers to a condition associated with cardiac muscle (e.g., cardiomyopathy, heart failure, arrhythmia, and inflammatory heart disease).

[0041] As used herein, "coding sequence" or "encoding nucleic acid" means a nucleic acid (RNA or DNA molecule) comprising a polynucleotide sequence that encodes a protein. The coding sequence can further comprise start and stop signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of inducing expression in a cell of an individual or mammal to which the nucleic acid is administered. The coding sequence can be codon optimized.

[0042] As used herein, "complement" or "complementary" means that a nucleic acid can effect Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between polynucleotides of a nucleic acid molecule or polynucleotide analogs. "Complementarity" refers to a property shared between two nucleic acid sequences such that when aligned antiparallel to each other, the polynucleotide bases at each position are complementary.

[0043] As used herein, "modifying," "genome editing," and "repairing" refer to changing a mutant gene that encodes a truncated protein or does not encode a protein at all so as to obtain full-length functionality or partially full-length functionality of the protein expression. Modifying or repairing a mutant gene can include replacing a region of the gene having a mutation, or replacing the entire mutant gene with a copy of the gene having no mutation using a repair mechanism such as homology-directed repair (HDR). Modifying or repairing a mutant gene can also include introducing a double-strand break in the gene and then repairing the gene using non-homologous end joining (NHEJ) to repair a frameshift mutation that results in a premature stop codon, an ectopic splice acceptor site, or an ectopic splice donor site. NHEJ can add or delete at least one base pair during repair, which can repair the appropriate reading frame and remove the premature stop codon. Modifying or repairing a mutant gene can also include disrupting an ectopic splice acceptor site or splice donor sequence. Modifying or repairing a mutant gene can also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand to repair the appropriate reading frame by removing the DNA between two nuclease target sites, and repairing the DNA cleavage by NHEJ.

[0044] As used interchangeably herein, "Cpf1 endonuclease" or "Cpf1" refers to a single RNA-guided endonuclease of a class 2 CRISPR-Cas system that is smaller and simpler than Cas9. The Cpf1 endonuclease targets and cleaves as a 5-nucleotide staggered cut distal to the 5'T-rich PAM.

[0045] As used interchangeably herein, "donor DNA", "donor template", and "repair template" refer to a double-stranded DNA fragment or molecule that contains at least a portion of a target gene. The donor DNA can encode a fully functional protein or a partially functional protein.

[0046] As used interchangeably herein, "Duchenne muscular dystrophy" or "DMD" refers to a lethal X-linked disorder of recessive inheritance that causes muscle degeneration and ultimately death. DMD is a common hereditary single-gene disease that occurs in 1 in 3,500 males. DMD is the result of a genetic or spontaneous mutation that causes a nonsense or frameshift mutation in the dystrophin gene. The majority of dystrophin mutations that cause DMD are deletions of exons that disrupt the reading frame in the dystrophin gene and cause premature translation termination. DMD patients generally lose the ability to support their own bodies in childhood, gradually lose muscle strength during their teens, and die in their twenties.

[0047] As used herein, "dystrophin" refers to a rod-shaped cytoplasmic protein that is part of a protein complex that links the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane, which is responsible for regulating the integrity and function of muscle cells. As used interchangeably herein, the dystrophin gene or "DMD gene" is 2.2 megabases at locus Xp21. The primary transcript is approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons encode a protein that is over 3,500 amino acids.

[0048] As used herein, "Exon 51" refers to the 51st exon of the dystrophin gene. Exon 51 is frequently adjacent to frameshift deletions in DMD patients and is targeted in clinical trials of oligonucleotide-based exon skipping. Clinical trials of the exon 51 skipping compound eteplirsen recently reported a significant functional benefit over 48 weeks with an average of 47% dystrophin-positive fibers compared to baseline. Mutations in Exon 51 are ideally suited for permanent correction by genome editing based on NHEJ.

[0049] As used interchangeably herein, "frameshift" or "frameshift mutation" refers to a type of genetic mutation in which the addition or deletion of one or more polynucleotides results in a shift in the reading frame of codons within the mRNA. The shift in the reading frame can lead to changes in the amino acid sequence in protein translation, such as missense mutations or premature stop codons.

[0050] As used herein, "functional" and "fully functional" describe a protein having biological activity. A "functional gene" refers to a gene that is transcribed into an mRNA that is translated into a functional protein.

[0051] As used herein, "gene construct" refers to a DNA or RNA molecule containing a polynucleotide sequence encoding a protein. The coding sequence includes start and stop signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of inducing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing an essential regulatory element operably linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present within the cells of an individual.

[0052] As used herein, "hereditary disease" refers to a disease, particularly a condition that is present at birth, that is caused, in whole or in part, directly or indirectly, by one or more abnormalities within the genome. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence or the regulatory sequence of a gene. Hereditary diseases can include, but are not limited to, DMD, Becker muscular dystrophy (BMD), hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, hereditary disorders of liver metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemias such as β-thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, and Tay-Sachs disease.

[0053] As used interchangeably herein, "homologous recombination repair" or "HDR" refers to a cellular mechanism for repairing double-strand DNA damage, typically in the G2 and S phases of the cell cycle, when homologous fragments of DNA are present in the nucleus. HDR uses a donor DNA template to induce repair and can also be used to create specific sequence changes to the genome, including the addition of targeted whole genes. When the donor template is provided with a CRISPR / Cpf1-based gene editing system, the cellular machinery will repair the break by homologous recombination, which is enhanced several-fold in the presence of DNA breaks. If homologous DNA fragments are not present, non-homologous end joining can occur instead.

[0054] As used herein, "genome editing" refers to changing a gene. Genome editing can include correcting or repairing a mutant gene. Genome editing can include knocking out a gene, such as a mutant gene or a normal gene. Genome editing can be used to treat a disease or enhance muscle repair by changing the gene of interest.

[0055] As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" means that these sequences have a certain percentage of residues that are identical over a particular region. That percentage can be calculated by optimally aligning the two sequences, comparing these two sequences over a particular region, determining the number of positions where identical residues exist in both sequences to obtain the number of match positions, dividing the number of match positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or if gaps are introduced by the alignment such that only a single sequence is included in the particular region being compared, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equal. Identity can be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0056] As used interchangeably herein, "mutated gene" or "mutated gene" refers to a gene that has undergone a detectable mutation. A mutated gene has undergone changes such as loss, gain, or exchange of genetic material that affect the normal transmission and expression of the gene. As used herein, "disrupted gene" refers to a mutated gene that has a mutation resulting in a premature stop codon. A disrupted gene product is truncated compared to the full-length non-disrupted gene product.

[0057] As used herein, the "non-homologous end joining (NHEJ) pathway" refers to a pathway that repairs double-strand breaks in DNA by directly ligating the cut ends without the need for a homologous template. The ligation of DNA ends independent of a template by NHEJ is a stochastic and error-prone repair process that introduces random microinsertions and microdeletions (indels) at the DNA break site. This method can be used to intentionally disrupt, delete, or change the reading frame of a target gene sequence. NHEJ generally uses short homologous DNA sequences called microhomologies to induce repair. These microhomologies are often present in the single-stranded overhangs at the ends of the double-strand break. When this overhang is perfectly matched, NHEJ usually repairs the cut accurately, while inaccurate repair resulting in the loss of polynucleotides can also occur, which is much more common when the overhangs do not match.

[0058] As used herein, the "normal gene" refers to a gene that has not undergone changes such as loss, gain, or exchange of genetic material. A normal gene undergoes normal gene transmission and gene expression.

[0059] As used herein, "nuclease-mediated NHEJ" refers to NHEJ that is initiated after a nuclease such as the Cpf1 endonuclease cleaves double-stranded DNA.

[0060] As used herein, "nucleic acid", "oligonucleotide", or "polynucleotide" means at least two polynucleotides linked together by covalent bonds. A single-stranded depiction also defines the sequence of the complementary strand. Thus, a nucleic acid includes the complementary strand of the depicted single strand. For the same purpose of a given nucleic acid, many variants of the nucleic acid can be used. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0061] A nucleic acid can be single-stranded or double-stranded, or can contain both double-stranded and single-stranded portions. A nucleic acid can be DNA, both genomic DNA and cDNA, RNA, or a hybrid, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. A nucleic acid can be obtained by chemical synthesis methods or by recombinant methods.

[0062] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter that is spatially linked to the gene. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene that the promoter controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0063] As used herein, "partially functional" describes a protein that is encoded by a mutant gene and has lower biological activity than the functional protein but higher biological activity than the non-functional protein.

[0064] As used interchangeably herein, "premature termination codon" or "out-of-frame termination codon" refers to a nonsense mutation within a DNA sequence that results in a termination codon at a position not normally found in the wild-type gene. A premature termination codon can result in a truncated or shorter protein compared to the full-length form.

[0065] As used herein, "promoter" means a synthetic or naturally occurring molecule that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences for further enhancing expression and / or altering the spatial and / or temporal expression thereof. A promoter can also contain distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site. Promoters can be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or variably regulate the expression of a genetic component in response to the cell, tissue, or organ in which expression occurs, or the developmental stage at which expression occurs, or external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, human U6 (hU6) promoter, and CMV IE promoter.

[0066] As used herein, "skeletal muscle" refers to a type of striated muscle that is under the control of the somatic nervous system and is attached to bone by bundles of collagen fibers known as tendons. Skeletal muscle is composed of individual components known as muscle cells (myocytes), sometimes colloquially referred to as "muscle fibers." Muscle cells are formed from the fusion of developmental myoblasts (a type of embryonic progenitor cell that gives rise to muscle cells) in a process known as myogenesis. These long, cylindrical, multinucleated cells are also referred to as myofibers.

[0067] As used herein, "skeletal muscle condition" refers to conditions associated with skeletal muscle, such as muscular dystrophy, aging, muscle degeneration, wound healing, and muscle weakness or atrophy.

[0068] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. The subject or patient may be receiving other forms of treatment.

[0069] "Target gene," as used herein, refers to any polynucleotide sequence that encodes a known or putative gene product. The target gene may be a mutant gene involved in a genetic disorder. In certain embodiments, the target gene is the human dystrophin gene or the human B-cell lymphoma / leukemia 11A gene. In certain embodiments, the target gene is a mutant human dystrophin gene.

[0070] "Target region," as used herein, refers to the region of a target gene that is designed to be bound and cleaved by a CRISPR / Cpf1-based gene editing system.

[0071] As used herein, "transgene" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into another organism. This non-natural segment of DNA can also retain the ability to produce RNA or protein in a transgenic organism, or can alter the normal function of the genetic code of a transgenic organism. The introduction of a transgene has the potential to change the phenotype of an organism.

[0072] As used herein with respect to nucleic acids, "variant" means (i) a part or fragment of a reference polynucleotide sequence; (ii) the complement of a reference polynucleotide sequence, or a part thereof; (iii) a nucleic acid that is substantially identical to the reference nucleic acid, or its complement; or (iv) a nucleic acid that hybridizes to the reference nucleic acid under stringent conditions, its complement, or a sequence substantially identical thereto.

[0073] A "variant" with respect to a peptide or polypeptide has an amino acid sequence that differs by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with another amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as being accompanied by minor changes. These minor changes can be somewhat characterized, as understood in the art, by considering the hydropathic index of the amino acid. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that protein function can still be retained even when amino acids with similar hydropathic indices are substituted. In one aspect, amino acids having a hydropathic index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide. Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydropathic index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with this finding, it will be understood that amino acid substitutions compatible with biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0074] As used herein, "vector" means a nucleic acid sequence containing an origin of replication. The vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be an extrachromosomal vector that self-replicates, preferably a DNA plasmid. For example, the vector can encode a Cpf1 endonuclease and at least one Cpf1 gRNA (e.g., a Cpf1 gRNA comprising any one of the polynucleotide sequences of SEQ ID NOs: 36 to 119) or its complement.

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the same meaning as commonly understood by one of ordinary skill in the art. For example, all academic terms and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, as well as protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The meaning and scope of the terms should be clear; however, in the case of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0076] 2. CRISPR System The gene construct (e.g., vector) of the present disclosure encodes a CRISPR / Cpf1-based gene editing system that is specific for the dystrophin gene (e.g., the human dystrophin gene). "Clustered regularly interspaced short palindromic repeats" and "CRISPR" when used interchangeably herein refer to a locus containing multiple short direct repeats found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. The CRISPR locus in a microbial host contains a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated between the CRISPR repeats of the genome and serve as a "memory" of past exposures.

[0077] Three classes of CRISPR systems (type I, type II, and type III effector systems) are known. The type II effector system uses a single effector enzyme, such as the Cpf1 endonuclease, to perform targeted DNA double-strand breakage and cleave dsDNA in four consecutive steps. Compared to type I and type III effector systems that require multiple different effectors acting as a complex, the type II effector system can function in alternative situations, such as in eukaryotic cells. The Cpf1 endonuclease mediates cleavage of the target DNA if the correct PAM is also present at the 5' end of the protospacer.

[0078] The activity of the CRISPR / Cpf1 system has three stages: modification, crRNA formation, and interference. During the modification stage, Cas1 and Cas2 proteins facilitate the modification of small DNA fragments into the CRISPR array. During crRNA formation, processing of pre-crRNA occurs, producing mature crRNA that recruits a Cas protein, namely, the Cpf1 endonuclease. During the interference stage, Cpf1 binds to the crRNA to form a binary complex, identifies and cleaves the target DNA sequence.

[0079] In this system, the Cpf1 endonuclease is directed to genomic target sites by synthetically reconstituted Cpf1 "guide RNA" ("Cpf1 gRNA"). The Cpf1 endonuclease generates "sticky" ends, e.g., sticky ends 4-5 nucleotides in length, unlike Cas9 which generates blunt ends by making one strand longer than the other. The Cpf1 endonuclease also cleaves target DNA further from the PAM compared to Cas9.

[0080] A target gene (e.g., the dystrophin gene, e.g., the human dystrophin gene) can be involved in cell differentiation or any other process where gene activation may be desired, or can have a mutation (e.g., a frameshift or nonsense mutation). If this target gene has a mutation that creates a premature stop codon, an ectopic splice acceptor site, or an ectopic splice donor site, a CRISPR / Cpf1-based gene editing system can be designed to recognize and bind to a polynucleotide sequence upstream or downstream of this premature stop codon, ectopic splice acceptor site, or ectopic splice donor site. Using this CRISPR / Cpf1-based system, normal gene splicing can also be disrupted by targeting splice acceptors and donors to induce skipping of the premature stop codon, or by restoring the disrupted reading frame. This CRISPR / Cpf1-based gene editing system may or may not mediate off-target changes to the protein-coding regions of the genome.

[0081] This specification provides a CRISPR / Cpf1-based gene editing system for use in genome editing and the treatment of genetic diseases. The unique ability of the CRISPR / Cpf1-based gene editing system is the direct ability to simultaneously target multiple different genomic loci by co-expressing a single Cpf1 endonuclease with two or more Cpf1 gRNAs. The CRISPR / Cpf1-based gene editing system can be designed to target any gene, including genes involved in genetic diseases, aging, tissue regeneration, or wound healing. The CRISPR / Cpf1-based gene editing system can include a Cpf1 endonuclease and at least one Cpf1 gRNA. In certain embodiments, the system includes two Cpf1 gRNAs.

[0082] a. Cpf1 endonuclease The gene editing system based on CRISPR / Cpf1 can include a Cpf1 endonuclease. The Cpf1 endonuclease is an endonuclease that cleaves nucleic acids. While Cas9 generates blunt-ended 3 nucleotides upstream of the PAM site, the Cpf1 endonuclease cleaves in a staggered manner, generating a 5 nucleotide 5’ overhang that is 18 - 23 bases away from the PAM.The Cpf1 endonuclease can be derived from any bacterial or archaeal species, including but not limited to: Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens or Porphyromonas macacae. In certain embodiments, the Cpf1 endonuclease is a Cpf1 endonuclease derived from Lachnospiraceae bacterium ND2006 ("LbCpf1") or Acidaminococcus ("AsCpf1").

[0083] In some embodiments, the Cpf1 endonuclease may comprise the following humanized AsCpf1 sequence (SEQ ID NO: 124):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0084] In some embodiments, the Cpf1 endonuclease may comprise the following humanized LbCpf1 sequence (SEQ ID NO: 125):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0085] Cpf1 endonuclease can interact with one or more Cpf1 gRNAs and, in conjunction with the Cpf1 gRNA, localize to a site that includes a target domain and, in certain embodiments, a PAM sequence. In certain embodiments, the ability of the Cpf1 endonuclease to interact with and cleave a target nucleic acid is dependent on the PAM sequence. The PAM sequence is a sequence within the target nucleic acid. In certain embodiments, cleavage of the target nucleic acid occurs upstream of the PAM sequence. Cpf1 endonucleases from various bacterial species can recognize various sequence motifs (e.g., PAM sequences). In certain embodiments, the Cpf1 endonuclease recognizes a PAM of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0086] In certain embodiments, the vector encodes at least one Cpf1 endonuclease that recognizes a PAM of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). In certain embodiments, the at least one Cpf1 endonuclease is a Cpf1 endonuclease from Lachnospiraceae bacterium ("LbCpf1") or Acidaminococcus ("AsCpf1"). In certain embodiments, the Cpf1 endonuclease is encoded by a polynucleotide sequence of SEQ ID NO: 124 or SEQ ID NO: 125.

[0087] The nucleic acid encoding the Cpf1 endonuclease can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. The synthetic nucleic acid sequence can be codon-optimized, e.g., at least one non-common codon or less common codon is replaced with a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system described herein.

[0088] In addition to, or alternatively, the nucleic acid encoding the Cpf1 endonuclease may include a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0089] b. Cpf1 gRNA The CRISPR / Cpf1-based gene editing system includes at least one Cpf1 gRNA, for example, one Cpf1 gRNA, two Cpf1 gRNAs, three gRNAs, etc. Targeting of the CRISPR / Cpf1-based gene editing system is achieved by this gRNA. This Cpf1 gRNA can target any desired DNA sequence by replacing the sequence encoding the protospacer that confers targeting specificity to the desired DNA target. When "target region", "target sequence" or "protospacer" are used interchangeably herein, they refer to the region of the target gene (e.g., the dystrophin gene) targeted by the CRISPR / Cpf1-based gene editing system. Prior to this target sequence or protospacer, a PAM sequence is present at the 5' end of this protospacer. In some embodiments, the PAM sequence may be TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0090] In some embodiments, the protospacer may be about 17 bp to about 23 bp. In some embodiments, the Cpf1 gRNA includes a polynucleotide sequence corresponding to the protospacer or a fragment thereof. In some embodiments, the Cpf1 gRNA may include a protospacer of about 17 bp to about 23 bp. In some embodiments, the protospacer of about 17 bp to about 23 bp is continuous.

[0091] In some embodiments, the target region may comprise any one of the polynucleotide sequences of SEQ ID NOs: 1-35, any one of the fragments of SEQ ID NOs: 1-35, or a complement thereof. In some embodiments, the Cpf1 gRNA may comprise any one of the polynucleotide sequences of SEQ ID NOs: 36-119, any one of the fragments of SEQ ID NOs: 36-119, or a complement thereof. In some embodiments, any one of the fragments of SEQ ID NOs: 36-119 is about 17 bp to about 23 bp in length. In some embodiments, about 17 bp to about 23 bp in the fragment are continuous.

[0092] The CRISPR / Cpf1-based gene editing system may include at least one Cpf1 gRNA, and these gRNAs target different DNA sequences. These target DNA sequences may overlap. The number of Cpf1 gRNAs encoded by the gene constructs (e.g., AAV vectors) of the present disclosure is at least 1 Cpf1 gRNA, at least 2 different Cpf1 gRNAs, at least 3 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs, at least 5 different Cpf1 gRNAs, at least 6 different Cpf1 gRNAs, at least 7 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs, at least 9 different Cpf1 gRNAs, at least 10 different Cpf1 gRNAs, at least 11 different Cpf1 gRNAs, at least 12 different Cpf1 gRNAs, at least 13 different Cpf1 gRNAs, at least 14 different Cpf1 gRNAs, at least 15 different Cpf1 gRNAs, at least 16 different Cpf1 gRNAs, at least 17 different Cpf1 gRNAs, at least 18 different Cpf1 gRNAs, at least 18 different Cpf1 gRNAs, at least 20 different Cpf1 gRNAs, at least 25 different Cpf1 gRNAs, at least 30 different Cpf1 gRNAs, at least 35 different Cpf1 gRNAs, at least 40 different Cpf1 gRNAs, at least 45 different Cpf1 gRNAs, or at least 50 different Cpf1 gRNAs. The number of Cpf1 gRNAs encoded by the vectors of the present disclosure is from at least 1 Cpf1 gRNA to at least 50 different Cpf1 gRNAs, from at least 1 Cpf1 gRNA to at least 45 different Cpf1 gRNAs, from at least 1 Cpf1 gRNA to at least 40 different Cpf1 gRNAs, from at least 1 Cpf1 gRNA to at least 35 different Cpf1 gRNAs, from at least 1 Cpf1 gRNA to at least 30 different Cpf1 gRNAs, from at least 1 Cpf1 gRNA to at least 25 different Cpf1 gRNAs, at least 1 Cpf1gRNA ~ at least 20 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 16 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 12 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 8 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 4 different Cpf1 gRNAs, at least 4 Cpf1 gRNAs ~ at least 50 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 45 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 40 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 35 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 30 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 25 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 20 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 16 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 12 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 8 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 50 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 45 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 40 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 35 different Cpf1 gRNAs, 8 different Cpf1 gRNAs ~ at least 30 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 25 different Cpf1 gRNAs, 8 different Cpf1 gRNAs ~ at least 20 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 16 different Cpf1 gRNAs, or 8 different Cpf1 gRNAs ~ at least 12 different Cpf1It may be a gRNA. In certain embodiments, this gene construct (e.g., an AAV vector) encodes one Cpf1 gRNA (i.e., the first gRNA) and optionally a Cpf1 endonuclease. In certain embodiments, the first gene construct (e.g., the first AAV vector) encodes one Cpf1 gRNA (i.e., the first Cpf1 gRNA) and optionally a Cpf1 endonuclease, and the second gene construct (e.g., the second AAV vector) encodes one Cpf1 gRNA (i.e., the second Cpf1 gRNA) and optionally a Cpf1 endonuclease.

[0093] 3. Gene construct of a CRISPR / Cpf1-based gene editing system for genome editing of the dystrophin gene The present invention is directed to gene constructs for genome editing, genome modification or modification of gene expression of the dystrophin gene (e.g., the human dystrophin gene). The gene construct comprises at least one Cpf1 gRNA targeting a human dystrophin gene sequence such as a Cpf1 endonuclease-compatible target. The disclosed gRNA can be incorporated into a CRISPR / Cpf1-based gene editing system and, as such, a system using a Cpf1 endonuclease to target regions within the dystrophin gene, e.g., intron regions around exons such as exon 51 in the human dystrophin gene, splice acceptor sites, and / or exon regions, to cause genomic deletions in this region and restore the expression of functional dystrophin in cells derived from DMD patients.

[0094] DMD is a severe muscle wasting disease caused by gene mutations in the dystrophin gene. Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix via the cell membrane. Dystrophin provides structural stability to the dystroglycan complex in the cell membrane. The dystrophin gene is 2.2 megabases at locus Xp21. Primary transcription measures approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons encode a protein of over 3,500 amino acids. Normal skeletal muscle tissue contains only a small amount of dystrophin, but the absence of abnormal expression of this dystrophin results in severe and incurable symptoms. Some mutations in the dystrophin gene produce an incomplete dystrophin and a severe dystrophy phenotype in affected patients. Some mutations in the dystrophin gene result in a partially functional dystrophin protein and a very mild dystrophy phenotype in affected patients.

[0095] DMD is the result of genetic or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Naturally occurring mutations and their consequences are relatively well understood with respect to DMD. Mutations are typically deletions or duplications of regions of the gene, which cause the protein to be out of frame and thus completely dysfunctional. Correction of the frame to restore a nearly functional protein allows single exon removal to be applied to 83% of patients. CPF1 can target dystrophin exons and can be used to knock out a single exon by targeting the splice acceptor or to delete gene regions to remove single or multiple exons.

[0096] In-frame deletions occurring in the region of exons 45-55 (e.g., exon 51) contained in the rod domain can produce a highly functional dystrophin protein, and many carriers are known to be asymptomatic or exhibit mild symptoms. Furthermore, targeting exons in this region of the dystrophin gene (e.g., targeting exons of the dystrophin gene such as exon 51) can theoretically treat more than 60% of patients. By skipping non-essential exons during mRNA splicing (e.g., skipping exon 51) and producing a dystrophin protein that is internally deleted but functional, efforts have been made to restore the disrupted dystrophin reading frame in DMD patients. Deletion of internal dystrophin exons (e.g., deletion of exon 51) causes a relatively mild Becker muscular dystrophy (i.e., BMD) although the proper reading frame is maintained. The Becker muscular dystrophy (i.e., BMD) genotype is similar to DMD in that there are deletions in the dystrophin gene. However, this deletion keeps the reading frame intact. Therefore, a dystrophin protein that is internally truncated but partially functional is made. BMD has a wide range of phenotypes, but often, when there is a deletion between exons 45-55 of dystrophin, this phenotype is much milder compared to DMD. Therefore, changing the DMD genotype to the BMD genotype is a common strategy for correcting dystrophin. There are many strategies for correcting dystrophin, and many of these rely on restoring the reading frame of endogenous dystrophin. This causes the genotype of the disease to shift from DMD to Becker muscular dystrophy. Many BMD patients have an intragenic deletion that maintains the translational reading frame, resulting in a shorter but mostly functional dystrophin protein.

[0097] In certain embodiments, a phenotype DMD subject such as a DMD subject having a deletion mutation is improved by modification of exon 51 to restore the reading frame (e.g., deletion or elimination of exon 51, e.g., by NHEJ). In certain embodiments, exon 51 of the dystrophin gene refers to the 51st exon of the dystrophin gene. Exon 51 is frequently adjacent to frameshift deletions in DMD patients and has been targeted in clinical trials of oligonucleotide-based exon skipping. In clinical trials of the exon 51 skipping compound eteplirsen, significant functional benefits were reported over 48 weeks with an average of 47% dystrophin-positive fibers compared to baseline. Mutations in exon 51 are ideally suited for permanent correction by NHEJ-based genome editing.

[0098] The vectors of the present disclosure can cause deletions in the dystrophin gene (e.g., the human dystrophin gene). In certain embodiments, the vector forms two double-strand breaks (a first double-strand break and a second double-strand break) in two introns (a first intron and a second intron) adjacent to the target position of the dystrophin gene, whereby a segment containing the dystrophin target position in the dystrophin gene is deleted. The "dystrophin target position" can be the exon target position of dystrophin or the intron target position within an exon of dystrophin as described herein. Deletion of the exon target position of dystrophin can optimize the dystrophin sequence in a patient suffering from Duchenne muscular dystrophy, e.g., the function or activity of the encoded dystrophin protein can be increased, or the disease state of the subject can be improved. In certain embodiments, the reading frame is restored by elimination of the exon target position of dystrophin. This exon target position of dystrophin can include one or more exons of the dystrophin gene. In certain embodiments, this dystrophin target position includes exon 51 of the dystrophin gene (e.g., the human dystrophin gene).

[0099] The gene constructs (e.g., vectors) of the present disclosure can mediate highly efficient gene editing at exon 51 of the dystrophin gene (e.g., the human dystrophin gene). The gene constructs (e.g., vectors) of the present disclosure restore the expression of dystrophin protein in cells derived from DMD patients. Exon 51 is frequently adjacent to frameshift deletions in DMD. Removal of exon 51 from the dystrophin transcript by exon skipping can be used to treat approximately 15% of all DMD patients. This class of dystrophin mutations is ideally suited for NHEJ-based genome editing and permanent correction by HDR. The gene constructs (e.g., vectors) described herein have been developed for targeted modification of exon 51 in the human dystrophin gene. The gene constructs (e.g., vectors) of the present disclosure are introduced into human DMD cells and mediate efficient gene modification and gene conversion to correct the reading frame. Protein restoration is concurrent with frame restoration and is detected in the majority of cells treated with the CRISPR / Cpf1-based gene editing system.

[0100] Single or multiple gRNAs can be designed to restore the dystrophin reading frame by targeting mutation hotspots at exon 51, and / or by introducing small insertions and deletions within the exon, and / or by exclusion of exon 51. After treatment with the vectors of the present disclosure, dystrophin expression can be restored in vitro in muscle cells of Duchenne patients. Human dystrophin was detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Significantly, the unique multiplex gene editing ability of the CRISPR / Cpf1-based gene editing system enables efficient generation of large deletions in this mutation hotspot region that can correct up to 62% of the patient's mutations by a universal or patient-specific gene editing approach. In some embodiments, candidate gRNAs are evaluated and selected based on off-target activity measured by surveyor, on-target activity, and distance from the exon.

[0101] Cpf1 gRNA can target a region of the dystrophin gene (DMD). In certain embodiments, the Cpf1 gRNA can target at least one of an exon, intron, promoter region, enhancer region, splice acceptor site, and / or transcription region of the dystrophin gene. In some embodiments, the target region comprises at least one polynucleotide sequence of SEQ ID NOs: 1-28. In certain embodiments, the Cpf1 gRNA targets intron 50 of the human dystrophin gene. In certain embodiments, the Cpf1 gRNA targets intron 51 of the human dystrophin gene. In certain embodiments, the Cpf1 gRNA targets exon 51 of the human dystrophin gene. The Cpf1 gRNA can comprise any one polynucleotide sequence of SEQ ID NOs: 36-64, 71-119, a fragment of any one of SEQ ID NOs: 36-64, 71-119, or its complement.

[0102] 4. CRISPR / Cpf1-based gene editing system gene construct for genome editing of B-cell lymphoma / leukemia 11A (BCL11a) gene Sickle cell anemia (SCA) is caused by a point mutation in the β-globin gene, while β-thalassemia is caused by other mutations that result in the loss of β-globin expression. BCL11a is a transcriptional repressor that suppresses embryonic and fetal globin genes. Complete loss of BCL11a is embryonically lethal; however, by disrupting the erythroid-specific enhancer region of BCL11a, the abundance of the transcriptional repressor can be reduced, fetal globin levels can be increased, and the disease phenotype can be improved. Similarly, specific mutations in the γ-globin (HBG1 / 2) promoter result in transcriptional repression and the loss of hereditary persistence of fetal hemoglobin (HPFH). The larger the indel footprint produced by Cpf1, the more efficiently it can disrupt the enhancer region of BCL11a or the inhibitory region of HBG1 / 2. In some embodiments, a Cpf1 gRNA is designed to disrupt the enhancer region of BCL11a, increase fetal globin levels, and improve the phenotype of SCA. In some embodiments, the enhancer region comprises at least one polynucleotide sequence of SEQ ID NOs: 29-35. In some embodiments, the Cpf1 gRNA comprises any one polynucleotide sequence of SEQ ID NOs: 65-70, a fragment of any one of SEQ ID NOs: 65-70, or its complement.

[0103] 5. DNA targeting compositions The present invention also targets DNA targeting compositions containing such gene constructs. This DNA targeting composition contains at least one Cpf1 gRNA (e.g., one Cpf1 gRNA, two Cpf1 gRNAs, three gRNAs, etc.) that targets the dystrophin gene (e.g., the human dystrophin gene) as described above. This at least one Cpf1 gRNA can bind to and recognize the target region. This target region can be selected immediately upstream of a possible out-of-frame stop codon, such that insertions or deletions during the repair process restore the dystrophin reading frame by frameshift. The target region can also be a splice acceptor site or a splice donor site, such that insertions or deletions during the repair process disrupt splicing and restore the dystrophin reading frame by disruption of the splice site and exclusion of the exon. The target region can also be a heterologous stop codon, such that insertions or deletions during the repair process restore the dystrophin reading frame by removing or disrupting this stop codon.

[0104] In certain embodiments, the DNA targeting composition of the present disclosure contains a first Cpf1 gRNA and a second Cpf1 gRNA, and the first Cpf1 gRNA and the second Cpf1 gRNA contain the polynucleotide sequences set forth in SEQ ID NOs: 36-119, or a complement thereof. In some embodiments, the polynucleotide sequence contains at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof. In some embodiments, the polynucleotide sequence contains at least one of SEQ ID NOs: 65-70, or a complement thereof. In certain embodiments, the first Cpf1 gRNA and the second Cpf1 gRNA contain the polynucleotide sequence.

[0105] In certain embodiments, the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0106] In certain embodiments, the DNA targeting composition may further comprise at least one Cpf1 endonuclease that recognizes a PAM of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). In some embodiments, the DNA targeting composition comprises a Cpf1 endonuclease encoded by the polynucleotide sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125. In certain embodiments, the vector is configured to form first and second double-strand breaks in the first and second introns adjacent to exon 51 of the human dystrophin gene, thereby deleting the segment containing exon 51 in the dystrophin gene.

[0107] The deletion efficiency of the vectors of the present disclosure can be related to the deletion size (i.e., the size of the segment deleted by this vector). In certain embodiments, the length or size of the specific deletion is determined by the distance between PAM sequences in the target gene (e.g., the dystrophin gene). In certain embodiments, the specific deletion of a segment of the dystrophin gene (defined with respect to the length of this segment and the sequences it contains, e.g., exon 51) is the result of a cleavage made adjacent to a specific PAM sequence within the target gene (e.g., the dystrophin gene).

[0108] In certain embodiments, this deletion size is from about 50 to about 2,000 base pairs (bp), for example, about 50 to about 1999 bp, about 50 to about 1900 bp, about 50 to about 1800 bp, about 50 to about 1700 bp, about 50 to about 1650 bp, about 50 to about 1600 bp, about 50 to about 1500 bp, about 50 to about 1400 bp, about 50 to about 1300 bp, about 50 to about 1200 bp, about 50 to about 1150 bp, about 50 to about 1100 bp, about 50 to about 1000 bp, about 50 to about 900 bp, about 50 to about 850 bp, about 50 to about 800 bp, about 50 to about 750 bp, about 50 to about 700 bp, about 50 to about 600 bp, about 50 to about 500 bp, about 50 to about 400 bp, about 50 to about 350 bp, about 50 to about 300 bp, about 50 to about 250 bp, about 50 to about 200 bp, about 50 to about 150 bp, about 50 to about 100 bp, about 100 to about 1999 bp, about 100 to about 1900 bp, about 100 to about 1800 bp, about 100 to about 1700 bp, about 100 to about 1650 bp, about 100 to about 1600 bp, about 100 to about 1500 bp, about 100 to about 1400 bp, about 100 to about 1300 bp, about 100 to about 1200 bp, about 100 to about 1150 bp, about 100 to about 1100 bp, about 100 to about 1000 bp, about 100 to about 900 bp, about 100 to about 850 bp, about 100 to about 800 bp, about 100 to about 750 bp, about 100 to about 700 bp, about 100 to about 600 bp, about 100 to about 1000 bp, about 100 to about 400 bp, about 100 to about 350 bp, about 100 to about 300 bp, about 100 to about 250 bp, about 100 to about 200 bp, about 100 to about 150 bp, about 200 to about 1999 bp, about 200 to about 1900 bp, about 200 to about 1800 bp, about 200 to about 1700 bp, about 200 to about 1650 bp, about 200 to about 1600 bp, about 200 to about 1500 bp, about 200 to about 1400 bp, about 200 to about 1300 bp, about 200 to about 1200 bp, about 200 to about 1150 bp, about 200 to about 1100 bp, about 200 to about 1000 bp, about 200 to about 900 bp, about 200 to about 850 bp, about 200 to about 800 bp, about 200 to about 750 bp, about 200 to about 700 bp, about 200 to about 600 bp, about 200 to about 2000 bp, about 200 to about 400 bp, about 200 to about 350 bp, about 200 to about 300 bp, about 200 to about 250 bp, about 300 to about 1999 bp,It is about 300 to about 1900 bp, about 300 to about 1800 bp, about 300 to about 1700 bp, about 300 to about 1650 bp, about 300 to about 1600 bp, about 300 to about 1500 bp, about 300 to about 1400 bp, about 300 to about 1300 bp, about 300 to about 1200 bp, about 300 to about 1150 bp, about 300 to about 1100 bp, about 300 to about 1000 bp, about 300 to about 900 bp, about 300 to about 850 bp, about 300 to about 800 bp, about 300 to about 750 bp, about 300 to about 700 bp, about 300 to about 600 bp, about 300 to about 3000 bp, about 300 to about 400 bp or about 300 to about 350 bp. In certain embodiments, this deletion size can be about 118 base pairs, about 233 base pairs, about 326 base pairs, about 766 base pairs, about 805 base pairs or about 1611 base pairs.,

[0109] 6. Composition for gene editing in muscle The present invention is directed to a gene construct (e.g., a vector) or a composition thereof for genomic editing of a target gene in the skeletal muscle or the cardiac muscle of a subject. This composition comprises a modified AAV vector and a polynucleotide sequence encoding a gene editing system based on CRISPR / Cpf1 (e.g., Cpf1 gRNA and Cpf1 endonuclease). This composition delivers an active CRISPR / Cpf1-based gene editing system to skeletal muscle or cardiac muscle. The gene construct (e.g., a vector) of the present disclosure can be used to correct or reduce the effects of mutations in the dystrophin gene involved in hereditary diseases and / or other skeletal muscle or cardiac muscle conditions (e.g., DMD). This composition can further comprise donor DNA or a transgene. This composition can be used for genomic editing, genomic manipulation, and for correcting or reducing the effects of mutations in genes involved in hereditary diseases and / or other skeletal muscle or cardiac muscle conditions.,

[0110] a. CRISPR / Cpf1-based gene editing system for targeting dystrophin Disclosed herein is a CRISPR / Cpf1-based gene editing system specific for the dystrophin gene. This CRISPR / Cpf1-based gene editing system can include a Cpf1 endonuclease and at least one Cpf1 gRNA for targeting the dystrophin gene. This CRISPR / Cpf1-based gene editing system can bind to and recognize a target region. This target region can be selected immediately upstream of a possible out-of-frame stop codon, such that insertions or deletions during the repair process restore the dystrophin reading frame by frame-shifting. The target region can also be a splice acceptor site or a splice donor site, such that insertions or deletions during the repair process disrupt splicing and restore the dystrophin reading frame by disruption of the splice site and exclusion of an exon. The target region can also be a heterologous stop codon, such that insertions or deletions during the repair process restore the dystrophin reading frame by removing or disrupting this stop codon.

[0111] This Cpf1 gRNA can target a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-35 or its complement. For example, the CRISPR / Cpf1-based gene editing system of the present disclosure was engineered to mediate more efficient gene editing at exon 51 of the dystrophin gene. This CRISPR / Cpf1-based gene editing system restored dystrophin protein expression in cells derived from DMD patients.

[0112] b. Adeno-associated virus vector This composition can also include a viral delivery system. In certain embodiments, the vector is an adeno-associated virus (AAV) vector. This AAV vector is a small virus belonging to the genus Dependovirus of the family Parvoviridae, which infects humans and several other primate species. The AAV vector can be used to deliver a CRISPR / Cpf1-based gene editing system using various construct structures. For example, the AAV vector can deliver the Cpf1 endonuclease and the Cpf1 gRNA expression cassette on separate vectors or on the same vector. Alternatively, both the Cpf1 endonuclease and up to two gRNA expression cassettes can be combined in a single AAV vector within the 4.7 kb packaging limit.

[0113] In certain embodiments, the AAV vector is a modified AAV vector. This modified AAV vector can have enhanced tissue tropism for cardiac muscle and skeletal muscle. This modified AAV vector can enable the delivery and expression of a CRISPR / Cpf1-based gene editing system in mammalian cells. For example, this modified AAV vector can be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). This modified AAV vector can deliver nucleases to skeletal muscle and cardiac muscle in vivo. This modified AAV vector can be based on one or more of several capsid types (e.g., AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9). This modified AAV vector can be based on an AAV2 pseudotype having an alternative muscle tropic AAV capsid, e.g., an AAV2 / 1 vector, an AAV2 / 6 vector, an AAV2 / 7 vector, an AAV2 / 8 vector, an AAV2 / 9 vector, an AAV2.5 vector, and an AAV / SASTG vector that can efficiently transduce skeletal muscle or cardiac muscle by systemic delivery and local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151). This modified AAV vector can be AAV2i8G9 (Shen et al. J. Biol. Chem. (2013) 288:28814-28823).

[0114] 7. Method for Gene Editing in Muscle The present disclosure is directed to a method for gene editing in the skeletal muscle or cardiac muscle of a subject. This method includes administering to the skeletal muscle or cardiac muscle of the subject a composition for gene editing in skeletal muscle or cardiac muscle as described above. This genome editing can include correcting a mutant gene or inserting a transgene. Correcting a mutant gene can include deleting, re-editing, or replacing this mutant gene. Correcting a mutant gene can include nuclease-mediated NHEJ or HDR.

[0115] 8. Method of Treating a Subject by Modifying a Mutated Gene The subject matter of the present disclosure provides a method of treating a subject suffering from a genetic disease (e.g., DMD) by modifying a mutated gene (e.g., a mutated dystrophin gene, e.g., a mutated human dystrophin gene) in a cell. The method can include administering to the cell or the subject the gene construct (e.g., vector) of the present disclosure described above or a composition comprising the gene construct. The method can include administering to the skeletal muscle or the myocardium of the subject the gene construct (e.g., vector) of the present disclosure for gene editing in skeletal muscle or myocardium described above or a composition comprising the gene construct. By use of the gene construct (e.g., vector) of the present disclosure or a composition comprising the gene construct for delivery of a CRISPR / Cpf1-based gene editing system to skeletal muscle or myocardium, expression of a fully functional or partially functional protein can be restored, along with a repair template or donor DNA, which can replace the entire gene or the region containing the mutation. A site-specific double-strand break can be introduced at a targeted genomic locus using a CRISPR / Cpf1-based gene editing system. A site-specific double-strand break occurs when the CRISPR / Cpf1-based gene editing system binds to a target DNA sequence, thereby enabling cleavage of the target DNA. This DNA cleavage can stimulate the natural DNA repair mechanism, resulting in one of two possible repair pathways: the homologous recombination repair (HDR) pathway or the non-homologous end joining (NHEJ) pathway.

[0116] The present disclosure is directed to gene editing by a CRISPR / Cpf1-based gene editing system without a repair template, which can efficiently correct a reading frame and restore the expression of a functional protein involved in a genetic disease by this gene editing. The CRISPR / Cpf1-based gene editing system of the present disclosure can include the use of correction methods based on homologous recombination repair or nuclease-mediated non-homologous end joining (NHEJ) (these enable efficient correction in primary cell lines with limited proliferation that may not accept gene correction based on homologous recombination or selection). This strategy integrates the rapid and robust assembly of an active CRISPR / Cpf1-based gene editing system with an efficient gene editing method for the treatment of genetic diseases caused by mutations in non-essential coding regions that result in frameshifts, premature termination codons, ectopic splice donor sites or ectopic splice acceptor sites.

[0117] a. Nuclease-mediated non-homologous end joining Restoration of protein expression from an endogenous mutant gene can be by NHEJ-mediated DNA repair without a template. In contrast to transient methods targeting target gene RNA, correction of the target gene reading frame in the genome by a transiently expressed CRISPR / Cpf1-based gene editing system can result in permanently restored target gene expression by all of the modified cells and their progeny. In certain embodiments, NHEJ is nuclease-mediated NHEJ, which in certain embodiments refers to NHEJ initiated by a Cpf1 endonuclease to cleave double-stranded DNA. This method includes administering a gene construct (e.g., a vector) of the present disclosure or a composition comprising this gene construct to the skeletal muscle or myocardium of a subject for gene editing in the skeletal muscle or myocardium.

[0118] Nuclease-mediated NHEJ gene correction can provide several potential advantages over the HDR pathway in correcting mutated target genes. For example, NHEJ does not require a donor template, which can introduce non-specific insertion mutations. In contrast to HDR, NHEJ operates efficiently in all phases of the cell cycle and can thus be used efficiently in both cycling and post-mitotic cells (such as muscle fibers). This provides robust and permanent gene repair, potentially requiring only one drug treatment in theory, as an alternative to oligonucleotide-based exon skipping or read-through forced by drugs at stop codons. NHEJ-based gene correction using CRISPR / Cpf1-based gene editing systems, as well as other engineered nucleases including meganucleases and zinc finger nucleases, can be combined with other existing ex vivo and in vivo platforms for cell- and gene-based therapies in addition to the plasmid electroporation approach described herein. For example, delivery of a CRISPR / Cpf1-based gene editing system by mRNA-based transfection or as a purified cell-permeable protein can enable DNA-free genome editing approaches that would avoid any potential for insertion mutations.

[0119] b. Homologous recombination repair Restoration of protein expression from an endogenous mutated gene can involve homologous recombination repair. The methods as described above further include administering a donor template to the cell. The donor template can include a polynucleotide sequence encoding a fully functional or partially functional protein. For example, the donor template can include a miniaturized dystrophin construct (referred to as "minidys"), a fully functional dystrophin construct for repairing the mutated dystrophin gene, or a fragment of the dystrophin gene that results in repair of the mutated dystrophin gene after homologous recombination repair.

[0120] c. A method of treating a subject using a CRISPR / Cpf1-based gene editing system by correcting a mutant gene The present disclosure also targets genome editing using a CRISPR / Cpf1-based gene editing system for restoring the expression of a fully functional or partially functional protein using a repair template or donor DNA, which can replace the entire gene or the region containing the mutation. The CRISPR / Cpf1-based gene editing system can be used to introduce a site-specific double-strand break at a targeted genomic locus. The site-specific double-strand break is brought about when the CRISPR / Cpf1-based gene editing system binds to a target DNA sequence using a gRNA, thereby enabling cleavage of the target DNA. The CRISPR / Cpf1-based gene editing system has the advantage of advancing genome editing due to its rapid and successful and efficient gene modification. This DNA cleavage can stimulate the natural DNA repair mechanism and result in one of two possible repair pathways: homologous recombination repair (HDR) or non-homologous end joining (NHEJ) pathway. For example, a CRISPR / Cpf1-based gene editing system induced in the dystrophin gene can comprise a Cpf1 gRNA having any one of the nucleic acid sequences of SEQ ID NOs: 36 to 64, 71 to 119, or a complement thereof.

[0121] The present disclosure is directed to genome editing using a CRISPR / Cpf1-based gene editing system without a repair template that can efficiently correct reading frames and restore the expression of functional proteins involved in genetic diseases. The disclosed CRISPR / Cpf1-based gene editing systems and methods can include using a correction method based on homologous recombination repair or nuclease-mediated non-homologous end joining (NHEJ) (which enables efficient correction in primary cell lines with limited proliferation where gene correction based on homologous recombination or selection may not be applicable). This strategy integrates the rapid and robust assembly of an active CRISPR / Cpf1-based gene editing system with an efficient gene editing method for the treatment of genetic diseases caused by mutations in non-essential coding regions that result in frameshifts, premature stop codons, ectopic splice donor sites, or ectopic splice acceptor sites.

[0122] The present disclosure provides a method for correcting mutant genes in cells and treating a subject suffering from a genetic disease such as DMD. This method can include administering to the cell or subject a CRISPR / Cpf1-based gene editing system, a polynucleotide or vector encoding the CRISPR / Cpf1-based gene editing system, or a composition of the CRISPR / Cpf1-based gene editing system, as described above. This method can include administering a CRISPR / Cpf1-based gene editing system, for example, a Cpf1 endonuclease, a polynucleotide sequence encoding the Cpf1 endonuclease, and / or at least one Cpf1 gRNA (wherein the gRNAs target different DNA sequences). These target DNA sequences can be overlapping. The number of gRNAs administered to the cell can be at least one gRNA, at least two different gRNAs, at least three different gRNAs, at least four different gRNAs, at least five different gRNAs, at least six different gRNAs, at least seven different gRNAs, at least eight different gRNAs, at least nine different gRNAs, at least ten different gRNAs, at least fifteen different gRNAs, at least twenty different gRNAs, at least thirty different gRNAs, or at least fifty different gRNAs, as described above. The gRNA can include at least one nucleic acid sequence of SEQ ID NOs: 36-64, 71-119, or a complement thereof. This method can include homologous recombination repair or non-homologous end joining.

[0123] 9. Method for treating a disease The present disclosure is directed to a method of treating a subject in need thereof. The method includes administering to the tissue of the subject the gene construct (e.g., vector) of the present disclosure as described above or a composition comprising the gene construct. In certain embodiments, the method can include administering to the skeletal muscle or cardiac muscle of the subject the gene construct (e.g., vector) of the present disclosure as described above or a composition comprising the gene construct. In certain embodiments, the method can include administering to a vein of the subject the gene construct (e.g., vector) of the present disclosure as described above or a composition comprising the gene construct. In certain embodiments, the subject is afflicted with a condition of skeletal muscle or cardiac muscle that causes degeneration or weakness or a genetic disease. For example, the subject is afflicted with Duchenne muscular dystrophy as described above.

[0124] a. Duchenne muscular dystrophy Using the methods described above, the dystrophin gene can be modified to restore the expression of a fully functional or partially functional protein of the mutant dystrophin gene. In some aspects and embodiments, the present disclosure provides a method of reducing the effects (e.g., clinical symptoms / indications) of DMD in a patient. In some aspects and embodiments, the present disclosure provides a method of treating DMD in a patient. In some aspects and embodiments, the present disclosure provides a method of preventing DMD in a patient. In some aspects and embodiments, the present disclosure provides a method of preventing further worsening of DMD in a patient.

[0125] 10. Constructs and plasmids The compositions described above can include a gene construct encoding a CRISPR / Cpf1-based gene editing system disclosed herein. This gene construct (e.g., a plasmid) can include a nucleic acid encoding a CRISPR / Cpf1-based gene editing system (e.g., at least one of a Cpf1 endonuclease and / or a Cpf1 gRNA). The compositions described above can include a gene construct encoding a modified AAV vector and a nucleic acid sequence encoding a CRISPR / Cpf1-based gene editing system disclosed herein. This gene construct (e.g., a plasmid) can include a nucleic acid encoding this CRISPR / Cpf1-based gene editing system. The compositions described above can include a gene construct encoding a modified lentiviral vector disclosed herein.

[0126] In some embodiments, the gene construct may include a promoter operably linked to a polynucleotide sequence encoding at least one Cpf1 gRNA and / or a Cpf1 endonuclease. In some embodiments, this promoter is operably linked to a polynucleotide sequence encoding a first Cpf1 gRNA, a second Cpf1 gRNA, and / or a Cpf1 endonuclease. This gene construct can exist in the cell as a functional extrachromosomal molecule. This gene construct can be a linear minichromosome, e.g., a centromere, telomere or plasmid or cosmid.

[0127] The gene construct can also be part of the genome of a recombinant viral vector, including recombinant lentivirus, recombinant adenovirus, and recombinant adeno-associated virus. The gene construct can be part of the genetic material in an attenuated live microorganism or recombinant microbial vector that survives within a cell. The gene construct can include regulatory elements for the gene expression of the coding sequence of the nucleic acid. The regulatory element can be a promoter, enhancer, start codon, stop codon, or polyadenylation signal.

[0128] In certain embodiments, this gene construct is a vector. This vector can be an adeno-associated virus (AAV) vector, which encodes at least one Cpf1 endonuclease and at least one Cpf1 gRNA; this vector can express at least one Cpf1 endonuclease and at least one Cpf1 gRNA in mammalian cells. This vector can be a plasmid. This vector can be used for gene therapy in vivo. This vector can be recombinant. This vector can include a heterologous nucleic acid encoding a CRISPR / Cpf1-based gene editing system. This vector can be a plasmid. This vector can be useful for transfecting a cell with a nucleic acid encoding a CRISPR / Cpf1-based gene editing system and culturing and maintaining the transformed host cell under conditions where expression of the CRISPR / Cpf1-based gene editing system occurs.

[0129] The coding sequence can be optimized for stability and high-level expression. In some cases, the codons are selected to reduce the formation of secondary structures of RNA, such as those formed due to intramolecular binding.

[0130] The vector can contain a heterologous nucleic acid encoding a CRISPR / Cpf1-based gene editing system, and can further contain a start codon that can be upstream of the CRISPR / Cpf1-based gene editing system coding sequence, and a stop codon that can be downstream of the CRISPR / Cpf1-based gene editing system coding sequence. The start and stop codons can be in-frame with the CRISPR / Cpf1-based gene editing system coding sequence. The vector can also contain a promoter operably linked to the CRISPR / Cpf1-based gene editing system coding sequence. The promoter operably linked to the CRISPR / Cpf1-based gene editing system coding sequence can be a promoter derived from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter, Epstein-Barr virus (EBV) promoter, U6 promoter, such as human U6 promoter, or Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter derived from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue-specific promoter, such as a natural or synthetic, muscle or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication Nos. 20040175727 and 20040192593, the contents of which are hereby incorporated by reference in their entirety.Examples of muscle-specific promoters include the Spc5-12 promoter (described in US Patent Application Publication No. 20040192593, which is hereby incorporated by reference in its entirety; Hakim et al. Mol. Ther. Methods Clin. Dev. (2014) 1:14002; and Lai et al. Hum Mol Genet. (2014) 23(12):3189-3199), the MHCK7 promoter (described in Salva et al., Mol. Ther. (2007) 15:320-329), the CK8 promoter (described in Park et al. PLoS ONE (2015) 10(4):e0124914), and the CK8e promoter (described in Muir et al., Mol. Ther. Methods Clin. Dev. (2014) 1:14025). In some embodiments, the expression of the gRNA and / or Cpf1 endonuclease is driven by a tRNA.

[0131] Each of the polynucleotide sequences encoding the Cpf1 gRNA and / or Cpf1 endonuclease can be operably linked to a promoter. The promoter operably linked to the Cpf1 gRNA and / or Cpf1 endonuclease may be the same promoter. The promoter operably linked to the Cpf1 gRNA and / or Cpf1 endonuclease may be different promoters. This promoter can be a constitutive promoter, an inducible promoter, a repressive promoter or a regulatory promoter.

[0132] The vector can also contain a polyadenylation signal that can be downstream of a CRISPR / Cpf1-based gene editing system. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal can be a polyadenylation signal derived from the pCEP4 vector (Invitrogen, San Diego, CA).

[0133] The vector can also contain a CRISPR / Cpf1-based gene editing system, i.e., a Cpf1 endonuclease coding sequence, an enhancer upstream of the Cpf1 gRNA, or a CRISPR / Cpf1-based gene editing system. The enhancer can be essential for DNA expression. The enhancer can be derived from human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as CMV, HA, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patent No. 5,593,972, U.S. Patent No. 5,962,428, and International Publication No. 94 / 016737, the contents of each of which are incorporated by reference in their entirety. The vector can also contain a mammalian origin of replication to maintain the vector episomally and can produce multiple copies of the vector in a cell. The vector can also contain regulatory sequences that can be adapted to be sufficient for gene expression in mammalian or human cells to which the vector is administered. The vector can also contain a reporter gene such as green fluorescent protein (“GFP”) and / or a selectable marker such as hygromycin (“Hygro”).

[0134] The vector can be an expression vector or system for producing proteins by conventional techniques and readily available starting materials (incorporated in its entirety by reference, Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). In some embodiments, the vector can contain a nucleic acid sequence encoding a CRISPR / Cpf1-based gene editing system, for example, a nucleic acid sequence encoding a Cpf1 endonuclease, and a nucleic acid sequence encoding at least one Cpf1 gRNA containing at least one nucleic acid sequence of SEQ ID NOs: 36-119 or its complement.

[0135] 11. Pharmaceutical Composition The subject matter of the present disclosure provides a composition containing the gene construct described above. The pharmaceutical composition according to the present invention can be formulated according to the mode of administration used. When the pharmaceutical composition is an injectable pharmaceutical composition, the pharmaceutical composition is sterile, pyrogen-free, and particulate-free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution (e.g., phosphate buffered saline) is preferred. Gelatin and albumin can be mentioned as stabilizers. In some embodiments, a vasoconstrictor is added to this formulation.

[0136] The composition can further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient can be a gene delivery promoter (which can include surfactants), such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, vesicles, such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known gene delivery promoters.

[0137] The gene delivery promoter is a polyanion, polycation (including poly-L-glutamic acid (LGS)), or lipid. The gene delivery promoter is poly-L-glutamic acid, and more preferably, poly-L-glutamic acid is present at a concentration of less than 6 mg / ml in the composition for genome editing in skeletal muscle or in cardiac muscle. The gene delivery promoter can also include surfactants, such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, and vesicles, such as squalene and squalene, and hyaluronic acid can also be used together with the gene construct. In some embodiments, the DNA vector encoding the composition can also include a gene delivery promoter, such as a lipid, liposome (including lecithin liposomes, or other liposomes known in the art), as a DNA-liposome mixture (see, for example, WO 93 / 24640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known gene delivery promoters. Preferably, the gene delivery promoter is a polyanion, polycation (including poly-L-glutamic acid (LGS)), or lipid.

[0138] 12. Method of Delivery Provided herein is a method for delivering a gene construct (e.g., vector) or composition of the present disclosure to a cell. Delivery of this composition can be transfection or electroporation of this composition as a nucleic acid molecule that is expressed in the cell and delivered to the surface of the cell. This nucleic acid molecule can be electroporated using a BioRad Gene Pulser Xcell device or an Amaxa Nucleofector IIb device. Several different buffers can be used, such as BioRad electroporation solution, Sigma phosphate buffered saline product number D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfection can include a transfection reagent such as Lipofectamine 2000.

[0139] When a gene construct or composition of the present disclosure is delivered to a tissue and as a result a vector is delivered into mammalian cells, the transfected cells express a Cpf1 gRNA and a Cpf1 endonuclease. This gene construct or composition can be administered to a mammal to alter gene expression or to re-edit or alter the genome. For example, this gene construct or composition can be administered to a mammal to correct the dystrophin gene in the mammal. The mammal can be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, guinea pig, bovid, deer, hamster, elephant, llama, alpaca, mouse, rat, or chicken, and preferably can be a human, cow, pig, or chicken.

[0140] A gene construct (e.g., a vector) encoding Cpf1 gRNA and Cpf1 endonuclease can be delivered to mammals by DNA injection (also referred to as DNA vaccination) with and without electroporation in vivo, liposome-mediated, nanoparticle-facilitated, and / or by a recombinant vector. This recombinant vector can be delivered by any viral type. This viral type can be a recombinant lentivirus, a recombinant adenovirus, and / or a recombinant adeno-associated virus.

[0141] The gene construct (e.g., a vector) of the present disclosure or a composition containing this gene construct can be introduced into cells to genetically modify the dystrophin gene (e.g., the human dystrophin gene). In certain embodiments, the gene construct (e.g., a vector) of the present disclosure or a composition containing this gene construct is introduced into myoblasts derived from DMD patients. In certain embodiments, the gene construct (e.g., a vector) or a composition containing this gene construct is introduced into fibroblasts derived from DMD patients, and the genetically modified fibroblasts can be treated with MyoD to induce differentiation into myoblasts, and these myoblasts can be transplanted into a subject (e.g., the damaged muscle of the subject) to verify that the modified dystrophin protein functions and / or to treat the subject. These modified cells can also be stem cells (e.g., induced pluripotent stem cells), bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts derived from DMD patients, CD133 + cells, mesoangioblasts, and MyoD-transduced cells or Pax7-transduced cells, or other myogenic progenitor cells. For example, a gene editing system based on CRISPR / Cpf1 can cause neuronal differentiation or myogenic differentiation of induced pluripotent stem cells.

[0142] 13. Route of Administration The gene construct (e.g., vector) of the present disclosure or a composition comprising this gene construct can be administered to a subject by various routes (e.g., oral, parenteral, sublingual, transdermal, intrarectal, transmucosal, topical, via inhalation, via buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof). In certain embodiments, the gene construct (e.g., vector) or composition of the present disclosure is administered intramuscularly, intravenously, or in combination thereof to a subject (e.g., a subject suffering from DMD). For veterinary use, the gene construct (e.g., vector) or composition of the present disclosure can be administered as a suitably acceptable formulation according to normal veterinary practice. A veterinarian can readily determine the most suitable dosing regimen and route of administration for a particular animal. This composition can be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gone gun", or other physical methods (e.g., electroporation ("EP"), "hydrodynamic methods" or ultrasound).

[0143] The gene construct (e.g., vector) or composition of the present disclosure can be delivered to mammals by several techniques (e.g., DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome-mediated, nanoparticle-facilitated, recombinant vectors (e.g., recombinant lentivirus, recombinant adenovirus and recombinant adeno-associated virus)). This composition can be injected into skeletal muscle or cardiac muscle. For example, this composition can be injected into the tibialis anterior muscle or the tail.

[0144] In some embodiments, the gene construct (e.g., vector) of the present disclosure or a composition comprising this gene construct is administered 1) by tail vein injection (systemic) into adult mice, 2) by intramuscular injection, e.g., by local injection into muscle (e.g., TA or gastrocnemius muscle) in adult mice, 3) by intraperitoneal injection into P2 mice, or 4) by facial vein injection (systemic) into P2 mice.

[0145] 14. Cell type Any of these delivery methods and / or routes of delivery can be utilized with numerous cell types (e.g., cell types currently under investigation for cell-based therapies for DMD), including but not limited to: immortalized myoblasts, such as wild-type and DMD patient-derived strains, e.g., Δ48-50 DMD, DMD6594 (del48-50), DMD8036 (del48-50), C25C14, and DMD-7796 cell lines, primary DMD skin fibroblasts, induced pluripotent stem cells, bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts from DMD patients, CD133 + cells, mesodermal angioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD-transduced or Pax7-transduced cells, or other myogenic progenitor cells. Immortalization of human myogenic cells can be used to derive clones of genetically modified myogenic cells. Cells can be modified ex vivo to isolate and expand a clonal population of immortalized DMD myoblasts that contain the genetically modified dystrophin gene and have no other nuclease-introduced mutations in the protein-coding regions of the genome. Alternatively, in vivo transient delivery of a CRISPR / Cpf1-based system by non-viral or non-integrating viral gene transfer or by direct delivery of gRNA containing purified proteins and cell-penetrating motifs can enable highly specific in situ modification with minimal or no risk of exogenous DNA integration.

[0146] 15. Kit Provided herein is a kit that can be used to correct a mutant dystrophin gene. This kit includes at least a Cpf1 gRNA for correcting the mutant dystrophin gene and instructions for using a CRISPR / Cpf1-based gene editing system. Also provided herein is a kit that can be used for genome editing of the dystrophin gene in skeletal muscle or in cardiac muscle. This kit includes a gene construct (e.g., a vector) for genome editing in skeletal muscle or in cardiac muscle as described above or a composition comprising this gene construct, and instructions for using said composition.

[0147] The instructions included in the kit can be attached to the packaging material or included as a package insert. The instructions are generally, but not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions.

[0148] A gene construct (e.g., a vector) for correcting mutant dystrophin or for genome editing of the dystrophin gene in skeletal muscle or in cardiac muscle, or a composition comprising this gene construct, can include a modified vector comprising the Cpf1 gRNA and Cpf1 endonuclease described above that specifically binds to and cleaves a region of the dystrophin gene. To specifically bind to and target a specific region in the mutant dystrophin gene, a CRISPR / Cpf1-based gene editing system as described above can be included in the kit. This kit can further include the donor DNA, another gRNA, or a transgene described above.

[0149] The kit may further optionally include one or more components necessary for the use of the disclosed composition or for facilitating quality control assessment, such as standards, buffers, diluents, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components such as buffers and solutions for cell isolation and / or processing can be included in the kit. The kit can further include one or more controls. One or more components of the kit can be lyophilized, in which case the kit may further include reagents suitable for reconstituting the lyophilized components.

Examples

[0150] 16. Examples Other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and recognizable, and can be made using appropriate equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein, which will be readily apparent to those skilled in the art. Although the present disclosure has been described in detail heretofore, it will be more clearly understood by reference to the following examples. These examples are merely intended to illustrate some aspects and embodiments of the present disclosure and should not be regarded as limiting the scope of the present disclosure. The disclosures of all academic journal references, U.S. patents and publications mentioned herein are hereby incorporated by reference in their entirety.

[0151] The present invention has multiple aspects and is illustrated by the following non-limiting examples.

[0152] Example 1 Guide RNA Design and Material Preparation Cpf1 derived from the genus Acidaminococcus was obtained from the Addgene non-profit plasmid repository (pY010 (pcDNA3.1-hAsCpf1; "AsCPF1 plasmid") from Feng Zhang (Addgene plasmid #69982)). The AsCPF1 plasmid was transformed into chemically competent Escherichia coli (E. coli), amplified, and then the sequence was confirmed. The Cpf1 guide RNA (also known as Cpf1 crRNA) was designed to target splice sites on dominant exon mutations in dystrophin and the enhancer of BCL11a using the University of California Santa Cruz Genome Browser program, ordered as oligomers from Integrated DNA Technologies (IDT), prepared by PCR, and then column purified as previously described (Zetsche et al., Cell 163(3):759-71 (2015)).

[0153] Confirmation of guide RNA. Transfection of HEK293 cells (ATCC) was performed using Lipofectamine 2000 in a 24-well plate according to the manufacturer's recommendations. Each well received 400 ng of the AsCPF1 plasmid and 100 ng of the U6::sgRNA PCR product. After 72 hours, the cells were isolated and genomic DNA was purified using a DNeasy column (QIAGEN). As previously described (Ousterout et al., Nature Communications 6:6244 (2015); Guschin et al., Methods Mol. Biol. 649:247-256 (2010)), Surveyor nuclease digestion (IDT) and deletion PCR were performed using primers flanking the genomic region of interest. The digested PCR products were electrophoresed in a TBE gel (Invitrogen) at 200 V for 30 minutes, stained with ethidium bromide (EtBr), and then imaged on a Gel Doc™ (Biorad). The deletion PCR products were electrophoresed in a 1% agarose gel at 120 V for 30 minutes, stained with EtBr, and then imaged on a Gel Doc™ (Biorad).

[0154] Example 2 Dystrophin splice acceptor guide RNA By targeting cleavage regions as close as possible to the splice acceptor, 15 guide RNAs targeting the top highly mutated dystrophin exons were designed, which was achieved by the presence of available PAMs (Table 1). If possible, multiple guide RNAs were made to target the same splice acceptor. Candidate guide RNAs were screened in vitro. Guide RNAs that showed immediate positive results included those targeting exon 44, exon 46, and exon 51 (Figures 2A - 2C). Surveyor nuclease digestion was detected in guide RNAs targeting the exon 44 splice acceptor (Figure 2A), the exon 46 splice acceptor (Figure 2B), and the 3’ end of exon 51 (Figure 2C). Figure 2D revealed that gene deletions could be caused using guide RNAs targeting the splice acceptor of exon 51 and the 3’ end of exon 51, indicating the activity of the guide RNA targeting exon 51.

[0155] Table 1 shows the design of guide RNAs targeting dystrophin exons. The PAM sequence (TTTN) is underlined. Sense guide RNAs have TTTN at the 5’ end. Antisense strand guide RNAs have NAAA PAM at the 3’ end.

[0156]

Table 1

[0157] Example 3 Exon 51 Compatible Overhang Deletion To determine whether guide RNAs with compatible overhang arrays promote seamless deletions, six guide RNAs were designed within intron 50 and seven guide RNAs were designed within intron 51 (Table 2), resulting in compatible overhang deletions. Forty-two unique gRNA pairs (6×7) were tested and screened for deletion activity, i.e., targeting exon 51 deletion. This set included three overhang-compatible pairs (see Table 2). Seven pairs were confirmed for activity. Figure 3 is a representative image showing smaller bands indicating deletion of exon 51. These results demonstrate disruption of the first Cpf1 target splice acceptor of the dystrophin gene and deletion of exon 51.

[0158]

Table 2

[0159] Example 4 Targeted deletion of exon 51 in patient-derived myoblasts Patient-derived myoblasts with deletions of exons 48 - 50 (Δ48 - 50) were cultured in skeletal muscle amplification medium. Electroporation was performed according to standard laboratory procedures. Cells were cultured for 3 days and evaluated for protein expression in patient-derived myoblasts (Figure 4) and genomic deletions induced by SaCas9 (Cas9 from Staphylococcus aureus) or LbCpf1 (CPF1 from Lachnospiraceae bacterium ND2006) (Figure 5). Figure 4 demonstrates that Western blots of HA-tagged SaCas9 and LbCpf1 show expression in extracted proteins 72 hours after plasmid transfection. Figure 5 demonstrates that PCR across the target genomic region shows smaller bands in bulk-treated myoblasts with SaCas9 gRNA or Cpf1 crRNA, which is consistent with removal of exon 51 and surrounding intronic regions.

[0160] Next, after differentiating myoblasts, dystrophin transcript expression and exon 51 deletion were evaluated (Figure 6). Figure 6 demonstrates that differentiated myoblasts expressed dystrophin transcripts lacking exon 51, as indicated by the small bands generated by RT-PCR, thus showing that SaCas9 or LbCpf1 targeting exon 51 removed the exon 51 exon from the transcript.

[0161] A large population of Cpf1 crRNAs was evaluated in HEK293 cells (Figure 7; see Table 3 for Cpf1 crRNA sequences). All Cpf1 crRNAs targeting exon 51 or the surrounding introns used are listed in Table 3. As shown in Figure 7, HEK293 cells treated with the population of crRNAs for 3 days showed variable activities by the Surveyor® nuclease assay. Cpf1 crRNAs #38, 41, 42, 43, 45, 46, 47, 49, 54, 55, 56, 59, 63, 64, and 65 showed the highest activities indicated by short bands.

[0162]

Table 3-1

Table 3-2

[0163] Example 5 BCL11a Enhancer Targeting Potent candidates were designed to increase fetal globin levels in sickle cell anemia (SCA). For the purpose of generating potent candidates to increase fetal globin levels in sickle cell anemia (SCA), guide RNAs for Cpf1 were designed to target the BCL11a enhancer region (Table 3). These reagents were designed to disrupt the BCL11a enhancer. These reagents were tested in an SCA cell model.

[0164]

Table 4

[0165] The foregoing detailed description and the accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0166] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Without limitation, such changes and modifications, including those related to the chemical structure, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

[0167] For the sake of completeness, various aspects of the invention are presented in the following numbered clauses.

[0168] Clause 1. A Cpf1 guide RNA (gRNA) that targets the dystrophin gene and contains a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0169] Clause 2. A DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA according to Clause 1.

[0170] Clause 3. A DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA, wherein each of the first Cpf1 gRNA and the second Cpf1 gRNA contains a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, where the first Cpf1 gRNA and the second Cpf1 gRNA contain different polynucleotide sequences, and the first Cpf1 gRNA and the second Cpf1 gRNA target the dystrophin gene.

[0171] Item 4. The DNA targeting composition according to Item 3, wherein the first Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, and the second Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 61.

[0172] Item 5. The DNA targeting composition according to Item 3 or 4, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0173] Item 6. The DNA targeting composition according to any one of Items 3 to 5, further comprising a Cpf1 endonuclease.

[0174] Item 7. The DNA targeting composition according to Item 2 or 6, wherein the Cpf1 endonuclease recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0175] Item 8. The DNA targeting composition according to Item 7, wherein the Cpf1 endonuclease is derived from a bacterial species selected from the group consisting of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae.

[0176] Item 9. The DNA targeting composition according to any one of Items 6 to 8, wherein the Cpf1 endonuclease is derived from Lachnospiraceae bacterium ND2006 (LbCpf1) or Acidaminococcus (AsCpf1).

[0177] Item 10. The DNA targeting composition according to any one of Items 6 to 9, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence containing SEQ ID NO: 124 or SEQ ID NO: 125.

[0178] Item 11. An isolated polynucleotide containing a polynucleotide sequence encoding the Cpf1 gRNA according to Item 1 or the DNA targeting composition according to any one of Items 2 to 10.

[0179] Item 12. A vector containing the Cpf1 gRNA according to Item 1, a polynucleotide sequence encoding the DNA targeting composition according to any one of Items 2 to 10, or the isolated polynucleotide according to Item 10.

[0180] Item 13. The vector according to Item 12, further containing a polynucleotide sequence encoding a Cpf1 endonuclease.

[0181] Item 14. A vector encoding (a) a first Cpf1 guide RNA (gRNA), (b) a second Cpf1 gRNA, and (c) at least one Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA contain a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36 to 64, 71 to 119, or a complement thereof, and the first Cpf1 gRNA and the second Cpf1 gRNA contain different polynucleotide sequences.

[0182] Item 15. The vector according to item 14, wherein the vector is configured to form first and second double strands in first and second introns adjacent to exon 51 of the human DMD gene.

[0183] Item 16. The vector according to item 14 or 15, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0184] Item 17. The vector according to any one of items 12 to 16, wherein the vector is a viral vector.

[0185] Item 18. The vector according to item 17, wherein the vector is an adeno-associated virus (AAV) vector.

[0186] Item 19. The vector according to any one of items 12 to 18, wherein the vector comprises a tissue-specific promoter operably linked to the polynucleotide sequence encoding the first Cpf1 gRNA, the second Cpf1 gRNA, and / or the Cpf1 endonuclease.

[0187] Item 20. The vector according to item 19, wherein the tissue-specific promoter is a muscle-specific promoter.

[0188] Item 21. A cell comprising the Cpf1 gRNA according to claim 1, a polynucleotide sequence encoding the DNA targeting composition according to any one of claims 2 to 10, the isolated polynucleotide according to claim 11, or the vector according to any one of claims 12 to 20.

[0189] Item 22. A kit comprising the Cpf1 gRNA according to claim 1, a polynucleotide sequence encoding the DNA targeting composition according to any one of claims 2 to 10, the isolated polynucleotide according to claim 11, the vector according to any one of claims 12 to 20, or the cell according to claim 21.

[0190] Item 23. A composition for deleting a segment containing exon 51 in the dystrophin gene, the composition comprising: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36 to 64, 71 to 119, or a complement thereof, the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first vector and the second vector are each configured to form first and second double-strand breaks in the first intron and the second intron adjacent to exon 51 of the human DMD gene, whereby a segment containing exon 51 in the dystrophin gene is deleted.

[0191] Item 24. The composition according to item 23, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0192] Item 25. The composition according to item 23 or 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are the same.

[0193] Item 26. The composition according to item 23 or 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are different.

[0194] Item 27. The composition according to item 25 or 26, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is a CPF1 endonuclease derived from Lachnospiraceae bacterium ND2006 (LbCpf1) and / or Acidaminococcus (AsCpf1).

[0195] Item 28. The composition according to any one of items 25 to 27, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO: 124 or SEQ ID NO: 125.

[0196] Item 29. The composition according to any one of Items 23 to 28, wherein the first vector and / or the second vector is a viral vector.

[0197] Item 30. The composition according to Item 29, wherein the first vector and / or the second vector is an adeno-associated virus (AAV) vector.

[0198] Item 31. The composition according to Item 30, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

[0199] Item 32. The composition according to any one of Items 23 to 31, wherein the dystrophin gene is a human dystrophin gene.

[0200] Item 33. The composition according to any one of Items 23 to 32, for use as a medicament.

[0201] Item 34. The composition according to any one of Items 23 to 32, for use in the treatment of Duchenne muscular dystrophy.

[0202] Item 35. A cell comprising the composition according to any one of Items 23 to 34.

[0203] Item 36. A modified adeno-associated virus vector for genome editing to edit a mutant dystrophin gene in a subject, the modified adeno-associated virus vector comprising a first polynucleotide sequence encoding the Cpf1 gRNA according to Item 1 and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0204] Item 37. A method for correcting a mutant dystrophin gene in a cell, the method comprising administering to the cell the Cpf1 gRNA according to Item 1, a polynucleotide sequence encoding the DNA targeting composition according to any one of Items 2 to 10, the isolated polynucleotide according to Item 11, the vector according to any one of Items 12 to 20, the composition according to any one of Items 23 to 34, or the modified adeno-associated virus vector according to Item 36.

[0205] Item 38. The method according to Item 37, wherein the correction of the mutant dystrophin gene involves nuclease-mediated non-homologous end joining or homologous recombination repair.

[0206] Item 39. A method for genome editing of a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising the Cpf1 gRNA according to Item 1, a polynucleotide sequence encoding the DNA targeting composition according to any one of Items 2 to 10, the isolated polynucleotide according to Item 11, the vector according to any one of Items 12 to 20, the composition according to any one of Items 23 to 34, or the modified adeno-associated virus vector according to Item 36.

[0207] Item 40. The method according to Item 39, wherein the genome editing composition is administered to the subject intramuscularly, intravenously, or a combination thereof.

[0208] Item 41. The method according to Item 39 or 40, wherein the genome editing involves nuclease-mediated non-homologous end joining or homologous recombination repair.

[0209] Item 42. A method of treating a subject in need thereof, having a mutant dystrophin gene, the method comprising administering to the subject a Cpf1 gRNA according to claim 1, a polynucleotide sequence encoding a DNA targeting composition according to any one of claims 2-10, an isolated polynucleotide according to claim 11, a vector according to any one of claims 12-20, a composition according to any one of claims 23-34, or a modified adeno-associated virus vector according to claim 36.

[0210] Item 43. A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to the cell: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the vectors are each configured to form first and second double-strand breaks in the first and second introns adjacent to exon 51 of the human dystrophin gene, whereby a segment containing exon 51 in the dystrophin gene is deleted, thereby correcting the mutant dystrophin in the cell.

[0211] Item 44. The method according to item 43, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0212] Item 45. The method according to item 43 or 44, wherein the mutant dystrophin gene comprises a premature stop codon, a disrupted reading frame, an ectopic splice acceptor site, or an ectopic splice donor site.

[0213] Item 46. The method according to item 45, wherein the mutant dystrophin gene comprises a premature stop codon and a frameshift mutation that results in a truncated gene product.

[0214] Item 47. The method according to item 43 or 44, wherein the mutant dystrophin gene comprises a deletion of one or more exons that disrupts the reading frame.

[0215] Item 48. The method according to any one of items 43 to 47, wherein the correction of the mutant dystrophin gene comprises deletion of a premature stop codon, correction of a disrupted reading frame, or regulation of splicing by disruption of a splice acceptor site or disruption of a splice donor sequence.

[0216] Item 49. The method according to item 48, wherein the correction of the mutant dystrophin gene comprises deletion of exon 51.

[0217] Item 50. The method according to any one of Items 43 to 49, wherein the correction of the mutant dystrophin gene includes nuclease-mediated non-homologous end joining or homologous recombination repair.

[0218] Item 51. The method according to any one of Items 43 to 50, wherein the cell is a myoblast.

[0219] Item 52. The method according to any one of Items 43 to 51, wherein the cell is derived from a subject suffering from Duchenne muscular dystrophy.

[0220] Item 53. A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36 to 64, 71 to 119, or a complement thereof, and the first vector and the second vector are each configured to form first and second double-strand breaks in the first and second introns adjacent to exon 51 of the human dystrophin gene, whereby a segment containing exon 51 is deleted in the dystrophin gene to treat the subject.

[0221] Item 54. The method according to item 53, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56 and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0222] Item 55. The method according to any one of items 53 or 54, wherein the subject has Duchenne muscular dystrophy.

[0223] Item 56. The method according to any one of items 53 to 55, wherein the first vector and the second vector are administered to the subject intramuscularly, intravenously, or in combination thereof.

[0224] Item 57. A Cpf1 guide RNA (gRNA) that targets an enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 65 to 70, or a complement thereof.

[0225] Item 58. A method for disrupting an enhancer of the B-cell lymphoma / leukemia 11A gene in a cell, the method comprising administering to the cell at least one Cpf1 gRNA and a Cpf1 endonuclease according to item 57.

Claims

1. A Cpf1 guide RNA (gRNA) that targets the dystrophin gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

2. A DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA described in claim 1.

3. A DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA, wherein the first Cpf1 gRNA and the second Cpf1 gRNA each comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and wherein the first Cpf1 gRNA and the second Cpf1 gRNA target a dystrophin gene.

4. The DNA targeting composition of claim 3, wherein the first Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56, and the second Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:

61.

5. The first Cpf1 gRNA and the second Cpf1 gRNA are (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:

61. The DNA targeting composition of claim 3 or 4, selected from the group consisting of:

6. The DNA targeting composition of any one of claims 3 to 5, further comprising Cpf1 endonuclease.

7. The DNA targeting composition of claim 2 or 6, wherein the Cpf1 endonuclease recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

8. The Cpf1 endonuclease is capable of inhibiting the activity of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albenzis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium MC2017 2, bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens 10. The DNA targeting composition of claim 7, derived from a bacterial species selected from the group consisting of: Leptospira elegens, Moraxella bovoculi 237, Leptospira inadais, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae.

9. 9. The DNA targeting composition of any one of claims 6 to 8, wherein the Cpf1 endonuclease is derived from Lachnospiraceae bacterium ND2006 (LbCpf1) or Acidaminococcus (AsCpf1).

10. The DNA targeting composition of any one of claims 6 to 9, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO:124 or SEQ ID NO:

125.

11. 11. An isolated polynucleotide comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1 or the DNA targeting composition of any one of claims 2 to 10.

12. A vector comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, a DNA targeting composition of any one of claims 2 to 10, or an isolated polynucleotide of claim 10.

13. The vector of claim 12 , further comprising a polynucleotide sequence encoding a Cpf1 endonuclease.

14. below: (a) a first Cpf1 guide RNA (gRNA); (b) a second Cpf1 gRNA, and (c) at least one Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). A vector encoding A vector, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences.

15. 15. The vector of claim 14, wherein the vector is configured to form first and second duplexes in first and second introns flanking exon 51 of the human DMD gene.

16. The first Cpf1 gRNA and the second Cpf1 gRNA are (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:

61.

16. The vector of claim 14 or 15, selected from the group consisting of:

17. The vector according to any one of claims 12 to 16, wherein the vector is a viral vector.

18. The vector of claim 17, wherein the vector is an adeno-associated virus (AAV) vector.

19. The vector of any one of claims 12 to 18, wherein the vector comprises a tissue-specific promoter operably linked to a polynucleotide sequence encoding the first Cpf1 gRNA, the second Cpf1 gRNA, and / or the Cpf1 endonuclease.

20. 20. The vector of claim 19, wherein the tissue-specific promoter is a muscle-specific promoter.

21. A cell comprising the Cpf1 gRNA of claim 1, a polynucleotide sequence encoding the DNA targeting composition of any one of claims 2 to 10, an isolated polynucleotide of claim 11, or a vector of any one of claims 12 to 20.

22. A kit comprising the Cpf1 gRNA of claim 1, a polynucleotide sequence encoding the DNA targeting composition of any one of claims 2 to 10, an isolated polynucleotide of claim 11, a vector of any one of claims 12 to 20, or a cell of claim 21.

23. 1. A composition for deleting a segment containing exon 51 in a dystrophin gene, comprising: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). Including, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first vector and the second vector are configured to form first and second double-stranded breaks in a first intron and a second intron, respectively, adjacent to exon 51 of the human DMD gene, thereby deleting a segment including exon 51 in the dystrophin gene.

24. The first Cpf1 gRNA and the second Cpf1 gRNA are (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:

61.

24. The composition of claim 23, selected from the group consisting of:

25. 25. The composition of claim 23 or 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are the same.

26. 25. The composition of claim 23 or 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are different.

27. 27. The composition of claim 25 or 26, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is a CPF1 endonuclease from Lachnospiraceae bacterium ND2006 (LbCpf1) and / or Acidaminococcus (AsCpf1).

28. 28. The composition of any one of claims 25 to 27, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO:124 or SEQ ID NO:

125.

29. The composition according to any one of claims 23 to 28, wherein the first vector and / or the second vector is a viral vector.

30. 30. The composition of claim 29, wherein the first vector and / or the second vector is an adeno-associated virus (AAV) vector.

31. The composition of claim 30, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

32. The composition according to any one of claims 23 to 31, wherein the dystrophin gene is a human dystrophin gene.

33. A composition according to any one of claims 23 to 32 for use in medicine.

34. A composition according to any one of claims 23 to 32 for use in the treatment of Duchenne muscular dystrophy.

35. A cell comprising the composition according to any one of claims 23 to 34.

36. A modified adeno-associated virus vector for genome editing of a mutant dystrophin gene in a subject, comprising a first polynucleotide sequence encoding the Cpf1 gRNA of claim 1 and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

37. 13. A method of correcting a mutated dystrophin gene in a cell, the method comprising administering to the cell a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, a DNA targeting composition of any one of claims 2-10, an isolated polynucleotide of claim 11, a vector of any one of claims 12-20, a composition of any one of claims 23-34, or a modified adeno-associated viral vector of claim 36.

38. 38. The method of claim 37, wherein the correction of the mutant dystrophin gene comprises nuclease-mediated non-homologous end joining or homology directed repair.

39. 13. A method of genome editing a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, a DNA targeting composition of any one of claims 2-10, an isolated polynucleotide of claim 11, a vector of any one of claims 12-20, a composition of any one of claims 23-34, or a modified adeno-associated viral vector of claim 36.

40. 40. The method of Claim 39, wherein the genome editing composition is administered to the subject intramuscularly, intravenously, or a combination thereof.

41. 41. The method of claim 39 or 40, wherein the genome editing comprises nuclease-mediated non-homologous end joining or homology directed repair.

42. 13. A method of treating a subject in need thereof having a mutated dystrophin gene, said method comprising administering to said subject a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, a DNA targeting composition of any one of claims 2-10, an isolated polynucleotide of claim 11, a vector of any one of claims 12-20, a composition of any one of claims 23-34, or a modified adeno-associated viral vector of claim 36.

43. 1. A method for correcting a mutant dystrophin gene in a cell, comprising administering to said cell: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). administering wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the vector is configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene and correcting the mutant dystrophin in a cell.

44. The first Cpf1 gRNA and the second Cpf1 gRNA are (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:

61.

44. The method of claim 43, selected from the group consisting of:

45. 45. The method of claim 43 or 44, wherein the mutated dystrophin gene comprises a premature stop codon, a disrupted reading frame, an ectopic splice acceptor site, or an ectopic splice donor site.

46. 46. ​​The method of claim 45, wherein the mutated dystrophin gene contains a premature stop codon and a frameshift mutation resulting in a truncated gene product.

47. 45. The method of claim 43 or 44, wherein the mutant dystrophin gene comprises a deletion of one or more exons that disrupts the reading frame.

48. 48. The method of any one of claims 43 to 47, wherein the correction of the mutant dystrophin gene comprises deleting a premature stop codon, correcting a disrupted reading frame, or modulating splicing by disrupting a splice acceptor site or disrupting a splice donor sequence.

49. 49. The method of claim 48, wherein the mutant dystrophin gene comprises a deletion of exon 51.

50. 50. The method of any one of claims 43 to 49, wherein the correction of the mutant dystrophin gene comprises nuclease-mediated non-homologous end joining or homology directed repair.

51. The method of any one of claims 43 to 50, wherein the cells are myoblasts.

52. The method of any one of claims 43 to 51, wherein the cells are derived from a subject suffering from Duchenne muscular dystrophy.

53. 1. A method of treating a subject in need thereof having a mutated dystrophin gene, the method comprising administering to the subject: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). administering wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first vector and the second vector are configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene to treat the subject.

54. The first Cpf1 gRNA and the second Cpf1 gRNA are (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO:

61.

54. The method of claim 53, selected from the group consisting of:

55. 55. The method of claim 53 or 54, wherein the subject is afflicted with Duchenne muscular dystrophy.

56. 56. The method of any one of claims 53-55, wherein the first and second vectors are administered to the subject intramuscularly, intravenously, or a combination thereof.

57. A Cpf1 guide RNA (gRNA) that targets the enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 65-70, or a complement thereof.

58. A method for disrupting an enhancer of the B cell lymphoma / leukemia 11A gene in a cell, the method comprising administering to the cell at least one Cpf1 gRNA and Cpf1 endonuclease described in claim 57.

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